Sensitive detection of alkaline phosphatase by switching on gold nanoclusters fluorescence quenched by pyridoxal phosphate.

PubMed

Halawa, Mohamed Ibrahim; Gao, Wenyue; Saqib, Muhammad; Kitte, Shimeles Addisu; Wu, Fengxia; Xu, Guobao

2017-09-15

In this work, we designed highly sensitive and selective luminescent detection method for alkaline phosphatase using bovine serum albumin functionalized gold nanoclusters (BSA-AuNCs) as the nanosensor probe and pyridoxal phosphate as the substrate of alkaline phosphatase. We found that pyridoxal phosphate can quench the fluorescence of BSA-AuNCs and pyridoxal has little effect on the fluorescence of BSA-AuNCs. The proposed mechanism of fluorescence quenching by PLP was explored on the basis of data obtained from high-resolution transmission electron microscopy (HRTEM), dynamic light scattering (DLS), UV-vis spectrophotometry, fluorescence spectroscopy, fluorescence decay time measurements and circular dichroism (CD) spectroscopy. Alkaline phosphatase catalyzes the hydrolysis of pyridoxal phosphate to generate pyridoxal, restoring the fluorescence of BSA-AuNCs. Therefore, a recovery type approach has been developed for the sensitive detection of alkaline phosphatase in the range of 1.0-200.0U/L (R 2 =0.995) with a detection limit of 0.05U/L. The proposed sensor exhibit excellent selectivity among various enzymes, such as glucose oxidase, lysozyme, trypsin, papain, and pepsin. The present switch-on fluorescence sensing strategy for alkaline phosphatase was successfully applied in human serum plasma with good recoveries (100.60-104.46%), revealing that this nanosensor probe is a promising tool for ALP detection. Copyright © 2017 Elsevier B.V. All rights reserved.

Quality and stability of extemporaneous pyridoxal phosphate preparations used in the treatment of paediatric epilepsy.

PubMed

Mohamed-Ahmed, Abeer H A; Wilson, Matthew P; Albuera, Maedelyn; Chen, Ting; Mills, Philippa B; Footitt, Emma J; Clayton, Peter T; Tuleu, Catherine

2017-04-01

To assess the pyridoxal 5'-phosphate (PLP) content and stability of extemporaneous PLP liquids prepared from dietary supplements used for the treatment of vitamin B 6 -dependent epilepsy. Pyridoxal 5'-phosphate liquids were prepared in accordance with the guidelines given to patients from marketed 50 mg PLP dietary capsules and tablets. The PLP content and its stability were evaluated under conditions resembling the clinical setting using reverse phase HPLC and mass spectrometry. Pyridoxal 5'-phosphate content in most of the extemporaneously prepared liquids from dietary supplements was found to be different from the expected amount (~16-60 mg). Most of these PLP extemporaneous liquids were stable at room temperature (protected from light) after 24 h but unstable after 4 h when exposed to light. A key photodegradation product of PLP in water was confirmed as 4-pyridoxic acid 5'-phosphate (PAP). Pyridoxal 5'-phosphate tablets from Solgar ® were found to be the most reliable product for the preparation of extemporaneous PLP liquids. This work highlighted the difference between the marketed PLP dietary supplements quality and the importance of proper storage of aqueous PLP. There is a need to develop pharmaceutical forms of PLP that ensure dose accuracy and avoid potentially unsafe impurities with the aim of enhancing safety and compliance. © 2017 Royal Pharmaceutical Society.

Biosynthesis of putrescine in the prostate gland of the rat

PubMed Central

Pegg, A. E.; Williams-Ashman, H. G.

1968-01-01

In the rat ventral prostate gland the biosynthesis of putrescine, a precursor of spermidine and spermine, is shown to occur by the direct decarboxylation of l-ornithine. Some properties of a soluble pyridoxal phosphate-dependent l-ornithine decarboxylase are described. The findings are discussed in relation to other enzymic reactions involved in the biosynthesis of polyamines by the prostate gland. PMID:5667265

Structural studies on the decameric S. typhimurium arginine decarboxylase (ADC): Pyridoxal 5'-phosphate binding induces conformational changes.

PubMed

Deka, G; Bharath, S R; Savithri, H S; Murthy, M R N

2017-09-02

Enteric pathogens such as Salmonella typhimurium colonize the human gut in spite of the lethal acidic pH environment (pH < 2.5) due to the activation of inducible acid tolerance response (ATR) systems. The pyridoxal 5'-phosphate (PLP)-dependent enzyme, biodegradative arginine decarboxylase (ADC, encoded by AdiA), is a component of an ATR system. The enzyme consumes a cytoplasmic proton in the process of arginine degradation to agmatine. Arginine-agmatine antiporter (AdiC) exchanges the product agmatine for arginine. In this manuscript, we describe the structure of Salmonella typhimurium ADC (StADC). The decameric structure assembled from five dimers related by a non crystallographic 5-fold symmetry represents the first apo-form of the enzyme. The structure suggests that PLP-binding is not a prerequisite for oligomerization. Comparison with E. coli ADC reveals that PLP-binding is accompanied by the movement and ordering of two loops (residues 150-159 and 191-197) and a few active site residues such as His256 and Lys257. A number of residues important for substrate binding are disordered in the apo-StADC structure indicating that PLP binding is important for substrate binding. Unlike the interactions between 5-fold related protomers, interactions that stabilize the dimeric structure are not pH dependent. Copyright © 2017 Elsevier Inc. All rights reserved.

Quantum mechanics/molecular mechanics studies on the mechanism of action of cofactor pyridoxal 5'-phosphate in ornithine 4,5-aminomutase.

PubMed

Pang, Jiayun; Scrutton, Nigel S; Sutcliffe, Michael J

2014-09-01

A computational study was performed on the experimentally elusive cyclisation step in the cofactor pyridoxal 5'-phosphate (PLP)-dependent D-ornithine 4,5-aminomutase (OAM)-catalysed reaction. Calculations using both model systems and a combined quantum mechanics/molecular mechanics approach suggest that regulation of the cyclic radical intermediate is achieved through the synergy of the intrinsic catalytic power of cofactor PLP and the active site of the enzyme. The captodative effect of PLP is balanced by an enzyme active site that controls the deprotonation of both the pyridine nitrogen atom (N1) and the Schiff-base nitrogen atom (N2). Furthermore, electrostatic interactions between the terminal carboxylate and amino groups of the substrate and Arg297 and Glu81 impose substantial "strain" energy on the orientation of the cyclic intermediate to control its trajectory. In addition the "strain" energy, which appears to be sensitive to both the number of carbon atoms in the substrate/analogue and the position of the radical intermediates, may play a key role in controlling the transition of the enzyme from the closed to the open state. Our results provide new insights into several aspects of the radical mechanism in aminomutase catalysis and broaden our understanding of cofactor PLP-dependent reactions. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Study of the hydrolysis and ionization constants of Schiff base from pyridoxal 5'-phosphate and n-hexylamine in partially aqueous solvents. An application to phosphorylase b.

PubMed Central

Donoso, J; Muñoz, F; García Del Vado, A; Echevarría, G; García Blanco, F

1986-01-01

Formation and hydrolysis rate constants as well as equilibrium constants of the Schiff base derived from pyridoxal 5'-phosphate and n-hexylamine were determined between pH 3.5 and 7.5 in ethanol/water mixtures (3:17, v/v, and 49:1, v/v). The results indicate that solvent polarity scarcely alters the values of these constants but that they are dependent on the pH. Spectrophotometric titration of this Schiff base was also carried out. We found that a pKa value of 6.1, attributed in high-polarity media to protonation of the pyridine nitrogen atom, is independent of solvent polarity, whereas the pKa of the monoprotonated form of the imine falls from 12.5 in ethanol/water (3:17) to 11.3 in ethanol/water (49:1). Fitting of the experimental results for the hydrolysis to a theoretical model indicates the existence of a group with a pKa value of 6.1 that is crucial in the variation of kinetic constant of hydrolysis with pH. Studies of the reactivity of the coenzyme (pyridoxal 5'-phosphate) of glycogen phosphorylase b with hydroxylamine show that this reaction only occurs when the pH value of solution is below 6.5 and the hydrolysis of imine bond has started. We propose that the decrease in activity of phosphorylase b when the pH value is less than 6.2 must be caused by the cleavage of enzyme-coenzyme binding and that this may be related with protonation of the pyridine nitrogen atom of pyridoxal 5'-phosphate. PMID:3099764

The chaperone role of the pyridoxal 5'-phosphate and its implications for rare diseases involving B6-dependent enzymes.

PubMed

Cellini, Barbara; Montioli, Riccardo; Oppici, Elisa; Astegno, Alessandra; Voltattorni, Carla Borri

2014-02-01

The biologically active form of the B6 vitamers is pyridoxal 5'-phosphate (PLP), which plays a coenzymatic role in several distinct enzymatic activities ranging from the synthesis, interconversion and degradation of amino acids to the replenishment of one-carbon units, synthesis and degradation of biogenic amines, synthesis of tetrapyrrolic compounds and metabolism of amino-sugars. In the catalytic process of PLP-dependent enzymes, the substrate amino acid forms a Schiff base with PLP and the electrophilicity of the PLP pyridine ring plays important roles in the subsequent catalytic steps. While the essential role of PLP in the acquisition of biological activity of many proteins is long recognized, the finding that some PLP-enzymes require the coenzyme for refolding in vitro points to an additional role of PLP as a chaperone in the folding process. Mutations in the genes encoding PLP-enzymes are causative of several rare inherited diseases. Patients affected by some of these diseases (AADC deficiency, cystathionuria, homocystinuria, gyrate atrophy, primary hyperoxaluria type 1, xanthurenic aciduria, X-linked sideroblastic anaemia) can benefit, although at different degrees, from the administration of pyridoxine, a PLP precursor. The effect of the coenzyme is not limited to mutations that affect the enzyme-coenzyme interaction, but also to those that cause folding defects, reinforcing the idea that PLP could play a chaperone role and improve the folding efficiency of misfolded variants. In this review, recent biochemical and cell biology studies highlighting the chaperoning activity of the coenzyme on folding-defective variants of PLP-enzymes associated with rare diseases are presented and discussed. Copyright © 2013 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Purification, properties, and N-terminal amino acid sequence of homogeneous Escherichia coli 2-amino-3-ketobutyrate CoA ligase, a pyridoxal phosphate-dependent enzyme.

PubMed

Mukherjee, J J; Dekker, E E

1987-10-25

Starting with 100 g (wet weight) of a mutant of Escherichia coli K-12 forced to grow on L-threonine as sole carbon source, we developed a 6-step procedure that provides 30-40 mg of homogeneous 2-amino-3-ketobutyrate CoA ligase (also called aminoacetone synthetase or synthase). This ligase, which catalyzes the cleavage/condensation reaction between 2-amino-3-ketobutyrate (the presumed product of the L-threonine dehydrogenase-catalyzed reaction) and glycine + acetyl-CoA, has an apparent molecular weight approximately equal to 85,000 and consists of two identical (or nearly identical) subunits with Mr = 42,000. Computer analysis of amino acid composition data, which gives the best fit nearest integer ratio for each residue, indicates a total of 387 amino acids/subunit with a calculated Mr = 42,093. Stepwise Edman degradation provided the N-terminal sequence of the first 21 amino acids. It is a pyridoxal phosphate-dependent enzyme since (a) several carbonyl reagents caused greater than 90% loss of activity, (b) dialysis against buffer containing hydroxylamine resulted in 89% loss of activity coincident with an 86% decrease in absorptivity at 428 nm, (c) incubation of the apoenzyme with 20 microM pyridoxal phosphate showed a parallel recovery (greater than 90%) of activity and 428-nm absorptivity, and (d) reduction of the holoenzyme with NaBH4 resulted in complete inactivation, disappearance of a new absorption maximum at 333 nm. Strict specificity for glycine is shown but acetyl-CoA (100%), n-propionyl-CoA (127%), or n-butyryl-CoA (16%) is utilized in the condensation reaction. Apparent Km values for acetyl-CoA, n-propionyl-CoA, and glycine are 59 microM, 80 microM, and 12 mM, respectively; the pH optimum = 7.5. Added divalent metal ions or sulfhydryl compounds inhibited catalysis of the condensation reaction.

Functional Evolution of PLP-dependent Enzymes based on Active-Site Structural Similarities

PubMed Central

Catazaro, Jonathan; Caprez, Adam; Guru, Ashu; Swanson, David; Powers, Robert

2014-01-01

Families of distantly related proteins typically have very low sequence identity, which hinders evolutionary analysis and functional annotation. Slowly evolving features of proteins, such as an active site, are therefore valuable for annotating putative and distantly related proteins. To date, a complete evolutionary analysis of the functional relationship of an entire enzyme family based on active-site structural similarities has not yet been undertaken. Pyridoxal-5’-phosphate (PLP) dependent enzymes are primordial enzymes that diversified in the last universal ancestor. Using the Comparison of Protein Active Site Structures (CPASS) software and database, we show that the active site structures of PLP-dependent enzymes can be used to infer evolutionary relationships based on functional similarity. The enzymes successfully clustered together based on substrate specificity, function, and three-dimensional fold. This study demonstrates the value of using active site structures for functional evolutionary analysis and the effectiveness of CPASS. PMID:24920327

Functional evolution of PLP-dependent enzymes based on active-site structural similarities.

PubMed

Catazaro, Jonathan; Caprez, Adam; Guru, Ashu; Swanson, David; Powers, Robert

2014-10-01

Families of distantly related proteins typically have very low sequence identity, which hinders evolutionary analysis and functional annotation. Slowly evolving features of proteins, such as an active site, are therefore valuable for annotating putative and distantly related proteins. To date, a complete evolutionary analysis of the functional relationship of an entire enzyme family based on active-site structural similarities has not yet been undertaken. Pyridoxal-5'-phosphate (PLP) dependent enzymes are primordial enzymes that diversified in the last universal ancestor. Using the comparison of protein active site structures (CPASS) software and database, we show that the active site structures of PLP-dependent enzymes can be used to infer evolutionary relationships based on functional similarity. The enzymes successfully clustered together based on substrate specificity, function, and three-dimensional-fold. This study demonstrates the value of using active site structures for functional evolutionary analysis and the effectiveness of CPASS. © 2014 Wiley Periodicals, Inc.

Rapid determination of the various native forms of vitamin B6 and B2 in cow's milk using ultra-high performance liquid chromatography.

PubMed

Schmidt, A; Schreiner, M G; Mayer, H K

2017-06-02

As the formation of pyridoxal phosphate, the active cofactor of vitamin B 6 , is dependent on riboflavin 5-phosphate, we propose a fast and simple ultra-high performance liquid chromatography method for the simultaneous determination of the native B 6 vitamers pyridoxal, pyridoxine, pyridoxamine, their mono phosphorus esters and 4-pyridoxic acid as well as vitamin B 2 as riboflavin and its phosphorus ester riboflavin 5-phosphate in milk. Separation was achieved under 6.0min by reversed-phase and pH gradient elution. Sample preparation was optimized regarding various acids and pH levels. Changes in those parameters led to significant deviations of sample matrix breakdown efficiency. The optimized method was then validated regarding specificity, accuracy, precision, linearity, range, detection and quantification limits. As the method performed satisfactory, is was used to study commercial liquid cow's milk (n=31), regarding effects of the employed preservation technique (pasteurization, extended shelf-life, ultra-high temperature) on the composition and content of B 6 and B 2 vitamers. In cow's milk, vitamin B 6 mostly consists of pyridoxal and its phosphate ester, with pyridoxal phosphate being the bulk component. The catabolite of the vitamin B 6 metabolism, 4-pyridoxic acid was present in significant amounts in all studied samples, with up to 2.69μmolL -1 . Vitamin B 2 was present as riboflavin and its phosphate ester up to 12.86μmolL -1 . Copyright © 2017 Elsevier B.V. All rights reserved.

Vitamin B6 metabolism in chronic alcohol abuse The effect of ethanol oxidation on hepatic pyridoxal 5'-phosphate metabolism.

PubMed Central

Vech, R L; Lumeng, L; Li, T K

1975-01-01

Individuals with chronic alcohol abuse frequently exhibit lowered plasma levels of pyridoxal 5'-phosphate, the coenzyme form of vitamin B6. Because the liver is the primary source of this coenzyme in plasma and also the principal organ that oxidizes ethanol, the effect of ethanol on hepatic pyridoxal phosphate metabolism was studied in the rat. The chronic feeding of ethanol (36 percent of the total dietary calories) for 6 wk significantly decreased the hepatic pyridoxal phosphate content both in animals given a sufficient amount of vitamin B6 in their diet and in those rendered vitamin B6 deficient. In isolated perfused livers, the addition of 18 mM ethanol lowered the pyridoxal phosphate content of livers from vitamin B6-sufficient animals and deceased the net synthesis of pyridoxal phosphate from pyridoxine by the livers of vitamin B6-deficient animals. Ethanol also diminished the rate of release of pyridoxal phosphate into the perfusate by the livers of vitamin B6-deficient rats. These effects of ethanol, in vitro, were abolished by 4-methyl pyrazole, an inhibitor of alcohol dehydrogenase. Thus the derangement of pyridoxal phosphate metabolism produced by ethanol is dependt upon its oxidation. These data support previous findings whic indicate that acetaldehyde is the responsible agent which acts by accelerating the degradation of intracellular pyridoxal phosphate. Images PMID:1168205

Role of the pyridine nitrogen in pyridoxal 5'-phosphate catalysis: activity of three classes of PLP enzymes reconstituted with deazapyridoxal 5'-phosphate.

PubMed

Griswold, Wait R; Toney, Michael D

2011-09-21

Pyridoxal 5'-phosphate (PLP; vitamin B(6))-catalyzed reactions have been well studied, both on enzymes and in solution, due to the variety of important reactions this cofactor catalyzes in nitrogen metabolism. Three functional groups are central to PLP catalysis: the C4' aldehyde, the O3' phenol, and the N1 pyridine nitrogen. In the literature, the pyridine nitrogen has traditionally been assumed to be protonated in enzyme active sites, with the protonated pyridine ring providing resonance stabilization of carbanionic intermediates. This assumption is certainly correct for some PLP enzymes, but the structures of other active sites are incompatible with protonation of N1, and, consequently, these enzymes are expected to use PLP in the N1-unprotonated form. For example, aspartate aminotransferase protonates the pyridine nitrogen for catalysis of transamination, while both alanine racemase and O-acetylserine sulfhydrylase are expected to maintain N1 in the unprotonated, formally neutral state for catalysis of racemization and β-elimination. Herein, kinetic results for these three enzymes reconstituted with 1-deazapyridoxal 5'-phosphate, an isosteric analogue of PLP lacking the pyridine nitrogen, are compared to those for the PLP enzyme forms. They demonstrate that the pyridine nitrogen is vital to the 1,3-prototropic shift central to transamination, but not to reactions catalyzed by alanine racemase or O-acetylserine sulfhydrylase. Not all PLP enzymes require the electrophilicity of a protonated pyridine ring to enable formation of carbanionic intermediates. It is proposed that modulation of cofactor electrophilicity plays a central role in controlling reaction specificity in PLP enzymes.

Identification, Purification, and Characterization of a Novel Amino Acid Racemase, Isoleucine 2-Epimerase, from Lactobacillus Species

PubMed Central

Mutaguchi, Yuta; Ohmori, Taketo; Wakamatsu, Taisuke; Doi, Katsumi

2013-01-01

Accumulation of d-leucine, d-allo-isoleucine, and d-valine was observed in the growth medium of a lactic acid bacterium, Lactobacillus otakiensis JCM 15040, and the racemase responsible was purified from the cells and identified. The N-terminal amino acid sequence of the purified enzyme was GKLDKASKLI, which is consistent with that of a putative γ-aminobutyrate aminotransferase from Lactobacillus buchneri. The putative γ-aminobutyrate aminotransferase gene from L. buchneri JCM 1115 was expressed in recombinant Escherichia coli and then purified to homogeneity. The enzyme catalyzed the racemization of a broad spectrum of nonpolar amino acids. In particular, it catalyzed at high rates the epimerization of l-isoleucine to d-allo-isoleucine and d-allo-isoleucine to l-isoleucine. In contrast, the enzyme showed no γ-aminobutyrate aminotransferase activity. The relative molecular masses of the subunit and native enzyme were estimated to be about 49 kDa and 200 kDa, respectively, indicating that the enzyme was composed of four subunits of equal molecular masses. The Km and Vmax values of the enzyme for l-isoleucine were 5.00 mM and 153 μmol·min−1·mg−1, respectively, and those for d-allo-isoleucine were 13.2 mM and 286 μmol·min−1·mg−1, respectively. Hydroxylamine and other inhibitors of pyridoxal 5′-phosphate-dependent enzymes completely blocked the enzyme activity, indicating the enzyme requires pyridoxal 5′-phosphate as a coenzyme. This is the first evidence of an amino acid racemase that specifically catalyzes racemization of nonpolar amino acids at the C-2 position. PMID:24039265

Typical and atypical phenotypes of PNPO deficiency with elevated CSF and plasma pyridoxamine on treatment.

PubMed

Ware, Tyson L; Earl, John; Salomons, Gajja S; Struys, Eduard A; Peters, Heidi L; Howell, Katherine B; Pitt, James J; Freeman, Jeremy L

2014-05-01

Pyridox(am)ine phosphate oxidase (PNPO) deficiency causes severe early infantile epileptic encephalopathy and has been characterized as responding to pyridoxal-5'-phosphate but not to pyridoxine. Two males with PNPO deficiency and novel PNPO mutations are reported and their clinical, metabolic, and video-electroencephalographic (EEG) findings described. The first child showed electro-clinical responses to pyridoxine and deterioration when pyridoxine was withheld. At last review, he has well-controlled epilepsy with pyridoxal-5'-phosphate monotherapy and an autism spectrum disorder. The second child had a perinatal middle cerebral artery infarct and a myoclonic encephalopathy. He failed to respond to pyridoxine but responded well to pyridoxal-5'-phosphate. At the age of 21 months he has global developmental delay and hemiparesis but is seizure-free with pyridoxal-5'-phosphate monotherapy. Plasma and cerebrospinal fluid pyridoxamine levels were increased in both children during treatment with pyridoxine or pyridoxal-5'-phosphate. These observations indicate that differential responses to pyridoxine and pyridoxal-5'-phosphate treatment cannot be relied upon to diagnose PNPO deficiency. © 2013 Mac Keith Press.

Arginase 2 Suppresses Renal Carcinoma Progression via Biosynthetic Cofactor Pyridoxal Phosphate Depletion and Increased Polyamine Toxicity.

PubMed

Ochocki, Joshua D; Khare, Sanika; Hess, Markus; Ackerman, Daniel; Qiu, Bo; Daisak, Jennie I; Worth, Andrew J; Lin, Nan; Lee, Pearl; Xie, Hong; Li, Bo; Wubbenhorst, Bradley; Maguire, Tobi G; Nathanson, Katherine L; Alwine, James C; Blair, Ian A; Nissim, Itzhak; Keith, Brian; Simon, M Celeste

2018-05-04

Kidney cancer, one of the ten most prevalent malignancies in the world, has exhibited increased incidence over the last decade. The most common subtype is "clear cell" renal cell carcinoma (ccRCC), which features consistent metabolic abnormalities, such as highly elevated glycogen and lipid deposition. By integrating metabolomics, genomic, and transcriptomic data, we determined that enzymes in multiple metabolic pathways are universally depleted in human ccRCC tumors, which are otherwise genetically heterogeneous. Notably, the expression of key urea cycle enzymes, including arginase 2 (ARG2) and argininosuccinate synthase 1 (ASS1), is strongly repressed in ccRCC. Reduced ARG2 activity promotes ccRCC tumor growth through at least two distinct mechanisms: conserving the critical biosynthetic cofactor pyridoxal phosphate and avoiding toxic polyamine accumulation. Pharmacological approaches to restore urea cycle enzyme expression would greatly expand treatment strategies for ccRCC patients, where current therapies only benefit a subset of those afflicted with renal cancer. Copyright © 2018 Elsevier Inc. All rights reserved.

Apoenzyme of aspartate aminotransferase isozymes in serum and its diagnostic usefullness for hepatic diseases.

PubMed

Kamei, S; Ohkubo, A; Yamanaka, M

1979-08-15

Aspartate aminotransferase in the sera of normal subjects and of patients with hepatic diseases has been immunologically separated into two isoenzymes, cytosolic aspartate aminotransferase and mitochondrial aspartate aminotransferase. The activity of the isoenzymes was measured in three different buffer solutions with or without pyridoxal 5'-phosphate. To attain maximal activation, the apoenzyme of mitochondrial fraction must be preincubated with pyridoxal 5'-phosphate longer than that of the cytosolic fraction in either of the three reaction mixtures. In most sera the activity of both isoenzymes increased substantially in the presence of pyridoxal 5'-phosphate regardless of the type of buffer solutions. Both the apoenzymatic activity and the ratio of apo- to holo-enzymatic activity of each of the isoenzymes varied among samples from the patients with hepatic diseases. However, significantly high ratios of apo- to holo-enzymatic activity of both isoenzymes were observed in the patients with hepatoma in contrast with those with other hepatic diseases. These findings suggest that the simultaneous measurement of both apo- and holo-enzyme activities of aspartate aminotransferase isoenzymes may be useful in the clinical assessment of hepatic diseases.

Catalytic and regulatory roles of species involved in metal-nucleotide equilibriums in human pyridoxal kinase.

PubMed

Navarro, Freddy; Ramírez-Sarmiento, César A; Guixé, Victoria

2013-10-01

Pyridoxal 5'-phosphate is the active form of vitamin B6 and its deficiency is directly related with several human disorders, which make human pyridoxal kinase (hPLK) an important pharmacologic target. In spite of this, a carefully kinetic characterization of hPLK including the main species that regulates the enzymatic activity is at date missing. Here we analyse the catalytic and regulatory mechanisms of hPLK as a function of a precise determination of the species involved in metal-nucleotide equilibriums and describe new regulatory mechanisms for this enzyme. hPLK activity is supported by several metals, being Zn(2+) the most effective, although the magnitude of the effect observed is highly dependent on the relative concentrations of metal and nucleotide used. The true substrate for the reaction catalyzed by hPLK is the metal nucleotide complex, while ATP(4-) and HATP(3-) did not affect the activity. The enzyme presents substrate inhibition by both pyridoxal (PL) and ZnATP(2-), although the latter behaves as a weakly inhibitor. Our study also established, for the first time, a dual role for free Zn(2+); as an activator at low concentrations (19 μM optimal concentration) and as a potent inhibitor with a IC50 of 37 μM. These results highlighted the importance of an accurate estimation of the actual concentration of the species involved in metal-nucleotide equilibriums in order to obtain reliable values for the kinetic parameters, and for determine the true regulators of the PLK activity. They also help to explain the dissimilar kinetic parameters reported in the literature for this enzyme.

A new role for coenzyme F420 in aflatoxin reduction by soil mycobacteria.

PubMed

Graham, David E

2010-11-01

Hepatotoxic aflatoxins have found a worthy adversary in two new families of bacterial oxidoreductases. These enzymes use the reduced coenzyme F420 to initiate the degradation of furanocoumarin compounds, including the major mycotoxin products of Aspergillus flavus. Along with pyridoxal 5'-phosphate synthases and aryl nitroreductases, these proteins form a large and versatile superfamily of flavin and deazaflavin-dependent oxidoreductases. F420-dependent members of this family appear to share a common mechanism of hydride transfer from the reduced, low-potential deazaflavin to the electron-deficient ring systems of their substrates. © 2010 Blackwell Publishing Ltd.

Molecular cloning and characterization of l-methionine γ-lyase from Streptomyces avermitilis.

PubMed

Kudou, Daizou; Yasuda, Eri; Hirai, Yoshiyuki; Tamura, Takashi; Inagaki, Kenji

2015-10-01

A pyridoxal 5'-phosphate-dependent methionine γ-lyase (MGL) was cloned from Streptomyces avermitilis catalyzed the degradation of methionine to α-ketobutyrate, methanethiol, and ammonia. The sav7062 gene (1,242 bp) was corresponded to 413 amino acid residues with a molecular mass of 42,994 Da. The deduced amino acid sequence showed a high degree of similarity to those of other MGL enzymes. The sav7062 gene was overexpressed in Escherichia coli. The enzyme was purified to homogeneity and exhibited the MGL catalytic activities. We cloned the enzyme that has the MGL activity in Streptomyces for the first time. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Methionine biosynthesis in higher plants. II. Purification and characterization of cystathionine beta-lyase from spinach chloroplasts.

PubMed

Droux, M; Ravanel, S; Douce, R

1995-01-10

Cystathionine beta-lyase, the second enzyme of the transsulfuration pathway leading to homocysteine synthesis was purified over 16,000-fold from spinach (Spinacia oleracea L.) leaf chloroplasts (soluble fraction). Enzyme activity was followed along the purification scheme by either a colorimetric method for the determination of cysteine or by fluorescence detection of the bimane derivative of L-homocysteine after reverse-phase HPLC. Cystathionine beta-lyase has a molecular mass of 170,000 +/- 5000 Da and consists of four identical subunits of 44,000 Da. The enzyme exhibits an absorption spectrum in the visible range with a maximum at 418 nm due to pyridoxal 5'-phosphate. The chloroplastic enzyme catalyzes alpha,beta-cleavage of the thioether L-cystathionine and the dithioacetal L-djenkolate with apparent Km values of 0.15 and 0.34 mM, respectively, and apparent Vm values corresponding to a specific activity of 13 Units mg-1. However, no activity was detected toward the disulfide L-cysteine. With either L-cystathionine and L-djenkolate as substrate, maximal activity was obtained between pH 8.3 and pH 9.0. Besides the chloroplastic enzyme form, anion exchange chromatography of a total spinach leaf extract allowed the detection of a second pool of cystathionine beta-lyase activity that is associated with the cytosolic compartment and eluted at a lower salt concentration than the chloroplastic isoform. Kinetics of inactivation of cystathionine beta-lyase by the L-alpha-(2-aminoethoxyvinyl) glycine (AVG), an analogue of L-cystathionine, are consistent with the existence of an intermediate reversible enzyme inhibitor complex (apparent inhibition constant Kappi of 110 microM) preceding the irreversible formation of a final inactivated state of the enzyme (kd = 4.8 x 10(-3) s-1). Pyridoxal 5'-phosphate free in solution binds AVG with an apparent dissociation constant Kapp in the order of 350 microM. The comparison between the Kapp (free pyridoxal 5'-phosphate) and Kappi (enzyme inactivation) values indicate that the prosthetic group of spinach chloroplast cystathionine beta-lyase is freely accessible to the inhibitor compound AVG.

The periplasmic transaminase PtaA of Pseudomonas fluorescens converts the glutamic acid residue at the pyoverdine fluorophore to α-ketoglutaric acid.

PubMed

Ringel, Michael T; Dräger, Gerald; Brüser, Thomas

2017-11-10

The periplasmic conversion of ferribactin to pyoverdine is essential for siderophore biogenesis in fluorescent pseudomonads, such as pathogenic Pseudomonas aeruginosa or plant growth-promoting Pseudomonas fluorescens The non-ribosomal peptide ferribactin undergoes cyclizations and oxidations that result in the fluorophore, and a strictly conserved fluorophore-bound glutamic acid residue is converted to a range of variants, including succinamide, succinic acid, and α-ketoglutaric acid residues. We recently discovered that the pyridoxal phosphate-containing enzyme PvdN is responsible for the generation of the succinamide, which can be hydrolyzed to succinic acid. Based on this, a distinct unknown enzyme was postulated to be responsible for the conversion of the glutamic acid to α-ketoglutaric acid. Here we report the identification and characterization of this enzyme in P. fluorescens strain A506. In silico analyses indicated a periplasmic transaminase in fluorescent pseudomonads and other proteobacteria that we termed PtaA for " p eriplasmic t ransaminase A " An in-frame-deleted ptaA mutant selectively lacked the α-ketoglutaric acid form of pyoverdine, and recombinant PtaA complemented this phenotype. The ptaA / pvdN double mutant produced exclusively the glutamic acid form of pyoverdine. PtaA is homodimeric and contains a pyridoxal phosphate cofactor. Mutation of the active-site lysine abolished PtaA activity and affected folding as well as Tat-dependent transport of the enzyme. In pseudomonads, the occurrence of ptaA correlates with the occurrence of α-ketoglutaric acid forms of pyoverdines. As this enzyme is not restricted to pyoverdine-producing bacteria, its catalysis of periplasmic transaminations is most likely a general tool for specific biosynthetic pathways. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Simultaneous Determination of Underivatized Vitamin B1 and B6 in Whole Blood by Reversed Phase Ultra High Performance Liquid Chromatography Tandem Mass Spectrometry

PubMed Central

Puts, Johan; de Groot, Monique; Haex, Martin; Jakobs, Bernadette

2015-01-01

Background Vitamin B1 (thiamine-diphosphate) and B6 (pyridoxal-5’phosphate) are micronutrients. Analysis of these micronutrients is important to diagnose potential deficiency which often occurs in elderly people due to malnutrition, in severe alcoholism and in gastrointestinal compromise due to bypass surgery or disease. Existing High Performance Liquid Chromatography (HPLC) based methods include the need for derivatization and long analysis time. We developed an Ultra High Performance Liquid Chromatography Tandem Mass spectrometry (UHPLC-MS/MS) assay with internal standards for simultaneous measurement of underivatized thiamine-diphosphate and pyridoxal-5’phosphate without use of ion pairing reagent. Methods Whole blood, deproteinized with perchloric acid, containing deuterium labelled internal standards thiamine-diphosphate(thiazole-methyl-D3) and pyridoxal-5’phosphate(methyl-D3), was analyzed by UHPLC-MS/MS. The method was validated for imprecision, linearity, recovery and limit of quantification. Alternate (quantitative) method comparisons of the new versus currently used routine HPLC methods were established with Deming regression. Results Thiamine-diphosphate and pyridoxal-5’phosphate were measured within 2.5 minutes instrumental run time. Limits of detection were 2.8 nmol/L and 7.8 nmol/L for thiamine-diphosphate and pyridoxal-5’phosphate respectively. Limit of quantification was 9.4 nmol/L for thiamine-diphosphate and 25.9 nmol/L for pyridoxal-5’phosphate. The total imprecision ranged from 3.5–7.7% for thiamine-diphosphate (44–157 nmol/L) and 6.0–10.4% for pyridoxal-5’phosphate (30–130 nmol/L). Extraction recoveries were 101–102% ± 2.5% (thiamine-diphosphate) and 98–100% ± 5% (pyridoxal-5’phosphate). Deming regression yielded slopes of 0.926 and 0.990 in patient samples (n = 282) and national proficiency testing samples (n = 12) respectively, intercepts of +3.5 and +3 for thiamine-diphosphate (n = 282 and n = 12) and slopes of 1.04 and 0.84, intercepts of -2.9 and +20 for pyridoxal-5’phosphate (n = 376 and n = 12). Conclusion The described UHPLC-MS/MS method allows simultaneous determination of underivatized thiamine-diphosphate and pyridoxal-5’phosphate in whole blood without intensive sample preparation. PMID:26134844

A hybrid QM/MM simulation study of intramolecular proton transfer in the pyridoxal 5'-phosphate in the active site of transaminase: influence of active site interaction on proton transfer.

PubMed

Dutta Banik, Sindrila; Chandra, Amalendu

2014-09-25

Pyridoxal 5'-phosphate (PLP) Schiff base, a versatile cofactor, exhibits a tautomeric equilibrium that involves an intramolecular proton transfer between the N-protonated zwitterionic ketoenamine tautomer and the O-protonated covalent enolimine tautomer. It has been postulated that for the catalytic activity, the PLP has to be in the zwitterionic ketoenamine tautomeric form. However, the exact position of the tautomeric equilibrium of Schiff base in the active site of PLP-dependent enzyme is not known yet. In the present work, we investigated the tautomeric equilibrium for the external aldimine state of PLP aspartate (PLP-Asp) Schiff base in the active site of aspartate aminotransferase (AspAT) using combined quantum mechanical and molecular mechanical simulations. The main focus of the present study is to analyze the factors that control the tautomeric equilibrium in the active sites of various PLP-dependent enzymes. The results show that the ketoenamine tautomer is more preferred than the enolimine tautomer both in the gas and aqueous phases as well as in the active site of AspAT. Current simulations show that the active site of AspAT is more suitable for the ketoenamine tautomer compared to the enolimine tautomer. Interestingly, the Tyr225 acts as a proton donor to the phenolic oxygen in the ketoenamine tautomer, while in the covalent enolimine tautomer, it acts as a proton acceptor to the phenolic oxygen. Finally, the metadynamics study confirms this result. The calculated free energy barrier is about 7.5 kcal/mol. A comparative analysis of the microenvironment created by the active site residues of three different PLP-dependent enzymes (aspartate aminotransferase, Dopa decarboxylase, and Ala-racemase) has been carried out to understand the controlling factor(s) of the tautomeric equilibrium. The analysis shows that the intermolecular hydrogen bonding between active site residues and the phenolic oxygen of PLP shifts the tautomeric equilibrium toward the N-protonated ketoenamine tautomeric form.

Measurement of pyridoxal 5'-phosphate, pyridoxal, and 4-pyridoxic acid in the cerebrospinal fluid of children.

PubMed

Akiyama, Tomoyuki; Akiyama, Mari; Hayashi, Yumiko; Shibata, Takashi; Hanaoka, Yoshiyuki; Toda, Soichiro; Imai, Katsumi; Hamano, Shin-Ichiro; Okanishi, Tohru; Yoshinaga, Harumi; Kobayashi, Katsuhiro

2017-03-01

We quantified pyridoxal 5'-phosphate (PLP), pyridoxal (PL), and 4-pyridoxic acid (PA) in the cerebrospinal fluid (CSF) of children and to investigate the effect of age, sex, epilepsy, and anti-epileptic drug (AED) therapy on these vitamers. CSF samples prospectively collected from 116 pediatric patients were analyzed. PLP, PL, and PA were measured using high-performance liquid chromatography with fluorescence detection, using pre-column derivatization by semicarbazide. Effects of age, sex, epilepsy, and AEDs on these vitamers and the PLP/PL ratio were evaluated using multiple linear regression models. The PLP, PL, and PA concentrations were correlated negatively with age and the PLP/PL ratio was correlated positively with age. Multiple regression analysis revealed that the presence of epilepsy was associated with lower PLP concentrations and PLP/PL ratios but sex and AED therapy had no influence on these values. The observed ranges of these vitamers in epileptic and non-epileptic patients were demonstrated. We showed the age dependence of PLP and PL in CSF from pediatric patients. Epileptic patients had lower PLP concentrations and PLP/PL ratios than non-epileptic patients, but it is unknown whether this is the cause, or a result, of epilepsy. Copyright © 2016 Elsevier B.V. All rights reserved.

Nodding syndrome in Tanzania may not be associated with circulating anti-NMDA-and anti-VGKC receptor antibodies or decreased pyridoxal phosphate serum levels-a pilot study.

PubMed

Dietmann, Anelia; Wallner, Bernd; König, Rebekka; Friedrich, Katrin; Pfausler, Bettina; Deisenhammer, Florian; Griesmacher, Andrea; Seger, Christoph; Matuja, William; JilekAall, Louise; Winkler, Andrea S; Schmutzhard, Erich

2014-06-01

Nodding syndrome (NS) is a seemingly progressive epilepsy disorder of unknown underlying cause. We investigated association of pyridoxal-phosphate serum levels and occurrence of anti-neuronal antibodies against N-methyl-D-aspartate (NMDA) receptor and voltage gated potassium channel (VGKC) complex in NS patients. Sera of a Tanzanian cohort of epilepsy and NS patients and community controls were tested for the presence of anti-NMDA-receptor and anti-VGKC complex antibodies by indirect immunofluorescence assay. Furthermore pyridoxal-phosphate levels were measured. Auto-antibodies against NMDA receptor or VGKC (LG1 or Caspr2) complex were not detected in sera of patients suffering from NS (n=6), NS plus other seizure types (n=16), primary generalized epilepsy (n=1) and community controls without epilepsy (n=7). Median Pyridoxal-phosphate levels in patients with NS compared to patients with primary generalized seizures and community controls were not significantly different. However, these median pyridoxal-phosphate levels are significantly lower compared to the range considered normal in Europeans. In this pilot study NS was not associated with serum anti-NMDA receptor or anti-VGKC complex antibodies and no association to pyridoxal-phosphate serum levels was found.

Thiamin Pyrimidine Biosynthesis in Candida albicans: A Remarkable Reaction between Histidine and Pyridoxal Phosphate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lai, Rung-Yi; Huang, Siyu; Fenwick, Michael K.

2012-06-26

In Saccharomyces cerevisiae, thiamin pyrimidine is formed from histidine and pyridoxal phosphate (PLP). The origin of all of the pyrimidine atoms has been previously determined using labeling studies and suggests that the pyrimidine is formed using remarkable chemistry that is without chemical or biochemical precedent. Here we report the overexpression of the closely related Candida albicans pyrimidine synthase (THI5p) and the reconstitution and preliminary characterization of the enzymatic activity. A structure of the C. albicans THI5p shows PLP bound at the active site via an imine with Lys62 and His66 in close proximity to the PLP. Our data suggest thatmore » His66 of the THI5 protein is the histidine source for pyrimidine formation and that the pyrimidine synthase is a single-turnover enzyme.« less

Integrating metabolomics and transcriptomics data to discover a biocatalyst that can generate the amine precursors for alkamide biosynthesis

PubMed Central

Rizhsky, Ludmila; Jin, Huanan; Shepard, Michael R.; Scott, Harry W.; Teitgen, Alicen M.; Perera, M. Ann; Mhaske, Vandana; Jose, Adarsh; Zheng, Xiaobin; Crispin, Matt; Wurtele, Eve S.; Jones, Dallas; Hur, Manhoi; Góngora-Castillo, Elsa; Buell, C. Robin; Minto, Robert E.; Nikolau, Basil J.

2016-01-01

Summary The Echinacea genus is exemplary of over 30 plant families that produce a set of bioactive amides, called alkamides. The Echinacea alkamides may be assembled from two distinct moieties, a branched-chain amine that is acylated with a novel polyunsaturated fatty acid. In this study we identified the potential enzymological source of the amine moiety as a pyridoxal phosphate dependent decarboxylating enzyme that uses branched chain amino acids as substrate. This identification was based on a correlative analysis of the transcriptomes and metabolomes of 36 different E. purpurea tissues and organs, which expressed distinct alkamide profiles. Although no correlation was found between the accumulation patterns of the alkamides and their putative metabolic precursors (i.e., fatty acids and branched chain amino acids), isotope-labeling analyses supported the transformation of valine and isoleucine to isobutylamine and 2-methylbutylamine as reactions of alkamide biosynthesis. Sequence homology identified the pyridoxal phosphate dependent decarboxylase-like proteins in the translated proteome of E. purpurea. These sequences were prioritized for direct characterization by correlating their transcript levels with alkamide accumulation patterns in different organs and tissues, and this multi-pronged approach led to the identification and characterization of a branched-chain amino acid decarboxylase, which would appear to be responsible for generating the amine moieties of naturally occurring alkamides. PMID:27497272

Comparative genomics of pyridoxal 5′-phosphate-dependent transcription factor regulons in Bacteria

PubMed Central

Suvorova, Inna A.

2016-01-01

The MocR-subfamily transcription factors (MocR-TFs) characterized by the GntR-family DNA-binding domain and aminotransferase-like sensory domain are broadly distributed among certain lineages of Bacteria. Characterized MocR-TFs bind pyridoxal 5′-phosphate (PLP) and control transcription of genes involved in PLP, gamma aminobutyric acid (GABA) and taurine metabolism via binding specific DNA operator sites. To identify putative target genes and DNA binding motifs of MocR-TFs, we performed comparative genomics analysis of over 250 bacterial genomes. The reconstructed regulons for 825 MocR-TFs comprise structural genes from over 200 protein families involved in diverse biological processes. Using the genome context and metabolic subsystem analysis we tentatively assigned functional roles for 38 out of 86 orthologous groups of studied regulators. Most of these MocR-TF regulons are involved in PLP metabolism, as well as utilization of GABA, taurine and ectoine. The remaining studied MocR-TF regulators presumably control genes encoding enzymes involved in reduction/oxidation processes, various transporters and PLP-dependent enzymes, for example aminotransferases. Predicted DNA binding motifs of MocR-TFs are generally similar in each orthologous group and are characterized by two to four repeated sequences. Identified motifs were classified according to their structures. Motifs with direct and/or inverted repeat symmetry constitute the majority of inferred DNA motifs, suggesting preferable TF dimerization in head-to-tail or head-to-head configuration. The obtained genomic collection of in silico reconstructed MocR-TF motifs and regulons in Bacteria provides a basis for future experimental characterization of molecular mechanisms for various regulators in this family. PMID:28348826

Allosteric communication between the pyridoxal 5'-phosphate (PLP) and heme sites in the H2S generator human cystathionine β-synthase.

PubMed

Yadav, Pramod Kumar; Xie, Peter; Banerjee, Ruma

2012-11-02

Human cystathionine β-synthase (CBS) is a unique pyridoxal 5'-phosphate (PLP)-dependent enzyme that has a regulatory heme cofactor. Previous studies have demonstrated the importance of Arg-266, a residue at the heme pocket end of α-helix 8, for communication between the heme and PLP sites. In this study, we have examined the role of the conserved Thr-257 and Thr-260 residues, located at the other end of α-helix 8 on the heme electronic environment and on activity. The mutations at the two positions destabilize PLP binding, leading to lower PLP content and ~2- to ~500-fold lower activity compared with the wild-type enzyme. Activity is unresponsive to PLP supplementation, consistent with the pyridoxine-nonresponsive phenotype of the T257M mutation in a homocystinuric patient. The H(2)S-producing activities, also impacted by the mutations, show a different pattern of inhibition compared with the canonical transsulfuration reaction. Interestingly, the mutants exhibit contrasting sensitivities to the allosteric effector, S-adenosylmethionine (AdoMet); whereas T257M and T257I are inhibited, the other mutants are hyperactivated by AdoMet. All mutants showed an increased propensity of the ferrous heme to form an inactive species with a 424 nm Soret peak and exhibited significantly reduced enzyme activity in the ferrous and ferrous-CO states. Our results provide the first evidence for bidirectional transmission of information between the cofactor binding sites, suggest the additional involvement of this region in allosteric communication with the regulatory AdoMet-binding domain, and reveal the potential for independent modulation of the canonical transsulfuration versus H(2)S-generating reactions catalyzed by CBS.

Methionine biosynthesis in higher plants. I. Purification and characterization of cystathionine gamma-synthase from spinach chloroplasts.

PubMed

Ravanel, S; Droux, M; Douce, R

1995-01-10

Cystathionine gamma-synthase, the first enzyme specific for the methionine biosynthetic pathway, was purified to apparent homogeneity from spinach leaf chloroplasts. A nonradioactive assay based on O-phthaldialdehyde derivatization of L-cystathionine and fluorescence detection was developed to determine the cystathionine gamma-synthase activity. A unique cystathionine gamma-synthase activity was located in the stromal fraction of chloroplasts while cystathionine beta-lyase, the second enzyme of the transsulfuration pathway, was associated with both the chloroplastic and cytosolic compartments (see companion manuscript). The purified enzyme exhibited a specific activity of 13 U mg-1. As estimated by gel filtration and polyacrylamide gel electrophoresis (PAGE) under nondenaturing conditions followed by activity staining, the native enzyme had an apparent M(r) of 215,000. On the basis of sodium dodecyl sulfate-PAGE, purified cystathionine gamma-synthase migrated as two molecular species of M(r) 53,000 and 50,000 that are identical in their N-termini. The absorption spectrum obtained at pH 7.5 exhibited a peak at 425 nm due to pyridoxal 5'-phosphate (PLP). The purified enzyme catalyzed the formation of L-cystathionine or L-homocysteine depending on the sulfur-containing substrate, L-cysteine or sulfide. Maximal cystathionine gamma-synthase activity was found at pH 7.4. The apparent Km values for O-phospho-L-homoserine (the unique homoserine ester synthesized in the chloroplast), L-cysteine, and sulfide were 1.4, 0.18, and 0.6 mM, respectively. Inactivation of cystathionine gamma-synthase by DL-propargylglycine (PAG) showed pseudo-first-order kinetics and data were consistent with the existence of an intermediate reversible enzyme-inhibitor complex (Kappi = 140 microM) preceding the formation of a final enzyme-inhibitor complex (kd = 24 x 10(-3) s-1). The irreversibility of the inhibition and the partial restoration of the activity by pyridoxal-phosphate suggest that PAG interacts with the PLP prosthetic group of the enzyme. Kinetic and equilibrium binding studies showed that PAG binding to PLP was considerably enhanced in the enzyme binding pocket compared to that with PLP free in solution.

Expression analysis of a novel pyridoxal kinase messenger RNA splice variant, PKL, in oil rape suffering abiotic stress and phytohormones.

PubMed

Yu, Shunwu; Luo, Lijun

2008-12-01

Pyridoxal kinase is key enzyme for the biosynthesis of pyridoxal 5'-phosphate, the biologically active form of vitamin B6, in the salvage pathway. A pyridoxal kinase gene, BnPKL (GenBank accession No. DQ463962), was isolated from oilseed rape (Brassica napus L.) following water stress through rapid amplification of complementary DNA (cDNA) ends. The results showed that the gene had two splice variants: PKL and PKL2. PKL, the long cDNA, encodes a 334 amino acid protein with a complete ATP-binding site, pyridoxal kinase-binding site and dimer interface site of a pyridoxal kinase, while PKL2, the short cDNA, lacked a partial domain. Southern blot showed that there were two copies in Brassica napus. The expression of BnPKL cDNA could rescue the mutant phenotype of Escherichia coli defective in pyridoxal kinase. Real-time reverse transcription-polymerase chain reaction revealed that the relative abundance of two transcripts are modulated by development and environmental stresses. Abscisic acid and NaCl were inclined to decrease PKL expression, but H2O2 and cold temperatures induced the PKL expression. In addition, the PKL expression could be transiently induced by jasmonate acid at an early stage, abscisic acid, salicylic acid and jasmonate acid enhanced the PKL expression in roots. Our results demonstrated that BnPKL was a pyridoxal kinase involved in responses to biotic and abiotic stresses.

Identification and characterization of a pyridoxal 5'-phosphate phosphatase in the silkworm (Bombyx mori).

PubMed

Huang, ShuoHao; Han, CaiYun; Ma, ZhenQiao; Zhou, Jie; Zhang, JianYun; Huang, LongQuan

2017-03-01

Vitamin B 6 comprises six interconvertible pyridine compounds, among which pyridoxal 5'-phosphate (PLP) is a coenzyme for over 140 enzymes. PLP is also a very reactive aldehyde. The most well established mechanism for maintaining low levels of free PLP is its dephosphorylation by phosphatases. A human PLP-specific phosphatase has been identified and characterized. However, very little is known about the phosphatase in other living organisms. In this study, a cDNA clone of putative PLP phosphatase was identified from B. mori and characterized. The cDNA encodes a polypeptide of 343 amino acid residues, and the recombinant enzyme purified from E. coli exhibited properties similar to that of human PLP phosphatase. B. mori has a single copy of the PLPP gene, which is located on 11th chromosome, spans a 5.7kb region and contains five exons and four introns. PLP phosphatase transcript was detected in every larva tissue except hemolymph, and was most highly represented in Malpighian tube. We further down-regulated the gene expression of the PLP phosphatase in 5th instar larvae with the RNA interference. However, no significant changes in the gene expression of PLP biosynthetic enzymes and composition of B 6 vitamers were detected as compared with the control. Copyright © 2017 Elsevier Inc. All rights reserved.

Effect of medium acidity on the thermodynamics and kinetics of the reaction of pyridoxal 5'-phosphate with isoniazid in an aqueous solution

NASA Astrophysics Data System (ADS)

Gamov, G. A.; Zavalishin, M. N.; Usacheva, T. R.; Sharnin, V. A.

2017-05-01

Thermodynamic characteristics of the formation of the Schiff base between isoniazid and pyridoxal 5'-phosphate in an aqueous solution at different pH values of a medium are determined by means of spectrophotometry and calorimetric titration. The process kinetics is studied spectrophotometrically, and the reaction rate constants for the formation of the imine at different acidities of a medium are determined. Biochemical aspects of the binding of pyridoxal 5'-phosphate into stable compounds are discussed.

Lysine Decarboxylase with an Enhanced Affinity for Pyridoxal 5-Phosphate by Disulfide Bond-Mediated Spatial Reconstitution.

PubMed

Sagong, Hye-Young; Kim, Kyung-Jin

2017-01-01

Lysine decarboxylase (LDC) catalyzes the decarboxylation of l-lysine to produce cadaverine, an important industrial platform chemical for bio-based polyamides. However, due to high flexibility at the pyridoxal 5-phosphate (PLP) binding site, use of the enzyme for cadaverine production requires continuous supplement of large amounts of PLP. In order to develop an LDC enzyme from Selenomonas ruminantium (SrLDC) with an enhanced affinity for PLP, we introduced an internal disulfide bond between Ala225 and Thr302 residues with a desire to retain the PLP binding site in a closed conformation. The SrLDCA225C/T302C mutant showed a yellow color and the characteristic UV/Vis absorption peaks for enzymes with bound PLP, and exhibited three-fold enhanced PLP affinity compared with the wild-type SrLDC. The mutant also exhibited a dramatically enhanced LDC activity and cadaverine conversion particularly under no or low PLP concentrations. Moreover, introduction of the disulfide bond rendered SrLDC more resistant to high pH and temperature. The formation of the introduced disulfide bond and the maintenance of the PLP binding site in the closed conformation were confirmed by determination of the crystal structure of the mutant. This study shows that disulfide bond-mediated spatial reconstitution can be a platform technology for development of enzymes with enhanced PLP affinity.

Multiple turnovers of the nicotino-enzyme PdxB require α-keto acids as co-substrates

PubMed Central

Rudolph, Johannes; Kim, Juhan; Copley, Shelley D.

2012-01-01

PdxB catalyzes the second step in the biosynthesis of pyridoxal phosphate by oxidizing 4-phospho-D-erythronate (4PE) to 2-oxo-3-hydroxy-4-phospho-butanoate (OHPB) with concomitant reduction of NAD+ to NADH. PdxB is a nicotino-enzyme wherein the NAD(H) cofactor remains tightly bound to PdxB. It has been a mystery how PdxB performs multiple turnovers since addition of free NAD+ does not re-oxidize the enzyme-bound NADH following conversion of 4PE to OHPB. We have solved this mystery by demonstrating that a variety of physiologically available α-ketoacids serve as oxidants of PdxB to sustain multiple turnovers. In a coupled assay using the next two enzymes of the biosynthetic pathway for pyridoxal phosphate (SerC and PdxA), we have found that α-ketoglutarate, oxaloacetic acid, and pyruvate are equally good substrates for PdxB (kcat/Km values ~ 1 × 104 M-1s-1). The kinetic parameters for the substrate 4PE include a kcat of 1.4 s-1, a Km of 2.9 μM, and a kcat/Km of 6.7 × 106 M-1s-1. Additionally, we have characterized the stereochemistry of α-ketoglutarate reduction by showing that D-2-HGA, but not L-2-HGA, is a competitive inhibitor vs. 4PE and a noncompetitive inhibitor vs. α-ketoglutarate. PMID:20831184

The Enantiomers of 4-Amino-3-fluorobutanoic Acid as Substrates for γ-Aminobutyric Acid Aminotransferase. Conformational Probes for GABA Binding†

PubMed Central

Clift, Michael; Ji, Haitao; Deniau, Gildas P.; O’Hagan, David; Silverman, Richard B.

2008-01-01

γ-Aminobutyric acid aminotransferase (GABA-AT), a pyridoxal 5’-phosphate dependent enzyme, catalyzes the degradation of the inhibitory neurotransmitter γ-aminobutyric acid (GABA) to succinic semialdehyde with concomitant conversion of pyridoxal 5’-phosphate (PLP) to pyridoxamine 5’-phosphate (PMP). The enzyme then catalyzes the conversion of α-ketoglutarate to the excitatory neurotransmitter L-glutamate. Racemic 4-amino-3-fluorobutanoic acid (3-F-GABA) was shown previously to act as a substrate for GABA-AT, not for transamination, but for HF elimination. Here we report studies of the reaction catalyzed by GABA-AT on (R)- and (S)-3-F-GABA. Neither enantiomer is a substrate for transamination. Very little elimination from the (S)-enantiomer was detected using a coupled enzyme assay; The rate of elimination of HF from the (R)-enantiomer is at least 10 times greater than that for the (S)-enantiomer. The (R)-enantiomer is about 20 times more efficient as a substrate for GABA-AT catalyzed HF elimination than GABA is a substrate for transamination. The (R)-enantiomer also inhibits the transamination of GABA 10 times more effectively than the (S)-enantiomer. Using a combination of computer modeling and the knowledge that vicinal C-F and C-NH3+ bonds have a strong preference to align gauche rather than anti to each other, it is concluded that on binding of free 3-F-GABA to GABA-AT the optimal conformation places the C-NH3+ and C-F bonds gauche in the (R)-enantiomer but anti in the (S)-enantiomer. Furthermore, the dynamic binding process and the bioactive conformation of GABA bound to GABA-AT have been inferred based on the different biological behavior of the two enantiomers of 3-F-GABA when they bind to the enzyme. The present study suggests that the C-F bond can be utilized as a conformational probe to explore the dynamic binding process and provide insight into the bioactive conformation of substrates, which cannot be easily determined by other biophysical approaches. PMID:17988152

Systems-Wide Prediction of Enzyme Promiscuity Reveals a New Underground Alternative Route for Pyridoxal 5’-Phosphate Production in E. coli

DOE PAGES

Oberhardt, Matthew A.; Zarecki, Raphy; Reshef, Leah; ...

2016-01-28

Recent insights suggest that non-specific and/or promiscuous enzymes are common and active across life. Understanding the role of such enzymes is an important open question in biology. Here we develop a genome-wide method, PROPER, that uses a permissive PSI-BLAST approach to predict promiscuous activities of metabolic genes. Enzyme promiscuity is typically studied experimentally using multicopy suppression, in which over-expression of a promiscuous ‘replacer’ gene rescues lethality caused by inactivation of a ‘target’ gene. We use PROPER to predict multicopy suppression in Escherichia coli, achieving highly significant overlap with published cases (hypergeometric p = 4.4e-13). We then validate three novel predictedmore » target-replacer gene pairs in new multicopy suppression experiments. We next go beyond PROPER and develop a network-based approach, GEM-PROPER, that integrates PROPER with genome-scale metabolic modeling to predict promiscuous replacements via alternative metabolic pathways. GEM-PROPER predicts a new indirect replacer (thiG) for an essential enzyme (pdxB) in production of pyridoxal 5’-phosphate (the active form of Vitamin B 6), which we validate experimentally via multicopy suppression. Here, we perform a structural analysis of thiG to determine its potential promiscuous active site, which we validate experimentally by inactivating the pertaining residues and showing a loss of replacer activity. Thus, this study is a successful example where a computational investigation leads to a network-based identification of an indirect promiscuous replacement of a key metabolic enzyme, which would have been extremely difficult to identify directly.« less

Infantile hypophosphatasia combined with vitamin B6-responsive seizures and reticular formation lesions on magnetic resonance imaging: A case report.

PubMed

Fukazawa, Mitsuharu; Tezuka, Junichiro; Sasazuki, Momoko; Masumoto, Natsuko; Baba, Haruhisa; Doi, Takehiko; Tsutsumi, Yasushi; Mizuno, Yuji; Mihara, Futoshi; Nakayama, Hideki

2018-02-01

Hypophosphatasia (HPP) is a rare genetic disorder characterized by rachitic bone manifestations and a low serum alkaline phosphatase (ALP) level. It is caused by mutations in the tissue non-specific alkaline phosphatase (TNSALP) gene, which encodes the tissue non-specific isozyme of ALP. HPP patients exhibit various presentations depending on their age at onset, such as infantile HPP combined with vitamin B6-responsive seizures. A newborn with infantile HPP presented with tonic convulsions from day 5 after birth and received intravenous vitamin B6 (10mg/kg/day pyridoxal phosphate). Eleven days later, frequent apneic episodes occurred, and head magnetic resonance imaging (MRI) showed bilateral reticular formation lesions in the brain stem, including the medulla oblongata. After the pyridoxal phosphate dose was increased (to 40mg/kg/day), the patient's seizures and apnea resolved, and her MRI findings also improved. Genetic testing revealed that she was homozygous for the 1559delT mutation of TNSALP. High-dose pyridoxal phosphate is a useful treatment for HPP-induced seizures and might improve reticular formation lesions. Copyright © 2017 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.

Isolation and characterization of racemase from Ensifer sp. 23-3 that acts on α-aminolactams and α-amino acid amides.

PubMed

Matsui, Daisuke; Fuhshuku, Ken-Ichi; Nagamori, Shingo; Takata, Momoko; Asano, Yasuhisa

2017-11-01

Limited information is available on α-amino-ε-caprolactam (ACL) racemase (ACLR), a pyridoxal 5'-phosphate-dependent enzyme that acts on ACL and α-amino acid amides. In the present study, eight bacterial strains with the ability to racemize α-amino-ε-caprolactam were isolated and one of them was identified as Ensifer sp. strain 23-3. The gene for ACLR from Ensifer sp. 23-3 was cloned and expressed in Escherichia coli. The recombinant ACLR was then purified to homogeneity from the E. coli transformant harboring the ACLR gene from Ensifer sp. 23-3, and its properties were characterized. This enzyme acted not only on ACL but also on α-amino-δ-valerolactam, α-amino-ω-octalactam, α-aminobutyric acid amide, and alanine amide.

Expression, purification, crystallization, data collection and preliminary biochemical characterization of methicillin-resistant Staphylococcus aureus Sar2028, an aspartate/tyrosine/phenylalanine pyridoxal-5′-phosphate-dependent aminotransferase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Seetharamappa, Jaldappagari; Department of Chemistry, Karnatak University, Pavate Nagar, Dharwad 580 003, Karnataka State; Oke, Muse

2007-05-01

As part of work on S. aureus, the crystallization of Sar2028, a protein that is upregulated in MRSA, is reported. Sar2028, an aspartate/tyrosine/phenylalanine pyridoxal-5′-phosphate-dependent aminotransferase with a molecular weight of 48 168 Da, was overexpressed in methicillin-resistant Staphylococcus aureus compared with a methicillin-sensitive strain. The protein was expressed in Escherichia coli, purified and crystallized. The protein crystallized in a primitive orthorhombic Laue group with unit-cell parameters a = 83.6, b = 91.3, c = 106.0 Å, α = β = γ = 90°. Analysis of the systematic absences along the three principal axes indicated the space group to be P2{submore » 1}2{sub 1}2{sub 1}. A complete data set was collected to 2.5 Å resolution.« less

Differences in the roles of a glutamine amidotransferase subunit of pyridoxal 5'-phosphate synthase between Bacillus circulans and Bacillus subtilis.

PubMed

Itagaki, Shiori; Haga, Minami; Oikawa, Yuji; Sakoda, Ayaka; Ohke, Yoshie; Sawada, Hiroshi; Eguchi, Tadashi; Tamegai, Hideyuki

2013-01-01

BtrC2 of the butirosin producer Bacillus circulans is a non-catalytic subunit of 2-deoxy-scyllo-inosose (DOI) synthase that is involved in butirosin biosynthesis, and also a homolog of glutamine amidotransferase subunit (PdxT) of pyridoxal 5'-phosphate (PLP) synthase of Bacillus subtilis. BtrC2 has been found to have functions in B. circulans both in primary and secondary metabolism. In this study, we investigated the properties of PdxT of B. subtilis in order to determine whether the property of enzyme stabilization is universal among PdxT homologs. Complementation with PdxT in the btrC2 disruptant of B. circulans restored the growth and short-term production of antibiotics, but long-term production of antibiotics cannot be restored. Additionally, PdxT did not bind physically with or stabilize BtrC. Our results indicate that the function of BtrC2 in secondary metabolism is specific properties, not universal among PdxT homologs.

Prediagnostic plasma vitamin B6 (pyridoxal 50-phosphate) and survival in patients with colorectal cancer

USDA-ARS?s Scientific Manuscript database

Higher plasma pyridoxal 5'-phosphate (PLP) levels are associated with a decreased incidence of colorectal cancer, but the influence of plasma PLP on survival of patients with colorectal cancer is unknown. We prospectively examined whether prediagnostic plasma PLP levels are associated with mortality...

Enzyme replacement therapy for murine hypophosphatasia.

PubMed

Millán, José Luis; Narisawa, Sonoko; Lemire, Isabelle; Loisel, Thomas P; Boileau, Guy; Leonard, Pierre; Gramatikova, Svetlana; Terkeltaub, Robert; Camacho, Nancy Pleshko; McKee, Marc D; Crine, Philippe; Whyte, Michael P

2008-06-01

Hypophosphatasia (HPP) is the inborn error of metabolism that features rickets or osteomalacia caused by loss-of-function mutation(s) within the gene that encodes the tissue-nonspecific isozyme of alkaline phosphatase (TNALP). Consequently, natural substrates for this ectoenzyme accumulate extracellulary including inorganic pyrophosphate (PPi), an inhibitor of mineralization, and pyridoxal 5'-phosphate (PLP), a co-factor form of vitamin B6. Babies with the infantile form of HPP often die with severe rickets and sometimes hypercalcemia and vitamin B6-dependent seizures. There is no established medical treatment. Human TNALP was bioengineered with the C terminus extended by the Fc region of human IgG for one-step purification and a deca-aspartate sequence (D10) for targeting to mineralizing tissue (sALP-FcD10). TNALP-null mice (Akp2-/-), an excellent model for infantile HPP, were treated from birth using sALP-FcD10. Short-term and long-term efficacy studies consisted of once daily subcutaneous injections of 1, 2, or 8.2 mg/kg sALP-FcD10 for 15, 19, and 15 or 52 days, respectively. We assessed survival and growth rates, circulating levels of sALP-FcD10 activity, calcium, PPi, and pyridoxal, as well as skeletal and dental manifestations using radiography, microCT, and histomorphometry. Akp2-/- mice receiving high-dose sALP-FcD10 grew normally and appeared well without skeletal or dental disease or epilepsy. Plasma calcium, PPi, and pyridoxal concentrations remained in their normal ranges. We found no evidence of significant skeletal or dental disease. Enzyme replacement using a bone-targeted, recombinant form of human TNALP prevents infantile HPP in Akp2-/- mice.

Enzyme Replacement Therapy for Murine Hypophosphatasia*

PubMed Central

Millán, José Luis; Narisawa, Sonoko; Lemire, Isabelle; Loisel, Thomas P; Boileau, Guy; Leonard, Pierre; Gramatikova, Svetlana; Terkeltaub, Robert; Camacho, Nancy Pleshko; McKee, Marc D; Crine, Philippe; Whyte, Michael P

2008-01-01

Introduction Hypophosphatasia (HPP) is the inborn error of metabolism that features rickets or osteomalacia caused by loss-of-function mutation(s) within the gene that encodes the tissue-nonspecific isozyme of alkaline phosphatase (TNALP). Consequently, natural substrates for this ectoenzyme accumulate extracellulary including inorganic pyrophosphate (PPi), an inhibitor of mineralization, and pyridoxal 5`-phosphate (PLP), a co-factor form of vitamin B6. Babies with the infantile form of HPP often die with severe rickets and sometimes hypercalcemia and vitamin B6-dependent seizures. There is no established medical treatment. Materials and Methods Human TNALP was bioengineered with the C terminus extended by the Fc region of human IgG for one-step purification and a deca-aspartate sequence (D10) for targeting to mineralizing tissue (sALP-FcD10). TNALP-null mice (Akp2−/−), an excellent model for infantile HPP, were treated from birth using sALP-FcD10. Short-term and long-term efficacy studies consisted of once daily subcutaneous injections of 1, 2, or 8.2 mg/kg sALP-FcD10 for 15, 19, and 15 or 52 days, respectively. We assessed survival and growth rates, circulating levels of sALP-FcD10 activity, calcium, PPi, and pyridoxal, as well as skeletal and dental manifestations using radiography, μCT, and histomorphometry. Results Akp2−/− mice receiving high-dose sALP-FcD10 grew normally and appeared well without skeletal or dental disease or epilepsy. Plasma calcium, PPi, and pyridoxal concentrations remained in their normal ranges. We found no evidence of significant skeletal or dental disease. Conclusions Enzyme replacement using a bone-targeted, recombinant form of human TNALP prevents infantile HPP in Akp2−/− mice. PMID:18086009

Direct evidence that an extended hydrogen-bonding network influences activation of pyridoxal 5'-phosphate in aspartate aminotransferase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dajnowicz, Steven; Parks, Jerry M.; Hu, Xiche

We used pyridoxal 5'-phosphate (PLP) is a fundamental, multifunctional enzyme cofactor to catalyze a wide variety of chemical reactions involved in amino acid metabolism. PLP-dependent enzymes optimize specific chemical reactions by modulating the electronic states of PLP through distinct active site environments. In aspartate aminotransferase (AAT), an extended hydrogen bond network is coupled to the pyridinyl nitrogen of the PLP, influencing the electrophilicity of the cofactor. This network, which involves residues Asp-222, His-143, Thr-139, His-189, and structural waters, is located at the edge of PLP opposite the reactive Schiff base. We demonstrate that this hydrogen bond network directly influences themore » protonation state of the pyridine nitrogen of PLP, which affects the rates of catalysis. We analyzed perturbations caused by single- and double-mutant variants using steady-state kinetics, high resolution X-ray crystallography, and quantum chemical calculations. Protonation of the pyridinyl nitrogen to form a pyridinium cation induces electronic delocalization in the PLP, which correlates with the enhancement in catalytic rate in AAT. Therefore, PLP activation is controlled by the proximity of the pyridinyl nitrogen to the hydrogen bond microenvironment. Quantum chemical calculations indicate that Asp-222, which is directly coupled to the pyridinyl nitrogen, increases the pKa of the pyridine nitrogen and stabilizes the pyridinium cation. His-143 and His-189 also increase the pKa of the pyridine nitrogen but, more significantly, influence the position of the proton that resides between Asp-222 and the pyridinyl nitrogen. Our findings indicate that the second shell residues directly enhance the rate of catalysis in AAT.« less

Direct evidence that an extended hydrogen-bonding network influences activation of pyridoxal 5'-phosphate in aspartate aminotransferase

DOE PAGES

Dajnowicz, Steven; Parks, Jerry M.; Hu, Xiche; ...

2017-02-23

We used pyridoxal 5'-phosphate (PLP) is a fundamental, multifunctional enzyme cofactor to catalyze a wide variety of chemical reactions involved in amino acid metabolism. PLP-dependent enzymes optimize specific chemical reactions by modulating the electronic states of PLP through distinct active site environments. In aspartate aminotransferase (AAT), an extended hydrogen bond network is coupled to the pyridinyl nitrogen of the PLP, influencing the electrophilicity of the cofactor. This network, which involves residues Asp-222, His-143, Thr-139, His-189, and structural waters, is located at the edge of PLP opposite the reactive Schiff base. We demonstrate that this hydrogen bond network directly influences themore » protonation state of the pyridine nitrogen of PLP, which affects the rates of catalysis. We analyzed perturbations caused by single- and double-mutant variants using steady-state kinetics, high resolution X-ray crystallography, and quantum chemical calculations. Protonation of the pyridinyl nitrogen to form a pyridinium cation induces electronic delocalization in the PLP, which correlates with the enhancement in catalytic rate in AAT. Therefore, PLP activation is controlled by the proximity of the pyridinyl nitrogen to the hydrogen bond microenvironment. Quantum chemical calculations indicate that Asp-222, which is directly coupled to the pyridinyl nitrogen, increases the pKa of the pyridine nitrogen and stabilizes the pyridinium cation. His-143 and His-189 also increase the pKa of the pyridine nitrogen but, more significantly, influence the position of the proton that resides between Asp-222 and the pyridinyl nitrogen. Our findings indicate that the second shell residues directly enhance the rate of catalysis in AAT.« less

High-dose vitamin B6 decreases homocysteine serum levels in patients with schizophrenia and schizoaffective disorders: a preliminary study.

PubMed

Miodownik, Chanoch; Lerner, Vladimir; Vishne, Tali; Sela, Ben-Ami; Levine, Joseph

2007-01-01

Vitamin B6 plays an essential role in the normal functioning of the central nervous system. Normal homocysteine (Hcy) serum level is maintained by remethylation of Hcy to methionine by enzymes that require folic acid and vitamin B12 and by catabolism to cysteine by a vitamin B6-dependent enzyme. These findings may be consistent with the hypothesis that the vitamin B6 status may influence plasma Hcy levels. The aims of this preliminary study were (1) to determine whether a correlation exists between Hcy and vitamin B6 levels in patients with schizophrenia and schizoaffective disorders and (2) to investigate whether treatment with high-dose vitamin B6 may reduce Hcy levels in these patients. In this preliminary study, we enrolled 11 patients with schizophrenia or schizoaffective disorders (7 men and 4 women; mean age +/- SD, 50 +/- 12 years) receiving high doses of vitamin B6 treatment (1200 mg/d) for 12 weeks. Blood samples for the assessment of pyridoxal-5-phosphate and Hcy serum levels were obtained at baseline and after 12 weeks of treatment. Age was significantly positively correlated with Hcy levels at baseline (r = 0.392, P = 0.004). All other parameters, including diagnosis, disease duration, and pyridoxal-5-phosphate serum level, were not correlated with Hcy serum levels at baseline. After vitamin B6 treatment, Hcy serum levels significantly decreased (14.2 +/- 3.4 vs. 11.8 +/- 2.0 micromol/L, respectively, t = 2.679, P = 0.023); this decrease being statistically significant in men but not in women. High doses of vitamin B6 lead to a decrease in Hcy serum level in male patients with schizophrenia or schizoaffective disorder.

Characterization of homologous sphingosine-1-phosphate lyase isoforms in the bacterial pathogen Burkholderia pseudomallei[S

PubMed Central

McLean, Christopher J.; Marles-Wright, Jon; Custodio, Rafael; Lowther, Jonathan; Kennedy, Amanda J.; Pollock, Jacob; Clarke, David J.; Brown, Alan R.; Campopiano, Dominic J.

2017-01-01

Sphingolipids (SLs) are ubiquitous elements in eukaryotic membranes and are also found in some bacterial and viral species. As well as playing an integral structural role, SLs also act as potent signaling molecules involved in numerous cellular pathways and have been linked to many human diseases. A central SL signaling molecule is sphingosine-1-phosphate (S1P), whose breakdown is catalyzed by S1P lyase (S1PL), a pyridoxal 5′-phosphate (PLP)-dependent enzyme that catalyzes the cleavage of S1P to (2E)-hexadecenal (2E-HEX) and phosphoethanolamine. Here, we show that the pathogenic bacterium, Burkholderia pseudomallei K96243, encodes two homologous proteins (S1PL2021 and S1PL2025) that display moderate sequence identity to known eukaryotic and prokaryotic S1PLs. Using an established MS-based methodology, we show that recombinant S1PL2021 is catalytically active. We also used recombinant human fatty aldehyde dehydrogenase to develop a spectrophotometric enzyme-coupled assay to detect 2E-HEX formation and measure the kinetic constants of the two B. pseudomallei S1PL isoforms. Furthermore, we determined the X-ray crystal structure of the PLP-bound form of S1PL2021 at 2.1 Å resolution revealing that the enzyme displays a conserved structural fold and active site architecture comparable with known S1PLs. The combined data suggest that B. pseudomallei has the potential to degrade host SLs in a S1PL-dependent manner. PMID:27784725

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhao, Xin-Yu; Zhu, Ying-Jie, E-mail: y.j.zhu@mail.sic.ac.cn; Lu, Bing-Qiang

Graphical abstract: Hydroxyapatite nanorods are synthesized using biocompatible biomolecule pyridoxal-5′-phosphate as a new organic phosphorus source by the hydrothermal method. - Highlights: • Hydrothermal synthesis of hydroxyapatite nanorods is reported. • Biocompatible pyridoxal-5′-phosphate is used as an organic phosphorus source. • This method is simple, surfactant-free and environmentally friendly. - Abstract: Hydroxyapatite nanorods are synthesized by the hydrothermal method using biocompatible biomolecule pyridoxal-5′-phosphate (PLP) as a new organic phosphorus source. In this method, PLP biomolecules are hydrolyzed to produce phosphate ions under hydrothermal conditions, and these phosphate ions react with pre-existing calcium ions to form hydroxyapatite nanorods. The effects ofmore » experimental conditions including hydrothermal temperature and time on the morphology and crystal phase of the products are investigated. This method is simple, surfactant-free and environmentally friendly. The products are characterized by X-ray powder diffraction, scanning electron microscopy, transmission electron microscopy, Fourier transform infrared (FTIR) spectroscopy, and thermogravimetric (TG) analysis.« less

Biochemical and functional characterization of MRA-1571 of Mycobacterium tuberculosis H37Ra and effect of its down-regulation on survival in macrophages

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sharma, Rishabh; Keshari, Deepa; Singh, Kumar Sachin

Amino acid biosynthesis has emerged as a source of new drug targets as many bacterial strains auxotrophic for amino acids fail to proliferate under in vivo conditions. Branch chain amino acids (BCAAs) are important for Mycobacterium tuberculosis (Mtb) survival and strains deficient in their biosynthesis were attenuated for growth in mice. Threonine dehydratase (IlvA) is a pyridoxal-5-phosphate (PLP) dependent enzyme that catalyzes the first step in isoleucine biosynthesis. The MRA-1571 of Mycobacterium tuberculosis H37Ra (Mtb-Ra), annotated to be coding for IlvA, was cloned, expressed and purified. Purified protein was subsequently used for developing enzyme assay and to study its biochemical properties.more » Also, E. coli BL21 (DE3) IlvA knockout (E. coli-ΔilvA) was developed and genetically complemented with Mtb-Ra ilvA expression construct (pET32a-ilvA) to make complemented E. coli strain (E. coli-ΔilvA + pET32a-ilvA). The E. coli-ΔilvA showed growth failure in minimal medium but growth restoration was observed in E. coli-ΔilvA + pET32a-ilvA. E. coli-ΔilvA growth was also restored in the presence of isoleucine. The IlvA localization studies detected its distribution in cell wall and membrane fractions with relatively minor presence in cytosolic fraction. Maximum IlvA expression was observed at 72 h in wild-type (WT) Mtb-Ra infecting macrophages. Also, Mtb-Ra IlvA knockdown (KD) showed reduced survival in macrophages compared to WT and complemented strain (KDC). - Highlights: • Mtb-Ra gene MRA-1571 codes for a functional threonine dehydratase (IlvA). • IlvA is pyridoxal 5’-phosphate dependent and is inhibited by isoleucine. • E. coli IlvA knockout growth can be supplemented by isoleucine or by Mtb-Ra IlvA. • The enzyme is primarily localized in cell wall and membrane fractions. • IlvA knockdown Mtb-Ra shows reduced growth in macrophages.« less

Mutant form C115H of Clostridium sporogenes methionine γ-lyase efficiently cleaves S-Alk(en)yl-l-cysteine sulfoxides to antibacterial thiosulfinates.

PubMed

Kulikova, Vitalia V; Anufrieva, Natalya V; Revtovich, Svetlana V; Chernov, Alexander S; Telegin, Georgii B; Morozova, Elena A; Demidkina, Tatyana V

2016-10-01

Pyridoxal 5'-phosphate-dependent methionine γ-lyase (MGL) catalyzes the β-elimination reaction of S-alk(en)yl-l-cysteine sulfoxides to thiosulfinates, which possess antimicrobial activity. Partial inactivation of the enzyme in the course of the reaction occurs due to oxidation of active site cysteine 115 conserved in bacterial MGLs. In this work, the C115H mutant form of Clostridium sporogenes MGL was prepared and the steady-state kinetic parameters of the enzyme were determined. The substitution results in an increase in the catalytic efficiency of the mutant form towards S-substituted l-cysteine sulfoxides compared to the wild type enzyme. We used a sulfoxide/enzyme system to generate antibacterial activity in situ. Two-component systems composed of the mutant enzyme and three S-substituted l-cysteine sulfoxides were demonstrated to be effective against Gram-positive and Gram-negative bacteria and three clinical isolates from mice. © 2016 IUBMB Life, 68(10):830-835, 2016. © 2016 International Union of Biochemistry and Molecular Biology.

Pyridoxal phosphate synthases PdxS/PdxT are required for Actinobacillus pleuropneumoniae viability, stress tolerance and virulence.

PubMed

Xie, Fang; Li, Gang; Wang, Yalei; Zhang, Yanhe; Zhou, Long; Wang, Chengcheng; Liu, Shuanghong; Liu, Siguo; Wang, Chunlai

2017-01-01

Pyridoxal 5'-phosphate (PLP) is an essential cofactor for numerous enzymes involved in a diversity of cellular processes in living organisms. Previous analysis of the Actinobacillus pleuropneumoniae S-8 genome sequence revealed the presence of pdxS and pdxT genes, which are implicated in deoxyxylulose 5-phosphate (DXP)-independent pathway of PLP biosynthesis; however, little is known about their roles in A. pleuropneumoniae pathogenicity. Our data demonstrated that A. pleuropneumoniae could synthesize PLP by PdxS and PdxT enzymes. Disruption of the pdxS and pdxT genes rendered the pathogen auxotrophic for PLP, and the defective growth as a result of these mutants was chemically compensated by the addition of PLP, suggesting the importance of PLP production for A. pleuropneumoniae growth and viability. Additionally, the pdxS and pdxT deletion mutants displayed morphological defects as indicated by irregular and aberrant shapes in the absence of PLP. The reduced growth of the pdxS and pdxT deletion mutants under osmotic and oxidative stress conditions suggests that the PLP synthases PdxS/PdxT are associated with the stress tolerance of A. pleuropneumoniae. Furthermore, disruption of the PLP biosynthesis pathway led to reduced colonization and attenuated virulence of A. pleuropneumoniae in the BALB/c mouse model. The data presented in this study reveal the critical role of PLP synthases PdxS/PdxT in viability, stress tolerance, and virulence of A. pleuropneumoniae.

Purification and characterization of O-acetylserine (thiol) lyase from spinach chloroplasts.

PubMed

Droux, M; Martin, J; Sajus, P; Douce, R

1992-06-01

O-Acetylserine (thiol) lyase, the last enzyme in the cysteine biosynthetic pathway, was purified to homogeneity from spinach leaf chloroplasts. The enzyme has a molecular mass of 68,000 and consists of two identical subunits of Mr 35,000. The absorption spectrum obtained at pH 7.5 exhibited a peak at 407 nm due to pyridoxal phosphate, and addition of O-acetylserine induced a considerable modification of the spectrum. The pyridoxal phosphate content was found to be 1.1 per subunit of 35,000, and the chromophore was displaced from the enzyme by O-acetylserine, leading to a progressive inactivation of the holoenzyme. Upon gel filtration chromatography on Superdex 200, part of the chloroplastic O-acetylserine (thiol) lyase eluted in association with serine acetyltransferase at a position corresponding to a molecular mass of 310,000 (such a complex called cysteine synthase has been characterized in bacteria). The activity of O-acetylserine (thiol) lyase was optimum between pH 7.5 and 8.5. The apparent Km for O-acetylserine was 1.3 mM and for sulfide was 0.25 mM. The calculated activation energy was 12.6 kcal/mol at 10 mM O-acetylserine. The overall amino-acid composition of spinach chloroplast O-acetylserine (thiol) lyase was different than that determined for the same enzyme (cytosolic?) obtained from a crude extract of spinach leaves. A polyclonal antibody prepared against the chloroplastic O-acetylserine (thiol) lyase exhibited a very low cross-reactivity with a preparation of mitochondrial matrix and cytosolic proteins suggesting that the chloroplastic isoform was distinct from the mitochondrial and cytosolic counterparts.

Analysis of amadori-glycated phosphatidylethanolamine in the plasma of healthy subjects and diabetic patients by liquid chromatography-tandem mass spectrometry.

PubMed

Miyazawa, Teruo; Ibusuki, Daigo; Yamashita, Shinji; Nakagawa, Kiyotaka

2008-04-01

Peroxidized phospholipid-mediated cytotoxity, the abnormal increase in the levels of phosphatidylcholine hydroperoxide (PCOOH) found in the plasma of type 2 diabetic patients, is involved in the pathophysiology of many diseases. PCOOH accumulation may be related to Amadori-glycated phosphatidylethanolamine (deoxy-D-fructosyl PE, or Amadori-PE) because Amadori-PE causes oxidative stress. However, the occurrence of lipid glycation products, including Amadori-PE, in vivo remains unclear. We developed a method to analyze Amadori-PE by using quadrupole/linear ion-trap mass spectrometry, the Applied Biosystems 4000 Q TRAP. We found that pyridoxals could easily be condensed with PE before the glucose-PE reaction occurred. The PE-pyridoxal 5'-phosphate adduct was detectable in human red blood cells, and the increased plasma Amadori-PE concentration in streptozotocin-induced diabetic rats was decreased by dietary supplementation with pyridoxal 5'-phosphate. Therefore, it is likely that pyridoxal 5'-phosphate acts as a lipid glycation inhibitor in vivo, and this may contribute to diabetes prevention.

NMR studies of the protonation states of pyridoxal-5‧-phosphate in water

NASA Astrophysics Data System (ADS)

Chan-Huot, Monique; Niether, Christiane; Sharif, Shasad; Tolstoy, Peter M.; Toney, Michael D.; Limbach, Hans-Heinrich

2010-07-01

We have measured the 13C NMR spectra of the cofactor pyridoxal-5'-phosphate (vitamin B 6, PLP) at 278 K in aqueous solution as a function of pH. By 13C enrichment of PLP in the C-4' and C-5' positions we were able to measure spectra down to pH 1. From the dependence of the 13C chemical shifts on pH, the p Ka values of PLP could be determined. In particular, the heretofore uncharacterized protonation state of PLP, in which the phosphate group as well as the pyridine ring and the phenolic groups are fully protonated, has been analyzed. The corresponding p Ka value of 2.4 indicates that the phosphate group is solely involved in the first deprotonation step. The 15N chemical shifts of the pyridine ring of PLP published previously are in good agreement with the new results. These shifts contain information about the tautomerism of the different protonation states of PLP. The implications of these findings for the biological function of PLP are discussed.

Studying the antiemetic effect of vitamin B6 for morning sickness: pyridoxine and pyridoxal are prodrugs.

PubMed

Matok, Ilan; Clark, Shannon; Caritis, Steve; Miodovnik, Menachem; Umans, Jason G; Hankins, Gary; Mattison, Donald R; Koren, Gideon

2014-12-01

Vitamin B6 has been known to possess antiemetic effects since 1942. This water soluble compound has several forms in the circulation including pyridoxine, pyridoxal, and pyridoxal phosphate. The active antiemetic form of vitamin B6 is unknown. This was a pre-specified substudy of a randomized, placebo-controlled trial comparing the antiemetic effect of the doxylamine-vitamin B6 combination (Diclectin®) (n = 131) to placebo (n = 126) in women with nausea and vomiting of pregnancy. Serum concentrations of pyridoxine, pyridoxal, and pyridoxal 5' phosphate (PLP) and doxylamine were measured on Days 4, 8, and 15. With Diclectin® exhibiting a significant antiemetic effect in pregnancy, serum concentrations of pyridoxine were unmeasurable in almost all patients and those of pyridoxal were undetectable in half of patients. In contrast, PLP was measurable at sustained, stable steady-state levels in all patients. Our data suggest that there is a correlation between PLP levels and PUQE score of morning sickness symptoms when pyridoxine and pyridoxal levels are undetectable, and hence they might be prodrugs of PLP, which may be the active antiemetic form of vitamin B6. © 2014, The American College of Clinical Pharmacology.

Characterization of Enzymes Catalyzing Transformations of Cysteine S-Conjugated Intermediates in the Lincosamide Biosynthetic Pathway.

PubMed

Ushimaru, Richiro; Lin, Chia-I; Sasaki, Eita; Liu, Hung-Wen

2016-09-02

Lincosamides such as lincomycin A, celesticetin, and Bu-2545, constitute an important group of antibiotics. These natural products are characterized by a thiooctose linked to a l-proline residue, but they differ with regards to modifications of the thioacetal moiety, the pyrrolidine ring, and the octose core. Here we report that the pyridoxal 5'-phosphate-dependent enzyme CcbF (celesticetin biosynthetic pathway) is a decarboxylating deaminase that converts a cysteine S-conjugated intermediate into an aldehyde. In contrast, the homologous enzyme LmbF (lincomycin biosynthetic pathway) catalyzes C-S bond cleavage of the same intermediate to afford a thioglycoside. We show that Ccb4 and LmbG (downstream methyltransferases) convert the aldehyde and thiol intermediates into a variety of methylated lincosamide compounds including Bu-2545. The substrates used in these studies are the β-anomers of the natural substrates. The findings not only provide insight into how the biosynthetic pathway of lincosamide antibiotics can bifurcate to generate different lincosamides, but also reveal the promiscuity of the enzymes involved. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Inhibition of the Induced Formation of Tryptophanase in Escherichia coli by Near-Ultraviolet Radiation

PubMed Central

Swenson, P. A.; Setlow, R. B.

1970-01-01

Induced formation of tryptophanase in Escherichia coli B/r is temporarily inhibited by near-ultraviolet (UV) irradiation. The inhibition is greater when irradiation is at 5 C than when at room temperature. Hence, the inhibition is the result of a photochemical, rather than photoenzymatic, alteration of some cellular component. The action spectrum has a peak in the region of 334 nm and is similar to that for growth delay. However, inhibition of tryptophanase formation is more sensitive to near-UV irradiation than are growth, respiration, and the induced formation of β-galactosidase. Thus, for tryptophanase the lack of formation cannot be due to general inhibition of metabolism. Pyridoxal phosphate absorbs in the near-UV region of the spectrum and is a cofactor for tryptophanase, but this enzyme in induced cells is not inactivated by near UV-radiations. An experiment in which toluene-treated suspensions from irradiated and unirradiated cells were mixed showed that irradiation does not cause the formation of an inhibitor of tryptophanase activity. The possibility remains that the absorption of radiant energy by pyridoxal phosphate interferes with the synthesis of tryptophanase. PMID:4914082

Coenzyme Q10 and Pyridoxal Phosphate Deficiency Is a Common Feature in Mucopolysaccharidosis Type III.

PubMed

Yubero, Dèlia; Montero, Raquel; O'Callaghan, Mar; Pineda, Mercè; Meavilla, Silvia; Delgadillo, Veronica; Sierra, Cristina; Altimira, Laura; Navas, Plácido; Pope, Simon; Oppenheim, Marcus; Neergheen, Viruna; Ghosh, Arunabha; Mills, Phillipa; Clayton, Peter; Footitt, Emma; Cleary, Maureen; Hargreaves, Iain; Jones, Simon A; Heales, Simon; Artuch, Rafael

2016-01-01

Mucopolysaccharidoses (MPS) are a group of lysosomal storage disorders caused by deficiencies of lysosomal enzymes catalyzing degradation of glycosaminoglycans (GAGs). Previously, we reported a secondary plasma coenzyme Q 10 (CoQ) deficiency in MPS patients. For this study, nine MPS patients were recruited in the Hospital Sant Joan de Déu (HSJD, Barcelona) and two patients in the Neurometabolic Unit, National Hospital (NMU, London), to explore the nutritional status of MPS type III patients by analyzing several vitamins and micronutrients in blood and in cerebrospinal fluid. Plasma CoQ and plasma and cerebrospinal fluid pyridoxal phosphate (PLP) content were analyzed by high-pressure liquid chromatography (HPLC) with electrochemical and fluorescence detection, respectively. We found that most MPS-III patients disclosed low plasma pyridoxal phosphate (PLP) values (seven out of nine) and also low plasma CoQ concentrations (eight out of nine). We observed significantly lower median values of PLP, tocopherol, and CoQ (Mann-Whitney U test, p = 0.006, p = 0.004, and p = 0.001, respectively) in MPS patients when compared with age-matched controls. Chi-square test showed a significant association between the fact of having low plasma PLP and CoQ values in the whole cohort of patients. Cerebrospinal fluid PLP values were clearly deficient in the two patients studied. In conclusion, we report a combined CoQ and PLP deficiency in MPS-III patients. These observations could be related to the complexity of the physiopathology of the disease. If our results are confirmed in larger series of patients, CoQ and PLP therapy could be trialed as coadjuvant therapy with the current MPS treatments.

Folding pathway of the pyridoxal 5′-phosphate C-S lyase MalY from Escherichia coli

PubMed Central

2005-01-01

MalY from Escherichia coli is a bifunctional dimeric PLP (pyridoxal 5′-phosphate) enzyme acting as a β-cystathionase and as a repressor of the maltose system. The spectroscopic and molecular properties of the holoenzyme, in the untreated and NaBH4-treated forms, and of the apoenzyme have been elucidated. A systematic study of the urea-induced unfolding of MalY has been monitored by gel filtration, cross-linking, ANS (8-anilino-1-naphthalenesulphonic acid) binding and by visible, near- and far-UV CD, fluorescence and NMR spectroscopies under equilibrium conditions. Unfolding proceeds in at least three stages. The first transition, occurring between 0 and 1 M urea, gives rise to a partially active dimeric species that binds PLP. The second equilibrium transition involving dimer dissociation, release of PLP and loss of lyase activity leads to the formation of a monomeric equilibrium intermediate. It is a partially unfolded molecule that retains most of the native-state secondary structure, binds significant amounts of ANS (a probe for exposed hydrophobic surfaces) and tends to self-associate. The self-associated aggregates predominate at urea concentrations of 2–4 M for holoMalY. The third step represents the complete unfolding of the enzyme. These results when compared with the urea-induced unfolding profiles of apoMalY and NaBH4-reduced holoenzyme suggest that the coenzyme group attached to the active-site lysine residue increases the stability of the dimeric enzyme. Both holo- and apo-MalY could be successfully refolded into the active enzyme with an 85% yield. Further refolding studies suggest that large misfolded soluble aggregates that cannot be refolded could be responsible for the incomplete re-activation. PMID:15823094

Racemization of alanine by the alanine racemases from Salmonella typhimurium and Bacillus stearothermophilus: energetic reaction profiles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Faraci, W.S.; Walsh, C.T.

1988-05-03

Alanine racemases are bacterial pyridoxal 5'-phosphate (PLP) dependent enzymes providing D-alanine as an essential building block for biosynthesis of the peptidoglycan layer of the cell wall. Two isozymic alanine racemases, encoded by the dadB gene and the alr gene, from the Gram-negative mesophilic Salmonella typhimurium and one from the Gram-positive thermophilic Bacillus stearothermophilus have been examined for the racemization mechanism. Substrate deuterium isotope effects and solvent deuterium isotope effects have been measured in both L ..-->.. D and D..-->.. L directions for all three enzymes to assess the degree to which abstraction of the ..cap alpha..-proton or protonation of substratemore » PLP carbanion is limiting in catalysis. Additionally, experiments measuring internal return of ..cap alpha..-/sup 3/H from substrate to product and solvent exchange/substrate conversion experiments in /sup 3/H/sub 2/O have been used with each enzyme to examine the partitioning of substrate PLP carbanion intermediates and to obtain the relative heights of kinetically significant energy barriers in alanine racemase catalysis.« less

Snapshots of C-S Cleavage in Egt2 Reveals Substrate Specificity and Reaction Mechanism.

PubMed

Irani, Seema; Naowarojna, Nathchar; Tang, Yang; Kathuria, Karan R; Wang, Shu; Dhembi, Anxhela; Lee, Norman; Yan, Wupeng; Lyu, Huijue; Costello, Catherine E; Liu, Pinghua; Zhang, Yan Jessie

2018-05-17

Sulfur incorporation in the biosynthesis of ergothioneine, a histidine thiol derivative, differs from other well-characterized transsulfurations. A combination of a mononuclear non-heme iron enzyme-catalyzed oxidative C-S bond formation and a subsequent pyridoxal 5'-phosphate (PLP)-mediated C-S lyase reaction leads to the net transfer of a sulfur atom from a cysteine to a histidine. In this study, we structurally and mechanistically characterized a PLP-dependent C-S lyase Egt2, which mediates the sulfoxide C-S bond cleavage in ergothioneine biosynthesis. A cation-π interaction between substrate and enzyme accounts for Egt2's preference of sulfoxide over thioether as a substrate. Using mutagenesis and structural biology, we captured three distinct states of the Egt2 C-S lyase reaction cycle, including a labile sulfenic intermediate captured in Egt2 crystals. Chemical trapping and high-resolution mass spectrometry were used to confirm the involvement of the sulfenic acid intermediate in Egt2 catalysis. Copyright © 2018 Elsevier Ltd. All rights reserved.

Cloning and characterization of d-threonine aldolase from the green alga Chlamydomonas reinhardtii.

PubMed

Hirato, Yuki; Tokuhisa, Mayumi; Tanigawa, Minoru; Ashida, Hiroyuki; Tanaka, Hiroyuki; Nishimura, Katsushi

2017-03-01

d-Threonine aldolase (DTA) catalyzes the pyridoxal 5'-phosphate (PLP)-dependent interconversion of d-threonine and glycine plus acetaldehyde. The enzyme is a powerful tool for the stereospecific synthesis of various β-hydroxy amino acids in synthetic organic chemistry. In this study, DTA from the green alga Chlamydomonas reinhardtii was discovered and characterized, representing the first report to describe the existence of eukaryotic DTA. DTA was overexpressed in recombinant Escherichia coli BL21 (DE3) cells; the specific activity of the enzyme in the cell-free extract was 0.8 U/mg. The recombinant enzyme was purified to homogeneity by ammonium sulfate fractionation, DEAE-Sepharose, and Mono Q column chromatographies (purified enzyme 7.0 U/mg). For the cleavage reaction, the optimal temperature and pH were 70 °C and pH 8.4, respectively. The enzyme demonstrated 90% of residual activity at 50 °C for 1 h. The enzyme catalyzed the synthesis of d- and d-allo threonine from a mixture of glycine and acetaldehyde (the diastereomer excess of d-threonine was 18%). DTA was activated by several divalent metal ions, including manganese, and was inhibited by PLP enzyme inhibitors and metalloenzyme inhibitors. Copyright © 2016 Elsevier Ltd. All rights reserved.

Evolution of Substrate Specificity within a Diverse Family of [beta/alpha]-Barrel-fold Basic Amino Acid Decarboxylases X-ray Structure Determination of Enzymes with Specificity for L-Arginine and Carboxynorspermidine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Deng, Xiaoyi; Lee, Jeongmi; Michael, Anthony J.

2010-08-26

Pyridoxal 5{prime}-phosphate (PLP)-dependent basic amino acid decarboxylases from the {beta}/{alpha}-barrel-fold class (group IV) exist in most organisms and catalyze the decarboxylation of diverse substrates, essential for polyamine and lysine biosynthesis. Herein we describe the first x-ray structure determination of bacterial biosynthetic arginine decarboxylase (ADC) and carboxynorspermidine decarboxylase (CANSDC) to 2.3- and 2.0-{angstrom} resolution, solved as product complexes with agmatine and norspermidine. Despite low overall sequence identity, the monomeric and dimeric structures are similar to other enzymes in the family, with the active sites formed between the {beta}/{alpha}-barrel domain of one subunit and the {beta}-barrel of the other. ADC contains bothmore » a unique interdomain insertion (4-helical bundle) and a C-terminal extension (3-helical bundle) and it packs as a tetramer in the asymmetric unit with the insertions forming part of the dimer and tetramer interfaces. Analytical ultracentrifugation studies confirmed that the ADC solution structure is a tetramer. Specificity for different basic amino acids appears to arise primarily from changes in the position of, and amino acid replacements in, a helix in the {beta}-barrel domain we refer to as the 'specificity helix.' Additionally, in CANSDC a key acidic residue that interacts with the distal amino group of other substrates is replaced by Leu{sup 314}, which interacts with the aliphatic portion of norspermidine. Neither product, agmatine in ADC nor norspermidine in CANSDC, form a Schiff base to pyridoxal 5{prime}-phosphate, suggesting that the product complexes may promote product release by slowing the back reaction. These studies provide insight into the structural basis for the evolution of novel function within a common structural-fold.« less

Vitamin B6 metabolism in microbes and approaches for fermentative production.

PubMed

Rosenberg, Jonathan; Ischebeck, Till; Commichau, Fabian M

Vitamin B6 is a designation for the six vitamers pyridoxal, pyridoxine, pyridoxamine, pyridoxal 5'-phosphate (PLP), pyridoxine 5'-phosphate, and pyridoxamine. PLP, being the most important B6 vitamer, serves as a cofactor for many proteins and enzymes. In contrast to other organisms, animals and humans have to ingest vitamin B6 with their food. Several disorders are associated with vitamin B6 deficiency. Moreover, pharmaceuticals interfere with metabolism of the cofactor, which also results in vitamin B6 deficiency. Therefore, vitamin B6 is a valuable compound for the pharmaceutical and the food industry. Although vitamin B6 is currently chemically synthesized, there is considerable interest on the industrial side to shift from chemical processes to sustainable fermentation technologies. Here, we review recent findings regarding biosynthesis and homeostasis of vitamin B6 and describe the approaches that have been made in the past to develop microbial production processes. Moreover, we will describe novel routes for vitamin B6 biosynthesis and discuss their potential for engineering bacteria that overproduce the commercially valuable substance. We also highlight bottlenecks of the vitamin B6 biosynthetic pathways and propose strategies to circumvent these limitations. Copyright © 2016 Elsevier Inc. All rights reserved.

Evolutionary analysis of a novel zinc ribbon in the N-terminal region of threonine synthase.

PubMed

Kaur, Gurmeet; Subramanian, Srikrishna

2017-10-18

Threonine synthase (TS) catalyzes the terminal reaction in the biosynthetic pathway of threonine and requires pyridoxal phosphate as a cofactor. TSs share a common catalytic domain with other fold type II PALP dependent enzymes. TSs are broadly grouped into two classes based on their sequence, quaternary structure, and enzyme regulation. We report the presence of a novel zinc ribbon domain in the N-terminal region preceding the catalytic core in TS. The zinc ribbon domain is present in TSs belonging to both classes. Our sequence analysis reveals that archaeal TSs possess all zinc chelating residues to bind a metal ion that are lacking in the structurally characterized homologs. Phylogenetic analysis suggests that TSs with an N-terminal zinc ribbon likely represents the ancestral state of the enzyme while TSs without a zinc ribbon must have diverged later in specific lineages. The zinc ribbon and its N- and C-terminal extensions are important for enzyme stability, activity and regulation. It is likely that the zinc ribbon domain is involved in higher order oligomerization or mediating interactions with other biomolecules leading to formation of larger metabolic complexes.

Chelation of UO(2)(2+) by vitamin B6 complex derivatives: synthesis and characterization of [UO2(beta-pyracinide)2(H2O)] and [UO2(Pyr2en)DMSO]Cl2{Pyr2en=N,N'-ethylenebis(pyridoxylideneiminato)}. A useful modeling of assimilation of uranium by living beings.

PubMed

Back, Davi Fernando; de Oliveira, Gelson Manzoni; Lang, Ernesto Schulz

2006-10-01

The vitamin B(6) derivatives 4-pyridoxic acid (anionic) and the Schiff base N,N'-ethylenebis(pyridoxylideneiminato) react with UO(2)(NO(3))(2) * 6H(2)O to give [UO(2)(beta-pyracinide)(2)(H(2)O)] (beta-pyracin=4-pyridoxic acid) and [UO(2)(Pyr(2)en)DMSO]Cl(2)(Pyr(2)en=N,N'-ethylenebis(pyridoxylideneiminato); DMSO=dimethyl sulfoxide). In both compounds the two uranyl oxo ligands set the axis of distorted pentagonal bipyramides. The ability of vitamin B(6) derivatives to react with UO(2)(2+) allowing the chelation of one uranium atom represents a very specific model of assimilation of uranium by living beings. It could also explain the serious damages caused by heavy or radioactive metals like uranium since their complexation "in vivo" by enzymatic systems like pyridoxal phosphate-containing enzymes would lead to a modification of the prosthetic groups of the metalloenzymes with loss of their catalytic activities.

Evolutionarily conserved regions and hydrophobic contacts at the superfamily level: The case of the fold-type I, pyridoxal-5′-phosphate-dependent enzymes

PubMed Central

Paiardini, Alessandro; Bossa, Francesco; Pascarella, Stefano

2004-01-01

The wealth of biological information provided by structural and genomic projects opens new prospects of understanding life and evolution at the molecular level. In this work, it is shown how computational approaches can be exploited to pinpoint protein structural features that remain invariant upon long evolutionary periods in the fold-type I, PLP-dependent enzymes. A nonredundant set of 23 superposed crystallographic structures belonging to this superfamily was built. Members of this family typically display high-structural conservation despite low-sequence identity. For each structure, a multiple-sequence alignment of orthologous sequences was obtained, and the 23 alignments were merged using the structural information to obtain a comprehensive multiple alignment of 921 sequences of fold-type I enzymes. The structurally conserved regions (SCRs), the evolutionarily conserved residues, and the conserved hydrophobic contacts (CHCs) were extracted from this data set, using both sequence and structural information. The results of this study identified a structural pattern of hydrophobic contacts shared by all of the superfamily members of fold-type I enzymes and involved in native interactions. This profile highlights the presence of a nucleus for this fold, in which residues participating in the most conserved native interactions exhibit preferential evolutionary conservation, that correlates significantly (r = 0.70) with the extent of mean hydrophobic contact value of their apolar fraction. PMID:15498941

Reaction Mechanism and Molecular Basis for Selenium/Sulfur Discrimination of Selenocysteine Lyase*

PubMed Central

Omi, Rie; Kurokawa, Suguru; Mihara, Hisaaki; Hayashi, Hideyuki; Goto, Masaru; Miyahara, Ikuko; Kurihara, Tatsuo; Hirotsu, Ken; Esaki, Nobuyoshi

2010-01-01

Selenocysteine lyase (SCL) catalyzes the pyridoxal 5′-phosphate-dependent removal of selenium from l-selenocysteine to yield l-alanine. The enzyme is proposed to function in the recycling of the micronutrient selenium from degraded selenoproteins containing selenocysteine residue as an essential component. The enzyme exhibits strict substrate specificity toward l-selenocysteine and no activity to its cognate l-cysteine. However, it remains unclear how the enzyme distinguishes between selenocysteine and cysteine. Here, we present mechanistic studies of selenocysteine lyase from rat. ESI-MS analysis of wild-type and C375A mutant SCL revealed that the catalytic reaction proceeds via the formation of an enzyme-bound selenopersulfide intermediate on the catalytically essential Cys-375 residue. UV-visible spectrum analysis and the crystal structure of SCL complexed with l-cysteine demonstrated that the enzyme reversibly forms a nonproductive adduct with l-cysteine. Cys-375 on the flexible loop directed l-selenocysteine, but not l-cysteine, to the correct position and orientation in the active site to initiate the catalytic reaction. These findings provide, for the first time, the basis for understanding how trace amounts of a selenium-containing substrate is distinguished from excessive amounts of its cognate sulfur-containing compound in a biological system. PMID:20164179

Applications of vitamin B6 cofactor pyridoxal 5‧-phosphate and pyridoxal 5‧-phosphate crowned gold nanoparticles for optical sensing of metal ions

NASA Astrophysics Data System (ADS)

Bothra, Shilpa; Upadhyay, Yachana; Kumar, Rajender; Sahoo, Suban K.

2017-03-01

Vitamin B6 cofactor pyridoxal 5‧-phosphate (PLP) and PLP crowned gold nanoparticles (PLP-AuNPs) was applied for the optical chemosensing of metal ions in aqueous medium. PLP showed a visually detectable colour change from colourless to yellow and 'turn-off' fluorescence in the presence of Fe3 +. The fluorescence intensity of PLP at 433 nm was also blue-shifted and enhanced at 395 nm upon addition of Al3 +. When the PLP was functionalized over AuNPs surface, the wine red colour of PLP-AuNPs was turned to purplish-blue and the SPR band at 525 nm was red-shifted upon addition of Al3 +, Cd2 + and Pb2 + due to the complexation-induced aggregation of nanoparticles. The developed sensing systems exhibited good selectivity and specificity for the detected analytes (Fe3 +, Al3 +, Cd2 + and Pb2 +).

Pyridoxal 5'-phosphate, pyridoxal, and 4-pyridoxic acid in the paired serum and cerebrospinal fluid of children.

PubMed

Akiyama, Tomoyuki; Hayashi, Yumiko; Hanaoka, Yoshiyuki; Shibata, Takashi; Akiyama, Mari; Tsuchiya, Hiroki; Yamaguchi, Tokito; Kobayashi, Katsuhiro

2017-09-01

We quantified pyridoxal 5'-phosphate (PLP), pyridoxal (PL), and 4-pyridoxic acid (PA) in paired serum and cerebrospinal fluid (CSF) samples from children and investigated the effect of age on the concentrations and CSF-to-serum ratios of these vitamers. Serum and CSF samples prospectively collected from 49 pediatric patients were analyzed. PLP, PL, and PA were measured using high-performance liquid chromatography with fluorescence detection, using pre-column derivatization by semicarbazide. Effects of age on these vitamers, the PLP-to-PL ratio, CSF-to-serum PLP ratio, and CSF-to-serum PL ratio were evaluated using correlation analysis. The PLP, PL, and PA concentrations in the serum and CSF were higher at younger ages, except for CSF PA concentrations that were mostly below the limit of detection (<1.2nmol/l). The PLP-to-PL ratios in the serum and CSF correlated positively with age. The CSF-to-serum PLP ratio and CSF-to-serum PL ratio were independent of age. Age-related changes in PLP, PL, and PA in serum and in CSF from pediatric patients and CSF-to-serum ratios of PLP and PL demonstrated in this study will provide valuable information for evaluating PLP supply to the central nervous system from the peripheral blood. Copyright © 2017 Elsevier B.V. All rights reserved.

Structures of Bacterial Biosynthetic Arginine Decarboxylases

DOE Office of Scientific and Technical Information (OSTI.GOV)

F Forouhar; S Lew; J Seetharaman

2011-12-31

Biosynthetic arginine decarboxylase (ADC; also known as SpeA) plays an important role in the biosynthesis of polyamines from arginine in bacteria and plants. SpeA is a pyridoxal-5'-phosphate (PLP)-dependent enzyme and shares weak sequence homology with several other PLP-dependent decarboxylases. Here, the crystal structure of PLP-bound SpeA from Campylobacter jejuni is reported at 3.0 {angstrom} resolution and that of Escherichia coli SpeA in complex with a sulfate ion is reported at 3.1 {angstrom} resolution. The structure of the SpeA monomer contains two large domains, an N-terminal TIM-barrel domain followed by a {beta}-sandwich domain, as well as two smaller helical domains. Themore » TIM-barrel and {beta}-sandwich domains share structural homology with several other PLP-dependent decarboxylases, even though the sequence conservation among these enzymes is less than 25%. A similar tetramer is observed for both C. jejuni and E. coli SpeA, composed of two dimers of tightly associated monomers. The active site of SpeA is located at the interface of this dimer and is formed by residues from the TIM-barrel domain of one monomer and a highly conserved loop in the {beta}-sandwich domain of the other monomer. The PLP cofactor is recognized by hydrogen-bonding, {pi}-stacking and van der Waals interactions.« less

Crystal Structure of Serine Racemase that Produces Neurotransmitter d-Serine for Stimulation of the NMDA Receptor

NASA Astrophysics Data System (ADS)

Goto, Masaru

d-Serine is an endogenous coagonist for the N-methyl-d-aspartate receptor and is involved in excitatory neurotransmission in the brain. Mammalian pyridoxal 5’-phosphate-dependent serine racemase, which is localized in the mammalian brain, catalyzes the racemization of l-serine to yield d-serine and vice versa. We have determined the structures of three forms of the mammalian enzyme homolog from Schizosaccharomyces pombe. Lys57 and Ser82 located on the protein and solvent sides, respectively, with respect to the cofactor plane, are acid-base catalysts that shuttle protons to the substrate. The modified enzyme, which has a unique lysino-d-alanyl residue at the active site, also binds the substrate serine in the active site, suggesting that the lysino-d-alanyl residue acts as a catalytic base in the same manner as Lys57 of the wild type enzyme.

Molecular dynamics simulations of apo, holo, and inactivator bound GABA-at reveal the role of active site residues in PLP dependent enzymes.

PubMed

Gökcan, Hatice; Monard, Gerald; Sungur Konuklar, F Aylin

2016-07-01

The pyridoxal 5-phosphate (PLP) cofactor is a significant organic molecule in medicinal chemistry. It is often found covalently bound to lysine residues in proteins to form PLP dependent enzymes. An example of this family of PLP dependent enzymes is γ-aminobutyric acid aminotransferase (GABA-AT) which is responsible for the degradation of the neurotransmitter GABA. Its inhibition or inactivation can be used to prevent the reduction of GABA concentration in brain which is the source of several neurological disorders. As a test case for PLP dependent enzymes, we have performed molecular dynamics simulations of GABA-AT to reveal the roles of the protein residues and its cofactor. Three different states have been considered: the apoenzyme, the holoenzyme, and the inactive state obtained after the suicide inhibition by vigabatrin. Different protonation states have also been considered for PLP and two key active site residues: Asp298 and His190. Together, 24 independent molecular dynamics trajectories have been simulated for a cumulative total of 2.88 µs. Our results indicate that, unlike in aqueous solution, the PLP pyridine moiety is protonated in GABA-AT. This is a consequence of a pKa shift triggered by a strong charge-charge interaction with an ionic "diad" formed by Asp298 and His190 that would help the activation of the first half-reaction of the catalytic mechanism in GABA-AT: the conversion of PLP to free pyridoxamine phosphate (PMP). In addition, our MD simulations exhibit additional strong hydrogen bond networks between the protein and PLP: the phosphate group is held in place by the donation of at least three hydrogen bonds while the carbonyl oxygen of the pyridine ring interacts with Gln301; Phe181 forms a π-π stacking interaction with the pyridine ring and works as a gate keeper with the assistance of Val300. All these interactions are hypothesized to help maintain free PMP in place inside the protein active site to facilitate the second half-reaction in GABA-AT: the regeneration of PLP-bound GABA-AT (i.e., the holoenzyme). Proteins 2016; 84:875-891. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lima,Santiago; Kumar,Sunil; Gawandi,Vijay

Homo sapiens kynureninase is a pyridoxal-5'-phosphate dependent enzyme that catalyzes the hydrolytic cleavage of 3-hydroxykynurenine to yield 3-hydroxyanthranilate and L-alanine as part of the tryptophan catabolic pathway leading to the de novo biosynthesis of NAD{sup +}. This pathway results in quinolinate, an excitotoxin that is an NMDA receptor agonist. High levels of quinolinate have been correlated with the etiology of neurodegenerative disorders such as AIDS-related dementia and Alzheimer's disease. We have synthesized a novel kynureninase inhibitor, 3-hydroxyhippurate, cocrystallized it with human kynureninase, and solved the atomic structure. On the basis of an analysis of the complex, we designed a seriesmore » of His-102, Ser-332, and Asn-333 mutants. The H102W/N333T and H102W/S332G/N333T mutants showed complete reversal of substrate specificity between 3-hydroxykynurenine and L-kynurenine, thus defining the primary residues contributing to substrate specificity in kynureninases.« less

Crystal structure and confirmation of the alanine:glyoxylate aminotransferase activity of the YFL030w yeast protein.

PubMed

Meyer, Philippe; Liger, Dominique; Leulliot, Nicolas; Quevillon-Cheruel, Sophie; Zhou, Cong-Zhao; Borel, Franck; Ferrer, Jean-Luc; Poupon, Anne; Janin, Joël; van Tilbeurgh, Herman

2005-12-01

We have determined the three-dimensional crystal structure of the protein encoded by the open reading frame YFL030w from Saccharomyces cerevisiae to a resolution of 2.6 A using single wavelength anomalous diffraction. YFL030w is a 385 amino-acid protein with sequence similarity to the aminotransferase family. The structure of the protein reveals a homodimer adopting the fold-type I of pyridoxal 5'-phosphate (PLP)-dependent aminotransferases. The PLP co-factor is covalently bound to the active site in the crystal structure. The protein shows close structural resemblance with the human alanine:glyoxylate aminotransferase (EC 2.6.1.44), an enzyme involved in the hereditary kidney stone disease primary hyperoxaluria type 1. In this paper we show that YFL030w codes for an alanine:glyoxylate aminotransferase, highly specific for its amino donor and acceptor substrates.

Molecular insights into primary hyperoxaluria type 1 pathogenesis.

PubMed

Cellini, Barbara; Oppici, Elisa; Paiardini, Alessandro; Montioli, Riccardo

2012-01-01

Primary hyperoxaluria type 1 (PH1) is a rare autosomal recessive disorder of glyoxylate metabolism caused by the deficiency of liver peroxisomal alanine:glyoxylate aminotransferase (AGT), a pyridoxal 5'-phosphate (PLP)-dependent enzyme. The PH1 pathogenesis is mostly due to single point mutations (more than 150 so far identified) on the AGXT gene, and is characterized by a marked heterogeneity in terms of genotype, enzymatic and clinical phenotypes. This article presents an up to date review of selected aspects of the biochemical properties of the two allelic forms of AGT and of some PH1-causing variants. These recent discoveries highlight the effects at the protein level of the pathogenic mutations, and, together with previous cell biology and clinical data, (i) improve the understanding of the molecular basis of PH1 pathogenesis, and (ii) help to delineate perspectives for predicting the response to pyridoxine treatment or for suggesting new strategies for PH1 patients bearing the analyzed mutations.

The value of plasma vitamin B6 profiles in early onset epileptic encephalopathies.

PubMed

Mathis, Déborah; Abela, Lucia; Albersen, Monique; Bürer, Céline; Crowther, Lisa; Beese, Karin; Hartmann, Hans; Bok, Levinus A; Struys, Eduard; Papuc, Sorina M; Rauch, Anita; Hersberger, Martin; Verhoeven-Duif, Nanda M; Plecko, Barbara

2016-09-01

Recent decades have unravelled the molecular background of a number of inborn errors of metabolism (IEM) causing vitamin B6-dependent epilepsy. As these defects interfere with vitamin B6 metabolism by different mechanisms, the plasma vitamin B6 profile can give important clues for further molecular work-up. This has so far been investigated in only a small number of patients. We evaluated the vitamin B6 vitamers pyridoxal 5'-phosphate (PLP), pyridoxal (PL), pyridoxamine (PM), pyridoxine (PN) and the catabolite pyridoxic acid (PA) in the so far largest patient cohort: reference (n = 50); pyridox(am)ine 5'-phosphate oxidase (PNPO) deficiency (n = 6); antiquitin (ATQ) deficiency (n = 21); tissue non-specific alkaline phosphatase (TNSALP) deficiency (n = 2) and epileptic encephalopathy (EE) of unknown etiology tested negative for ATQ and PNPO deficiency (n = 64). High plasma PM concentration was found in all patients with PNPO deficiency irrespective of vitamin B6 supplementation. Their PM concentration and the PM/PA ratio was significantly higher (p < 0.0001), compared to any other patients analysed. One patient with TNSALP deficiency and sampling prior to PN supplementation had markedly elevated plasma PLP concentration. On PN supplementation, patients with TNSALP deficiency, ATQ deficiency and patients of the EE cohort had similar plasma vitamin B6 profiles that merely reflect the intake of supra-physiological doses of vitamin B6. The interval of sampling to the last PN intake strongly affected the plasma concentrations of PN, PL and PA. PM concentrations and the PM/PA ratio clearly separated PNPO-deficient patients from the other cohorts. The plasma PM/PA ratio thus represents a robust biomarker for the selective screening of PNPO deficiency.

The Parkinson's disease death rate: carbidopa and vitamin B6.

PubMed

Hinz, Marty; Stein, Alvin; Cole, Ted

2014-01-01

The only indication for carbidopa and benserazide is the management of L-3,4-dihydroxyphenylalanine (L-dopa)-induced nausea. Both drugs irreversibly bind to and permanently deactivate pyridoxal 5'-phosphate (PLP), the active form of vitamin B6, and PLP-dependent enzymes. PLP is required for the function of over 300 enzymes and proteins. Virtually every major system in the body is impacted directly or indirectly by PLP. The administration of carbidopa and benserazide potentially induces a nutritional catastrophe. During the first 15 years of prescribing L-dopa, a decreasing Parkinson's disease death rate was observed. Then, in 1976, 1 year after US Food and Drug Administration approved the original L-dopa/carbidopa combination drug, the Parkinson's disease death rate started increasing. This trend has continued to the present, for 38 years and counting. The previous literature documents this increasing death rate, but no hypothesis has been offered concerning this trend. Carbidopa is postulated to contribute to the increasing Parkinson's disease death rate and to the classification of Parkinson's as a progressive neurodegenerative disease. It may contribute to L-dopa tachyphylaxis.

The Parkinson’s disease death rate: carbidopa and vitamin B6

PubMed Central

Hinz, Marty; Stein, Alvin; Cole, Ted

2014-01-01

The only indication for carbidopa and benserazide is the management of L-3,4-dihydroxyphenylalanine (L-dopa)-induced nausea. Both drugs irreversibly bind to and permanently deactivate pyridoxal 5′-phosphate (PLP), the active form of vitamin B6, and PLP-dependent enzymes. PLP is required for the function of over 300 enzymes and proteins. Virtually every major system in the body is impacted directly or indirectly by PLP. The administration of carbidopa and benserazide potentially induces a nutritional catastrophe. During the first 15 years of prescribing L-dopa, a decreasing Parkinson’s disease death rate was observed. Then, in 1976, 1 year after US Food and Drug Administration approved the original L-dopa/carbidopa combination drug, the Parkinson’s disease death rate started increasing. This trend has continued to the present, for 38 years and counting. The previous literature documents this increasing death rate, but no hypothesis has been offered concerning this trend. Carbidopa is postulated to contribute to the increasing Parkinson’s disease death rate and to the classification of Parkinson’s as a progressive neurodegenerative disease. It may contribute to L-dopa tachyphylaxis. PMID:25364278

Crystal structure of Escherichia coli diaminopropionate ammonia-lyase reveals mechanism of enzyme activation and catalysis.

PubMed

Bisht, Shveta; Rajaram, Venkatesan; Bharath, Sakshibeedu R; Kalyani, Josyula Nitya; Khan, Farida; Rao, Appaji N; Savithri, Handanahal S; Murthy, Mathur R N

2012-06-08

Pyridoxal 5'-phosphate (PLP)-dependent enzymes utilize the unique chemistry of a pyridine ring to carry out diverse reactions involving amino acids. Diaminopropionate (DAP) ammonia-lyase (DAPAL) is a prokaryotic PLP-dependent enzyme that catalyzes the degradation of d- and l-forms of DAP to pyruvate and ammonia. Here, we report the first crystal structure of DAPAL from Escherichia coli (EcDAPAL) in tetragonal and monoclinic forms at 2.0 and 2.2 Å resolutions, respectively. Structures of EcDAPAL soaked with substrates were also determined. EcDAPAL has a typical fold type II PLP-dependent enzyme topology consisting of a large and a small domain with the active site at the interface of the two domains. The enzyme is a homodimer with a unique biological interface not observed earlier. Structure of the enzyme in the tetragonal form had PLP bound at the active site, whereas the monoclinic structure was in the apo-form. Analysis of the apo and holo structures revealed that the region around the active site undergoes transition from a disordered to ordered state and assumes a conformation suitable for catalysis only upon PLP binding. A novel disulfide was found to occur near a channel that is likely to regulate entry of ligands to the active site. EcDAPAL soaked with dl-DAP revealed density at the active site appropriate for the reaction intermediate aminoacrylate, which is consistent with the observation that EcDAPAL has low activity under crystallization conditions. Based on the analysis of the structure and results of site-directed mutagenesis, a two-base mechanism of catalysis involving Asp(120) and Lys(77) is suggested.

Efficient Production of γ-GABA Using Recombinant E. coli Expressing Glutamate Decarboxylase (GAD) Derived from Eukaryote Saccharomyces cerevisiae.

PubMed

Xiong, Qiang; Xu, Zheng; Xu, Lu; Yao, Zhong; Li, Sha; Xu, Hong

2017-12-01

γ-Aminobutyric acid (γ-GABA) is a non-proteinogenic amino acid, which acts as a major regulator in the central nervous system. Glutamate decarboxylase (namely GAD, EC 4.1.1.15) is known to be an ideal enzyme for γ-GABA production using L-glutamic acid as substrate. In this study, we cloned and expressed GAD gene from eukaryote Saccharomyces cerevisiae (ScGAD) in E. coli BL21(DE3). This enzyme was further purified and its optimal reaction temperature and pH were 37 °C and pH 4.2, respectively. The cofactor of ScGAD was verified to be either pyridoxal 5'-phosphate (PLP) or pyridoxal hydrochloride. The optimal concentration of either cofactor was 50 mg/L. The optimal medium for E. coli-ScGAD cultivation and expression were 10 g/L lactose, 5 g/L glycerol, 20 g/L yeast extract, and 10 g/L sodium chloride, resulting in an activity of 55 U/mL medium, three times higher than that of using Luria-Bertani (LB) medium. The maximal concentration of γ-GABA was 245 g/L whereas L-glutamic acid was near completely converted. These findings provided us a good example for bio-production of γ-GABA using recombinant E. coli expressing a GAD enzyme derived from eukaryote.

The proteins of Fusobacterium spp. involved in hydrogen sulfide production from L-cysteine.

PubMed

Basic, Amina; Blomqvist, Madeleine; Dahlén, Gunnar; Svensäter, Gunnel

2017-03-14

Hydrogen sulfide (H 2 S) is a toxic foul-smelling gas produced by subgingival biofilms in patients with periodontal disease and is suggested to be part of the pathogenesis of the disease. We studied the H 2 S-producing protein expression of bacterial strains associated with periodontal disease. Further, we examined the effect of a cysteine-rich growth environment on the synthesis of intracellular enzymes in F. nucleatum polymorphum ATCC 10953. The proteins were subjected to one-dimensional (1DE) and two-dimensional (2DE) gel electrophoresis An in-gel activity assay was used to detect the H 2 S-producing enzymes; Sulfide from H 2 S, produced by the enzymes in the gel, reacted with bismuth forming bismuth sulfide, illustrated as brown bands (1D) or spots (2D) in the gel. The discovered proteins were identified with liquid chromatography - tandem mass spectrometry (LC-MS/MS). Cysteine synthase and proteins involved in the production of the coenzyme pyridoxal 5'phosphate (that catalyzes the production of H 2 S) were frequently found among the discovered enzymes. Interestingly, a higher expression of H 2 S-producing enzymes was detected from bacteria incubated without cysteine prior to the experiment. Numerous enzymes, identified as cysteine synthase, were involved in the production of H 2 S from cysteine and the expression varied among Fusobacterium spp. and strains. No enzymes were detected with the in-gel activity assay among the other periodontitis-associated bacteria tested. The expression of the H 2 S-producing enzymes was dependent on environmental conditions such as cysteine concentration and pH but less dependent on the presence of serum and hemin.

A periplasmic, pyridoxal-5'-phosphate-dependent amino acid racemase in Pseudomonas taetrolens.

PubMed

Matsui, Daisuke; Oikawa, Tadao; Arakawa, Noriaki; Osumi, Shintaro; Lausberg, Frank; Stäbler, Norma; Freudl, Roland; Eggeling, Lothar

2009-07-01

The pyridoxal-5'-phosphate (PLP)-dependent amino acid racemases occur in almost every bacterium but may differ considerably with respect to substrate specificity. We here isolated the cloned broad substrate specificity racemase ArgR of Pseudomonas taetrolens from Escherichia coli by classical procedures. The racemase was biochemically characterized and amongst other aspects it was confirmed that it is mostly active with lysine, arginine and ornithine, but merely weakly active with alanine, whereas the alanine racemase of the same organism studied in comparison acts on alanine only. Unexpectedly, sequencing the amino-terminal end of ArgR revealed processing of the protein, with a signal peptide cleaved off. Subsequent localization studies demonstrated that in both P. taetrolens and E. coli ArgR activity was almost exclusively present in the periplasm, a feature so far unknown for any amino acid racemase. An ArgR-derivative carrying a carboxy-terminal His-tag was made and this was demonstrated to localize even in an E. coli mutant devoid of the twin-arginine translocation (Tat) pathway in the periplasm. These data indicate that ArgR is synthesized as a prepeptide and translocated in a Tat-independent manner. We therefore propose that ArgR translocation depends on the Sec system and a post-translocational insertion of PLP occurs. As further experiments showed, ArgR is necessary for the catabolism of D: -arginine and D: -lysine by P. taetrolens.

Mechanistic deductions from kinetic isotope effects and pH studies of pyridoxal phosphate dependent carbon-carbon lyases: Erwinia herbicola and Citrobacter freundii tyrosine phenol-lyase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kiick, D.M.; Phillips, R.S.

1988-09-20

The pH dependence of the kinetic parameters and primary deuterium isotope effects have been determined for tyrosine phenol-lyase from both Erwinia herbicola and Citrobacter freundii. The primary deuterium isotope effects indicate that proton abstraction from the 2-position of the substrate is partially rate-limiting for both enzymes. The C. freundii enzyme primary deuterium isotope effects (DV = 3.5 and D(V/Ktyr) = 2.5) are pH independent, indicating that tyrosine is not sticky (i.e., does not dissociate slower than it reacts to give products). Since Vmax for both tyrosine and the alternate substrate S-methyl-L-cysteine is also pH independent, substrate binds only to themore » correctly protonated form of the enzyme. For the E. herbicola enzyme, both Vmax and V/K for tyrosine or S-methyl-L-cysteine are pH dependent, as well as both DV and D(V/Ktyr). Thus, while both the protonated and unprotonated enzyme can bind substrate, and may be interconverted directly, only the unprotonated Michaelis complex is catalytically competent. At pH 9.5, DV = 2.5 and D(V/Ktyr) = 1.5. However, at pH 6.4 the isotope effect on both parameters is equal to 4.1. From these data, the forward commitment factor (cf = 5.2) and catalytic ratio (cvf = 1.1) for tyrosine and S-methyl-L-cysteine (cf = 2.2, cvf = 24) are calculated. Also, the Michaelis complex partition ratio (cf/cvf) for substrate and products is calculated to be 4.7 for tyrosine and 0.1 for S-methyl-L-cysteine.« less

4′-CyanoPLP presents better prospect for the experimental detection of elusive cyclic intermediate radical in the reaction of lysine 5,6-aminomutase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Maity, Amarendra Nath; Ke, Shyue-Chu, E-mail: ke@mail.ndhu.edu.tw

2015-02-06

Graphical abstract: The results of our calculations suggest that the reaction of 4′-cyanoPLP with lysine 5,6-aminomutase offers better prospect for the experimental detection of elusive cyclic azacyclopropylcarbinyl radical, which is proposed to be a key intermediate in the reaction of pyridoxal-5′-phosphate dependent radical aminomutases. - Highlights: • 4′-CyanoI{sup ·} is the lowest energy radical intermediate in the reaction of 5,6-LAM. • 4′-CyanoPLP offers good prospect for the experimental observation of elusive I{sup ·}. • The calculated HFCCs would help to characterize 4′-cyanoI{sup ·} by EPR. - Abstract: The results of our calculations suggest that the reaction of 4′-cyanoPLP with lysinemore » 5,6-aminomutase offers better prospect for the experimental detection of elusive cyclic azacyclopropylcarbinyl radical (I{sup ·}), which is proposed to be a key intermediate in the reaction of pyridoxal-5′-phosphate dependent radical aminomutases. We have calculated the corresponding hyperfine coupling constants (HFCCs) for {sup 14}N and {sup 13}C of cyano group using several basis sets to help the characterization of 4′-cyanoI{sup ·}.« less

Multiphoton manipulations of enzymatic photoactivity in aspartate aminotransferase.

PubMed

Hill, Melissa P; Freer, Lucy H; Vang, Mai C; Carroll, Elizabeth C; Larsen, Delmar S

2011-04-21

The aspartate aminotransferase (AAT) enzyme utilizes the chromophoric pyridoxal 5'-phosphate (PLP) cofactor to facilitate the transamination of amino acids. Recently, we demonstrated that, upon exposure to blue light, PLP forms a reactive triplet state that rapidly (in microseconds) generates the high-energy quinonoid intermediate when bound to PLP-dependent enzymes [J. Am. Chem. Soc.2010, 132 (47), 16953-16961]. This increases the net catalytic activity (k(cat)) of AAT, since formation of the quinonoid is partially rate limiting via the thermally activated enzymatic pathway. The magnitude of observed photoenhancement initially scales linearly with pump fluence; however when a critical threshold is exceeded, the photoactivity saturates and is even suppressed at greater excitation fluences. The photodynamic mechanisms associated with this suppression behavior are characterized with the use of ultrafast multipulse pump-dump-probe and pump-repump-probe transient absorption techniques in combination with complementary two-color, steady-state excitation assays. Via multistate kinetic modeling of the transient ultrafast data and the steady-state assay data, the nonmonotonic incident power dependence of the photoactivty in AAT is decomposed into contributions from high-intensity dumping of the excited singlet state and repumping of the excited triplet state with induces the repopulation of the ground state via rapid intersystem crossing in the higher-lying triplet electronic manifold.

Evidence that the metabolite repair enzyme NAD(P)HX epimerase has a moonlighting function.

PubMed

Niehaus, Thomas D; Elbadawi-Sidhu, Mona; Huang, Lili; Prunetti, Laurence; Gregory, Jesse F; de Crécy-Lagard, Valérie; Fiehn, Oliver; Hanson, Andrew D

2018-06-29

NAD(P)H-hydrate epimerase (EC 5.1.99.6) is known to help repair NAD(P)H hydrates (NAD(P)HX), which are damage products existing as R and S epimers. The S epimer is reconverted to NAD(P)H by a dehydratase; the epimerase facilitates epimer interconversion. Epimerase deficiency in humans causes a lethal disorder attributed to NADHX accumulation. However, bioinformatic evidence suggest caution about this attribution by predicting that the epimerase has a second function connected to vitamin B 6 (pyridoxal 5'-phosphate and related compounds). Specifically, (i) the epimerase is fused to a B 6 salvage enzyme in plants, (ii) epimerase genes cluster on the chromosome with B 6 -related genes in bacteria, and (iii) epimerase and B 6 -related genes are coexpressed in yeast and Arabidopsis The predicted second function was explored in Escherichia coli , whose epimerase and dehydratase are fused and encoded by yjeF The putative NAD(P)HX epimerase active site has a conserved lysine residue (K192 in E. coli YjeF). Changing this residue to alanine cut in vitro epimerase activity by ≥95% but did not affect dehydratase activity. Mutant cells carrying the K192A mutation had essentially normal NAD(P)HX dehydratase activity and NAD(P)HX levels, showing that the mutation had little impact on NAD(P)HX repair in vivo However, these cells showed metabolome changes, particularly in amino acids, which exceeded those in cells lacking the entire yjeF gene. The K192A mutant cells also had reduced levels of 'free' (i.e. weakly bound or unbound) pyridoxal 5'-phosphate. These results provide circumstantial evidence that the epimerase has a metabolic function beyond NAD(P)HX repair and that this function involves vitamin B 6 . © 2018 The Author(s).

Evidence that the metabolite repair enzyme NAD(P)HX epimerase has a moonlighting function

PubMed Central

Niehaus, Thomas D.; Elbadawi-Sidhu, Mona; Huang, Lili; Prunetti, Laurence; Gregory, Jesse F.; de Crécy-Lagard, Valérie; Fiehn, Oliver; Hanson, Andrew D.

2018-01-01

NAD(P)H-hydrate epimerase (EC 5.1.99.6) is known to help repair NAD(P)H hydrates (NAD(P)HX), which are damage products existing as R and S epimers. The S epimer is reconverted to NAD(P)H by a dehydratase; the epimerase facilitates epimer interconversion. Epimerase deficiency in humans causes a lethal disorder attributed to NADHX accumulation. However, bioinformatic evidence suggest caution about this attribution by predicting that the epimerase has a second function connected to vitamin B6 (pyridoxal 5′-phosphate and related compounds). Specifically, (i) the epimerase is fused to a B6 salvage enzyme in plants, (ii) epimerase genes cluster on the chromosome with B6-related genes in bacteria, and (iii) epimerase and B6-related genes are coexpressed in yeast and Arabidopsis. The predicted second function was explored in Escherichia coli, whose epimerase and dehydratase are fused and encoded by yjeF. The putative NAD(P)HX epimerase active site has a conserved lysine residue (K192 in E. coli YjeF). Changing this residue to alanine cut in vitro epimerase activity by ≥95% but did not affect dehydratase activity. Mutant cells carrying the K192A mutation had essentially normal NAD(P)HX dehydratase activity and NAD(P)HX levels, showing that the mutation had little impact on NAD(P)HX repair in vivo. However, these cells showed metabolome changes, particularly in amino acids, which exceeded those in cells lacking the entire yjeF gene. The K192A mutant cells also had reduced levels of ‘free’ (i.e. weakly bound or unbound) pyridoxal 5'-phosphate. These results provide circumstantial evidence that the epimerase has a metabolic function beyond NAD(P)HX repair and that this function involves vitamin B6. PMID:29654173

Dry entrapment of enzymes by epoxy or polyester resins hardened on different solid supports.

PubMed

Barig, Susann; Funke, Andreas; Merseburg, Andrea; Schnitzlein, Klaus; Stahmann, K-Peter

2014-06-10

Embedding of enzymes was performed with epoxy or polyester resin by mixing in a dried enzyme preparation before polymerization was started. This fast and low-cost immobilization method produced enzymatically active layers on different solid supports. As model enzymes the well-characterized Thermomyces lanuginosus lipase and a new threonine aldolase from Ashbya gossypii were used. It was shown that T. lanuginosus lipase recombinantly expressed in Aspergillus oryzae is a monomeric enzyme with a molecular mass of 34kDa, while A. gossypii threonine aldolase expressed in Escherichia coli is a pyridoxal-5'-phosphate binding homotetramer with a mass of 180kDa. The enzymes were used freeze dried, in four different preparations: freely diffusing, adsorbed on octyl sepharose, as well as cross-linked enzyme aggregates or as suspensions in organic solvent. They were mixed with standard two-component resins and prepared as layers on solid supports made of different materials e.g. metal, glass, polyester. Polymerization led to encapsulated enzyme preparations showing activities comparable to literature values. Copyright © 2014 Elsevier Inc. All rights reserved.

Neurospora tryptophan synthase: N-terminal analysis and the sequence of the pyridoxal phosphate active site peptide

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pratt, M.L.; Hsu, P.Y.; DeMoss, J.A.

1986-05-01

Tryptophan synthase (TS), which catalyzes the final step of tryptophan biosynthesis, is a multifunctional protein requiring pyridoxal phosphate (B6P) for two of its three distinct enzyme activities. TS from Neurospora has a blocked N-terminal, is a homodimer of 150 KDa and binds one mole of B6P per mole of subunit. The authors shown the N-terminal residue to be acyl-serine. The B6P-active site of holoenzyme was labelled by reduction of the B6P-Schiff base with (/sup 3/H)-NaBH/sub 4/, and resulted in a proportionate loss of activity in the two B6P-requiring reactions. SDS-polyacrylamide gel electrophoresis of CNBr-generated peptides showed the labelled, active sitemore » peptide to be 6 KDa. The sequence of this peptide, purified to apparent homogeneity by a combination of C-18 reversed phase and TSK gel filtration HPLC is: gly-arg-pro-gly-gln-leu-his-lys-ala-glu-arg-leu-thr-glu-tyr-ala-gly-gly-ala-gln-ile-xxx-leu-lys-arg-glu-asp-leu-asn-his-xxx-gly-xxx-his-/sub ***/-ile-asn-asn-ala-leu. Although four residues (xxx, /sub ***/) are unidentified, this peptide is minimally 78% homologous with the corresponding peptide from yeast TS, in which residue (/sub ***/) is the lysine that binds B6P.« less

Acetate selective fluorescent turn-on sensors derived using vitamin B6 cofactor pyridoxal-5-phosphate

NASA Astrophysics Data System (ADS)

Sharma, Darshna; Kuba, Aman; Thomas, Rini; Ashok Kumar, S. K.; Kuwar, Anil; Choi, Heung-Jin; Sahoo, Suban K.

2016-03-01

Two new Schiff base receptors have been synthesized by condensation of pyridoxal-5-phosphate with 2-aminophenol (L1) or aniline (L2). In DMSO, the receptors showed both chromogenic and 'turn-on' fluorescence responses selectively in the presence of AcO- and F-. However, in mixed DMSO-H2O medium, the receptors showed AcO- selective 'turn-on' fluorescence without any interference from other tested anions including F-. The detection limit for AcO- was found to be 7.37 μM and 22.9 μM using the receptors L1 and L2, respectively.

Determination of copper (II) in foodstuffs based on its quenching effect on the fluorescence of N,N'-bis(pyridoxal phosphate)-o-phenylenediamine.

PubMed

Xu, Canhui; Liao, Lifu; He, Yunfei; Wu, Rurong; Li, Shijun; Yang, Yanyan

2015-01-01

A Schiff base-type fluorescence probe was prepared for the detection of copper (II) in foodstuffs. The probe is N,N'-bis(pyridoxal phosphate)-o-phenylenediamine (BPPP). It was synthesized by utilizing the Schiff base condensation reaction of pyridoxal 5-phosphate with 1,2-phenylenediamine. BPPP has the properties of high fluorescence stability, good water solubility and low toxicity. Its maximum excitation wavelength and maximum fluorescence emission wavelength are at 389 and 448 nm, respectively. When BPPP coexists with copper (II), its fluorescence is dramatically quenched. Under a certain condition, the fluorescence intensity decreased proportionally to the concentration of copper (II) by the quenching effect. Based on this fact, we established a fluorescence quenching method for the determination of copper (II). Under optimal conditions a linear range was found to be 0.5-50 ng/mL with a detection limit of 0.2 ng/mL. The method has been applied to determine copper (II) in foodstuff samples and the analytical results show good agreement with that obtained from atomic absorption spectrometry method. Copyright © 2015 Elsevier B.V. All rights reserved.

Two Site-Directed Mutations are Required for the Conversion of a Sugar Dehydratase into an Aminotransferase¶

PubMed Central

Cook, Paul D.; Kubiak, Rachel L.; Toomey, Daniel P.; Holden, Hazel M.

2009-01-01

l-colitose and d-perosamine are unusual sugars found in the O-antigens of some Gram-negative bacteria such as Escherichia coli, Vibrio cholerae, and Salmonella enterica, among others. The biosynthetic pathways for these two sugars begin with the formation of GDP-mannose from d-mannose-1-phosphate and GTP followed by the subsequent dehydration and oxidation of GDP-mannose to yield GDP-4-keto-6-deoxymannose. Following the production of GDP-4-keto-6-deoxymannose, the two pathways diverge. In the case of GDP-perosamine biosynthesis, the next step involves an amination reaction at the C-4′ position of the sugar, whereas in GDP-colitose production, the 3′-hydroxyl group is removed. The enzymes catalyzing these reactions are GDP-perosamine synthase and GDP-4-keto-6-deoxymannose-3-dehydratase (ColD), respectively. Both of these enzymes are pyridoxal-5′-phosphate (PLP)-dependent and their three-dimensional structures place them into the well-characterized aspartate aminotransferase superfamily. A comparison of the active site architecture of ColD from Escherichia coli (Strain 5a, type O55:H7) to that of GDP-perosamine synthase from Caulobacter crescentus CB15, suggested that only two mutations would be required to convert ColD into an aminotransferase. Here we present a combined structural and functional analysis of the ColD S187N/H188K mutant protein that, indeed, has been converted from a dehydratase into an aminotransferase. PMID:19402712

Engineering Saccharomyces cerevisiae To Release 3-Mercaptohexan-1-ol during Fermentation through Overexpression of an S. cerevisiae Gene, STR3, for Improvement of Wine Aroma▿

PubMed Central

Holt, Sylvester; Cordente, Antonio G.; Williams, Simon J.; Capone, Dimitra L.; Jitjaroen, Wanphen; Menz, Ian R.; Curtin, Chris; Anderson, Peter A.

2011-01-01

Sulfur-containing aroma compounds are key contributors to the flavor of a diverse range of foods and beverages. The tropical fruit characters of Vitis vinifera L. cv. Sauvignon blanc wines are attributed to the presence of the aromatic thiols 3-mercaptohexan-1-ol (3MH), 3-mercaptohexan-1-ol-acetate, and 4-mercapto-4-methylpentan-2-one (4MMP). These volatile thiols are found in small amounts in grape juice and are formed from nonvolatile cysteinylated precursors during fermentation. In this study, we overexpressed a Saccharomyces cerevisiae gene, STR3, which led to an increase in 3MH release during fermentation of a V. vinifera L. cv. Sauvignon blanc juice. Characterization of the enzymatic properties of Str3p confirmed it to be a pyridoxal-5′-phosphate-dependent cystathionine β-lyase, and we demonstrated that this enzyme was able to cleave the cysteinylated precursors of 3MH and 4MMP to release the free thiols. These data provide direct evidence for a yeast enzyme able to release aromatic thiols in vitro that can be applied in the development of self-cloned yeast to enhance wine flavor. PMID:21478306

The Vibrio cholerae quorum-sensing autoinducer CAI-1: analysis of the biosynthetic enzyme CqsA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kelly, R.; Bolitho, M; Higgins, D

2009-01-01

Vibrio cholerae, the bacterium that causes the disease cholera, controls virulence factor production and biofilm development in response to two extracellular quorum-sensing molecules, called autoinducers. The strongest autoinducer, called CAI-1 (for cholera autoinducer-1), was previously identified as (S)-3-hydroxytridecan-4-one. Biosynthesis of CAI-1 requires the enzyme CqsA. Here, we determine the CqsA reaction mechanism, identify the CqsA substrates as (S)-2-aminobutyrate and decanoyl coenzyme A, and demonstrate that the product of the reaction is 3-aminotridecan-4-one, dubbed amino-CAI-1. CqsA produces amino-CAI-1 by a pyridoxal phosphate-dependent acyl-CoA transferase reaction. Amino-CAI-1 is converted to CAI-1 in a subsequent step via a CqsA-independent mechanism. Consistent with this,more » we find cells release {ge}100 times more CAI-1 than amino-CAI-1. Nonetheless, V. cholerae responds to amino-CAI-1 as well as CAI-1, whereas other CAI-1 variants do not elicit a quorum-sensing response. Thus, both CAI-1 and amino-CAI-1 have potential as lead molecules in the development of an anticholera treatment.« less

The Mycobacterium tuberculosis Complex has a Pathway for the Biosynthesis of 4-Formamido-4,6-Dideoxy-d-Glucose.

PubMed

Brown, Haley A; Vinogradov, Evgeny; Gilbert, Michel; Holden, Hazel M

2018-05-15

Recent studies have demonstrated that the O-antigens of some pathogenic bacteria such as Brucella abortus, Francisella tularensis, and Campylobacter jejuni contain quite unusual N-formylated sugars (3-formamido-3,6-dideoxy-d-glucose or 4-formamido-4,6-dideoxy-d-glucose). Typically, four enzymes are required for the formation of such sugars: a thymidylyltransferase, a 4,6-dehydratase, a pyridoxal 5'-phosphate or PLP-dependent aminotransferase, and an N-formyltransferase. To date, there have been no published reports of N-formylated sugars associated with Mycobacterium tuberculosis. A recent investigation from our laboratories, however, has demonstrated that one gene product from M. tuberculosis, Rv3404c, functions as a sugar N-formyltransferase. Given that M. tuberculosis produces l-rhamnose, both a thymidylyltransferase (Rv0334) and a 4,6-dehydratase (Rv3464) required for its formation have been identified. Thus, there is one remaining enzyme needed for the production of an N-formylated sugar in M. tuberculosis, namely a PLP-dependent aminotransferase. Here we demonstrate that the M. tuberculosis rv3402c gene encodes such an enzyme. Our data prove that M. tuberculosis contains all of the enzymatic activities required for the formation of dTDP-4-formamido-4,6-dideoxy-d-glucose. Indeed, the rv3402c gene product likely contributes to virulence or persistence during infection, though its temporal expression and location remain to be determined. This article is protected by copyright. All rights reserved. © 2018 The Protein Society.

Magnetic field effects on coenzyme B12- and B6-dependent lysine 5,6-aminomutase: switching of the J-resonance through a kinetically competent radical-pair intermediate.

PubMed

Chen, Jun-Ru; Ke, Shyue-Chu

2018-05-09

The environmental magnetic field is beneficial to migratory bird navigation through the radical-pair mechanism. One of the continuing challenges in understanding how magnetic fields may perturb biological processes is that only a very few field-sensitive examples have been explored despite the prevalence of radical pairs in enzymatic reactions. We show that the reaction of adenosylcobalamin- and pyridoxal-5'-phosphate-dependent lysine 5,6-aminomutase proceeds via radical-pair intermediates and is magnetic field dependent. The 5'-deoxyadenosyl radical from adenosylcobalamin abstracts a C5(H) from the substrate to yield a {cob(ii)alamin - substrate} radical pair wherein the large spin-spin interaction (2J = 8000 gauss) locks the radical pair in a triplet state, as evidenced by electron paramagnetic resonance spectroscopy. Application of an external magnetic field in the range of 6500 to 8500 gauss triggers intersystem crossing to the singlet {cob(ii)alamin - substrate} radical-pair state. Spin-conserved H back-transfer from deoxyadenosine to the substrate radical yields a singlet {cob(ii)alamin-5'-deoxyadenosyl} radical pair. Spin-selective recombination to adenosylcobalamin decreased the enzyme catalytic efficiency kcat/Km by 16% at 7600 gauss. As a mechanistic probe, observation of magnetic field effects successfully demonstrates the presence of a kinetically significant radical pair in this enzyme. The study of a pronounced high-field level-crossing characteristic through an immobilized radical pair with a constant exchange interaction deepens our understanding of how a magnetic field may interact with an enzyme.

Identification of a novel amino acid racemase from a hyperthermophilic archaeon Pyrococcus horikoshii OT-3 induced by D-amino acids.

PubMed

Kawakami, Ryushi; Ohmori, Taketo; Sakuraba, Haruhiko; Ohshima, Toshihisa

2015-08-01

To date, there have been few reports analyzing the amino acid requirement for growth of hyperthermophilic archaea. We here found that the hyperthermophilic archaeon Pyrococcus horikoshii OT-3 requires Thr, Leu, Val, Phe, Tyr, Trp, His and Arg in the medium for growth, and shows slow growth in medium lacking Met or Ile. This largely corresponds to the presence, or absence, of genes related to amino acid biosynthesis in its genome, though there are exceptions. The amino acid requirements were dramatically lost by addition of D-isomers of Met, Leu, Val, allo-Ile, Phe, Tyr, Trp and Arg. Tracer analysis using (14)C-labeled D-Trp showed that D-Trp in the medium was used as a protein component in the cells, suggesting the presence of D-amino acid metabolic enzymes. Pyridoxal 5'-phosphate (PLP)-dependent racemase activity toward Met, Leu and Phe was detected in crude extract of P. horikoshii and was enhanced in cells grown in the medium supplemented with D-amino acids, especially D-allo-Ile. The gene encoding the racemase was narrowed down to one open reading frame on the basis of enzyme purification from P. horikoshii cells, and the recombinant enzyme exhibited PLP-dependent racemase activity toward several amino acids, including Met, Leu and Phe, but not Pro, Asp or Glu. This is the first report showing the presence in a hyperthermophilic archaeon of a PLP-dependent amino acid racemase with broad substrate specificity that is likely responsible for utilization of D-amino acids for growth.

Structure And Mutagenic Conversion of E(1) Dehydrase: at the Crossroads of Dehydration, Amino Transfer, And Epimerization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Smith, P.; Szu, P.-H.; Bui, C.

2009-05-26

Pyridoxal 5'-phosphate (PLP) and pyridoxamine 5'-phosphate (PMP) are highly versatile coenzymes whose importance is well recognized. The capability of PLP/PMP-dependent enzymes to catalyze a diverse array of chemical reactions is attributed to fine-tuning of the cofactor-substrate interactions in the active site. CDP-6-deoxy-l-threo-d-glycero-4-hexulose 3-dehydrase (E1), along with its reductase (E{sub 3}), catalyzes the C-3 deoxygenation of CDP-4-keto-6-deoxy-d-glucose to form the dehydrated product, CDP-4-keto-3,6-dideoxy-d-glucose, in the ascarylose biosynthetic pathway. This product is the progenitor to most 3,6-dideoxyhexoses, which are the major antigenic determinants of many Gram-negative pathogens. The dimeric [2Fe-2S] protein, E{sub 1}, cloned from Yersinia pseudotuberculosis, is the only known enzymemore » whose catalysis involves the direct participation of PMP in one-electron redox chemistry. E{sub 1} also contains an unusual [2Fe-2S] cluster with a previously unknown binding motif (C-X{sub 57}-C-X{sub 1}-C-X{sub 7}-C). Herein we report the first X-ray crystal structure of E{sub 1}, which exhibits an aspartate aminotransferase (AAT) fold. A comparison of the E{sub 1} active site architecture with homologous structures uncovers residues critical for the dehydration versus transamination activity. Site-directed mutagenesis of four E{sub 1} residues, D194H, Y217H, H220K, and F345H, converted E{sub 1} from a PMP-dependent dehydrase to a PLP/glutamate-dependent aminotransferase. The E{sub 1} quadruple mutant, having been conferred this altered enzyme activity, can transaminate the natural substrate to CDP-4,6-dideoxy-4-amino-d-galactose without E{sub 3}. Taken together, these results provide the molecular basis of the functional switch of E{sub 1} toward dehydration, epimerization, and transamination. The insights gained from these studies can be used for the development of inhibitors of disease-relevant PLP/PMP-dependent enzymes.« less

NAD(P)H-dependent aldose reductase from the xylose-assimilating yeast Candida tenuis. Isolation, characterization and biochemical properties of the enzyme.

PubMed Central

Neuhauser, W; Haltrich, D; Kulbe, K D; Nidetzky, B

1997-01-01

During growth on d-xylose the yeast Candida tenuis produces one aldose reductase that is active with both NADPH and NADH as coenzyme. This enzyme has been isolated by dye ligand and anion-exchange chromatography in yields of 76%. Aldose reductase consists ofa single 43 kDa polypeptide with an isoelectric point of 4.70. Initial velocity, product inhibition and binding studies are consistent with a compulsory-ordered, ternary-complex mechanism with coenzyme binding first and leaving last. The catalytic efficiency (kcat/Km) in d-xylose reduction at pH 7 is more than 60-fold higher than that in xylitol oxidation and reflects significant differences in the corresponding catalytic centre activities as well as apparent substrate-binding constants. The enzyme prefers NADP(H) approx. 2-fold to NAD(H), which is largely due to better apparent binding of the phosphorylated form of the coenzyme. NADP+ is a potent competitive inhibitor of the NADH-linked aldehyde reduction (Ki 1.5 microM), whereas NAD+ is not. Unlike mammalian aldose reductase, the enzyme from C. tenuis is not subject to oxidation-induced activation. Evidence of an essential lysine residue located in or near the coenzyme binding site has been obtained from chemical modification of aldose reductase with pyridoxal 5'-phosphate. The results are discussed in the context of a comparison of the enzymic properties of yeast and mammalian aldose reductase. PMID:9307017

Glutamate 338 is an electrostatic facilitator of C–Co bond breakage in a dynamic/electrostatic model of catalysis by ornithine aminomutase

PubMed Central

Menon, Binuraj R K; Menon, Navya; Fisher, Karl; Rigby, Stephen E J; Leys, David; Scrutton, Nigel S

2015-01-01

How cobalamin-dependent enzymes promote C–Co homolysis to initiate radical catalysis has been debated extensively. For the pyridoxal 5′-phosphate and cobalamin-dependent enzymes lysine 5,6-aminomutase and ornithine 4,5-aminomutase (OAM), large-scale re-orientation of the cobalamin-binding domain linked to C–Co bond breakage has been proposed. In these models, substrate binding triggers dynamic sampling of the B12-binding Rossmann domain to achieve a catalytically competent ‘closed’ conformational state. In ‘closed’ conformations of OAM, Glu338 is thought to facilitate C–Co bond breakage by close association with the cobalamin adenosyl group. We investigated this using stopped-flow continuous-wave photolysis, viscosity dependence kinetic measurements, and electron paramagnetic resonance spectroscopy of a series of Glu338 variants. We found that substrate-induced C–Co bond homolysis is compromised in Glu388 variant forms of OAM, although photolysis of the C–Co bond is not affected by the identity of residue 338. Electrostatic interactions of Glu338 with the 5′-deoxyadenosyl group of B12 potentiate C–Co bond homolysis in ‘closed’ conformations only; these conformations are unlocked by substrate binding. Our studies extend earlier models that identified a requirement for large-scale motion of the cobalamin domain. Our findings indicate that large-scale motion is required to pre-organize the active site by enabling transient formation of ‘closed’ conformations of OAM. In ‘closed’ conformations, Glu338 interacts with the 5′-deoxyadenosyl group of cobalamin. This interaction is required to potentiate C–Co homolysis, and is a crucial component of the approximately 1012 rate enhancement achieved by cobalamin-dependent enzymes for C–Co bond homolysis. PMID:25627283

S-Adenosylmethionine decarboxylase from human prostate. Activation by putrescine

PubMed Central

Zappia, Vincenzo; Cartenì-Farina, Maria; Pietra, Gennaro Della

1972-01-01

1. The presence of S-adenosylmethionine decarboxylase in human prostate gland is reported. A satisfactory radiochemical enzymic assay was developed and the enzyme was partially characterized. 2. Putrescine stimulates the reaction rate by up to 6-fold at pH7.5: the apparent activation constant was estimated to be 0.13mm. The stimulation is pH-dependent and a maximal effect is observed at acid pH values. 3. Putrescine activation is rather specific: other polyamines, such as spermidine and spermine, did not show any appreciable effect. 4. The apparent Km for the substrate is 4×10−5m. The calculated S-adenosylmethionine content of human prostate (0.18μmol/g wet wt. of tissue) demonstrates that the cellular amounts of sulphonium compound are saturating with respect to the enzyme. 5. The enzyme is moderately stable at 0°C and is rapidly inactivated at 40°C. The optimum pH is about 7.5, with one-half of the maximal activity occurring at pH6.6. 6. Several carboxy-14C-labelled analogues and derivatives of S-adenosylmethionine were tested as substrates. The enzyme appears to be highly specific: the replacement of the 6′-amino group of the sulphonium compound alone results in a complete loss of activity. 7. Inhibition of the enzyme activity by several carbonyl reagents suggests an involvement of either pyridoxal phosphate or pyruvate in the catalytic process. 8. The inhibitory effect of thiol reagents indicates the presence of `essential' thiol groups. PMID:4658995

NMR Crystallography of a Carbanionic Intermediate in Tryptophan Synthase: Chemical Structure, Tautomerization, and Reaction Specificity.

PubMed

Caulkins, Bethany G; Young, Robert P; Kudla, Ryan A; Yang, Chen; Bittbauer, Thomas J; Bastin, Baback; Hilario, Eduardo; Fan, Li; Marsella, Michael J; Dunn, Michael F; Mueller, Leonard J

2016-11-23

Carbanionic intermediates play a central role in the catalytic transformations of amino acids performed by pyridoxal-5'-phosphate (PLP)-dependent enzymes. Here, we make use of NMR crystallography-the synergistic combination of solid-state nuclear magnetic resonance, X-ray crystallography, and computational chemistry-to interrogate a carbanionic/quinonoid intermediate analogue in the β-subunit active site of the PLP-requiring enzyme tryptophan synthase. The solid-state NMR chemical shifts of the PLP pyridine ring nitrogen and additional sites, coupled with first-principles computational models, allow a detailed model of protonation states for ionizable groups on the cofactor, substrates, and nearby catalytic residues to be established. Most significantly, we find that a deprotonated pyridine nitrogen on PLP precludes formation of a true quinonoid species and that there is an equilibrium between the phenolic and protonated Schiff base tautomeric forms of this intermediate. Natural bond orbital analysis indicates that the latter builds up negative charge at the substrate C α and positive charge at C4' of the cofactor, consistent with its role as the catalytic tautomer. These findings support the hypothesis that the specificity for β-elimination/replacement versus transamination is dictated in part by the protonation states of ionizable groups on PLP and the reacting substrates and underscore the essential role that NMR crystallography can play in characterizing both chemical structure and dynamics within functioning enzyme active sites.

NMR Crystallography of a Carbanionic Intermediate in Tryptophan Synthase: Chemical Structure, Tautomerization, and Reaction Specificity

PubMed Central

2016-01-01

Carbanionic intermediates play a central role in the catalytic transformations of amino acids performed by pyridoxal-5′-phosphate (PLP)-dependent enzymes. Here, we make use of NMR crystallography—the synergistic combination of solid-state nuclear magnetic resonance, X-ray crystallography, and computational chemistry—to interrogate a carbanionic/quinonoid intermediate analogue in the β-subunit active site of the PLP-requiring enzyme tryptophan synthase. The solid-state NMR chemical shifts of the PLP pyridine ring nitrogen and additional sites, coupled with first-principles computational models, allow a detailed model of protonation states for ionizable groups on the cofactor, substrates, and nearby catalytic residues to be established. Most significantly, we find that a deprotonated pyridine nitrogen on PLP precludes formation of a true quinonoid species and that there is an equilibrium between the phenolic and protonated Schiff base tautomeric forms of this intermediate. Natural bond orbital analysis indicates that the latter builds up negative charge at the substrate Cα and positive charge at C4′ of the cofactor, consistent with its role as the catalytic tautomer. These findings support the hypothesis that the specificity for β-elimination/replacement versus transamination is dictated in part by the protonation states of ionizable groups on PLP and the reacting substrates and underscore the essential role that NMR crystallography can play in characterizing both chemical structure and dynamics within functioning enzyme active sites. PMID:27779384

Vitamin B6 Prevents IL-1β Protein Production by Inhibiting NLRP3 Inflammasome Activation.

PubMed

Zhang, Peipei; Tsuchiya, Kohsuke; Kinoshita, Takeshi; Kushiyama, Hiroko; Suidasari, Sofya; Hatakeyama, Mizuki; Imura, Hisanori; Kato, Norihisa; Suda, Takashi

2016-11-18

Vitamin B6 includes six water-soluble vitamers: pyridoxal (PL), pyridoxamine (PM), pyridoxine (PN), and their phosphorylated forms. Pyridoxal 5'-phosphate (PLP) is an important cofactor for many metabolic enzymes. Several lines of evidence demonstrate that blood levels of PLP are significantly lower in patients with inflammation than in control subjects and that vitamin B6 has anti-inflammatory effects, with therapeutic potential for a variety of inflammatory diseases. Although one of our group previously demonstrated that PL inhibits the NF-κB pathway, the molecular mechanism by which vitamin B6 suppresses inflammation is not well understood. Here, we showed that both PL and PLP suppressed the expression of cytokine genes in macrophages by inhibiting Toll-like receptor (TLR)-mediated TAK1 phosphorylation and the subsequent NF-κB and JNK activation. Furthermore, PL and PLP abolished NLRP3-dependent caspase-1 processing and the subsequent secretion of mature IL-1β and IL-18 in LPS-primed macrophages. In contrast, PM and PN had little effect on IL-1β production. PLP, but not PL, markedly reduced the production of mitochondrial reactive oxygen species (ROS) in peritoneal macrophages. Importantly, PL and PLP reduced IL-1β production induced by LPS and ATP, or by LPS alone, in mice. Moreover, PL and PLP protected mice from lethal endotoxic shock. Collectively, these findings reveal novel anti-inflammatory activities for vitamin B6 and suggest its potential for preventing inflammatory diseases driven by the NLRP3 inflammasome. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Mechanistic Enzyme Models: Pyridoxal and Metal Ions.

ERIC Educational Resources Information Center

Hamilton, S. E.; And Others

1984-01-01

Background information, procedures, and results are presented for experiments on the pyridoxal/metal ion model system. These experiments illustrate catalysis through Schiff's base formation between aldehydes/ketones and primary amines, catalysis by metal ions, and the predictable manner in which metal ions inhibit or catalyze reactions. (JN)

Partial trypsin digestion as an indicator of mis-folding of mutant alanine:glyoxylate aminotransferase and chaperone effects of specific ligands. Study of a spectrum of missense mutants.

PubMed

Coulter-Mackie, M B; Lian, Q

2008-07-01

Alanine:glyoxylate aminotransferase (AGT) is a liver peroxisomal enzyme whose deficiency results in primary hyperoxaluria type 1 (PH1). More than 75 PH1 mutations are now documented in the AGT gene (AGXT), of which about 50% are missense. We have previously demonstrated that many such mutants expressed by transcription/translation are subject to generalized degradation by the proteasome and a specific limited trimming by an endogenous ATP-independent protease activity. Here, we report the results of partial digestion using trypsin as a mimic for the endogenous non-proteasomal protease and the use of N-terminal protein sequencing to determine the sensitive site. Partial trypsin digestion also provided an indicator of proper folding of the mutant enzyme. For selected mutations the sensitivity to trypsin could be ameliorated by addition of pyridoxal phosphate or aminooxy acetic acid as specific pharmacological chaperones.

Crystal structure and characterization of a novel L-serine ammonia-lyase from Rhizomucor miehei

DOE Office of Scientific and Technical Information (OSTI.GOV)

Qin, Zhen; Yan, Qiaojuan; Ma, Qingjun

L-serine ammonia-lyase, as a member of the β-family of pyridoxal-5′-phosphate (PLP) dependent enzymes, catalyzes the conversion of L-serine (L-threonine) to pyruvate (α-ketobutyrate) and ammonia. The crystal structure of L-serine ammonia-lyase from Rhizomucor miehei (RmSDH) was solved at 1.76 Å resolution by X-ray diffraction method. The overall structure of RmSDH had the characteristic β-family PLP dependent enzyme fold. It consisted of two distinct domains, both of which show the typical open twisted α/β structure. A PLP cofactor was located in the crevice between the two domains, which was attached to Lys52 by a Schiff-base linkage. Unique residue substitutions (Gly78, Pro79, Ser146, Ser147more » and Thr312) were discovered at the catalytic site of RmSDH by comparison of structures of RmSDH and other reported eukaryotic L-serine ammonia-lyases. Optimal pH and temperature of the purified RmSDH were 7.5 and 40 °C, respectively. It was stable in the pH range of 7.0–9.0 and at temperatures below 40 °C. This is the first crystal structure of a fungal L-serine ammonia-lyase. It will be useful to study the catalytic mechanism of β-elimination enzymes and will provide a basis for further enzyme engineering. - Highlights: • The crystal structure of a fungal L-serine ammonia-lyase (RmSDH) was solved. • Five unique residue substitutions are found at the catalytic site of RmSDH. • RmSDH was expressed in Pichia. pastoris and biochemically characterized. • RmSDH has potential application in splitting D/L-serine.« less

Red cell aspartate aminotransferase saturation with oral pyridoxine intake.

PubMed

Oshiro, Marilena; Nonoyama, Kimiyo; Oliveira, Raimundo Antônio Gomes; Barretto, Orlando Cesar de Oliveira

2005-03-02

The coenzyme of aspartate aminotransferase is pyridoxal phosphate, generated from fresh vegetables containing pyridoxine. Vitamin B6-responsive sideroblastic anemia, myelofibrosis and Peyronies syndrome respond to high pyridoxine doses. The objective was to investigate the oral pyridoxine oral dose that would lead to maximized pyridoxal phosphate saturation of red cell aspartate aminotransferase. Controlled trial, in Hematology Division of Instituto Adolfo Lutz. Red cell aspartate aminotransferase activity was assayed (before and after) in normal volunteers who were given oral pyridoxine for 15-18 days (30 mg, 100 mg and 200 mg daily). In vitro study of blood from seven normal volunteers was also performed, with before and after assaying of aspartate aminotransferase activity. The in vivo study showed increasing aspartate aminotransferase saturation with increasing pyridoxine doses. 83% saturation was reached with 30 mg daily, 88% with 100 mg, and 93% with 200 mg after 20 days of oral supplementation. The in vitro study did not reach 100% saturation. Neither in vivo nor in vitro study demonstrated thorough aspartate aminotransferase saturation with its coenzyme pyridoxal phosphate in red cells, from increasing pyridoxine supplementation. However, the 200-mg dose could be employed safely in vitamin B6-responsive sideroblastic anemia, myelofibrosis and Peyronies syndrome treatment. Although maximum saturation in circulating red cells is not achieved, erythroblasts and other nucleated and cytoplasmic organelles containing cells certainly will reach thorough saturation, which possibly explains the results obtained in these diseases.

[Importance of toxicological studies in animals for elaboration of pathogenetic therapy of hydrazine intoxication].

PubMed

Baryshnikov, I I

1997-01-01

Pathogenesis of poisoning with hydrazine seems complicated. Hydrazines inhibit pyridoxal-dependent enzymes, interfere with carbohydrates transformation and lipid metabolism, alter the processes of energy metabolism. Treatment of poisoning with hydrazine is effective only when containing drug combinations. Based experimentally, recommendations on the combination (phenazepam, be methyl, piracetam, ionol) were supported by chemical testing of the drugs.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yan, Wupeng; Stone, Everett; Zhang, Yan Jessie

Enzyme therapeutics that can degrade l-methionine (l-Met) are of great interest as numerous malignancies are exquisitely sensitive to l-Met depletion. To exhaust the pool of methionine in human serum, we previously engineered an l-Met-degrading enzyme based on the human cystathionine-γ-lyase scaffold (hCGL-NLV) to circumvent immunogenicity and stability issues observed in the preclinical application of bacterially derived methionine-γ-lyases. To gain further insights into the structure–activity relationships governing the chemistry of the hCGL-NLV lead molecule, we undertook a biophysical characterization campaign that captured crystal structures (2.2 Å) of hCGL-NLV with distinct reaction intermediates, including internal aldimine, substrate-bound, gem-diamine, and external aldimine forms.more » Curiously, an alternate form of hCGL-NLV that crystallized under higher-salt conditions revealed a locally unfolded active site, correlating with inhibition of activity as a function of ionic strength. Subsequent mutational and kinetic experiments pinpointed that a salt bridge between the phosphate of the essential cofactor pyridoxal 5'-phosphate (PLP) and residue R62 plays an important role in catalyzing β- and γ-eliminations. Our study suggests that solvent ions such as NaCl disrupt electrostatic interactions between R62 and PLP, decreasing catalytic efficiency.« less

A novel C-S lyase from the latex-producing plant Taraxacum brevicorniculatum displays alanine aminotransferase and l-cystine lyase activity.

PubMed

Munt, Oliver; Prüfer, Dirk; Schulze Gronover, Christian

2013-01-01

We isolated a novel pyridoxal-5-phosphate-dependent l-cystine lyase from the dandelion Taraxacum brevicorniculatum. Real time qPCR analysis showed that C-S lyase from Taraxacum brevicorniculatum (TbCSL) mRNA is expressed in all plant tissues, although at relatively low levels in the latex and pedicel. The 1251 bp TbCSL cDNA encodes a protein with a calculated molecular mass of 46,127 kDa. It is homologous to tyrosine and alanine aminotransferases (AlaATs) as well as to an Arabidopsis thaliana carbon-sulfur lyase (C-S lyase) (SUR1), which has a role in glucosinolate metabolism. TbCSL displayed in vitrol-cystine lyase and AlaAT activities of 4 and 19nkatmg(-1) protein, respectively. However, we detected no in vitro tyrosine aminotransferase (TyrAT) activity and RNAi knockdown of the enzyme had no effect on phenotype, showing that TbCSL substrates might be channeled into redundant pathways. TbCSL is in vivo localized in the cytosol and functions as a C-S lyase or an aminotransferase in planta, but the purified enzyme converts at least two substrates specifically, and can thus be utilized for further in vitro applications. Copyright © 2012 Elsevier GmbH. All rights reserved.

Kinetics of Nitrite Reduction and Peroxynitrite Formation by Ferrous Heme in Human Cystathionine β-Synthase*

PubMed Central

Carballal, Sebastián; Cuevasanta, Ernesto; Yadav, Pramod K.; Gherasim, Carmen; Ballou, David P.; Alvarez, Beatriz; Banerjee, Ruma

2016-01-01

Cystathionine β-synthase (CBS) is a pyridoxal phosphate-dependent enzyme that catalyzes the condensation of homocysteine with serine or with cysteine to form cystathionine and either water or hydrogen sulfide, respectively. Human CBS possesses a noncatalytic heme cofactor with cysteine and histidine as ligands, which in its oxidized state is relatively unreactive. Ferric CBS (Fe(III)-CBS) can be reduced by strong chemical and biochemical reductants to Fe(II)-CBS, which can bind carbon monoxide (CO) or nitric oxide (NO•), leading to inactive enzyme. Alternatively, Fe(II)-CBS can be reoxidized by O2 to Fe(III)-CBS, forming superoxide radical anion (O2˙̄). In this study, we describe the kinetics of nitrite (NO2−) reduction by Fe(II)-CBS to form Fe(II)NO•-CBS. The second order rate constant for the reaction of Fe(II)-CBS with nitrite was obtained at low dithionite concentrations. Reoxidation of Fe(II)NO•-CBS by O2 showed complex kinetic behavior and led to peroxynitrite (ONOO−) formation, which was detected using the fluorescent probe, coumarin boronic acid. Thus, in addition to being a potential source of superoxide radical, CBS constitutes a previously unrecognized source of NO• and peroxynitrite. PMID:26867575

Crystal structure of a photolysis product of vitamin B6: A pyridodihydrofuran-condensed skeleton compound of pyridoxal 5‧-phosphate

NASA Astrophysics Data System (ADS)

Aoki, Katsuyuki; Nakamura, Hideyuki; Hattori, Toshiaki; Hu, Ning-Hai; Onishi, Masayoshi

2017-11-01

An aqueous solution dissolving pyridoxal 5‧-phosphate (PLP) was exposed to sun-light at room temperature to yield a photolysis product, 4b,9b-dihydro-4b,9b-dihydroxy-1,6-dimethyl-4,9-bis(phosphonooxymethyl){pyrido[3‧,4‧:2,3]furo[4,5-b]}pyrido[4,3-d]furan (1), whose structure was crystallographically determined. The product 1 was found to be a novel C(sp3)-C(sp3) side-sharing pyridodihydrofuran-condensed skeleton compound with the two pyridodihydrofuran planes taking a 'V-shape'-like molecular configuration. Hydrogen bonding patterns of molecules of 1 in the crystal lattice are analyzed by the graph set approach. The most probable mechanism for the formation of 1 is described.

Oxidative phosphorylation in Debaryomyces hansenii: physiological uncoupling at different growth phases.

PubMed

Cabrera-Orefice, Alfredo; Guerrero-Castillo, Sergio; Díaz-Ruíz, Rodrigo; Uribe-Carvajal, Salvador

2014-07-01

Physiological uncoupling of mitochondrial oxidative phosphorylation (OxPhos) was studied in Debaryomyces hansenii. In other species, such as Yarrowia lipolytica and Saccharomyces cerevisiae, OxPhos can be uncoupled through differential expression of branched respiratory chain enzymes or by opening of a mitochondrial unspecific channel (ScMUC), respectively. However D. hansenii mitochondria, which contain both a branched respiratory chain and a mitochondrial unspecific channel (DhMUC), selectively uncouple complex I-dependent rate of oxygen consumption in the stationary growth phase. The uncoupled complex I-dependent respiration was only 20% of the original activity. Inhibition was not due to inactivation of complex I, lack of protein expression or to differential expression of alternative oxidoreductases. Furthermore, all other respiratory chain activities were normal. Decrease of complex I-dependent respiration was due to NAD(+) loss from the matrix, probably through an open of DhMUC. When NAD(+) was added back, coupled complex I-activity was recovered. NAD(+) re-uptake was independent of DhMUC opening and seemed to be catalyzed by a NAD(+)-specific transporter, which was sensitive to bathophenanthroline, bromocresol purple or pyridoxal-5'-phosphate as described for S. cerevisiae mitochondrial NAD(+) transporters. Loss of NAD(+) from the matrix through an open MUC is proposed as an additional mechanism to uncouple OxPhos. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Saccharomyces cerevisiae Differential Functionalization of Presumed ScALT1 and ScALT2 Alanine Transaminases Has Been Driven by Diversification of Pyridoxal Phosphate Interactions

PubMed Central

Rojas-Ortega, Erendira; Aguirre-López, Beatriz; Reyes-Vivas, Horacio; González-Andrade, Martín; Campero-Basaldúa, Jose C.; Pardo, Juan P.; González, Alicia

2018-01-01

Saccharomyces cerevisiae arose from an interspecies hybridization (allopolyploidiza-tion), followed by Whole Genome Duplication. Diversification analysis of ScAlt1/ScAlt2 indicated that while ScAlt1 is an alanine transaminase, ScAlt2 lost this activity, constituting an example in which one of the members of the gene pair lacks the apparent ancestral physiological role. This paper analyzes structural organization and pyridoxal phosphate (PLP) binding properties of ScAlt1 and ScAlt2 indicating functional diversification could have determined loss of ScAlt2 alanine transaminase activity and thus its role in alanine metabolism. It was found that ScAlt1 and ScAlt2 are dimeric enzymes harboring 67% identity and intact conservation of the catalytic residues, with very similar structures. However, tertiary structure analysis indicated that ScAlt2 has a more open conformation than that of ScAlt1 so that under physiological conditions, while PLP interaction with ScAlt1 allows the formation of two tautomeric PLP isomers (enolimine and ketoenamine) ScAlt2 preferentially forms the ketoenamine PLP tautomer, indicating a modified polarity of the active sites which affect the interaction of PLP with these proteins, that could result in lack of alanine transaminase activity in ScAlt2. The fact that ScAlt2 forms a catalytically active Schiff base with PLP and its position in an independent clade in “sensu strictu” yeasts suggests this protein has a yet undiscovered physiological function. PMID:29867852

C-terminal splicing of NTPDase2 provides distinctive catalytic properties, cellular distribution and enzyme regulation

PubMed Central

2004-01-01

The present study provides functional characterization of alternative splicing of the NTPDase2 (ecto-nucleoside triphosphate diphosphohydrolase-2) involved in the regulation of extracellular nucleotide concentrations in a range of organ systems. A novel NTPDase2β isoform produced by alternative splicing of the rat NTPDase2 gene provides an extended intracellular C-terminus and distinguishes itself from NTPDase2α isoform in gaining several intracellular protein kinase CK2 (casein kinase 2) phosphorylation sites and losing the intracellular protein kinase C motif. The plasmids containing NTPDase2α or NTPDase2β cDNA were used to stably transfect Chinese-hamster ovary-S cells. Imaging studies showed that NTPDase2α was predominantly membrane-bound, whereas NTPDase2β had combined cell surface and intracellular localization. α and β isoforms showed variations in divalent cation dependence and substrate specificity for nucleoside-5′-triphosphates and nucleoside-5′-diphosphates. NTPDase2β exhibited reduced ATPase activity and no apparent ADPase activity. NTPDase2 isoforms demonstrated similar sensitivity to inhibitors such as suramin and pyridoxal phosphate-6-azophenyl-2′,4′-disulphonic acid, and differential regulation by protein kinases. NTPDase2β was up-regulated by intracellular protein kinase CK2 phosphorylation, whereas NTPDase2α activity was down-regulated by protein kinase C phosphorylation. The results demonstrate that alternative coding of the intracellular C-terminal domain contributes distinctive phenotypic variation with respect to extracellular nucleotide specificity, hydrolysis kinetics, protein kinase-dependent intracellular regulation and protein trafficking. These findings advance the molecular physiology of this enzyme system by characterizing the contribution of the C-terminal domain to many of the enzyme's signature properties. PMID:15362980

A simple high-performance liquid chromatography (HPLC) method for the measurement of pyridoxal-5-phosphate and 4-pyridoxic acid in human plasma.

PubMed

Cabo, Rona; Kozik, Karolina; Milanowski, Maciej; Hernes, Sigrunn; Slettan, Audun; Haugen, Margaretha; Ye, Shu; Blomhoff, Rune; Mansoor, M Azam

2014-06-10

Low concentration of plasma pyridoxal-5-phosphate (PLP) is associated with hyperhomocysteinemia and inflammation. Most methods for the measurement of plasma PLP require large specimen volume and involve the use of toxic reagents. We have developed a HPLC method for the measurement of PLP and 4-pyridoxic acid (4-PA) in plasma, which requires small specimen volume. The samples are prepared without adding any toxic reagents. Furthermore, we have examined whether intake of vitamin B6 affects the concentration of plasma PLP and 4-PA. The coefficient of variation of the method was 6% and the recovery of the added vitamin in plasma was about 100%. The concentrations of plasma PLP and 4-PA in 168 healthy subjects were 40.6 (8.4-165.0) nmol/L, median and (range) and 17.5 (3.7-114.79) nmol/L, median and (range) respectively. In the multiple regression analyses, the concentration of plasma PLP was associated with the concentration of plasma 4-PA (p<0.0001), BMI, (p=0.02) and sex, (p=0.0008). The concentration of plasma 4-PA was associated with plasma PLP (p<0.0001), serum folate (p=0.004), smoking (p=0.03) and vitamin B6 intake (p=0.01). The present method is suitable for large clinical studies for the measurement of plasma PLP and 4-PA. Our findings demonstrate that plasma 4-PA, BMI and sex are the major determinants of plasma PLP in healthy individuals. Copyright © 2014 Elsevier B.V. All rights reserved.

Identification of proteins regulated by ferulic acid in a middle cerebral artery occlusion animal model-a proteomics approach.

PubMed

Sung, Jin-Hee; Cho, Eun-Hae; Cho, Jae-Hyeon; Won, Chung-Kil; Kim, Myeong-Ok; Koh, Phil-Ok

2012-11-01

Ferulic acid plays a neuroprotective role in cerebral ischemia. The aim of this study was to identify the proteins that are differentially expressed following ferulic acid treatment during ischemic brain injury using a proteomics technique. Middle cerebral artery occlusion (MCAO) was performed to induce a focal cerebral ischemic injury in adult male rats, and ferulic acid (100 mg/kg) or vehicle was administered immediately after MCAO. Brain tissues were collected 24 hr after MCAO. The proteins in the cerebral cortex were separated using two-dimensional gel electrophoresis and were identified by mass spectrometry. We detected differentially expressed proteins between vehicle- and ferulic acid-treated animals. Adenosylhomocysteinase, isocitrate dehydrogenase [NAD(+)], mitogen-activated protein kinase kinase 1 and glyceraldehyde-3-phosphate dehydrogenase were decreased in the vehicle-treated group, and ferulic acid prevented the injury-induced decreases in these proteins. However, pyridoxal phosphate phosphatase and heat shock protein 60 were increased in the vehicle-treated group, while ferulic acid prevented the injury-induced increase in these proteins. It is accepted that these enzymes are involved in cellular metabolism and differentiation. Thus, these findings suggest evidence that ferulic acid plays a neuroprotective role against focal cerebral ischemia through the up- and down-modulation of specific enzymes.

Structure of the Mitochondrial Aminolevulinic Acid Synthase, a Key Heme Biosynthetic Enzyme.

PubMed

Brown, Breann L; Kardon, Julia R; Sauer, Robert T; Baker, Tania A

2018-04-03

5-Aminolevulinic acid synthase (ALAS) catalyzes the first step in heme biosynthesis. We present the crystal structure of a eukaryotic ALAS from Saccharomyces cerevisiae. In this homodimeric structure, one ALAS subunit contains covalently bound cofactor, pyridoxal 5'-phosphate (PLP), whereas the second is PLP free. Comparison between the subunits reveals PLP-coupled reordering of the active site and of additional regions to achieve the active conformation of the enzyme. The eukaryotic C-terminal extension, a region altered in multiple human disease alleles, wraps around the dimer and contacts active-site-proximal residues. Mutational analysis demonstrates that this C-terminal region that engages the active site is important for ALAS activity. Our discovery of structural elements that change conformation upon PLP binding and of direct contact between the C-terminal extension and the active site thus provides a structural basis for investigation of disruptions in the first step of heme biosynthesis and resulting human disorders. Copyright © 2018 Elsevier Ltd. All rights reserved.

[Characteristics of the glutamate decarboxylase reaction in homogenates of various regions of the rat brain].

PubMed

Rozanov, V A

1987-01-01

The glutamate decarboxylase activity in rough homogenates of cerebellum, cortex and truncal part of the rat brain was studied under different conditions of incubation: in the presence of 25 mM glutamate sodium, 0.4 mM pyridoxal-5'-phosphate and both these components. It is found that the initial glutamate decarboxylase activity in cerebellum homogenates is approximately twice as high as in the cortex and trunk homogenates. Addition of the substrate and cofactor, especially in the combination, stimulates considerably the yield of gamma-aminobutyric acid (GABA) in the glutamate decarboxylase reaction, the most pronounced activation being observed in the truncal homogenates. The glutamate/GABA relation both initial and after the completion of the reaction is the maximal in the cortex and minimal in the truncal part of the brain. The data obtained evidence for the differences in the content of the GABA-producing enzyme rather than for the presence of the specific mechanisms of the enzyme regulation in different brain areas.

Improvement of kynurenine aminotransferase-II inhibitors guided by mimicking sulfate esters.

PubMed

Jayawickrama, Gayan S; Nematollahi, Alireza; Sun, Guanchen; Church, William Bret

2018-01-01

The mammalian kynurenine aminotransferase (KAT) enzymes are a family of related isoforms that are pyridoxal 5'-phosphate-dependent, responsible for the irreversible transamination of kynurenine to kynurenic acid. Kynurenic acid is implicated in human diseases such as schizophrenia where it is found in elevated levels and consequently KAT-II, as the isoform predominantly responsible for kynurenic acid production in the brain, has been targeted for the development of specific inhibitors. One class of compounds that have also shown inhibitory activity towards the KAT enzymes are estrogens and their sulfate esters. Estradiol disulfate in particular is very strongly inhibitory and it appears that the 17-sulfate makes a significant contribution to its potency. The work here demonstrates that the effect of this moiety can be mirrored in existing KAT-II inhibitors, from the development of two novel inhibitors, JN-01 and JN-02. Both inhibitors were based on NS-1502 (IC50: 315 μM), but the deliberate placement of a sulfonamide group significantly improved the potency of JN-01 (IC50: 73.8 μM) and JN-02 (IC50: 112.8 μM) in comparison to the parent compound. This 3-4 fold increase in potency shows the potential of these moieties to be accommodated in the KAT-II active site and the effect they can have on improving inhibitors, and the environments in the KAT-II have been suitably modelled using docking calculations.

Interaction between vitamin B6 metabolism, nitrogen metabolism and autoimmunity.

PubMed

Colinas, Maite; Fitzpatrick, Teresa B

2016-01-01

The essential micronutrient vitamin B6 is best known in its enzymatic cofactor form, pyridoxal 5'-phosphate (PLP). However, vitamin B6 comprises the amine pyridoxamine 5'-phosphate (PMP) and the alcohol pyridoxine 5'-phosphate (PNP) in addition to PLP, as well as their corresponding non-phosphorylated forms. The different B6 forms (called vitamers) are enzymatically interconverted in a ubiquitous salvage pathway. Recently, we have shown that balancing the ratio of the different B6 vitamers in particular PMP by the PMP/PNP oxidase PDX3 is essential for growth and development in Arabidopsis thaliana. Intriguingly, nitrate to ammonium conversion is impaired in pdx3 mutants, such that the mutants become ammonium-dependent, suggesting an interaction between vitamin B6 and nitrogen metabolism. In addition, we found a strong up-regulation of genes related to plant defense. Here, we further show that pdx3 mutants display a temperature-sensitive phenotype that is typical of autoimmune mutants and is possibly connected to the impaired nitrogen metabolism.

Novel homozygous missense mutation in ALDH7A1 causes neonatal pyridoxine dependent epilepsy.

PubMed

Coci, Emanuele G; Codutti, Luca; Fink, Christian; Bartsch, Sophie; Grüning, Gunnar; Lücke, Thomas; Kurth, Ingo; Riedel, Joachim

2017-04-01

Pyridoxine dependent epilepsy (PDE) (OMIM#266100) is a neonatal form of epilepsy, caused by dysfunction of the enzyme α-aminoadipic semialdehyde dehydrogenase (ALDH7A1 or Antiquitin). This enzyme converts α-aminoadipic semialdehyde (α-AASA) into α-aminoadipate (AAA), a critical step in the lysine metabolism of the brain. ALDH7A1 dysfunction causes an accumulation of α-AASA and δ 1 -piperideine-6-carboxylic acid (P6C), which are in equilibrium with each other. P6C binds and inactivates pyridoxal 5'-phosphate (PLP), the active form of pyridoxine. Individuals affected by ALDH7A1 deficiency show pre-natal and post-natal seizures, which respond to oral pyridoxine but not to other pediatric anti-epileptic drugs. We discovered a novel missense mutation (c.566G > A, p.Gly189Glu) in homozygous state residing in the NAD+ binding domain coding region of exon 6 and affecting an highly conserved amino acid residue. The seizures stopped under post-natal pyridoxine therapy, nevertheless a longer follow-up is needed to evaluate the intellectual development of the child, who is additionally treated with oral l-arginine since the 13th month of life. Developmental delay with or without structural cortex abnormalities were reported in several patients. A brain MRI scan revealed hyperintense white matter in the right cerebellum compatible with cerebellar gliosis. Taken together, our studies enlarge the group of missense pathogenic mutations of ALDH7A1 gene and reveal a novel cerebellar finding within the PDE patients cohort. Copyright © 2016 Elsevier Ltd. All rights reserved.

Functional Analysis of PDX2 from Arabidopsis, a Glutaminase Involved in Vitamin B6 Biosynthesis1[W][OA

PubMed Central

Tambasco-Studart, Marina; Tews, Ivo; Amrhein, Nikolaus; Fitzpatrick, Teresa B.

2007-01-01

Vitamin B6 is an essential metabolite in all organisms, being required as a cofactor for a wide variety of biochemical reactions. De novo biosynthesis of the vitamin occurs in microorganisms and plants, but animals must obtain it from their diet. Two distinct and mutually exclusive de novo pathways have been identified to date, namely deoxyxylulose 5-phosphate dependent, which is restricted to a subset of eubacteria, and deoxyxylulose 5-phosphate independent, present in archaea, fungi, plants, protista, and most eubacteria. In these organisms, pyridoxal 5′-phosphate (PLP) formation is catalyzed by a single glutamine amidotransferase (PLP synthase) composed of a glutaminase domain, PDX2, and a synthase domain, PDX1. Despite plants being an important source of vitamin B6, very little is known about its biosynthesis. Here, we provide information for Arabidopsis thaliana. The functionality of PDX2 is demonstrated, using both in vitro and in vivo analyses. The expression pattern of PDX2 is assessed at both the RNA and protein level, providing insight into the spatial and temporal pattern of vitamin B6 biosynthesis. We then provide a detailed biochemical analysis of the plant PLP synthase complex. While the active sites of PDX1 and PDX2 are remote from each other, coordination of catalysis is much more pronounced with the plant proteins than its bacterial counterpart, Bacillus subtilis. Based on a model of the PDX1/PDX2 complex, mutation of a single residue uncouples enzyme coordination and in turn provides tangible evidence for the existence of the recently proposed ammonia tunnel through the core of PDX1. PMID:17468224

Tryptophan metabolism via transamination. In vitro aminotransferase assay using dinitrophenylhydrazine method.

PubMed

Drsata, Jaroslav

2003-01-01

Transamination of tryptophan belongs to minor pathways of amino acid metabolism. The present paper describes conditions for application of dinitrophenylhydrazine method, originally prepared for alanine aminortansferase and aspartate aminotransferase assay, to the measurement of tryptophan transamination catalysed by any of the enzymes mentioned above. The method was tested using purified pig heart AST. While the free enzyme showed a characteristic absorption profile with the maxima at 360 and 430 nm, the course of transamination of tryptophan was confirmed by the measurement of UV-VIS spectral changes of the coenzyme in the active site of the enzyme in the presence of the amino acid substrate only, when tryptophan caused a shift of the peak from 360 nm to 330 nm due to a change of the pyridoxal form to the pyridoxamine form (= the first step of ping-pong transaminating reaction). A general limitation of dinitrophenylhydrazine method is the interference of hydrazones formed from the coenzyme pyridoxal-5'-phosphate and from the oxo- substrate 2-oxoglutarate, showing the absorption maxima at 492 nm and 388 nm, respectively with the hydrazones formed by the oxo- products (pyruvate and/or oxaloacetate in the case of ALT/AST, the absorption maxima at 443 nm in our measurements). In the case of tryptophan transamination, indolepyruvate as the oxo- product of a catalysed reaction forms dinitrophenylhydrazone, which has, besides a maximum at 435 nm, a distinct peak at 542 nm, convenient for the product concentration measurement. This is favourable for resolution from other (interfering) hydrazones. Suitable conditions for tryptophan transamination in tissue and enzyme preparations were found. Reaching optimal conditions for tryptophan transamination measurements in vitro is generally limited by low solubility of the amino acid in water solutions: With AST preparation, the velocity of catalysed reaction at 5-50 x 10(-3) M tryptophan concentration was of 1st order to the amino acid substrate. Km for tryptophan was found > or = 2 x 10(-1) M. Therefore the enzyme activity measurement at two different tryptophan concentrations is recommended for unknown samples. Tryptophan transamination by purified pig AST was compared with that catalysed by preparations obtained from mammalian tissues.

Mechanism of Inactivation of GABA Aminotransferase by (E)- and (Z)-(1S,3S)-3-Amino-4-fluoromethylenyl-1-cyclopentanoic Acid

PubMed Central

Lee, Hyunbeom; Le, Hoang V.; Wu, Rui; Doud, Emma; Sanishvili, Ruslan; Kellie, John F.; Compton, Phillip D.; Pachaiyappan, Boobalan; Liu, Dali; Kelleher, Neil L.

2015-01-01

When γ-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the mammalian central nervous system, falls below a threshold level, seizures occur. One approach to raise GABA concentrations is to inhibit GABA aminotransferase (GABA-AT), a pyridoxal 5’-phosphate-dependent enzyme that degrades GABA. We have previously developed (1S,3S)-3-amino-4-difluoromethylene-1-cyclopentanoic acid (CPP-115), which is 186 times more efficient in inactivating GABA-AT than vigabatrin, the only FDA-approved inactivator of GABA-AT. We also developed (E)- and (Z)-(1S,3S)-3-amino-4-fluoromethylenyl-1-cyclopentanoic acid (1 and 2, respectively), monofluorinated analogs of CPP-115, which are comparable to vigabatrin in inactivating GABA-AT. Here we report the mechanism of inactivation of GABA-AT by 1 and 2. Both produce a metabolite that induces disruption of the Glu270-Arg445 salt bridge to accommodate interaction between the metabolite formyl group and Arg445. This is the second time that Arg445 has interacted with a ligand and is involved in GABA-AT inactivation, thereby confirming the importance of Arg445 in future inactivator design. PMID:26110556

Tyramine and phenylethylamine biosynthesis by food bacteria.

PubMed

Marcobal, Angela; De las Rivas, Blanca; Landete, José María; Tabera, Laura; Muñoz, Rosario

2012-01-01

Tyramine poisoning is caused by the ingestion of food containing high levels of tyramine, a biogenic amine. Any foods containing free tyrosine are subject to tyramine formation if poor sanitation and low quality foods are used or if the food is subject to temperature abuse or extended storage time. Tyramine is generated by decarboxylation of the tyrosine through tyrosine decarboxylase (TDC) enzymes derived from the bacteria present in the food. Bacterial TDC have been only unequivocally identified and characterized in Gram-positive bacteria, especially in lactic acid bacteria. Pyridoxal phosphate (PLP)-dependent TDC encoding genes (tyrDC) appeared flanked by a similar genetic organization in several species of lactic acid bacteria, suggesting a common origin by a single mobile genetic element. Bacterial TDC are also able to decarboxylate phenylalanine to produce phenylethylamine (PEA), another biogenic amine. The molecular knowledge of the genes involved in tyramine production has led to the development of molecular methods for the detection of bacteria able to produce tyramine and PEA. These rapid and simple methods could be used for the analysis of the ability to form tyramine by bacteria in order to evaluate the potential risk of tyramine biosynthesis in food products.

Mechanism of Inactivation of GABA Aminotransferase by (E)- and (Z)-(1S,3S)-3-Amino-4-fluoromethylenyl-1-cyclopentanoic Acid.

PubMed

Lee, Hyunbeom; Le, Hoang V; Wu, Rui; Doud, Emma; Sanishvili, Ruslan; Kellie, John F; Compton, Phillip D; Pachaiyappan, Boobalan; Liu, Dali; Kelleher, Neil L; Silverman, Richard B

2015-09-18

When γ-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the mammalian central nervous system, falls below a threshold level, seizures occur. One approach to raise GABA concentrations is to inhibit GABA aminotransferase (GABA-AT), a pyridoxal 5'-phosphate-dependent enzyme that degrades GABA. We have previously developed (1S,3S)-3-amino-4-difluoromethylene-1-cyclopentanoic acid (CPP-115), which is 186 times more efficient in inactivating GABA-AT than vigabatrin, the only FDA-approved inactivator of GABA-AT. We also developed (E)- and (Z)-(1S,3S)-3-amino-4-fluoromethylenyl-1-cyclopentanoic acid (1 and 2, respectively), monofluorinated analogs of CPP-115, which are comparable to vigabatrin in inactivating GABA-AT. Here, we report the mechanism of inactivation of GABA-AT by 1 and 2. Both produce a metabolite that induces disruption of the Glu270-Arg445 salt bridge to accommodate interaction between the metabolite formyl group and Arg445. This is the second time that Arg445 has interacted with a ligand and is involved in GABA-AT inactivation, thereby confirming the importance of Arg445 in future inactivator design.

A Canonical Biotin Synthesis Enzyme, 8-Amino-7-Oxononanoate Synthase (BioF), Utilizes Different Acyl Chain Donors in Bacillus subtilis and Escherichia coli.

PubMed

Manandhar, Miglena; Cronan, John E

2018-01-01

BioF (8-amino-7-oxononanoate synthase) is a strictly conserved enzyme that catalyzes the first step in assembly of the fused heterocyclic rings of biotin. The BioF acyl chain donor has long been thought to be pimeloyl-CoA. Indeed, in vitro the Escherichia coli and Bacillus sphaericus enzymes have been shown to condense pimeloyl-CoA with l-alanine in a pyridoxal 5'-phosphate-dependent reaction with concomitant CoA release and decarboxylation of l-alanine. However, recent in vivo studies of E. coli and Bacillus subtilis suggested that the BioF proteins of the two bacteria could have different specificities for pimelate thioesters in that E. coli BioF may utilize either pimeloyl coenzyme A (CoA) or the pimelate thioester of the acyl carrier protein (ACP) of fatty acid synthesis. In contrast, B. subtilis BioF seemed likely to be specific for pimeloyl-CoA and unable to utilize pimeloyl-ACP. We now report genetic and in vitro data demonstrating that B. subtilis BioF specifically utilizes pimeloyl-CoA. IMPORTANCE Biotin is an essential vitamin required by mammals and birds because, unlike bacteria, plants, and some fungi, these organisms cannot make biotin. Currently, the biotin included in vitamin tablets and animal feeds is made by chemical synthesis. This is partly because the biosynthetic pathways in bacteria are incompletely understood. This paper defines an enzyme of the Bacillus subtilis pathway and shows that it differs from that of Escherichia coli in the ability to utilize specific precursors. These bacteria have been used in biotin production and these data may aid in making biotin produced by biotechnology commercially competitive with that produced by chemical synthesis. Copyright © 2017 American Society for Microbiology.

Catalytically active alkaline molten globular enzyme: Effect of pH and temperature on the structural integrity of 5-aminolevulinate synthase*

PubMed Central

Stojanovski, Bosko M.; Breydo, Leonid; Hunter, Gregory A.; Uversky, Vladimir N.; Ferreira, Gloria C.

2014-01-01

5-Aminolevulinate synthase (ALAS), a pyridoxal-5′phosphate (PLP)-dependent enzyme, catalyzes the first step of heme biosynthesis in mammals. Circular dichroism (CD) and fluorescence spectroscopies were used to examine the effects of pH (1.0–3.0 and 7.5–10.5) and temperature (20 and 37 °C) on the structural integrity of ALAS. The secondary structure, as deduced from far-UV CD, is mostly resilient to pH and temperature changes. Partial unfolding was observed at pH 2.0, but further decreasing pH resulted in acid-induced refolding of the secondary structure to nearly native levels. The tertiary structure rigidity, monitored by near-UV CD, is lost under acidic and specific alkaline conditions (pH 10.5 and pH 9.5/37 °C), where ALAS populates a molten globule state. As the enzyme becomes less structured with increased alkalinity, the chiral environment of the internal aldimine is also modified, with a shift from a 420 nm to 330 nm dichroic band. Under acidic conditions, the PLP cofactor dissociates from ALAS. Reaction with 8-anilino-1-naphtalenesulfonic acid corroborates increased exposure of hydrophobic clusters in the alkaline and acidic molten globules, although the reaction is more pronounced with the latter. Furthermore, quenching the intrinsic fluorescence of ALAS with acrylamide at pH 1.0 and 9.5 yielded subtly different dynamic quenching constants. The alkaline molten globule state of ALAS is catalytically active (pH 9.5/37 °C), although the kcat value is significantly decreased. Finally, the binding of 5-aminolevulinate restricts conformational fluctuations in the alkaline molten globule. Overall, our findings prove how the structural plasticity of ALAS contributes to reaching a functional enzyme. PMID:25240868

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Hyunbeom; Doud, Emma H.; Wu, Rui

γ-Aminobutyric acid aminotransferase (GABA-AT) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that degrades GABA, the principal inhibitory neurotransmitter in mammalian cells. When the concentration of GABA falls below a threshold level, convulsions can occur. Inhibition of GABA-AT raises GABA levels in the brain, which can terminate seizures as well as have potential therapeutic applications in treating other neurological disorders, including drug addiction. Among the analogues that we previously developed, (1S,3S)-3-amino-4-difluoromethylene-1-cyclopentanoic acid (CPP-115) showed 187 times greater potency than that of vigabatrin, a known inactivator of GABA-AT and approved drug (Sabril) for the treatment of infantile spasms and refractory adult epilepsy. Recently,more » CPP-115 was shown to have no adverse effects in a Phase I clinical trial. Here we report a novel inactivation mechanism for CPP-115, a mechanism-based inactivator that undergoes GABA-AT-catalyzed hydrolysis of the difluoromethylene group to a carboxylic acid with concomitant loss of two fluoride ions and coenzyme conversion to pyridoxamine 5'-phosphate (PMP). The partition ratio for CPP-115 with GABA-AT is about 2000, releasing cyclopentanone-2,4-dicarboxylate (22) and two other precursors of this compound (20 and 21). Time-dependent inactivation occurs by a conformational change induced by the formation of the aldimine of 4-aminocyclopentane-1,3-dicarboxylic acid and PMP (20), which disrupts an electrostatic interaction between Glu270 and Arg445 to form an electrostatic interaction between Arg445 and the newly formed carboxylate produced by hydrolysis of the difluoromethylene group in CPP-115, resulting in a noncovalent, tightly bound complex. Ultimately, this represents a novel mechanism for inactivation of GABA-AT and a new approach for the design of mechanism-based inactivators in general.« less

Characterization of C-S lyase from Lactobacillus delbrueckii subsp. bulgaricus ATCC BAA-365 and its potential role in food flavour applications.

PubMed

Allegrini, Alessandra; Astegno, Alessandra; La Verde, Valentina; Dominici, Paola

2017-04-01

Volatile thiols have substantial impact on the aroma of many beverages and foods. Thus, the control of their formation, which has been linked to C-S lyase enzymatic activities, is of great significance in industrial applications involving food flavours. Herein, we have carried out a spectroscopic and functional characterization of a putative pyridoxal 5'-phosphate (PLP)-dependent C-S lyase from the lactic acid bacterium Lactobacillus delbrueckii subsp. bulgaricus ATCC BAA-365 (LDB C-S lyase). Recombinant LDB C-S lyase exists as a tetramer in solution and shows spectral properties of enzymes containing PLP as cofactor. The enzyme has a broad substrate specificity toward sulphur-containing amino acids with aminoethyl-L-cysteine and L-cystine being the most effective substrates over L-cysteine and L-cystathionine. Notably, the protein also reveals cysteine-S-conjugate β-lyase activity in vitro, and is able to cleave a cysteinylated substrate precursor into the corresponding flavour-contributing thiol, with a catalytic efficiency higher than L-cystathionine. Contrary to similar enzymes of other lactic acid bacteria however, LDB C-S lyase is not capable of α,γ-elimination activity towards L-methionine to produce methanethiol, which is a significant compound in flavour development. Based on our results, future developments can be expected regarding the flavour-forming potential of Lactobacillus C-S lyase and its use in enhancing food flavours. © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Ophthalmic acid accumulation in an Escherichia coli mutant lacking the conserved pyridoxal 5'-phosphate-binding protein YggS.

PubMed

Ito, Tomokazu; Yamauchi, Ayako; Hemmi, Hisashi; Yoshimura, Tohru

2016-12-01

Escherichia coli YggS is a highly conserved pyridoxal 5'-phosphate (PLP)-binding protein whose biochemical function is currently unknown. A previous study with a yggS-deficient E. coli strain (ΔyggS) demonstrated that YggS controls l-Ile- and l-Val-metabolism by modulating 2-ketobutyrate (2-KB), l-2-aminobutyrate (l-2-AB), and/or coenzyme A (CoA) availability in a PLP-dependent fashion. In this study, we found that ΔyggS accumulates an unknown metabolite as judged by amino acid analyses. LC/MS and MS/MS analyses of the compound with propyl chloroformate derivatization, and co-chromatography analysis identified this compound as γ-l-glutamyl-l-2-aminobutyryl-glycine (ophthalmic acid), a glutathione (GSH) analogue in which the l-Cys moiety is replaced by l-2-AB. We also determine the metabolic consequence of the yggS mutation. Absence of YggS initially increases l-2-AB availability, and then causes ophthalmic acid accumulation and CoA limitation in the cell. The expression of a γ-glutamylcysteine synthetase and a glutathione synthetase in a ΔyggS background causes high-level accumulation of ophthalmic acid in the cells (∼1.2 nmol/mg cells) in a minimal synthetic medium. This opens the possibility of a first fermentative production of ophthalmic acid. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Phosphate-dependent glutaminase in enterocyte mitochondria and its regulation by ammonium and other ions.

PubMed

Masola, B; Zvinavashe, E

2003-06-01

The effects of ammonium and other ions on phosphate dependent glutaminase (PDG) activity in intact rat enterocyte mitochondria were investigated. Sulphate and bicarbonate activated the enzyme in absence and presence of added phosphate. In presence of 10 mM phosphate, ammonium at concentrations <1 mM inhibited the enzyme. This inhibition was reversed by increased concentration of phosphate or sulphate. The inhibition of PDG by ammonium in presence of 10 mM phosphate was biphasic with respect to glutamine concentration, its effect being through a lowering of V(max) at glutamine concentration of

Lysine Restriction and Pyridoxal Phosphate Administration in a NADK2 Patient.

PubMed

Tort, Frederic; Ugarteburu, Olatz; Torres, Maria Angeles; García-Villoria, Judit; Girós, Marisa; Ruiz, Angeles; Ribes, Antonia

2016-11-01

We report the case of a 10-year-old Spanish girl with mutations in NADK2 Prenatal central nervous system abnormalities showed ventriculomegaly, colpocephaly, and hypoplasia of the corpus callosum. At birth, axial hypotonia, uncoordinated movements, microcephaly, and generalized cerebellar atrophy were detected. Metabolic investigations revealed high lysine, lactate, and pipecolic acid levels in blood and cerebrospinal fluid. Pyruvate carboxylase and pyruvate dehydrogenase activity in fibroblasts were normal. Beginning at birth she received biotin, thiamine, and carnitine supplementation. A lysine-restricted diet was started when she was 1 month old. Because pipecolic acid was high, pyridoxine was added to treatment. At 3 years old, astatic myoclonic epilepsy appeared, with no response to levetiracetam. We switched pyridoxine to pyridoxal phosphate, with electroclinical improvement. Because the activity of mitochondrial respiratory chain complexes III and IV was slightly low in muscle, other cofactors such as ubidecarenone, idebenone, vitamin E, and creatine were added to the treatment. At 8 years old, plasma acylcarnitine testing was performed, and high levels of 2-trans, 4-cis-decadienoylcarnitine were found. Whole exome sequencing identified a homozygous splice site mutation in NADK2 (c.956+6T>C; p.Trp319Cysfs*21). This substitution generates exon skipping, leading to a truncated protein. In fact, NADK2 messenger RNA and the corresponding protein were almost absent. Now, at 10 years of age she presents with ataxia and incoordination. She has oromotor dysphasia but is able to understand fluid language and is a very friendly girl. We hypothesize that the patient's clinical improvement could be due to her lysine-restricted diet together with cofactors and pyridoxal phosphate administration. Copyright © 2016 by the American Academy of Pediatrics.

Vitamin B6 in plasma and cerebrospinal fluid of children.

PubMed

Albersen, Monique; Bosma, Marjolein; Jans, Judith J M; Hofstede, Floris C; van Hasselt, Peter M; de Sain-van der Velden, Monique G M; Visser, Gepke; Verhoeven-Duif, Nanda M

2015-01-01

Over the past years, the essential role of vitamin B6 in brain development and functioning has been recognized and genetic metabolic disorders resulting in functional vitamin B6 deficiency have been identified. However, data on B6 vitamers in children are scarce. B6 vitamer concentrations in simultaneously sampled plasma and cerebrospinal fluid (CSF) of 70 children with intellectual disability were determined by ultra performance liquid chromatography-tandem mass spectrometry. For ethical reasons, CSF samples could not be obtained from healthy children. The influence of sex, age, epilepsy and treatment with anti-epileptic drugs, were investigated. The B6 vitamer composition of plasma (pyridoxal phosphate (PLP) > pyridoxic acid > pyridoxal (PL)) differed from that of CSF (PL > PLP > pyridoxic acid > pyridoxamine). Strong correlations were found for B6 vitamers in and between plasma and CSF. Treatment with anti-epileptic drugs resulted in decreased concentrations of PL and PLP in CSF. We provide concentrations of all B6 vitamers in plasma and CSF of children with intellectual disability (±epilepsy), which can be used in the investigation of known and novel disorders associated with vitamin B6 metabolism as well as in monitoring of the biochemical effects of treatment with vitamin B6.

Crystal Structure of Ll-Diaminopimelate Aminotransferase From 'Arabidopsis Thaliana': a Recently-Discovered Enzyme in the Biosynthesis of L-Lysine By Plants And 'Chlamydia'

DOE Office of Scientific and Technical Information (OSTI.GOV)

Watanabe, N.; Cherney, M.M.; van Belkum, M.J.

2007-07-13

The essential biosynthetic pathway to l-Lysine in bacteria and plants is an attractive target for the development of new antibiotics or herbicides because it is absent in humans, who must acquire this amino acid in their diet. Plants use a shortcut of a bacterial pathway to l-Lysine in which the pyridoxal-5-phosphate (PLP)-dependent enzyme ll-diaminopimelate aminotransferase (LL-DAP-AT) transforms l-tetrahydrodipicolinic acid (L-THDP) directly to LL-DAP. In addition, LL-DAP-AT was recently found in Chlamydia sp., suggesting that inhibitors of this enzyme may also be effective against such organisms. In order to understand the mechanism of this enzyme and to assist in the designmore » of inhibitors, the three-dimensional crystal structure of LL-DAP-AT was determined at 1.95 Angstroms resolution. The cDNA sequence of LL-DAP-AT from Arabidopsis thaliana (AtDAP-AT) was optimized for expression in bacteria and cloned in Escherichia coli without its leader sequence but with a C-terminal hexahistidine affinity tag to aid protein purification. The structure of AtDAP-AT was determined using the multiple-wavelength anomalous dispersion (MAD) method with a seleno-methionine derivative. AtDAP-AT is active as a homodimer with each subunit having PLP in the active site. It belongs to the family of type I fold PLP-dependent enzymes. Comparison of the active site residues of AtDAP-AT and aspartate aminotransferases revealed that the PLP binding residues in AtDAP-AT are well conserved in both enzymes. However, Glu97* and Asn309* in the active site of AtDAP-AT are not found at similar positions in aspartate aminotransferases, suggesting that specific substrate recognition may require these residues from the other monomer. A malate-bound structure of AtDAP-AT allowed LL-DAP and L-glutamate to be modeled into the active site. These initial three-dimensional structures of LL-DAP-AT provide insight into its substrate specificity and catalytic mechanism.« less

Non-enzymic beta-decarboxylation of aspartic acid.

NASA Technical Reports Server (NTRS)

Doctor, V. M.; Oro, J.

1972-01-01

Study of the mechanism of nonenzymic beta-decarboxylation of aspartic acid in the presence of metal ions and pyridoxal. The results suggest that aspartic acid is first converted to oxalacetic acid by transamination with pyridoxal which in turn is converted to pyridoxamine. This is followed by decarboxylation of oxalacetic acid to form pyruvic acid which transaminates with pyridoxamine to form alanine. The possible significance of these results to prebiotic molecular evolution is briefly discussed.

DNA damage during glycation of lysine by methylglyoxal: assessment of vitamins in preventing damage.

PubMed

Suji, G; Sivakami, S

2007-11-01

Amino acids react with methylglyoxal to form advanced glycation end products. This reaction is known to produce free radicals. In this study, cleavage to plasmid DNA was induced by the glycation of lysine with methylglyoxal in the presence of iron(III). This system was found to produce superoxide as well as hydroxyl radicals. The abilities of various vitamins to prevent damage to plasmid DNA were evaluated. Pyridoxal-5-phosphate showed maximum protection, while pyridoxamine showed no protection. The protective abilities could be directly correlated to inhibition of production of hydroxyl and superoxide radicals. Pyridoxal-5-phosphate exhibited low radical scavenging ability as evaluated by its TEAC, but showed maximum protection probably by interfering in free radical production. Pyridoxamine did not inhibit free radical production. Thiamine and thiamine pyrophosphate, both showed protective effects albeit to different extents. Tetrahydrofolic acid showed better antioxidant activity than folic acid but was found to damage DNA by itself probably by superoxide generation.

Higher Plasma Pyridoxal Phosphate Is Associated with Increased Antioxidant Enzyme Activities in Critically Ill Surgical Patients

PubMed Central

Cheng, Chien-Hsiang; Huang, Shih-Chien; Chiang, Ting-Yu; Wong, Yueching

2013-01-01

Critically ill patients experience severe stress, inflammation and clinical conditions which may increase the utilization and metabolic turnover of vitamin B-6 and may further increase their oxidative stress and compromise their antioxidant capacity. This study was conducted to examine the relationship between vitamin B-6 status (plasma and erythrocyte PLP) oxidative stress, and antioxidant capacities in critically ill surgical patients. Thirty-seven patients in surgical intensive care unit of Taichung Veterans General Hospital, Taiwan, were enrolled. The levels of plasma and erythrocyte PLP, serum malondialdehyde, total antioxidant capacity, and antioxidant enzyme activities (i.e., superoxide dismutase (SOD), glutathione S-transferase, and glutathione peroxidase) were determined on the 1st and 7th days of admission. Plasma PLP was positively associated with the mean SOD activity level on day 1 (r = 0.42, P < 0.05), day 7 (r = 0.37, P < 0.05), and on changes (Δ (day 7 − day 1)) (r = 0.56, P < 0.01) after adjusting for age, gender, and plasma C-reactive protein concentration. Higher plasma PLP could be an important contributing factor in the elevation of antioxidant enzyme activity in critically ill surgical patients. PMID:23819116

Zinc-ion-dependent acid phosphatase exhibits magnesium-ion-dependent myo-inositol-1-phosphatase activity.

PubMed

Fujimoto, S; Okano, I; Tanaka, Y; Sumida, Y; Tsuda, J; Kawakami, N; Shimohama, S

1996-06-01

We have purified bovine brain Zn(2+)-dependent acid phosphatase (Zn(2+)-APase), which requires Zn2+ ions to hydrolyze the substrate p-nitrophenyl phosphate (pNPP) in an acidic environment. The substrate specificity and metal requirement of Zn(2+)-APase at a physiological pH was also studied. The enzyme exhibited hydrolytic activity on myo-inositol-1- and -2-monophosphates, 2'-adenosine monophosphate, 2'-guanosine monophosphate, and the alpha- and beta-glycerophosphates, glucose-1-phosphate, and fructose-6-phosphate in 50 mM Tris-HCl buffer (pH 7.4) in the presence of Mg2+ ions, but not on pNPP and phosphotyrosine. Zn2+, Mn2+ and Co2+ ions were less effective for activation. Among the above substrates, myo-inositol-1-phosphate was the most susceptible to hydrolysis by the enzyme in the presence of 3 mM Mg2+ ions. The enzyme exhibited an optimum pH at around 8 for myo-inositol-1-phosphate in the presence of 3 mM Mg2+ ions. The Mg(2+)-dependent myo-inositol-1-phosphatase activity of the enzyme was significantly inhibited by Li+ ions. The Zn(2+)-dependent p-nitrophenyl phosphatase activity and Mg(2+)-dependent myo-inositol-1-phosphatase activity of the purified enzyme fraction exhibited similar behavior on Sephadex G-100 and Mono Q colomns. These findings suggest that Zn(2+)-APase also exhibits Mg(2+)-dependent myo-inositol-1-phosphatase activity under physiological conditions.

[19F]fluorine nuclear-magnetic-resonance study of the interaction of difluoro-oxaloacetate with aspartate transaminase.

PubMed Central

Briley, P A; Eisenthal, R; Harrison, R; Smith, G D

1977-01-01

Difluoro-oxaloacetate interacts with the aldimine form of aspartate transaminase to give a complex, the dissociation constant of which has been determined spectrophotometrically and by 19F n.m.r. (nuclear magnetic resonance). The 19F n.m.r. line-width-pH and chemical-shift-pH profiles of difluoro-oxaloacetate in the presence of the aldimine form of the enzyme both show inflexion points in the pH5 and pH8 regions, which may arise from variations in the binding of difluoro-oxaloacetate as specific groups on the enzyme are successively protonated. Difluoro-oxaloacetate also interacts with apoenzyme to form a complex, the dissociation constant of which was determined by 19F n.m.r. The 19F n.m.r. line-width-pH and chemical-shift-pH profiles of difluoro-oxaloacetate in the presence of apoenzyme show a single inflexion point in the region of pH8. The absence, in this case, of an inflexion in the pH5 region indicates that the latter, present in the corresponding profiles for the aldimine form of the enzyme, results from ionization of an enzyme group associated with the pyridoxal phosphate cofactor. PMID:17399

MRA_1571 is required for isoleucine biosynthesis and improves Mycobacterium tuberculosis H37Ra survival under stress

PubMed Central

Sharma, Rishabh; Keshari, Deepa; Singh, Kumar Sachin; Yadav, Shailendra; Singh, Sudheer Kumar

2016-01-01

Threonine dehydratase is a pyridoxal 5-phosphate dependent enzyme required for isoleucine biosynthesis. Threonine dehydratase (IlvA) participates in conversion of threonine to 2-oxobutanoate and ammonia is released as a by-product. MRA_1571 is annotated to be coding for IlvA in Mycobacterium tuberculosis H37Ra (Mtb-Ra). We developed a recombinant (KD) Mtb-Ra strain by down-regulating IlvA. The growth studies on different carbon sources suggested reduced growth of KD compared to wild-type (WT), also, isoleucine concentration dependent KD growth restoration was observed. The expression profiling of IlvA suggested increased expression of IlvA during oxygen, acid and oxidative stress. In addition, KD showed reduced survival under pH, starvation, nitric oxide and peroxide stresses. KD was more susceptible to antimycobacterial agents such as streptomycin (STR), rifampicin (RIF) and levofloxacin (LVF), while, no such effect was noticeable when exposed to isoniazid. Also, an increase in expression of IlvA was observed when exposed to STR, RIF and LVF. The dye accumulation studies suggested increased permeability of KD to ethidium bromide and Nile Red as compared to WT. TLC and Mass studies confirmed altered lipid profile of KD. In summary down-regulation of IlvA affects Mtb growth, increases its susceptibility to stress and leads to altered cell wall lipid profile. PMID:27353854

A Clinically Relevant Variant of the Human Hydrogen Sulfide-Synthesizing Enzyme Cystathionine β-Synthase: Increased CO Reactivity as a Novel Molecular Mechanism of Pathogenicity?

PubMed Central

Malagrinò, Francesca; Santo, Paulo E.; Gutierres, André; Bandeiras, Tiago M.; Leandro, Paula

2017-01-01

The human disease classical homocystinuria results from mutations in the gene encoding the pyridoxal 5′-phosphate- (PLP-) dependent cystathionine β-synthase (CBS), a key enzyme in the transsulfuration pathway that controls homocysteine levels, and is a major source of the signaling molecule hydrogen sulfide (H2S). CBS activity, contributing to cellular redox homeostasis, is positively regulated by S-adenosyl-L-methionine (AdoMet) but fully inhibited upon CO or NO• binding to a noncatalytic heme moiety. Despite extensive studies, the molecular basis of several pathogenic CBS mutations is not yet fully understood. Here we found that the ferrous heme of the reportedly mild p.P49L CBS variant has altered spectral properties and markedly increased affinity for CO, making the protein much more prone than wild type (WT) CBS to inactivation at physiological CO levels. The higher CO affinity could result from the slightly higher flexibility in the heme surroundings revealed by solving at 2.80-Å resolution the crystallographic structure of a truncated p.P49L. Additionally, we report that p.P49L displays impaired H2S-generating activity, fully rescued by PLP supplementation along the purification, despite a minor responsiveness to AdoMet. Altogether, the results highlight how increased propensity to CO inactivation of an otherwise WT-like variant may represent a novel pathogenic mechanism in classical homocystinuria. PMID:28421128

Deficiency of PdxR in Streptococcus mutans affects vitamin B6 metabolism, acid tolerance response and biofilm formation.

PubMed

Liao, S; Bitoun, J P; Nguyen, A H; Bozner, D; Yao, X; Wen, Z T

2015-08-01

Streptococcus mutans, a key etiological agent of the human dental caries, lives primarily on the tooth surface in tenacious biofilms. The SMU864 locus, designated pdxR, is predicted to encode a member of the novel MocR/GabR family proteins, which are featured with a winged helix DNA-binding N-terminal domain and a C-terminal domain highly homologous to the pyridoxal phosphate-dependent aspartate aminotransferases. A pdxR-deficient mutant, TW296, was constructed using allelic exchange. PdxR deficiency in S. mutans had little effect on cell morphology and growth when grown in brain heart infusion. However, when compared with its parent strain, UA159, the PdxR-deficient mutant displayed major defects in acid tolerance response and formed significantly fewer biofilms (P < 0.01). When analyzed by real-time polymerase chain reaction, PdxR deficiency was found to drastically reduce expression of an apparent operon encoding a pyridoxal kinase (SMU865) and a pyridoxal permease (SMU866) of the salvage pathway of vitamin B6 biosynthesis. In addition, PdxR deficiency also altered the expression of genes for ClpL protease, glucosyltransferase B and adhesin SpaP, which are known to play important roles in stress tolerance and biofilm formation. Consistently, PdxR-deficiency affected the growth of the deficient mutant when grown in defined medium with and without vitamin B6 . Further studies revealed that although S. mutans is known to require vitamin B6 to grow in defined medium, B6 vitamers, especially pyridoxal, were strongly inhibitory at millimolar concentrations, against S. mutans growth and biofilm formation. Our results suggest that PdxR in S. mutans plays an important role in regulation of vitamin B6 metabolism, acid tolerance response and biofilm formation. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

GLUTAMIC DECARBOXYLASE OF ERGOT, CLAVICEPS PURPUREA

PubMed Central

Anderson, John A.; Cheldelin, Vernon H.; King, Tsoo E.

1961-01-01

Anderson, John A. (Oregon State University, Corvallis), Vernon H. Cheldelin, and Tsoo E. King. Glutamic decarboxylase of ergot, Claviceps purpurea. J. Bacteriol. 82:354–358. 1961.—l-Glutamic acid is the only naturally occurring amino acid which can be decarboxylated by cell-free extracts of Claviceps purpurea. This decarboxylase was partially purified and the properties of the enzyme studied. The specific activity of the purified preparation was 111 μliters per 10 min per mg of protein. The products formed, stability, inhibition, stimulation of activity with pyridoxal phosphate, and pH activity curve were typical of l-glutamic decarboxylase in Escherichia coli and other microorganisms. The substrate constants at pH 4.6, 5.25, and 5.65 were 0.0169 m, 0.0174 m, and 0.0139 m, respectively. The respective maximal velocities at these pH values were 104, 104, and 90 μliters per 10 min. The pH optimum was 4.8 to 5.2. The enzyme was unstable below pH 4.5 and it was suggested that the fall in activity at the lower end of the pH curve was due to inactivation of the enzyme. The decrease in activity above pH 5.2 did not appear to be due to a change in affinity of enzyme for substrate but to a change of the enzyme-substrate complex into an inactive form. PMID:13683214

Frataxin Directly Stimulates Mitochondrial Cysteine Desulfurase by Exposing Substrate-binding Sites, and a Mutant Fe-S Cluster Scaffold Protein with Frataxin-bypassing Ability Acts Similarly*♦

PubMed Central

Pandey, Alok; Gordon, Donna M.; Pain, Jayashree; Stemmler, Timothy L.; Dancis, Andrew; Pain, Debkumar

2013-01-01

For iron-sulfur (Fe-S) cluster synthesis in mitochondria, the sulfur is derived from the amino acid cysteine by the cysteine desulfurase activity of Nfs1. The enzyme binds the substrate cysteine in the pyridoxal phosphate-containing site, and a persulfide is formed on the active site cysteine in a manner depending on the accessory protein Isd11. The persulfide is then transferred to the scaffold Isu, where it combines with iron to form the Fe-S cluster intermediate. Frataxin is implicated in the process, although it is unclear where and how, and deficiency causes Friedreich ataxia. Using purified proteins and isolated mitochondria, we show here that the yeast frataxin homolog (Yfh1) directly and specifically stimulates cysteine binding to Nfs1 by exposing substrate-binding sites. This novel function of frataxin does not require iron, Isu1, or Isd11. Once bound to Nfs1, the substrate cysteine is the source of the Nfs1 persulfide, but this step is independent of frataxin and strictly dependent on Isd11. Recently, a point mutation in Isu1 was found to bypass many frataxin functions. The data presented here show that the Isu1 suppressor mimics the frataxin effects on Nfs1, explaining the bypassing activity. We propose a regulatory mechanism for the Nfs1 persulfide-forming activity. Specifically, at least two separate conformational changes must occur in the enzyme for optimum activity as follows: one is mediated by frataxin interaction that exposes the “buried” substrate-binding sites, and the other is mediated by Isd11 interaction that brings the bound substrate cysteine and the active site cysteine in proximity for persulfide formation. PMID:24217246

Frataxin directly stimulates mitochondrial cysteine desulfurase by exposing substrate-binding sites, and a mutant Fe-S cluster scaffold protein with frataxin-bypassing ability acts similarly.

PubMed

Pandey, Alok; Gordon, Donna M; Pain, Jayashree; Stemmler, Timothy L; Dancis, Andrew; Pain, Debkumar

2013-12-27

For iron-sulfur (Fe-S) cluster synthesis in mitochondria, the sulfur is derived from the amino acid cysteine by the cysteine desulfurase activity of Nfs1. The enzyme binds the substrate cysteine in the pyridoxal phosphate-containing site, and a persulfide is formed on the active site cysteine in a manner depending on the accessory protein Isd11. The persulfide is then transferred to the scaffold Isu, where it combines with iron to form the Fe-S cluster intermediate. Frataxin is implicated in the process, although it is unclear where and how, and deficiency causes Friedreich ataxia. Using purified proteins and isolated mitochondria, we show here that the yeast frataxin homolog (Yfh1) directly and specifically stimulates cysteine binding to Nfs1 by exposing substrate-binding sites. This novel function of frataxin does not require iron, Isu1, or Isd11. Once bound to Nfs1, the substrate cysteine is the source of the Nfs1 persulfide, but this step is independent of frataxin and strictly dependent on Isd11. Recently, a point mutation in Isu1 was found to bypass many frataxin functions. The data presented here show that the Isu1 suppressor mimics the frataxin effects on Nfs1, explaining the bypassing activity. We propose a regulatory mechanism for the Nfs1 persulfide-forming activity. Specifically, at least two separate conformational changes must occur in the enzyme for optimum activity as follows: one is mediated by frataxin interaction that exposes the "buried" substrate-binding sites, and the other is mediated by Isd11 interaction that brings the bound substrate cysteine and the active site cysteine in proximity for persulfide formation.

The X-ray structure of Paramecium bursaria Chlorella virus arginine decarboxylase: insight into the structural basis for substrate specificity

PubMed Central

Shah, Rahul; Akella, Radha; Goldsmith, Elizabeth J.; Phillips, Margaret A.

2008-01-01

The group IV pyridoxal-5′-phosphate (PLP)-dependent decarboxylases belong to the β/α barrel structural family, and include enzymes with substrate specificity for a range of basic amino acids. A unique homolog of this family, the Paramecium bursaria Chlorella virus arginine decarboxylase (cvADC), shares about 40% amino acid sequence identity with the eukaryotic ornithine decarboxylases (ODCs). The X-ray structure of cvADC has been solved to 1.95 and 1.8 Å resolution for the free and agmatine (product)-bound enzymes. The global structural differences between cvADC and eukaryotic ODC are minimal (rmsd of 1.2 – 1.4 Å), however, the active site has significant structural rearrangements. The key “specificity element,” is identified as the 310-helix that contains and positions substrate-binding residues such as E296 cvADC (D332 in T. brucei ODC). In comparison to the ODC structures, the 310-helix in cvADC is shifted over 2 Å away from the PLP cofactor, thus accommodating the larger arginine substrate. Within the context of this conserved fold, the protein is designed to be flexible in the positioning and amino acid sequence of the 310-helix, providing a mechanism to evolve different substrate preferences within the family without large structural rearrangements. Also, in the structure, the “K148-loop” (homologous to the “K169-loop” of ODC) is observed in a closed, substrate-bound conformation for the first time. Apparently the K148 loop is a mobile loop, analogous to those observed in triose phosphate isomerase and tryptophan synthetase. In conjunction with prior structural studies these data predict that this loop adopts different conformations throughout the catalytic cycle, and that loop movement may be kinetically linked to the rate-limiting step of product release. PMID:17305368

The chemical properties and functional role of a lysine residue within the active site of native sodium and potassium ion-activated adenosinetriphosphatase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, K.Y.

1988-01-01

The peptide, HLLVMKGAPER, which contains Lysine 501 of the {alpha} polypeptide can be released from intact sodium and potassium ion activated adenosinetriphosphatase by tryptic digestion. An immunoadsorbent directed against the carboxy-terminal, -GAPER, has been constructed. Sealed, right-side-out vesicles, prepared from canine renal kidneys, were labeled with pyridoxal phosphate and sodium ({sup 3}H)borohydride in the absence or presence of saponin, respectively. Large increases in the incorporation of radioactivity into the peptides bound by the immunoadsorbent were observed in the digest obtained from the vesicles exposed to saponin. From the results of several control experiments examining the labeling reaction it could bemore » concluded that the increase in the extent of modification was due to the cytoplasmic disposition of this segment in the native enzyme.« less

Chlamydia trachomatis dapF Encodes a Bifunctional Enzyme Capable of Both d-Glutamate Racemase and Diaminopimelate Epimerase Activities

PubMed Central

2018-01-01

ABSTRACT Peptidoglycan is a sugar/amino acid polymer unique to bacteria and essential for division and cell shape maintenance. The d-amino acids that make up its cross-linked stem peptides are not abundant in nature and must be synthesized by bacteria de novo. d-Glutamate is present at the second position of the pentapeptide stem and is strictly conserved in all bacterial species. In Gram-negative bacteria, d-glutamate is generated via the racemization of l-glutamate by glutamate racemase (MurI). Chlamydia trachomatis is the leading cause of infectious blindness and sexually transmitted bacterial infections worldwide. While its genome encodes a majority of the enzymes involved in peptidoglycan synthesis, no murI homologue has ever been annotated. Recent studies have revealed the presence of peptidoglycan in C. trachomatis and confirmed that its pentapeptide includes d-glutamate. In this study, we show that C. trachomatis synthesizes d-glutamate by utilizing a novel, bifunctional homologue of diaminopimelate epimerase (DapF). DapF catalyzes the final step in the synthesis of meso-diaminopimelate, another amino acid unique to peptidoglycan. Genetic complementation of an Escherichia coli murI mutant demonstrated that Chlamydia DapF can generate d-glutamate. Biochemical analysis showed robust activity, but unlike canonical glutamate racemases, activity was dependent on the cofactor pyridoxal phosphate. Genetic complementation, enzymatic characterization, and bioinformatic analyses indicate that chlamydial DapF shares characteristics with other promiscuous/primordial enzymes, presenting a potential mechanism for d-glutamate synthesis not only in Chlamydia but also numerous other genera within the Planctomycetes-Verrucomicrobiae-Chlamydiae superphylum that lack recognized glutamate racemases. PMID:29615498

Structural and Functional Adaptation of Vancomycin Resistance VanT Serine Racemases

PubMed Central

Meziane-Cherif, Djalal; Stogios, Peter J.; Evdokimova, Elena; Egorova, Olga

2015-01-01

ABSTRACT Vancomycin resistance in Gram-positive bacteria results from the replacement of the d-alanyl–d-alanine target of peptidoglycan precursors with d-alanyl–d-lactate or d-alanyl–d-serine (d-Ala-d-Ser), to which vancomycin has low binding affinity. VanT is one of the proteins required for the production of d-Ala-d-Ser-terminating precursors by converting l-Ser to d-Ser. VanT is composed of two domains, an N-terminal membrane-bound domain, likely involved in l-Ser uptake, and a C-terminal cytoplasmic catalytic domain which is related to bacterial alanine racemases. To gain insight into the molecular function of VanT, the crystal structure of the catalytic domain of VanTG from VanG-type resistant Enterococcus faecalis BM4518 was determined. The structure showed significant similarity to type III pyridoxal 5′-phosphate (PLP)-dependent alanine racemases, which are essential for peptidoglycan synthesis. Comparative structural analysis between VanTG and alanine racemases as well as site-directed mutagenesis identified three specific active site positions centered around Asn696 which are responsible for the l-amino acid specificity. This analysis also suggested that VanT racemases evolved from regular alanine racemases by acquiring additional selectivity toward serine while preserving that for alanine. The 4-fold-lower relative catalytic efficiency of VanTG against l-Ser versus l-Ala implied that this enzyme relies on its membrane-bound domain for l-Ser transport to increase the overall rate of d-Ser production. These findings illustrate how vancomycin pressure selected for molecular adaptation of a housekeeping enzyme to a bifunctional enzyme to allow for peptidoglycan remodeling, a strategy increasingly observed in antibiotic-resistant bacteria. PMID:26265719

Structural and functional adaptation of vancomycin resistance VanT serine racemases

DOE PAGES

Meziane-Cherif, Djalal; Stogios, Peter J.; Evdokimova, Elena; ...

2015-08-11

Vancomycin resistance in Gram-positive bacteria results from the replacement of the D-alanyl–D-alanine target of peptidoglycan precursors with D-alanyl–D-lactate or D-alanyl–D-serine (D-Ala-D-Ser), to which vancomycin has low binding affinity. VanT is one of the proteins required for the production of D-Ala-D-Ser-terminating precursors by converting L-Ser to D-Ser. VanT is composed of two domains, an N-terminal membrane-bound domain, likely involved in L-Ser uptake, and a C-terminal cytoplasmic catalytic domain which is related to bacterial alanine racemases. To gain insight into the molecular function of VanT, the crystal structure of the catalytic domain of VanT G from VanG-type resistant Enterococcus faecalis BM4518more » was determined. The structure showed significant similarity to type III pyridoxal 5'-phosphate (PLP)-dependent alanine racemases, which are essential for peptidoglycan synthesis. Comparative structural analysis between VanT G and alanine racemases as well as site-directed mutagenesis identified three specific active site positions centered around Asn 696 which are responsible for theL-amino acid specificity. This analysis also suggested that VanT racemases evolved from regular alanine racemases by acquiring additional selectivity toward serine while preserving that for alanine. The 4-fold-lower relative catalytic efficiency of VanT G against L-Ser versus L-Ala implied that this enzyme relies on its membrane-bound domain for L-Ser transport to increase the overall rate of D-Ser production. These findings illustrate how vancomycin pressure selected for molecular adaptation of a housekeeping enzyme to a bifunctional enzyme to allow for peptidoglycan remodeling, a strategy increasingly observed in antibiotic-resistant bacteria.« less

Fragment Screening of Human Kynurenine Aminotransferase-II.

PubMed

Jayawickrama, Gayan S; Nematollahi, Alireza; Sun, Guanchen; Church, W Bret

2018-03-01

Kynurenine aminotransferase-II (KAT-II) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that acts in the tryptophan metabolic pathway by catalyzing the transamination of kynurenine into kynurenic acid (KYNA). It is one of four isoforms in the KAT family, of which it is the primary homologue responsible for KYNA production in the mammalian brain. KAT-II is targeted for inhibition as KYNA is implicated in diseases such as schizophrenia, where it is found in elevated concentrations. Previously, many different approaches have been taken to develop KAT-II inhibitors, and herein fragment-based drug design (FBDD) approaches have been exploited to provide further lead compounds that can be designed into novel inhibitors. Surface plasmon resonance (SPR) was used to screen a fragment library containing 1000 compounds, of which 41 hits were identified. These hits were further evaluated with SPR, and 18 were selected for inhibition studies. From these hits, two fragments, F6037-0164 and F0037-7280, were pursued and determined to have an IC 50 of 524.5 (± 25.6) μM and 115.2 (± 4.5) μM, respectively. This strategy shows the viability of using FBDD in gleaning knowledge about KAT-II inhibition and generating leads for the production of KAT-II inhibitors.

Endogenous Synthesis of 2-Aminoacrylate Contributes to Cysteine Sensitivity in Salmonella enterica

PubMed Central

Ernst, Dustin C.; Lambrecht, Jennifer A.; Schomer, Rebecca A.

2014-01-01

RidA, the archetype member of the widely conserved RidA/YER057c/UK114 family of proteins, prevents reactive enamine/imine intermediates from accumulating in Salmonella enterica by catalyzing their hydrolysis to stable keto acid products. In the absence of RidA, endogenous 2-aminoacrylate persists in the cellular environment long enough to damage a growing list of essential metabolic enzymes. Prior studies have focused on the dehydration of serine by the pyridoxal 5′-phosphate (PLP)-dependent serine/threonine dehydratases, IlvA and TdcB, as sources of endogenous 2-aminoacrylate. The current study describes an additional source of endogenous 2-aminoacrylate derived from cysteine. The results of in vivo analysis show that the cysteine sensitivity of a ridA strain is contingent upon CdsH, the predominant cysteine desulfhydrase in S. enterica. The impact of cysteine on 2-aminoacrylate accumulation is shown to be unaffected by the presence of serine/threonine dehydratases, revealing another mechanism of endogenous 2-aminoacrylate production. Experiments in vitro suggest that 2-aminoacrylate is released from CdsH following cysteine desulfhydration, resulting in an unbound aminoacrylate substrate for RidA. This work expands our understanding of the role played by RidA in preventing enamine stress resulting from multiple normal metabolic processes. PMID:25002544

From protein structure to function via single crystal optical spectroscopy

PubMed Central

Ronda, Luca; Bruno, Stefano; Bettati, Stefano; Storici, Paola; Mozzarelli, Andrea

2015-01-01

The more than 100,000 protein structures determined by X-ray crystallography provide a wealth of information for the characterization of biological processes at the molecular level. However, several crystallographic “artifacts,” including conformational selection, crystallization conditions and radiation damages, may affect the quality and the interpretation of the electron density maps, thus limiting the relevance of structure determinations. Moreover, for most of these structures, no functional data have been obtained in the crystalline state, thus posing serious questions on their validity in infereing protein mechanisms. In order to solve these issues, spectroscopic methods have been applied for the determination of equilibrium and kinetic properties of proteins in the crystalline state. These methods are UV-vis spectrophotometry, spectrofluorimetry, IR, EPR, Raman, and resonance Raman spectroscopy. Some of these approaches have been implemented with on-line instruments at X-ray synchrotron beamlines. Here, we provide an overview of investigations predominantly carried out in our laboratory by single crystal polarized absorption UV-vis microspectrophotometry, the most applied technique for the functional characterization of proteins in the crystalline state. Studies on hemoglobins, pyridoxal 5′-phosphate dependent enzymes and green fluorescent protein in the crystalline state have addressed key biological issues, leading to either straightforward structure-function correlations or limitations to structure-based mechanisms. PMID:25988179

A hydrogen-bonding network modulating enzyme function: asparagine-194 and tyrosine-225 of Escherichia coli aspartate aminotransferase.

PubMed

Yano, T; Mizuno, T; Kagamiyama, H

1993-02-23

The electron distribution within the coenzyme or coenzyme-substrate conjugate needs to be properly regulated during the catalytic process of aspartate aminotransferase (AspAT). Asn194 and Tyr225 may function in regulating the electron distribution through hydrogen-bonding to O(3') of the coenzyme, pyridoxal 5'-phosphate (PLP) or pyridoxamine 5'-phosphate (PMP). The roles of Tyr225 have already been explored by site-directed mutagenesis (Inoue et al., 1991; Goldberg et al., 1991). In the present studies, the mutant enzymes Asn194-->Ala and Asn194-->Ala + Tyr225-->Phe were analyzed kinetically and spectroscopically and were compared with the wild-type and Tyr225-->Phe enzymes. The kinetic studies showed that Asn194 is not essential for AspAT catalysis, although the Kd values for the substrates were increased by 10- to 50-fold upon the replacement of Asn194. The measurements of the absorption and fluorescence excitation spectra revealed that the ratio of an enolimine to a ketoenamine form was considerably increased as a tautomeric form of the protonated PLP in the active site of the double mutant enzyme. The pH-pKd relationship for the binding of maleate to AspAT could be explained by a simple thermodynamic cycle where only one ionizing group (the imine nitrogen of the internal aldimine bond) affects the binding of maleate. The analyses of the pH-pKd curves for the wild-type and mutant enzymes showed that (i) the hydrogen bond between O(3') of PLP and Asn194 is weakened by the binding of maleate to AspAT, while the hydrogen bond between O(3') and Tyr225 is not changed, and that (ii) the replacement of Asn194 causes some effect hampering the binding of maleate.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantification of vitamin B6 vitamers in human cerebrospinal fluid by ultra performance liquid chromatography-tandem mass spectrometry.

PubMed

van der Ham, M; Albersen, M; de Koning, T J; Visser, G; Middendorp, A; Bosma, M; Verhoeven-Duif, N M; de Sain-van der Velden, M G M

2012-01-27

Since vitamin B6 is essential for normal functioning of the central nervous system, there is growing need for sensitive analysis of B6 vitamers in cerebrospinal fluid (CSF). This manuscript describes the development and validation of a rapid, sensitive and accurate method for quantification of the vitamin B6 vitamers pyridoxal (PL), pyridoxamine (PM), pyridoxine (PN), pyridoxic acid (PA), pyridoxal 5'-phosphate (PLP), pyridoxamine 5'-phosphate (PMP) and pyridoxine 5'-phosphate (PNP) in human CSF. The method is based on ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) with a simple sample preparation procedure of protein precipitation using 50 g L(-1) trichloroacetic acid containing stable isotope labeled internal standards: PL-D(3) for PL and PM, PN-(13)C(4) for PN, PA-D(2) for PA and PLP-D(3) for the phosphorylated vitamers. B6 vitamers were separated (Acquity HSS-T3 UPLC column) with a buffer containing acetic acid, heptafluorobutyric acid and acetonitrile. Positive electrospray ionization was used to monitor transitions m/z 168.1→150.1 (PL), 169.1→134.1 (PM), 170.1→134.1 (PN), 184.1→148.1 (PA), 248.1→150.1 (PLP), 249.1→232.1 (PMP) and 250.1→134.1 (PNP). The method was validated at three concentration levels for each B6 vitamer in CSF. Recoveries of the internal standards were between 93% and 96%. Intra- and inter-assay variations were below 20%. Accuracy tests showed deviations from 3% (PN) to 39% (PMP). Limits of quantification were in the range of 0.03-5.37 nM. Poor results were obtained for quantification of PNP. The method was applied to CSF samples of 20 subjects and two patients on pyridoxine supplementation. Using minimal CSF volumes this method is suitable for implementation in a routine diagnostic setting. Copyright © 2011 Elsevier B.V. All rights reserved.

Intravenous Vitamin B6 Increases Resistance to Erythropoiesis-Stimulating Agents in Hemodialysis Patients: A Randomized Controlled Trial.

PubMed

Obi, Yoshitsugu; Mikami, Satoshi; Hamano, Takayuki; Obi, Yasue; Tanaka, Hirotaka; Shimomura, Akihiro; Rakugi, Hiromi; Inoue, Toru; Isaka, Yoshitaka

2016-11-01

Vitamin B6 deficiency is common in hemodialysis patients and may contribute to anemia and abnormal bone metabolism in this population. 6-month, open-label, randomized controlled parallel-group study in hemodialysis centers. Fifty-six maintenance hemodialysis patients with relatively high resistance to erythropoiesis-stimulating agents (ESA). Intravenous vitamin B6 (60 mg of intravenous pyridoxal 5'-phosphate after each thrice-weekly hemodialysis session). The primary and secondary outcomes were changes over time in ESA resistance index and bone turnover markers, respectively. The prevalence of vitamin B6 deficiency was 40% overall. Compared with the control group, the B6 group showed an upward change in ESA resistance index over time (P interaction  = .038). At week 13 (a priori-defined time point), pyridoxal 5'-phosphate administration was associated with higher ESA resistance index by 0.97 (95% confidence interval, 0.02-1.92) ×10 -2  μg ⋅darbepoetin-α/kg per g/dL⋅hemoglobin after baseline adjustment, which was not modified by baseline vitamin B6 status. There was a trend toward increase in serum erythropoietin concentrations in the B6 group after adjustment for baseline values, hemoglobin, and weekly ESA dose (P interaction  = .06). The downward changes of bone-specific alkaline phosphatase and tartrate-resistant acid phosphatase 5b in the B6 group relative to the control group were pronounced in patients without vitamin B6 deficiency (P interaction < .001 and .017, respectively), despite nonsignificant between-group difference in 1-84 parathyroid hormone. Thrice-weekly intravenous vitamin B6 (60 mg pyridoxal 5'-phosphate hydrate) worsens the response to ESA and may blunt the response of bone to parathyroid hormone in hemodialysis patients. Copyright © 2016 National Kidney Foundation, Inc. Published by Elsevier Inc. All rights reserved.

Inflammation, vitamin B6 and related pathways.

PubMed

Ueland, Per Magne; McCann, Adrian; Midttun, Øivind; Ulvik, Arve

2017-02-01

The active form of vitamin B6, pyridoxal 5'-phosphate (PLP), serves as a co-factor in more than 150 enzymatic reactions. Plasma PLP has consistently been shown to be low in inflammatory conditions; there is a parallel reduction in liver PLP, but minor changes in erythrocyte and muscle PLP and in functional vitamin B6 biomarkers. Plasma PLP also predicts the risk of chronic diseases like cardiovascular disease and some cancers, and is inversely associated with numerous inflammatory markers in clinical and population-based studies. Vitamin B6 intake and supplementation improve some immune functions in vitamin B6-deficient humans and experimental animals. A possible mechanism involved is mobilization of vitamin B6 to the sites of inflammation where it may serve as a co-factor in pathways producing metabolites with immunomodulating effects. Relevant vitamin B6-dependent inflammatory pathways include vitamin B6 catabolism, the kynurenine pathway, sphingosine 1-phosphate metabolism, the transsulfuration pathway, and serine and glycine metabolism. Copyright © 2016 Elsevier Ltd. All rights reserved.

Sphingolipid Long-Chain Base Synthesis in Plants (Characterization of Serine Palmitoyltransferase Activity in Squash Fruit Microsomes).

PubMed

Lynch, D. V.; Fairfield, S. R.

1993-12-01

The activity of serine palmitoyltransferase (palmitoyl-coenzyme A [CoA]:L-serine [Ser]-C-palmitoyltransferase [decarboxylating], EC 2.3.1.50), the enzyme catalyzing the first step in the synthesis of the long-chain base required for sphingolipid assembly, has been characterized in a plant system. Enzyme activity in a microsomal membrane fraction from summer squash fruit (Cucurbita pepo L. cv Early Prolific Straightneck) was assayed by monitoring the incorporation of L-[3H]Ser into the chloroform-soluble product, 3-ketosphinganine. Addition of NADPH to the assay system resulted in the conversion of 3-ketosphinganine to sphinganine. The apparent Km for Ser was approximately 1.8 mM. The enzyme exhibited a strong preference for palmitoyl-CoA, with optimal activity at a substrate concentration of 200 [mu]M. Pyridoxal 5[prime]-phosphate was required as a coenzyme. The pH optimum was 7.6, and the temperature optimum was 36 to 40[deg]C. Enzyme activity was greatest in the microsomal fraction obtained by differential centrifugation and was localized to the endoplasmic reticulum using marker enzymes. Two known mechanism-based inhibitors of the mammalian enzyme, L-cycloserine and [beta]-chloro-L-alanine, were effective inhibitors of enzyme activity in squash microsomes. Changes in enzyme activity with size (age) of squash fruit were observed. The results from this study suggest that the properties and catalytic mechanism of Ser palmitoyltransferase from squash are similar to those of the animal, fungal, and bacterial enzyme in most respects. The specific activity of the enzyme in squash microsomes ranged from 0.57 to 0.84 nmol min-1 mg-1 of protein, values 2- to 20-fold higher than those previously reported for preparations from animal tissues.

Bis-(3'-5')-cyclic dimeric GMP-linked quorum sensing controls swarming in Vibrio parahaemolyticus.

PubMed

Trimble, Michael J; McCarter, Linda L

2011-11-01

Movement over and colonization of surfaces are important survival strategies for bacteria, and many find it advantageous to perform these activities as a group, using quorum sensing to sample population size and synchronize behavior. It is puzzling however, that swarming-proficient and virulent strains of Vibrio parahaemolyticus are silenced for the vibrio archetypal pathway of quorum sensing. Here we describe the S-signal, a pheromone that can be communicated between cells in coculture to regulate surface colonization. This signal was harvested in cell-free supernatants and demonstrated to stimulate swarming gene expression at low cell density. The S-signal was generated by the pyridoxal phosphate-dependent aminotransferase ScrA; signal reception required the periplasmic binding protein ScrB and the membrane-bound GGDEF-EAL domain-containing protein ScrC. ScrC is a bifunctional enzyme that has the ability to form and degrade the second messenger bis-(3'-5') cyclic dimeric GMP (c-di-GMP). ScrA in neighboring cells was able to alter the activity of ScrC in a ScrB-dependent manner, transforming ScrC's repressing ability to inducing activity with respect to swarming. Conversely, cell-cell signaling repressed capsule gene expression. In summary, we report that quorum sensing can stimulate swarming in V. parahaemolyticus; it does so via an alternative pathway capable of generating an autoinducing signal that influences c-di-GMP, thereby expanding the lexicon and language of cell-cell communication.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oberhardt, Matthew A.; Zarecki, Raphy; Reshef, Leah

Recent insights suggest that non-specific and/or promiscuous enzymes are common and active across life. Understanding the role of such enzymes is an important open question in biology. Here we develop a genome-wide method, PROPER, that uses a permissive PSI-BLAST approach to predict promiscuous activities of metabolic genes. Enzyme promiscuity is typically studied experimentally using multicopy suppression, in which over-expression of a promiscuous ‘replacer’ gene rescues lethality caused by inactivation of a ‘target’ gene. We use PROPER to predict multicopy suppression in Escherichia coli, achieving highly significant overlap with published cases (hypergeometric p = 4.4e-13). We then validate three novel predictedmore » target-replacer gene pairs in new multicopy suppression experiments. We next go beyond PROPER and develop a network-based approach, GEM-PROPER, that integrates PROPER with genome-scale metabolic modeling to predict promiscuous replacements via alternative metabolic pathways. GEM-PROPER predicts a new indirect replacer (thiG) for an essential enzyme (pdxB) in production of pyridoxal 5’-phosphate (the active form of Vitamin B 6), which we validate experimentally via multicopy suppression. Here, we perform a structural analysis of thiG to determine its potential promiscuous active site, which we validate experimentally by inactivating the pertaining residues and showing a loss of replacer activity. Thus, this study is a successful example where a computational investigation leads to a network-based identification of an indirect promiscuous replacement of a key metabolic enzyme, which would have been extremely difficult to identify directly.« less

Catalytically active alkaline molten globular enzyme: Effect of pH and temperature on the structural integrity of 5-aminolevulinate synthase.

PubMed

Stojanovski, Bosko M; Breydo, Leonid; Hunter, Gregory A; Uversky, Vladimir N; Ferreira, Gloria C

2014-12-01

5-Aminolevulinate synthase (ALAS), a pyridoxal-5'phosphate (PLP)-dependent enzyme, catalyzes the first step of heme biosynthesis in mammals. Circular dichroism (CD) and fluorescence spectroscopies were used to examine the effects of pH (1.0-3.0 and 7.5-10.5) and temperature (20 and 37°C) on the structural integrity of ALAS. The secondary structure, as deduced from far-UV CD, is mostly resilient to pH and temperature changes. Partial unfolding was observed at pH2.0, but further decreasing pH resulted in acid-induced refolding of the secondary structure to nearly native levels. The tertiary structure rigidity, monitored by near-UV CD, is lost under acidic and specific alkaline conditions (pH10.5 and pH9.5/37°C), where ALAS populates a molten globule state. As the enzyme becomes less structured with increased alkalinity, the chiral environment of the internal aldimine is also modified, with a shift from a 420nm to 330nm dichroic band. Under acidic conditions, the PLP cofactor dissociates from ALAS. Reaction with 8-anilino-1-naphthalenesulfonic acid corroborates increased exposure of hydrophobic clusters in the alkaline and acidic molten globules, although the reaction is more pronounced with the latter. Furthermore, quenching the intrinsic fluorescence of ALAS with acrylamide at pH1.0 and 9.5 yielded subtly different dynamic quenching constants. The alkaline molten globule state of ALAS is catalytically active (pH9.5/37°C), although the kcat value is significantly decreased. Finally, the binding of 5-aminolevulinate restricts conformational fluctuations in the alkaline molten globule. Overall, our findings prove how the structural plasticity of ALAS contributes to reaching a functional enzyme. Copyright © 2014 Elsevier B.V. All rights reserved.

Salmonella typhimurium PtsJ is a novel MocR-like transcriptional repressor involved in regulating the vitamin B6 salvage pathway.

PubMed

Tramonti, Angela; Milano, Teresa; Nardella, Caterina; di Salvo, Martino L; Pascarella, Stefano; Contestabile, Roberto

2017-02-01

The vitamin B 6 salvage pathway, involving pyridoxine 5'-phosphate oxidase (PNPOx) and pyridoxal kinase (PLK), recycles B 6 vitamers from nutrients and protein turnover to produce pyridoxal 5'-phosphate (PLP), the catalytically active form of the vitamin. Regulation of this pathway, widespread in living organisms including humans and many bacteria, is very important to vitamin B 6 homeostasis but poorly understood. Although some information is available on the enzymatic regulation of PNPOx and PLK, little is known on their regulation at the transcriptional level. In the present work, we identified a new MocR-like regulator, PtsJ from Salmonella typhimurium, which controls the expression of the pdxK gene encoding one of the two PLKs expressed in this organism (PLK1). Analysis of pdxK expression in a ptsJ knockout strain demonstrated that PtsJ acts as a transcriptional repressor. This is the first case of a MocR-like regulator acting as repressor of its target gene. Expression and purification of PtsJ allowed a detailed characterisation of its effector and DNA-binding properties. PLP is the only B 6 vitamer acting as effector molecule for PtsJ. A DNA-binding region composed of four repeated nucleotide sequences is responsible for binding of PtsJ to its target promoter. Analysis of binding stoichiometry revealed that protein subunits/DNA molar ratio varies from 4 : 1 to 2 : 1, depending on the presence or absence of PLP. Structural characteristics of DNA transcriptional factor-binding sites suggest that PtsJ binds DNA according to a different model with respect to other characterised members of the MocR subgroup. © 2016 Federation of European Biochemical Societies.

Molecular mechanisms of the non-coenzyme action of thiamin in brain: biochemical, structural and pathway analysis

PubMed Central

Mkrtchyan, Garik; Aleshin, Vasily; Parkhomenko, Yulia; Kaehne, Thilo; Luigi Di Salvo, Martino; Parroni, Alessia; Contestabile, Roberto; Vovk, Andrey; Bettendorff, Lucien; Bunik, Victoria

2015-01-01

Thiamin (vitamin B1) is a pharmacological agent boosting central metabolism through the action of the coenzyme thiamin diphosphate (ThDP). However, positive effects, including improved cognition, of high thiamin doses in neurodegeneration may be observed without increased ThDP or ThDP-dependent enzymes in brain. Here, we determine protein partners and metabolic pathways where thiamin acts beyond its coenzyme role. Malate dehydrogenase, glutamate dehydrogenase and pyridoxal kinase were identified as abundant proteins binding to thiamin- or thiazolium-modified sorbents. Kinetic studies, supported by structural analysis, revealed allosteric regulation of these proteins by thiamin and/or its derivatives. Thiamin triphosphate and adenylated thiamin triphosphate activate glutamate dehydrogenase. Thiamin and ThDP regulate malate dehydrogenase isoforms and pyridoxal kinase. Thiamin regulation of enzymes related to malate-aspartate shuttle may impact on malate/citrate exchange, responsible for exporting acetyl residues from mitochondria. Indeed, bioinformatic analyses found an association between thiamin- and thiazolium-binding proteins and the term acetylation. Our interdisciplinary study shows that thiamin is not only a coenzyme for acetyl-CoA production, but also an allosteric regulator of acetyl-CoA metabolism including regulatory acetylation of proteins and acetylcholine biosynthesis. Moreover, thiamin action in neurodegeneration may also involve neurodegeneration-related 14-3-3, DJ-1 and β-amyloid precursor proteins identified among the thiamin- and/or thiazolium-binding proteins. PMID:26212886

The mechanism of action of serine transhydroxymethylase

PubMed Central

Jordan, P. M.; Akhtar, M.

1970-01-01

1. The preparation of stereospecifically tritiated glycines and the determination of their absolute configurations by the use of d-amino acid oxidase are described. 2. The reaction catalysed by serine transhydroxymethylase, which results in the conversion of glycine into serine, has been separated into at least four partial reactions. It is suggested that the first event in this conversion is the formation of a Schiff base intermediate of glycine and pyridoxal phosphate. The next important step involves the removal of the 2S-hydrogen atom of glycine to give a carbanion intermediate. Experiments pertinent to the mechanism of conversion of this carbanion intermediate into serine are described. 3. The enzyme preparation catalysing the conversion of glycine into serine also participates in the conversion of glycine into threonine and allothreonine. In both these conversions, glycine → serine and glycine → threonine, the 2S-hydrogen atom of glycine is eliminated and the 2R-hydrogen atom of glycine is retained. 4. In the light of these experiments the mechanism of action of serine transhydroxymethylase is discussed. It is suggested that methylenetetrahydrofolate is the carrier of formaldehyde, from which formaldehyde may be liberated at the active site of the enzyme, thus allowing the overall reaction to take place. PMID:5414101

Mechanism of Inactivation of γ-Aminobutyric Acid Aminotransferase by (1S,3S)-3-Amino-4-difluoromethylene-1-cyclopentanoic Acid (CPP-115)

PubMed Central

2016-01-01

γ-Aminobutyric acid aminotransferase (GABA-AT) is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that degrades GABA, the principal inhibitory neurotransmitter in mammalian cells. When the concentration of GABA falls below a threshold level, convulsions can occur. Inhibition of GABA-AT raises GABA levels in the brain, which can terminate seizures as well as have potential therapeutic applications in treating other neurological disorders, including drug addiction. Among the analogues that we previously developed, (1S,3S)-3-amino-4-difluoromethylene-1-cyclopentanoic acid (CPP-115) showed 187 times greater potency than that of vigabatrin, a known inactivator of GABA-AT and approved drug (Sabril) for the treatment of infantile spasms and refractory adult epilepsy. Recently, CPP-115 was shown to have no adverse effects in a Phase I clinical trial. Here we report a novel inactivation mechanism for CPP-115, a mechanism-based inactivator that undergoes GABA-AT-catalyzed hydrolysis of the difluoromethylene group to a carboxylic acid with concomitant loss of two fluoride ions and coenzyme conversion to pyridoxamine 5′-phosphate (PMP). The partition ratio for CPP-115 with GABA-AT is about 2000, releasing cyclopentanone-2,4-dicarboxylate (22) and two other precursors of this compound (20 and 21). Time-dependent inactivation occurs by a conformational change induced by the formation of the aldimine of 4-aminocyclopentane-1,3-dicarboxylic acid and PMP (20), which disrupts an electrostatic interaction between Glu270 and Arg445 to form an electrostatic interaction between Arg445 and the newly formed carboxylate produced by hydrolysis of the difluoromethylene group in CPP-115, resulting in a noncovalent, tightly bound complex. This represents a novel mechanism for inactivation of GABA-AT and a new approach for the design of mechanism-based inactivators in general. PMID:25616005

Mechanism of Inactivation of γ-Aminobutyric Acid Aminotransferase by (1 S ,3 S )-3-Amino-4-difluoromethylene-1-cyclopentanoic Acid (CPP-115)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Hyunbeom; Doud, Emma H.; Wu, Rui

gamma-Aminobutyric acid aminotransferase (GABA-AT) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that degrades GABA, the principal inhibitory neurotransmitter in mammalian cells. When the concentration of GABA falls below a threshold level, convulsions can occur. Inhibition of GABA-AT raises GABA levels in the brain, which can terminate seizures as well as have potential therapeutic applications in treating other neurological disorders, including drug addiction. Among the analogues that we previously developed, (1S,3S)-3-amino-4-difluoromethylene-1-cyclopentanoic acid (CPP-115) showed 187 times greater potency than that of vigabatrin, a known inactivator of GABA-AT and approved drug (Sabril) for the treatment of infantile spasms and refractory adult epilepsy. Recently,more » CPP-115 was shown to have no adverse effects in a Phase I clinical trial. Here we report a novel inactivation mechanism for CPP-115, a mechanism-based inactivator that undergoes GABA-AT-catalyzed hydrolysis of the difluoromethylene group to a carboxylic acid with concomitant loss of two fluoride ions and coenzyme conversion to pyridoxamine 5'-phosphate (PMP). The partition ratio for CPP-115 with GABA-AT is about 2000, releasing cyclopentanone-2,4-dicarboxylate (22) and two other precursors of this compound (20 and 21). Time-dependent inactivation occurs by a conformational change induced by the formation of the aldimine of 4-aminocyclopentane-1,3-dicarboxylic acid and PMP (20), which disrupts an electrostatic interaction between Glu270 and Arg445 to form an electrostatic interaction between Arg445 and the newly formed carboxylate produced by hydrolysis of the difluoromethylene group in CPP-115, resulting in a noncovalent, tightly bound complex. This represents a novel mechanism for inactivation of GABA-AT and a new approach for the design of mechanism-based inactivators in general.« less

Mechanism of inactivation of γ-aminobutyric acid aminotransferase by (1S,3S)-3-amino-4-difluoromethylene-1-cyclopentanoic acid (CPP-115).

PubMed

Lee, Hyunbeom; Doud, Emma H; Wu, Rui; Sanishvili, Ruslan; Juncosa, Jose I; Liu, Dali; Kelleher, Neil L; Silverman, Richard B

2015-02-25

γ-Aminobutyric acid aminotransferase (GABA-AT) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that degrades GABA, the principal inhibitory neurotransmitter in mammalian cells. When the concentration of GABA falls below a threshold level, convulsions can occur. Inhibition of GABA-AT raises GABA levels in the brain, which can terminate seizures as well as have potential therapeutic applications in treating other neurological disorders, including drug addiction. Among the analogues that we previously developed, (1S,3S)-3-amino-4-difluoromethylene-1-cyclopentanoic acid (CPP-115) showed 187 times greater potency than that of vigabatrin, a known inactivator of GABA-AT and approved drug (Sabril) for the treatment of infantile spasms and refractory adult epilepsy. Recently, CPP-115 was shown to have no adverse effects in a Phase I clinical trial. Here we report a novel inactivation mechanism for CPP-115, a mechanism-based inactivator that undergoes GABA-AT-catalyzed hydrolysis of the difluoromethylene group to a carboxylic acid with concomitant loss of two fluoride ions and coenzyme conversion to pyridoxamine 5'-phosphate (PMP). The partition ratio for CPP-115 with GABA-AT is about 2000, releasing cyclopentanone-2,4-dicarboxylate (22) and two other precursors of this compound (20 and 21). Time-dependent inactivation occurs by a conformational change induced by the formation of the aldimine of 4-aminocyclopentane-1,3-dicarboxylic acid and PMP (20), which disrupts an electrostatic interaction between Glu270 and Arg445 to form an electrostatic interaction between Arg445 and the newly formed carboxylate produced by hydrolysis of the difluoromethylene group in CPP-115, resulting in a noncovalent, tightly bound complex. This represents a novel mechanism for inactivation of GABA-AT and a new approach for the design of mechanism-based inactivators in general.

Mechanism of Inactivation of γ-Aminobutyric Acid Aminotransferase by (1 S ,3 S)-3-Amino-4-difluoromethylene-1-cyclopentanoic Acid (CPP-115)

DOE PAGES

Lee, Hyunbeom; Doud, Emma H.; Wu, Rui; ...

2015-01-23

γ-Aminobutyric acid aminotransferase (GABA-AT) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that degrades GABA, the principal inhibitory neurotransmitter in mammalian cells. When the concentration of GABA falls below a threshold level, convulsions can occur. Inhibition of GABA-AT raises GABA levels in the brain, which can terminate seizures as well as have potential therapeutic applications in treating other neurological disorders, including drug addiction. Among the analogues that we previously developed, (1S,3S)-3-amino-4-difluoromethylene-1-cyclopentanoic acid (CPP-115) showed 187 times greater potency than that of vigabatrin, a known inactivator of GABA-AT and approved drug (Sabril) for the treatment of infantile spasms and refractory adult epilepsy. Recently,more » CPP-115 was shown to have no adverse effects in a Phase I clinical trial. Here we report a novel inactivation mechanism for CPP-115, a mechanism-based inactivator that undergoes GABA-AT-catalyzed hydrolysis of the difluoromethylene group to a carboxylic acid with concomitant loss of two fluoride ions and coenzyme conversion to pyridoxamine 5'-phosphate (PMP). The partition ratio for CPP-115 with GABA-AT is about 2000, releasing cyclopentanone-2,4-dicarboxylate (22) and two other precursors of this compound (20 and 21). Time-dependent inactivation occurs by a conformational change induced by the formation of the aldimine of 4-aminocyclopentane-1,3-dicarboxylic acid and PMP (20), which disrupts an electrostatic interaction between Glu270 and Arg445 to form an electrostatic interaction between Arg445 and the newly formed carboxylate produced by hydrolysis of the difluoromethylene group in CPP-115, resulting in a noncovalent, tightly bound complex. Ultimately, this represents a novel mechanism for inactivation of GABA-AT and a new approach for the design of mechanism-based inactivators in general.« less

Influence of folic acid, pyridoxal phosphate and cobalamin on plasma homocyst(e)ine levels and the susceptibility of low-density lipoprotein to ex-vivo oxidation.

PubMed

Weiss, N; Feussner, A; Hailer, S; Spengel, F A; Keller, C; Wolfram, G

1999-10-15

Mild hyperhomocyst(e)inaemia is a risk factor for atherosclerotic vascular disease. In-vitro studies have shown that autooxidation of homocyst(e)ine is accompanied by the generation of oxygen radicals. This may lead to oxidative modification of low-density lipoproteins (LDL) and promote atherosclerotic vascular lesions. In male patients with peripheral arterial occlusive disease we determined fasting and post methionine load homocyst(e)ine levels by high performance liquid chromatography and the susceptibility of their LDL particles to ex-vivo oxidation by continously measuring the conjugated diene production induced by incubation with copper ions. Oxidation resistance (expressed as lag time), maximal oxidation rate, and extent of oxidation (expressed of total diene production) of LDL from patients with normal or mildly elevated homocyst(e)ine levels did not differ significantly. Folic acid, pyridoxal phosphate and cobalamin supplementation significantly decreased plasma homocyst(e)ine levels in hyperhomocyst(e)inaemic patients. This went along with a significant decrease in the extent of LDL oxidation and additionally increased HDL-cholesterol levels. The clinical relevance of these findings for the long-term course of atherosclerotic vascular disorders has to be determined by intervention studies.

Glucose-6-phosphate dehydrogenase-6-phosphogluconolactonase: a unique bifunctional enzyme from Plasmodium falciparum.

PubMed

Jortzik, Esther; Mailu, Boniface M; Preuss, Janina; Fischer, Marina; Bode, Lars; Rahlfs, Stefan; Becker, Katja

2011-06-15

The survival of malaria parasites in human RBCs (red blood cells) depends on the pentose phosphate pathway, both in Plasmodium falciparum and its human host. G6PD (glucose-6-phosphate dehydrogenase) deficiency, the most common human enzyme deficiency, leads to a lack of NADPH in erythrocytes, and protects from malaria. In P. falciparum, G6PD is combined with the second enzyme of the pentose phosphate pathway to create a unique bifunctional enzyme named GluPho (glucose-6-phosphate dehydrogenase-6-phosphogluconolactonase). In the present paper, we report for the first time the cloning, heterologous overexpression, purification and kinetic characterization of both enzymatic activities of full-length PfGluPho (P. falciparum GluPho), and demonstrate striking structural and functional differences with the human enzymes. Detailed kinetic analyses indicate that PfGluPho functions on the basis of a rapid equilibrium random Bi Bi mechanism, where the binding of the second substrate depends on the first substrate. We furthermore show that PfGluPho is inhibited by S-glutathionylation. The availability of recombinant PfGluPho and the major differences to hG6PD (human G6PD) facilitate studies on PfGluPho as an excellent drug target candidate in the search for new antimalarial drugs.

Longevity of major coenzymes allows minimal de novo synthesis in microorganisms.

PubMed

Hartl, Johannes; Kiefer, Patrick; Meyer, Fabian; Vorholt, Julia A

2017-05-15

Coenzymes are vital for cellular metabolism and act on the full spectrum of enzymatic reactions. Intrinsic chemical reactivity, enzyme promiscuity and high flux through their catalytic cycles make coenzymes prone to damage. To counteract such compromising factors and ensure stable levels of functional coenzymes, cells use a complex interplay between de novo synthesis, salvage, repair and degradation. However, the relative contribution of these factors is currently unknown, as is the overall stability of coenzymes in the cell. Here, we use dynamic 13 C-labelling experiments to determine the half-life of major coenzymes of Escherichia coli. We find that coenzymes such as pyridoxal 5-phosphate, flavins, nicotinamide adenine dinucleotide (phosphate) and coenzyme A are remarkably stable in vivo and allow biosynthesis close to the minimal necessary rate. In consequence, they are essentially produced to compensate for dilution by growth and passed on over generations of cells. Exceptions are antioxidants, which are short-lived, suggesting an inherent requirement for increased renewal. Although the growth-driven turnover of stable coenzymes is apparently subject to highly efficient end-product homeostasis, we exemplify that coenzyme pools are propagated in excess in relation to actual growth requirements. Additional testing of Bacillus subtilis and Saccharomyces cerevisiae suggests that coenzyme longevity is a conserved feature in biology.

Regulation of Maltodextrin Phosphorylase Synthesis in Escherichia coli by Cyclic Adenosine 3′, 5′-Monophosphate and Glucose1

PubMed Central

Chao, Julie; Weathersbee, Carolyn J.

1974-01-01

Cyclic adenosine 3′, 5′-monophosphate (AMP) stimulates maltodextrin phosphorylase synthesis in Escherichia coli cells induced with maltose. A maximal effect occurs at 2 to 3 mM cyclic AMP. The action of cyclic AMP is specific, inasmuch as adenosine triphosphate, 3′-AMP, 5′-AMP, adenosine, and dibutyryl cyclic AMP are inactive. Glucose, α-methyl glucoside, 2-deoxyglucose, and pyridoxal 5′-phosphate repress maltodextrin phosphorylase synthesis. This repression is reversed by cyclic AMP. The action of cyclic AMP appears to be at the transcriptional level, since cyclic AMP fails to stimulate phosphorylase production in induced cells in which messenger ribonucleic acid synthesis has been arrested by rifampin or by inducer removal. The two other enzymes involved in the metabolism of maltose, amylomaltase and maltose permease, are also induced in this strain of E. coli and affected by glucose and cyclic AMP in a manner similar to phosphorylase. PMID:4358043

Utilization of barley or wheat bran to bioconvert glutamate to γ-aminobutyric acid (GABA).

PubMed

Jin, Wen-Jie; Kim, Min-Ju; Kim, Keun-Sung

2013-09-01

This study deals with the utilization of agro-industrial wastes created by barley and wheat bran in the production of a value-added product, γ-aminobutyric acid (GABA). The simple and eco-friendly reaction requires no pretreatment or microbial fermentation steps but uses barley or wheat bran as an enzyme source, glutamate as a substrate, and pyridoxal 5'-phosphate (PLP) as a cofactor. The optimal reaction conditions were determined on the basis of the temperatures and times used for the decarboxylation reactions and the initial concentrations of barley or wheat bran, glutamate, and PLP. The optimal reactions produced 9.2 mM of GABA from 10 mM glutamate, yielding a 92% GABA conversion rate, when barley bran was used and 6.0 mM of GABA from 10 mM glutamate, yielding a 60% GABA conversion rate, when wheat bran was used. The results imply that barley bran is more efficient than wheat bran in the production of GABA. © 2013 Institute of Food Technologists®

Protein Homeostasis Defects of Alanine-Glyoxylate Aminotransferase: New Therapeutic Strategies in Primary Hyperoxaluria Type I

PubMed Central

Pey, Angel L.; Albert, Armando; Salido, Eduardo

2013-01-01

Alanine-glyoxylate aminotransferase catalyzes the transamination between L-alanine and glyoxylate to produce pyruvate and glycine using pyridoxal 5′-phosphate (PLP) as cofactor. Human alanine-glyoxylate aminotransferase is a peroxisomal enzyme expressed in the hepatocytes, the main site of glyoxylate detoxification. Its deficit causes primary hyperoxaluria type I, a rare but severe inborn error of metabolism. Single amino acid changes are the main type of mutation causing this disease, and considerable effort has been dedicated to the understanding of the molecular consequences of such missense mutations. In this review, we summarize the role of protein homeostasis in the basic mechanisms of primary hyperoxaluria. Intrinsic physicochemical properties of polypeptide chains such as thermodynamic stability, folding, unfolding, and misfolding rates as well as the interaction of different folding states with protein homeostasis networks are essential to understand this disease. The view presented has important implications for the development of new therapeutic strategies based on targeting specific elements of alanine-glyoxylate aminotransferase homeostasis. PMID:23956997

Expression analysis and clinical utility of L-Dopa decarboxylase (DDC) in prostate cancer.

PubMed

Avgeris, Margaritis; Koutalellis, Georgios; Fragoulis, Emmanuel G; Scorilas, Andreas

2008-10-01

L-Dopa decarboxylase (DDC) is a pyridoxal 5'-phosphate-dependent enzyme that was found to be involved in many malignancies. The aim of this study was to investigate the mRNA expression levels of DDC in prostate tissues and to evaluate its clinical utility in prostate cancer (CaP). Total RNA was isolated from 118 tissue specimens from benign prostate hyperplasia (BPH) and CaP patients and a highly sensitive quantitative real-time RT-PCR (qRT-PCR) method for DDC mRNA quantification has been developed using the SYBR Green chemistry. LNCaP prostate cancer cell line was used as a calibrator and GAPDH as a housekeeping gene. DDC was found to be overexpressed, at the mRNA level, in the specimens from prostate cancer patients, in comparison to those from benign prostate hyperplasia patients (p<0.001). Logistic regression and ROC analysis have demonstrated that the DDC expression has significant discriminatory value between CaP and BPH (p<0.001). DDC expression status was compared with other established prognostic factors, in prostate cancer. High expression levels of DDC were found more frequently in high Gleason's score tumors (p=0.022) as well as in advanced stage patients (p=0.032). Our data reveal the potential of DDC expression, at the mRNA level, as a novel biomarker in prostate cancer.

Conserved water molecules in bacterial serine hydroxymethyltransferases.

PubMed

Milano, Teresa; Di Salvo, Martino Luigi; Angelaccio, Sebastiana; Pascarella, Stefano

2015-10-01

Water molecules occurring in the interior of protein structures often are endowed with key structural and functional roles. We report the results of a systematic analysis of conserved water molecules in bacterial serine hydroxymethyltransferases (SHMTs). SHMTs are an important group of pyridoxal-5'-phosphate-dependent enzymes that catalyze the reversible conversion of l-serine and tetrahydropteroylglutamate to glycine and 5,10-methylenetetrahydropteroylglutamate. The approach utilized in this study relies on two programs, ProACT2 and WatCH. The first software is able to categorize water molecules in a protein crystallographic structure as buried, positioned in clefts or at the surface. The other program finds, in a set of superposed homologous proteins, water molecules that occur approximately in equivalent position in each of the considered structures. These groups of molecules are referred to as 'clusters' and represent structurally conserved water molecules. Several conserved clusters of buried or cleft water molecules were found in the set of 11 bacterial SHMTs we took into account for this work. The majority of these clusters were not described previously. Possible structural and functional roles for the conserved water molecules are envisaged. This work provides a map of the conserved water molecules helpful for deciphering SHMT mechanism and for rational design of molecular engineering experiments. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Characterization of Two Mitochondrial Flavin Adenine Dinucleotide-Dependent Glycerol-3-Phosphate Dehydrogenases in Trypanosoma brucei

PubMed Central

Škodová, Ingrid; Verner, Zdeněk; Bringaud, Fréderic; Fabian, Peter

2013-01-01

Glycerol-3-phosphate dehydrogenases (G3PDHs) constitute a shuttle that serves for regeneration of NAD+ reduced during glycolysis. This NAD-dependent enzyme is employed in glycolysis and produces glycerol-3-phosphate from dihydroxyacetone phosphate, while its flavin adenine dinucleotide (FAD)-dependent homologue catalyzes a reverse reaction coupled to the respiratory chain. Trypanosoma brucei possesses two FAD-dependent G3PDHs. While one of them (mitochondrial G3PDH [mtG3PDH]) has been attributed to the mitochondrion and seems to be directly involved in G3PDH shuttle reactions, the function of the other enzyme (putative G3PDH [putG3PDH]) remains unknown. In this work, we used RNA interference and protein overexpression and tagging to shed light on the relative contributions of both FAD-G3PDHs to overall cellular metabolism. Our results indicate that mtG3PDH is essential for the bloodstream stage of T. brucei, while in the procyclic stage the enzyme is dispensable. Expressed putG3PDH-V5 was localized to the mitochondrion, and the data obtained by digitonin permeabilization, Western blot analysis, and immunofluorescence indicate that putG3PDH is located within the mitochondrion. PMID:24142106

BSPS Program (ESI-Mass Spectrometry) Biological Sample Data Analysis; Disruption of Bacteria Spores

DTIC Science & Technology

2005-10-01

the original usage of the translational as a broad description of the entire process by which the polymer of the three-letter code in the mRNA is...translated. There is extensive review of post transnational modifications of proteins by Finn Wold(1981)24, given as in vivo chemical modifications... thiolation , biotin, bromination, carbamylation, deamidation, methylation, glu- cosylation, lipoyl, phosphorylation,, pyridoxal phosphate

Bone Alkaline Phosphatase and Tartrate-Resistant Acid Phosphatase: Potential Co-regulators of Bone Mineralization.

PubMed

Halling Linder, Cecilia; Ek-Rylander, Barbro; Krumpel, Michael; Norgård, Maria; Narisawa, Sonoko; Millán, José Luis; Andersson, Göran; Magnusson, Per

2017-07-01

Phosphorylated osteopontin (OPN) inhibits hydroxyapatite crystal formation and growth, and bone alkaline phosphatase (BALP) promotes extracellular mineralization via the release of inorganic phosphate from the mineralization inhibitor inorganic pyrophosphate (PPi). Tartrate-resistant acid phosphatase (TRAP), produced by osteoclasts, osteoblasts, and osteocytes, exhibits potent phosphatase activity towards OPN; however, its potential capacity as a regulator of mineralization has not previously been addressed. We compared the efficiency of BALP and TRAP towards the endogenous substrates for BALP, i.e., PPi and pyridoxal 5'-phosphate (PLP), and their impact on mineralization in vitro via dephosphorylation of bovine milk OPN. TRAP showed higher phosphatase activity towards phosphorylated OPN and PPi compared to BALP, whereas the activity of TRAP and BALP towards PLP was comparable. Bovine milk OPN could be completely dephosphorylated by TRAP, liberating all its 28 phosphates, whereas BALP dephosphorylated at most 10 phosphates. OPN, dephosphorylated by either BALP or TRAP, showed a partially or completely attenuated phosphorylation-dependent inhibitory capacity, respectively, compared to native OPN on the formation of mineralized nodules. Thus, there are phosphorylations in OPN important for inhibition of mineralization that are removed by TRAP but not by BALP. In conclusion, our data indicate that both BALP and TRAP can alleviate the inhibitory effect of OPN on mineralization, suggesting a potential role for TRAP in skeletal mineralization. Further studies are warranted to explore the possible physiological relevance of TRAP in bone mineralization.

Glutamine-dependent carbamoyl-phosphate synthetase and other enzyme activities related to the pyrimidine pathway in spleen of Squalus acanthias (spiny dogfish).

PubMed Central

Anderson, P M

1989-01-01

The first two steps of urea synthesis in liver of marine elasmobranchs involve formation of glutamine from ammonia and of carbamoyl phosphate from glutamine, catalysed by glutamine synthetase and carbamoyl-phosphate synthetase, respectively [Anderson & Casey (1984) J. Biol. Chem. 259, 456-462]; both of these enzymes are localized exclusively in the mitochondrial matrix. The objective of this study was to establish the enzymology of carbamoyl phosphate formation and utilization for pyrimidine nucleotide biosynthesis in Squalus acanthias (spiny dogfish), a representative elasmobranch. Aspartate carbamoyltransferase could not be detected in liver of dogfish. Spleen extracts, however, had glutamine-dependent carbamoyl-phosphate synthetase, aspartate carbamoyltransferase, dihydro-orotase, and glutamine synthetase activities, all localized in the cytosol; dihydro-orotate dehydrogenase, orotate phosphoribosyltransferase, and orotidine-5'-decarboxylase activities were also present. Except for glutamine synthetase, the levels of all activities were very low. The carbamoyl-phosphate synthetase activity is inhibited by UTP and is activated by 5-phosphoribosyl 1-pyrophosphate. The first three enzyme activities of the pyrimidine pathway were eluted in distinctly different positions during gel filtration chromatography under a number of different conditions; although complete proteolysis of inter-domain regions of a multifunctional complex during extraction cannot be excluded, the evidence suggests that in dogfish, in contrast to mammalian species, these three enzymes of the pyrimidine pathway exist as individual polypeptide chains. These results: (1) establish that dogfish express two different glutamine-dependent carbamoyl-phosphate synthetase activities, (2) confirm the report [Smith, Ritter & Campbell (1987) J. Biol. Chem. 262, 198-202] that dogfish express two different glutamine synthetases, and (3) provide indirect evidence that glutamine may not be available in liver for biosynthetic reactions other than urea formation. Images Fig. 1. PMID:2570570

Expression and characterization of recombinant pyruvate phosphate dikinase from Entamoeba histolytica.

PubMed

Saavedra-Lira, E; Ramirez-Silva, L; Perez-Montfort, R

1998-01-15

The parasite Entamoeba histolytica is an organism whose main energetic source comes from glycolysis. It has the singularity that several of its glycolytic enzymes use pyrophosphate as an alternative phosphate donor. Thus, pyruvate phosphate dikinase (PPDK), an inorganic pyrophosphate (PPi)-dependent enzyme, substitutes pyruvate kinase present in humans. We previously cloned and sequenced the gene that codifies for PPDK in E. histolytica. We now report its expression in a bacterial system and its purification to 98% homogeneity. We determined its K(m) for phosphoenolpyruvate, AMP and PPi (21, < 5 and 100 microM, respectively). Unlike PPDK from maize and bacteria and pyruvate kinase from other cells, EhPPDk is dependent on divalent cations but does not require monovalent cations for activity. The enzyme has an optimum pH of 6.0, it is labile to low temperatures and has a tetrameric structure. Since EhPPDK is a PPi-dependent enzyme, we also tested the effect of some pyrophosphate analogs as inhibitors of activity. Studies on the function and structure of this enzyme may be important for therapeutic research in several parasitic diseases, since it has no counterpart in humans.

Novel methods for the quantification of (2E)-hexadecenal by liquid chromatography with detection by either ESI QTOF tandem mass spectrometry or fluorescence measurement.

PubMed

Lüth, Anja; Neuber, Corinna; Kleuser, Burkhard

2012-04-13

Sphingosine-1-phosphate lyase (SPL) is the only known enzyme that irreversibly cleaves sphingosine-1-phosphate (S1P) into phosphoethanolamine and (2E)-hexadecenal during the final step of sphingolipid catabolism. Because S1P is involved in a wide range of physiological and diseased processes, determining the activity of the degrading enzyme is of great interest. Therefore, we developed two procedures based on liquid chromatography (LC) for analysing (2E)-hexadecenal, which is one of the two S1P degradation products. After separation, two different quantification methods were performed, tandem mass spectrometry (MS) and fluorescence detection. However, (2E)-hexadecenal as a long-chain aldehyde is not ionisable by electrospray ionisation (ESI) for MS quantification and has an insufficient number of corresponding double bonds for fluorescence detection. Therefore, we investigated 2-diphenylacetyl-1,3-indandione-1-hydrazone (DAIH) as a derivatisation reagent. DAIH transforms the aldehyde into an ionisable and fluorescent analogue for quantitative analysis. Our conditions were optimised to obtain the outstanding limit of detection (LOD) of 1 fmol per sample (30 μL) for LC-MS/MS and 0.75 pmol per sample (200 μL) for LC determination with fluorescence detection. We developed an extraction procedure to separate and concentrate (2E)-hexadecenal from biological samples for these measurements. To confirm our new methods, we analysed the (2E)-hexadecenal level of different cell lines and human plasma for the first time ever. Furthermore, we treated HT-29 cells with different concentrations of 4-deoxypyridoxine (DOP), which competitively inhibits pyridoxal-5-phosphate (P5P), an essential cofactor for SPL activity, and observed a significant decrease in (2E)-hexadecenal relative to the untreated cells. Copyright © 2012 Elsevier B.V. All rights reserved.

Sugar analog synthesis by in vitro biocatalytic cascade: A comparison of alternative enzyme complements for dihydroxyacetone phosphate production as a precursor to rare chiral sugar synthesis

PubMed Central

French, Nigel G.; Scoble, Judith A.; Williams, Charlotte C.; Churches, Quentin I.; Frazer, Andrew R.; Taylor, Matthew C.; Coia, Greg; Simpson, Gregory; Turner, Nicholas J.; Scott, Colin

2017-01-01

Carbon-carbon bond formation is one of the most challenging reactions in synthetic organic chemistry, and aldol reactions catalysed by dihydroxyacetone phosphate-dependent aldolases provide a powerful biocatalytic tool for combining C-C bond formation with the generation of two new stereo-centres, with access to all four possible stereoisomers of a compound. Dihydroxyacetone phosphate (DHAP) is unstable so the provision of DHAP for DHAP-dependent aldolases in biocatalytic processes remains complicated. Our research has investigated the efficiency of several different enzymatic cascades for the conversion of glycerol to DHAP, including characterising new candidate enzymes for some of the reaction steps. The most efficient cascade for DHAP production, comprising a one-pot four-enzyme reaction with glycerol kinase, acetate kinase, glycerophosphate oxidase and catalase, was coupled with a DHAP-dependent fructose-1,6-biphosphate aldolase enzyme to demonstrate the production of several rare chiral sugars. The limitation of batch biocatalysis for these reactions and the potential for improvement using kinetic modelling and flow biocatalysis systems is discussed. PMID:29112947

Sugar analog synthesis by in vitro biocatalytic cascade: A comparison of alternative enzyme complements for dihydroxyacetone phosphate production as a precursor to rare chiral sugar synthesis.

PubMed

Hartley, Carol J; French, Nigel G; Scoble, Judith A; Williams, Charlotte C; Churches, Quentin I; Frazer, Andrew R; Taylor, Matthew C; Coia, Greg; Simpson, Gregory; Turner, Nicholas J; Scott, Colin

2017-01-01

Carbon-carbon bond formation is one of the most challenging reactions in synthetic organic chemistry, and aldol reactions catalysed by dihydroxyacetone phosphate-dependent aldolases provide a powerful biocatalytic tool for combining C-C bond formation with the generation of two new stereo-centres, with access to all four possible stereoisomers of a compound. Dihydroxyacetone phosphate (DHAP) is unstable so the provision of DHAP for DHAP-dependent aldolases in biocatalytic processes remains complicated. Our research has investigated the efficiency of several different enzymatic cascades for the conversion of glycerol to DHAP, including characterising new candidate enzymes for some of the reaction steps. The most efficient cascade for DHAP production, comprising a one-pot four-enzyme reaction with glycerol kinase, acetate kinase, glycerophosphate oxidase and catalase, was coupled with a DHAP-dependent fructose-1,6-biphosphate aldolase enzyme to demonstrate the production of several rare chiral sugars. The limitation of batch biocatalysis for these reactions and the potential for improvement using kinetic modelling and flow biocatalysis systems is discussed.

Enzyme-substrate and enzyme-inhibitor complexes of triose phosphate isomerase studied by 31P nuclear magnetic resonance.

PubMed Central

Campbell, I D; Jones, R B; Kiener, P A; Waley, S G

1979-01-01

The complex formed between the enzyme triose phosphate isomerase (EC 5.3.1.1.), from rabbit and chicken muscle, and its substrate dihydroxyacetone phosphate was studied by 31P n.m.r. Two other enzyme-ligant complexes examined were those formed by glycerol 3-phosphate (a substrate analogue) and by 2-phosphoglycollate (potential transition-state analogue). Separate resonances were observed in the 31P n.m.r. spectrum for free and bound 2-phosphoglycollate, and this sets an upper limit to the rate constant for dissociation of the enzyme-inhibitor complex; the linewidth of the resonance assigned to the bound inhibitor provided further kinetic information. The position of this resonance did not vary with pH but remained close to that of the fully ionized form of the free 2-phosphoglycollate. It is the fully ionized form of this ligand that binds to the enzyme. The proton uptake that accompanies binding shows protonation of a group on the enzyme. On the basis of chemical and crystallographic information [Hartman (1971) Biochemistry 10, 146--154; Miller & Waley (1971) Biochem. J. 123, 163--170; De la Mare, Coulson, Knowles, Priddle & Offord )1972) Biochem. J. 129, 321--331; Phillips, Rivers, Sternberg, Thornton & Wilson (1977) Biochem. Soc. Trans. 5, 642--647] this group is believed to be glutamate-165. On the other hand, the position of the resonance of D-glycerol 3 phosphate (sn-glycerol 1-phosphate) in the enzyme-ligand complex changes with pH, and both monoanion and dianon of the ligand bind, although dianion binds better. The substrate, dihydroxyacetone phosphate, behaves essentially like glycerol 3-phosphate. The experiments with dihydroxy-acetone phosphate and triose phosphate isomerase have to be carried out at 1 degree C because at 37 degrees C there is conversion into methyl glyoxal and orthophosphate. The mechanismof the enzymic reaction and the reasons for rate-enhancement are considered, and aspects of the pH-dependence are discussed in an Appendix. PMID:38777

L-cysteine desulfidase: an [4Fe-4S] enzyme isolated from Methanocaldococcus jannaschii that catalyzes the breakdown of L-cysteine into pyruvate, ammonia, and sulfide.

PubMed

Tchong, Shih-I; Xu, Huimin; White, Robert H

2005-02-08

A [4Fe-4S] enzyme that decomposes L-cysteine to hydrogen sulfide, ammonia, and pyruvate has been isolated and characterized from Methanocaldococcus jannaschii. The sequence of the isolated enzyme demonstrated that the protein was the product of the M. jannaschii MJ1025 gene. The protein product of this gene was recombinantly produced in Escherichia coli and purified to homogeneity. Both the isolated and recombinant enzymes are devoid of pyridoxal phosphate (PLP) and are rapidly inactivated upon exposure to air. The air-inactivated enzyme is activated by reaction with Fe2+ and dithiothreitol in the absence of air. The air-inactivated enzyme contains 3 mol of iron per subunit (43 kDa, SDS gel electrophoresis), and the native enzyme has a measured molecular mass of 135 kDa (gel filtration), indicating it is a trimer. The enzyme is very specific for L-cysteine, with no activity being detected with D-cysteine, L-homocysteine, 3-mercaptopropionic acid (cysteine without the amino group), cysteamine (cysteine without the carboxylic acid), or mercaptolactate (the hydroxyl analogue of cysteine). The activity of the enzyme was stimulated by 40% when the enzyme was assayed in the presence of methyl viologen (4 mM) and inhibited by 70% when the enzyme was assayed in the presence of EDTA (7.1 mM). Preincubation of the enzyme with iodoacetamide (17 mM) completely abolishes activity. The enzymatic activity has a half-life of 8 or 12 min when the enzyme is treated at room temperature with 0.42 mM N-ethylmaleimide (NEM) or 0.42 mM iodoacetamide, respectively. MALDI analysis of the NEM-inactivated enzyme showed Cys25 as the site of alkylation. Site-directed mutagenesis of each of four of the cysteines conserved in the orthologues of the enzyme reduced the catalytic efficiency and thermal stability of the enzyme. The enzyme was found to catalyze exchange of the C-2 hydrogen of the L-cysteine with solvent. These results are consistent with three of the conserved cysteines being involved in the formation of the [4Fe-4S] center and the thiolate of Cys25 serving as a base to abstract the alpha-hydrogen in the first step of the elimination. Although the enzyme has no sequence homology to any known enzymes, including the non-PLP-dependent serine/threonine dehydratases or aconitases, the mechanisms of action of all of these enzymes are similar, in that each catalyzes an alpha,beta-elimination reaction adjacent to a carboxylate group. It is proposed that the enzyme may be responsible for the production of sulfide required for the biosynthesis of iron-sulfur centers in this archaea. A mechanism of action of the enzyme is proposed.

Biochemical characterization, homology modeling and docking studies of ornithine delta-aminotransferase--an important enzyme in proline biosynthesis of plants.

PubMed

Sekhar, P Nataraj; Amrutha, R Naga; Sangam, Shubhada; Verma, D P S; Kishor, P B Kavi

2007-11-01

Ornithine delta-aminotransferase (OAT) is an important enzyme in proline biosynthetic pathway and is implicated in salt tolerance in higher plants. OAT transaminates ornithine to pyrroline 5-carboxylate, which is further catalyzed to proline by pyrroline 5-carboxylate reductase. The Vigna aconitifolia OAT cDNA, encoding a polypeptide of 48.1 kDa, was expressed in Escherichia coli and the enzyme was partially characterized following its purification using (NH(4))(2)SO(4) precipitation and gel filtration techniques. Optimal activity of the enzyme was observed at a temperature of 25 degrees C and pH 8.0. The enzyme appeared to be a monomer and exhibited high activity at 4mM ornithine. Proline did not show any apparent effect but isoleucine, valine and serine inhibited the activity when added into the assay mixture along with ornithine. Omission of pyridoxal 5'-phosphate from the reaction mixture reduced the activity of this enzyme by 60%. To further evaluate these biochemical observations, homology modeling of the OAT was performed based on the crystal structure of the ornithine delta-aminotransferase from humans (PDB code 1OAT) by using the software MODELLER6v2. With the aid of the molecular mechanics and dynamics methods, the final model was obtained and assessed subsequently by PROCHECK and VERIFY-3D graph. With this model, a flexible docking study with the substrate and inhibitors was performed and the results indicated that Gly106 and Lys256 in OAT are the important determinant residues in binding as they have strong hydrogen bonding contacts with the substrate and inhibitors. These observations are in conformity with the results obtained from experimental investigations.

Pseudomonas aeruginosa 4-Amino-4-Deoxychorismate Lyase: Spatial Conservation of an Active Site Tyrosine and Classification of Two Types of Enzyme

PubMed Central

O'Rourke, Patrick E. F.; Eadsforth, Thomas C.; Fyfe, Paul K.; Shepherd, Sharon M.; Hunter, William N.

2011-01-01

4-Amino-4-deoxychorismate lyase (PabC) catalyzes the formation of 4-aminobenzoate, and release of pyruvate, during folate biosynthesis. This is an essential activity for the growth of Gram-negative bacteria, including important pathogens such as Pseudomonas aeruginosa. A high-resolution (1.75 Å) crystal structure of PabC from P. aeruginosa has been determined, and sequence-structure comparisons with orthologous structures are reported. Residues around the pyridoxal 5′-phosphate cofactor are highly conserved adding support to aspects of a mechanism generic for enzymes carrying that cofactor. However, we suggest that PabC can be classified into two groups depending upon whether an active site and structurally conserved tyrosine is provided from the polypeptide that mainly forms an active site or from the partner subunit in the dimeric assembly. We considered that the conserved tyrosine might indicate a direct role in catalysis: that of providing a proton to reduce the olefin moiety of substrate as pyruvate is released. A threonine had previously been suggested to fulfill such a role prior to our observation of the structurally conserved tyrosine. We have been unable to elucidate an experimentally determined structure of PabC in complex with ligands to inform on mechanism and substrate specificity. Therefore we constructed a computational model of the catalytic intermediate docked into the enzyme active site. The model suggests that the conserved tyrosine helps to create a hydrophobic wall on one side of the active site that provides important interactions to bind the catalytic intermediate. However, this residue does not appear to participate in interactions with the C atom that undergoes an sp 2 to sp 3 conversion as pyruvate is produced. The model and our comparisons rather support the hypothesis that an active site threonine hydroxyl contributes a proton used in the reduction of the substrate methylene to pyruvate methyl in the final stage of the mechanism. PMID:21935381

A novel cysteine desulfurase influencing organosulfur compounds in Lentinula edodes

PubMed Central

Liu, Ying; Lei, Xiao-Yu; Chen, Lian-Fu; Bian, Yin-Bing; Yang, Hong; Ibrahim, Salam A.; Huang, Wen

2015-01-01

Organosulfur compounds are the basis for the unique aroma of Lentinula edodes, and cysteine sulfoxide lyase (C-S lyase) is the key enzyme in this trait. The enzyme from Alliium sativum has been crystallized and well-characterized; however, there have been no reports of the characterization of fungi C-S lyase at the molecular level. We identified a L. edodes C-S lyase (Lecsl), cloned a gene of Csl encoded Lecsl and then combined modeling, simulations, and experiments to understand the molecular basis of the function of Lecsl. Our analysis revealed Lecsl to be a novel cysteine desulfurase and not a type of cysteine sulfoxide lyase. The pyridoxal-5-phosphate (PLP) molecule bonded tightly to Lecsl to form a Lecsl-PLP complex. Moreover, the Lecsl had one active center that served to bind two kinds of substrates, S-methyl-L-cysteine sulfoxide and L-cysteine, and had both cysteine sulfoxide lyase and cysteine desulfurase activity. We found that the amino acid residue Asn393 was essential for the catalytic activity of Lecsl and that the gene Csl encoded a novel cysteine desulfurase to influence organosulfur compounds in L. edodes. Our results provide a new insight into understanding the formation of the unique aroma of L. edodes. PMID:26054293

A novel cysteine desulfurase influencing organosulfur compounds in Lentinula edodes.

PubMed

Liu, Ying; Lei, Xiao-Yu; Chen, Lian-Fu; Bian, Yin-Bing; Yang, Hong; Ibrahim, Salam A; Huang, Wen

2015-06-09

Organosulfur compounds are the basis for the unique aroma of Lentinula edodes, and cysteine sulfoxide lyase (C-S lyase) is the key enzyme in this trait. The enzyme from Alliium sativum has been crystallized and well-characterized; however, there have been no reports of the characterization of fungi C-S lyase at the molecular level. We identified a L. edodes C-S lyase (Lecsl), cloned a gene of Csl encoded Lecsl and then combined modeling, simulations, and experiments to understand the molecular basis of the function of Lecsl. Our analysis revealed Lecsl to be a novel cysteine desulfurase and not a type of cysteine sulfoxide lyase. The pyridoxal-5-phosphate (PLP) molecule bonded tightly to Lecsl to form a Lecsl-PLP complex. Moreover, the Lecsl had one active center that served to bind two kinds of substrates, S-methyl-L-cysteine sulfoxide and L-cysteine, and had both cysteine sulfoxide lyase and cysteine desulfurase activity. We found that the amino acid residue Asn393 was essential for the catalytic activity of Lecsl and that the gene Csl encoded a novel cysteine desulfurase to influence organosulfur compounds in L. edodes. Our results provide a new insight into understanding the formation of the unique aroma of L. edodes.

Fructose-2,6-bisphosphatase and 6-phosphofructo-2-kinase are separable in yeast.

PubMed Central

Kretschmer, M; Schellenberger, W; Otto, A; Kessler, R; Hofmann, E

1987-01-01

Fructose-2,6-bisphosphatase was purified from yeast and separated from 6-phosphofructo-2-kinase and alkaline phosphatase. The enzyme released Pi from the 2-position of fructose 2,6-bisphosphate and formed fructose 6-phosphate in stoichiometric amounts. The enzyme displays hyperbolic kinetics towards fructose 2,6-bisphosphate, with a Km value of 0.3 microM. It is strongly inhibited by fructose 6-phosphate. The inhibition is counteracted by L-glycerol 3-phosphate. Phosphorylation of the enzyme by cyclic-AMP-dependent protein kinase causes inactivation, which is reversible by the action of protein phosphatase 2A. PMID:2825652

Tyrosine aminotransferase: biochemical and structural properties and molecular dynamics simulations.

PubMed

Mehere, Prajwalini; Han, Qian; Lemkul, Justin A; Vavricka, Christopher J; Robinson, Howard; Bevan, David R; Li, Jianyong

2010-11-01

Tyrosine aminotransferase (TAT) catalyzes the transamination of tyrosine and other aromatic amino acids. The enzyme is thought to play a role in tyrosinemia type II, hepatitis and hepatic carcinoma recovery. The objective of this study is to investigate its biochemical and structural characteristics and substrate specificity in order to provide insight regarding its involvement in these diseases. Mouse TAT (mTAT) was cloned from a mouse cDNA library, and its recombinant protein was produced using Escherichia coli cells and purified using various chromatographic techniques. The recombinant mTAT is able to catalyze the transamination of tyrosine using α-ketoglutaric acid as an amino group acceptor at neutral pH. The enzyme also can use glutamate and phenylalanine as amino group donors and p-hydroxy-phenylpyruvate, phenylpyruvate and alpha-ketocaproic acid as amino group acceptors. Through macromolecular crystallography we have determined the mTAT crystal structure at 2.9 Å resolution. The crystal structure revealed the interaction between the pyridoxal-5'-phosphate cofactor and the enzyme, as well as the formation of a disulphide bond. The detection of disulphide bond provides some rational explanation regarding previously observed TAT inactivation under oxidative conditions and reactivation of the inactive TAT in the presence of a reducing agent. Molecular dynamics simulations using the crystal structures of Trypanosoma cruzi TAT and human TAT provided further insight regarding the substrate-enzyme interactions and substrate specificity. The biochemical and structural properties of TAT and the binding of its cofactor and the substrate may help in elucidation of the mechanism of TAT inhibition and activation.

Protective Therapies for Monomethylhydrazine: Comparison of Pyridoxine and Physical Restraint in the Monkey

DTIC Science & Technology

1979-04-01

Pharmaco1, Exp. Ther. 140:133. 14. Meldrum , B. S. (1975). Epilepsy and y-aminobutyric acid -mediated inhibition. Int. Rev. Neurobiol. 17:1-36. 15. Rougeul, A...within the context of established neurochemical influences of the hydrazines on synthesis of the inhibitory neurotransmitter, gamma amino- butyric acid ...pyridoxal phosphate, a reaction which absorbs this coenzyme in the synthesis of glutamic acid decar- boxylase (GAD) and gamma-aminobutyric acid (GABA

Fungal trehalose phosphorylase: kinetic mechanism, pH-dependence of the reaction and some structural properties of the enzyme from Schizophyllum commune.

PubMed Central

Eis, C; Watkins, M; Prohaska, T; Nidetzky, B

2001-01-01

Initial-velocity measurements for the phospholysis and synthesis of alpha,alpha-trehalose catalysed by trehalose phosphorylase from Schizophyllum commune and product and dead-end inhibitor studies show that this enzyme has an ordered Bi Bi kinetic mechanism, in which phosphate binds before alpha,alpha-trehalose, and alpha-D-glucose is released before alpha-D-glucose 1-phosphate. The free-energy profile for the enzymic reaction at physiological reactant concentrations displays its largest barriers for steps involved in reverse glucosyl transfer to D-glucose, and reveals the direction of phospholysis to be favoured thermodynamically. The pH dependence of kinetic parameters for all substrates and the dissociation constant of D-glucal, a competitive dead-end inhibitor against D-glucose (K(i)=0.3 mM at pH 6.6 and 30 degrees C), were determined. Maximum velocities and catalytic efficiencies for the forward and reverse reactions decrease at high and low pH, giving apparent pK values of 7.2--7.8 and 5.5--6.0 for two groups whose correct protonation state is required for catalysis. The pH dependences of k(cat)/K are interpreted in terms of monoanionic phosphate and alpha-D-glucose 1-phosphate being the substrates, and of the pK value seen at high pH corresponding to the phosphate group in solution or bound to the enzyme. The K(i) value for the inhibitor decreases outside the optimum pH range for catalysis, indicating that binding of D-glucal is tighter with incorrectly ionized forms of the complex between the enzyme and alpha-D-glucose 1-phosphate. Each molecule of trehalose phosphorylase contains one Mg(2+) that is non-dissociable in the presence of metal chelators. Measurements of the (26)Mg(2+)/(24)Mg(2+) ratio in the solvent and on the enzyme by using inductively coupled plasma MS show that exchange of metal ion between protein and solution does not occur at measurable rates. Tryptic peptide mass mapping reveals close structural similarity between trehalose phosphorylases from basidiomycete fungi. PMID:11389683

Vitamin B6 is essential for serine de novo biosynthesis.

PubMed

Ramos, Rúben J; Pras-Raves, Mia L; Gerrits, Johan; van der Ham, Maria; Willemsen, Marcel; Prinsen, Hubertus; Burgering, Boudewijn; Jans, Judith J; Verhoeven-Duif, Nanda M

2017-11-01

Pyridoxal 5'-phosphate (PLP), the metabolically active form of vitamin B6, plays an essential role in brain metabolism as a cofactor in numerous enzyme reactions. PLP deficiency in brain, either genetic or acquired, results in severe drug-resistant seizures that respond to vitamin B6 supplementation. The pathogenesis of vitamin B6 deficiency is largely unknown. To shed more light on the metabolic consequences of vitamin B6 deficiency in brain, we performed untargeted metabolomics in vitamin B6-deprived Neuro-2a cells. Significant alterations were observed in a range of metabolites. The most surprising observation was a decrease of serine and glycine, two amino acids that are known to be elevated in the plasma of vitamin B6 deficient patients. To investigate the cause of the low concentrations of serine and glycine, a metabolic flux analysis on serine biosynthesis was performed. The metabolic flux results showed that the de novo synthesis of serine was significantly reduced in vitamin B6-deprived cells. In addition, formation of glycine and 5-methyltetrahydrofolate was decreased. Thus, vitamin B6 is essential for serine de novo biosynthesis in neuronal cells, and serine de novo synthesis is critical to maintain intracellular serine and glycine. These findings suggest that serine and glycine concentrations in brain may be deficient in patients with vitamin B6 responsive epilepsy. The low intracellular 5-mTHF concentrations observed in vitro may explain the favourable but so far unexplained response of some patients with pyridoxine-dependent epilepsy to folinic acid supplementation.

A new strategy for the cloning, overexpression and one step purification of three DHAP-dependent aldolases: rhamnulose-1-phosphate aldolase, fuculose-1-phosphate aldolase and tagatose-1,6-diphosphate aldolase.

PubMed

Garcia-Junceda, E; Shen, G J; Sugai, T; Wong, C H

1995-07-01

Three DHAP-dependent aldolases, rhamnulose-1-phosphate aldolase (Rham-1PA), fuculose-1-phosphate aldolase (Fuc-1PA) and tagatose-1,6-diphosphate aldolase (TDPA) have been cloned and overexpressed in Escherichia coli using two different expression vectors: pTrcHis for the expression of Rham-1PA and Fuc-1PA and pRSET for the expression of TDPA. In each case the recombinant enzyme is synthesized as a fusion protein with a hexahistidine tag on the N-terminus. The three enzymes have been purified in only one step by chelation affinity chromatography. The effects of cultivation temperature and concentration of inducer have been studied in order to optimize the expression of the recombinant proteins and to avoid the formation of inclusion bodies.

Pyridoxal-5'-phosphate deficiency is associated with hyperhomocysteinemia regardless of antioxidant, thiamine, riboflavin, cobalamine, and folate status in critically ill patients.

PubMed

Molina-López, Jorge; Florea, Daniela; Quintero-Osso, Bartolomé; de la Cruz, Antonio Pérez; Rodríguez-Elvira, Manuel; Del Pozo, Elena Planells

2016-06-01

Critically ill patients develop severe stress, inflammation and a clinical state that may raise the utilization and metabolic replacement of pyridoxal-5'-phosphate decreasing their body reserves. This study was designed to assess the nutritional pyridoxal-5'-phosphate status in critical care patients with systemic inflammatory response syndrome, comparing them with a group of healthy people, and studying it's association with factors involved in the pyridoxine and other B vitamins metabolism, as the total antioxidant capacity and Hcy as cardiovascular risk biomarker. Prospective, multicentre, comparative, observational and analytic study. One hundred and three critically ill patients from different hospitals, and eighty four healthy subjects from Granada, Spain, all with informed consent. Data from daily nutritional assessment, ICU severity scores, clinical and nutritional parameters, antioxidant status and homocysteine levels was taken at admission and at the seventh day of the ICU stay. Thiamine, riboflavin, pyridoxine and folate status proved deficient in a large number of patients, being significantly lower in comparison with control group, and significantly decreased at 7th day of ICU stay. Higher homocysteine was observed in patients compared with control group (p < 0.05) where 31.5 and 26.8 percent of subjects presented hyperhomocysteinemia at initial and final of study, respectively. Antioxidant status was lower than control group in two periods analysed, and decreased at 7th day of ICU stay (p < 0.05) being associated with PLP deficiency. PLP deficiency was also correlated with hyperhomocysteinemia at two times measured (r. -0.73, p < 0.001; r. -0.69, p < 0.001, respectively), showing at day 7 an odds ratio of 6.62 in our multivariate model. Critically ill patients with SIRS show deficient B vitamin and low antioxidant statuses. Despite association found between PLP deficiency and low antioxidant status in critically ill patients, PLP deficiency was only associated with hyperhomocysteinemia regardless of antioxidant, riboflavin, cobalamine, and folate statuses in critically ill patients with SIRS at seventh day of ICU stay. PLP deficient patients presented about six times more risk of cardiovascular disease than non deficients. Copyright © 2015 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

A Critical Examination of the Reaction of Pyridoxal 5-Phosphate with Human Hemoglobin Ao

DTIC Science & Technology

1989-01-01

sodium borohydride gives unacceptable levels of methemoglobin (i.e., > 10%). Excessive foaming and methemoglobin formation can be partially avoided using...a biochemical level . By using new advances in HPLC column technology, we could better determine hetero- geneity in the product mixture due solely to... diphosphoglycerate (2,3-DPG). 6 SFH, which had been stripped of 2,3-DPG, was deoxygenated with nitrogen and treated with a solution of PLP in Tris

Impact of Pre-Pregnancy BMI on B Vitamin and Inflammatory Status in Early Pregnancy: An Observational Cohort Study

PubMed Central

Bjørke-Monsen, Anne-Lise; Ulvik, Arve; Nilsen, Roy M.; Midttun, Øivind; Roth, Christine; Magnus, Per; Stoltenberg, Camilla; Vollset, Stein Emil; Reichborn-Kjennerud, Ted; Ueland, Per Magne

2016-01-01

Maternal nutrition and inflammation have been suggested as mediators in the development of various adverse pregnancy outcomes associated with maternal obesity. We have investigated the relation between pre-pregnancy BMI, B vitamin status, and inflammatory markers in a group of healthy pregnant women. Cobalamin, folate, pyridoxal 5′-phosphate, and riboflavin; and the metabolic markers homocysteine, methylmalonic acid, and 3-hydroxykynurenine/xanthurenic acid ratio (HK/XA); and markers of cellular inflammation, neopterin and kynurenine/tryptophan ratio (KTR) were determined in pregnancy week 18 and related to pre-pregnancy body mass index (BMI), in 2797 women from the Norwegian Mother and Child Cohort Study (MoBa). Pre-pregnancy BMI was inversely related to folate, cobalamin, pyridoxal 5′-phosphate (PLP), and riboflavin (p < 0.001), and associated with increased neopterin and KTR levels (p < 0.001). Inflammation seemed to be an independent predictor of low vitamin B6 status, as verified by low PLP and high HK/XA ratio. A high pre-pregnancy BMI is a risk factor for low B vitamin status and increased cellular inflammation. As an optimal micronutrient status is vital for normal fetal development, the observed lower B vitamin levels may contribute to adverse pregnancy outcomes associated with maternal obesity and B vitamin status should be assessed in women with high BMI before they get pregnant. PMID:27916904

Drosophila selenophosphate synthetase 1 regulates vitamin B6 metabolism: prediction and confirmation

PubMed Central

2011-01-01

Background There are two selenophosphate synthetases (SPSs) in higher eukaryotes, SPS1 and SPS2. Of these two isotypes, only SPS2 catalyzes selenophosphate synthesis. Although SPS1 does not contain selenophosphate synthesis activity, it was found to be essential for cell growth and embryogenesis in Drosophila. The function of SPS1, however, has not been elucidated. Results Differentially expressed genes in Drosophila SL2 cells were identified using two-way analysis of variance methods and clustered according to their temporal expression pattern. Gene ontology analysis was performed against differentially expressed genes and gene ontology terms related to vitamin B6 biosynthesis were found to be significantly affected at the early stage at which megamitochondria were not formed (day 3) after SPS1 knockdown. Interestingly, genes related to defense and amino acid metabolism were affected at a later stage (day 5) following knockdown. Levels of pyridoxal phosphate, an active form of vitamin B6, were decreased by SPS1 knockdown. Treatment of SL2 cells with an inhibitor of pyridoxal phosphate synthesis resulted in both a similar pattern of expression as that found by SPS1 knockdown and the formation of megamitochondria, the major phenotypic change observed by SPS1 knockdown. Conclusions These results indicate that SPS1 regulates vitamin B6 synthesis, which in turn impacts various cellular systems such as amino acid metabolism, defense and other important metabolic activities. PMID:21864351

Synthesis and Physicochemical Characterization of D-Tagatose-1-phosphate: The Substrate of the Tagatose-1-Phosphate Kinase TagK in the PTS-mediated D-Tagatose Catabolic Pathway of Bacillus licheniformis

PubMed Central

Van der Heiden, Edwige; Delmarcelle, Michaël; Simon, Patricia; Counson, Melody; Galleni, Moreno; Freedberg, Darón I.; Thompson, John; Joris, Bernard; Battistel, Marcos D.

2015-01-01

We report the first enzymatic synthesis of D-tagatose-1-phosphate (Tag-1P) by the multi-component PEP-dependent:tag-PTS present in tagatose-grown cells of Klebsiella pneumoniae. Physicochemical characterization by 31P and 1H NMR spectroscopy reveals that, in solution, this derivative is primarily in the pyranose form. Tag-1P was used to characterize the putative tagatose-1-phosphate kinase (TagK) of the Bacillus licheniformis PTS-mediated D-Tagatose catabolic Pathway (Bli-TagP). For this purpose, a soluble protein fusion was obtained with the 6 His-tagged trigger factor (TFHis6) of Escherichia coli. The active fusion enzyme was named TagK-TFHis6. Tag-1P and D-fructose-1-phosphate (Fru-1P) are substrates for the TagK-TFHis6 enzyme, whereas the isomeric derivatives D-tagatose-6-phosphate (Tag-6P) and D-fructose-6-phosphate (Fru-6P) are inhibitors. Studies of catalytic efficiency (kcat/Km) reveal that the enzyme specificity is markedly in favor of Tag-1P as substrate. Importantly, we show in vivo that the transfer of the phosphate moiety from PEP to the B. licheniformis tagatose-specific enzyme II (EIITag) in E.coli is inefficient. The capability of the PTS general cytoplasmic components of B. subtilis, HPr and EI, to restore the phosphate transfer is demonstrated. PMID:26159072

Sphingomyelinase D activity in model membranes: structural effects of in situ generation of ceramide-1-phosphate.

PubMed

Stock, Roberto P; Brewer, Jonathan; Wagner, Kerstin; Ramos-Cerrillo, Blanca; Duelund, Lars; Jernshøj, Kit Drescher; Olsen, Lars Folke; Bagatolli, Luis A

2012-01-01

The toxicity of Loxosceles spider venom has been attributed to a rare enzyme, sphingomyelinase D, which transforms sphingomyelin to ceramide-1-phosphate. The bases of its inflammatory and dermonecrotic activity, however, remain unclear. In this work the effects of ceramide-1-phosphate on model membranes were studied both by in situ generation of this lipid using a recombinant sphingomyelinase D from the spider Loxosceles laeta and by pre-mixing it with sphingomyelin and cholesterol. The systems of choice were large unilamellar vesicles for bulk studies (enzyme kinetics, fluorescence spectroscopy and dynamic light scattering) and giant unilamellar vesicles for fluorescence microscopy examination using a variety of fluorescent probes. The influence of membrane lateral structure on the kinetics of enzyme activity and the consequences of enzyme activity on the structure of target membranes containing sphingomyelin were examined. The findings indicate that: 1) ceramide-1-phosphate (particularly lauroyl ceramide-1-phosphate) can be incorporated into sphingomyelin bilayers in a concentration-dependent manner and generates coexistence of liquid disordered/solid ordered domains, 2) the activity of sphingomyelinase D is clearly influenced by the supramolecular organization of its substrate in membranes and, 3) in situ ceramide-1-phosphate generation by enzymatic activity profoundly alters the lateral structure and morphology of the target membranes.

Engineering of GlcNAc-1-Phosphotransferase for Production of Highly Phosphorylated Lysosomal Enzymes for Enzyme Replacement Therapy.

PubMed

Liu, Lin; Lee, Wang-Sik; Doray, Balraj; Kornfeld, Stuart

2017-06-16

Several lysosomal enzymes currently used for enzyme replacement therapy in patients with lysosomal storage diseases contain very low levels of mannose 6-phosphate, limiting their uptake via mannose 6-phosphate receptors on the surface of the deficient cells. These enzymes are produced at high levels by mammalian cells and depend on endogenous GlcNAc-1-phosphotransferase α/β precursor to phosphorylate the mannose residues on their glycan chains. We show that co-expression of an engineered truncated GlcNAc-1-phosphotransferase α/β precursor and the lysosomal enzyme of interest in the producing cells resulted in markedly increased phosphorylation and cellular uptake of the secreted lysosomal enzyme. This method also results in the production of highly phosphorylated acid β-glucocerebrosidase, a lysosomal enzyme that normally has just trace amounts of this modification.

Isolation of a Mutant of Salmonella typhimurium Dependent on D-Arabinose-5-phosphate for Growth and Synthesis of 3-Deoxy-D-mannoctulosonate (Ketodeoxyoctonate)

PubMed Central

Rick, P. D.; Osborn, M. J.

1972-01-01

A new type of auxotrophic mutant of Salmonella typhimurium has been isolated that is defective in the synthesis of the 3-deoxy-D-mannooctulosonate (ketodeoxyoctonate) region of the lipopolysaccharide and requires D-arabinose-5-phosphate for growth. Genetic and biochemical evidence indicated that the mutant defect was due to an altered ketodeoxyoctonate-8-phosphate synthetase with an apparent Km for D-arabinose-5-phosphate 35-fold higher than that of the parental enzyme. As a result of this enzymatic lesion, the mutant strain was dependent on exogenous D-arabinose-5-phosphate both for growth and for synthesis of a complete lipopolysaccharide. PMID:4566459

Trypanosoma evansi contains two auxiliary enzymes of glycolytic metabolism: Phosphoenolpyruvate carboxykinase and pyruvate phosphate dikinase.

PubMed

Rivero, Luz Amira; Concepción, Juan Luis; Quintero-Troconis, Ender; Quiñones, Wilfredo; Michels, Paul A M; Acosta, Héctor

2016-06-01

Trypanosoma evansi is a monomorphic protist that can infect horses and other animal species of economic importance for man. Like the bloodstream form of the closely related species Trypanosoma brucei, T. evansi depends exclusively on glycolysis for its free-energy generation. In T. evansi as in other kinetoplastid organisms, the enzymes of the major part of the glycolytic pathway are present within organelles called glycosomes, which are authentic but specialized peroxisomes. Since T. evansi does not undergo stage-dependent differentiations, it occurs only as bloodstream forms, it has been assumed that the metabolic pattern of this parasite is identical to that of the bloodstream form of T. brucei. However, we report here the presence of two additional enzymes, phosphoenolpyruvate carboxykinase and PPi-dependent pyruvate phosphate dikinase in T. evansi glycosomes. Their colocalization with glycolytic enzymes within the glycosomes of this parasite has not been reported before. Both enzymes can make use of PEP for contributing to the production of ATP within the organelles. The activity of these enzymes in T. evansi glycosomes drastically changes the model assumed for the oxidation of glucose by this parasite. Copyright © 2016 Elsevier Inc. All rights reserved.

Characterization of recombinant PPi-dependent 6-phosphofructokinases from Methylosinus trichosporium OB3b and Methylobacterium nodulans ORS 2060.

PubMed

Rozova, O N; Khmelenina, V N; Trotsenko, Y A

2012-03-01

The properties of the purified recombinant PPi-dependent 6-phosphofructokinases (PPi-PFKs) from the methanotroph Methylosinus trichosporium OB3b and rhizospheric phytosymbiont Methylobacterium nodulans ORS 2060 were determined. The dependence of activities of PPi-PFK-His(6)-tag from Ms. trichosporium OB3b (6 × 45 kDa) and PPi-PFK from Mb. nodulans ORS 2060 (4 × 43 kDa) on the concentrations of substrates of forward and reverse reactions conformed to Michaelis-Menten kinetics. Besides fructose-6-phosphate, the enzymes also phosphorylated sedoheptulose-7-phosphate. ADP or AMP (1 mM each) inhibited activity of the Ms. trichosporium PPi-PFK but did not affect the activity of the Mb. nodulans enzyme. Preference of PPi-PFKs to fructose-1,6-bisphosphate implied a predominant function of the enzymes in hexose phosphate synthesis in these bacteria. PPi-PFKs from the methylotrophs have low similarity of translated amino acid sequences (17% identity) and belong to different phylogenetic subgroups of type II 6-phosphofructokinases. The relationship of PPi-PFKs with microaerophilic character of Ms. trichosporium OB3b and adaptation of Mb. nodulans ORS 2060 to anaerobic phase of phytosymbiosis are discussed.

Extracellular Ca2(+)-dependent inducible alkaline phosphatase from extremely halophilic archaebacterium Haloarcula marismortui.

PubMed Central

Goldman, S; Hecht, K; Eisenberg, H; Mevarech, M

1990-01-01

When starved of inorganic phosphate, the extremely halophilic archaebacterium Haloarcula marismortui produces the enzyme alkaline phosphatase and secretes it to the medium. This inducible extracellular enzyme is a glycoprotein whose subunit molecular mass is 160 kDa, as estimated by sodium dodecyl sulfate-gel electrophoresis. The native form of the enzyme is heterogeneous and composed of multiple oligomeric forms. The enzymatic activity of the halophilic alkaline phosphatase is maximal at pH 8.5, and the enzyme is inhibited by phosphate. Unlike most alkaline phosphatases, the halobacterial enzyme requires Ca2+ and not Zn2+ ions for its activity. Both calcium ions (in the millimolar range) and NaCl (in the molar range) are required for the stability of the enzyme. Images PMID:2123861

Direct Comparison of the Enzymatic Characteristics and Superoxide Production of the Four Aldehyde Oxidase Enzymes Present in Mouse.

PubMed

Kücükgöze, Gökhan; Terao, Mineko; Garattini, Enrico; Leimkühler, Silke

2017-08-01

Aldehyde oxidases (AOXs) are molybdoflavoenzymes with an important role in the metabolism and detoxification of heterocyclic compounds and aliphatic as well as aromatic aldehydes. The enzymes use oxygen as the terminal electron acceptor and produce reduced oxygen species during turnover. Four different enzymes, mAOX1, mAOX3, mAOX4, and mAOX2, which are the products of distinct genes, are present in the mouse. A direct and simultaneous comparison of the enzymatic properties and characteristics of the four enzymes has never been performed. In this report, the four catalytically active mAOX enzymes were purified after heterologous expression in Escherichia coli The kinetic parameters of the four mouse AOX enzymes were determined and compared with the use of six predicted substrates of physiologic and toxicological interest, i.e., retinaldehyde, N 1 -methylnicotinamide, pyridoxal, vanillin, 4-(dimethylamino)cinnamaldehyde ( p- DMAC), and salicylaldehyde. While retinaldehyde, vanillin, p- DMAC, and salycilaldehyde are efficient substrates for the four mouse AOX enzymes, N 1 -methylnicotinamide is not a substrate of mAOX1 or mAOX4, and pyridoxal is not metabolized by any of the purified enzymes. Overall, mAOX1, mAOX2, mAOX3, and mAOX4 are characterized by significantly different K M and k cat values for the active substrates. The four mouse AOXs are also characterized by quantitative differences in their ability to produce superoxide radicals. With respect to this last point, mAOX2 is the enzyme generating the largest rate of superoxide radicals of around 40% in relation to moles of substrate converted, and mAOX1, the homolog to the human enzyme, produces a rate of approximately 30% of superoxide radicals with the same substrate. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

Polyamine biosynthesis in Phytomonas: biochemical characterisation of a very unstable ornithine decarboxylase.

PubMed

Marcora, M Silvina; Cejas, Silvina; González, Nélida S; Carrillo, Carolina; Algranati, Israel D

2010-10-01

The metabolism of polyamines as well as their functions as growth regulators in plants have been extensively studied for many years. However, almost nothing is known about the biosynthesis and roles of these substances in Phytomonas spp., parasites of several plants. We have used HPLC and electrophoretic analyses to investigate the presence and metabolism of polyamines in Phytomonas Jma strain, detecting both putrescine and spermidine but not spermine. Experiments carried out by incubation of intact parasites with labelled ornithine or putrescine showed the formation of radioactive putrescine or spermidine, respectively. These results indicated that Phytomonas Jma can synthesise these polyamines through the action of ornithine decarboxylase (ODC) and spermidine synthase. On the other hand, we could not detect the conversion of arginine to agmatine, suggesting the absence of arginine decarboxylase (ADC) in Phytomonas. However, we cannot ensure the complete absence of this enzymatic activity in the parasite. Phytomonas ODC required pyridoxal 5'-phosphate for maximum activity and was specifically inhibited by α-difluoromethylornithine. The metabolic turnover of the enzyme was very high, with a half-life of 10-15 min, one of the shortest found among all ODC enzymes studied to date. The parasite proteasome seems to be involved in degradation of the enzyme, since Phytomonas ODC can be markedly stabilized by MG-132, a well known proteasome inhibitor. The addition of polyamines to Phytomonas cultures did not decrease ODC activity, strongly suggesting the possible absence of antizyme in this parasite. Copyright © 2010 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

The contribution of two isozymes to the pyruvate kinase activity of Vibrio cholerae: One K+-dependent constitutively active and another K+-independent with essential allosteric activation

PubMed Central

Guerrero-Mendiola, Carlos; García-Trejo, José J.; Encalada, Rusely; Saavedra, Emma

2017-01-01

In a previous phylogenetic study of the family of pyruvate kinase EC (2.7.1.40), a cluster with Glu117 and another with Lys117 were found (numbered according to the rabbit muscle enzyme). The sequences with Glu117 have been found to be K+-dependent, whereas those with Lys117 were K+-independent. Interestingly, only γ-proteobacteria exhibit sequences in both branches of the tree. In this context, it was explored whether these phylogenetically distinct pyruvate kinases were both expressed and contribute to the pyruvate kinase activity in Vibrio cholerae. The main findings of this work showed that the isozyme with Glu117 is an active K+-dependent enzyme. At the same substrate concentration, its Vmax in the absence of fructose 1,6 bisphosphate was 80% of that with its effector. This result is in accordance with the non-essential activation described by allosteric ligands for most pyruvate kinases. In contrast, the pyruvate kinase with Lys117 was a K+-independent enzyme displaying an allosteric activation by ribose 5-phosphate. At the same substrate concentration, its activity without the effector was 0.5% of the one obtained in the presence of ribose 5-phosphate, indicating that this sugar monophosphate is a strong activator of this enzyme. This absolute allosteric dependence is a novel feature of pyruvate kinase activity. Interestingly, in the K+-independent enzyme, Mn2+ may “mimic” the allosteric effect of Rib 5-P. Despite their different allosteric behavior, both isozymes display a rapid equilibrium random order kinetic mechanism. The intracellular concentrations of fructose 1,6-bisphosphate and ribose 5-phosphate in Vibrio cholerae have been experimentally verified to be sufficient to induce maximal activation of both enzymes. In addition, Western blot analysis indicated that both enzymes were co-expressed. Therefore, it is concluded that VcIPK and VcIIPK contribute to the activity of pyruvate kinase in this γ-proteobacterium. PMID:28686591

Synergic effects of mycoplasmal lipopeptides and extracellular ATP on activation of macrophages.

PubMed

Into, Takeshi; Fujita, Mari; Okusawa, Tsugumi; Hasebe, Akira; Morita, Manabu; Shibata, Ken-Ichiro

2002-07-01

Mycoplasmal lipopeptides S-(2,3-bispalmitoyloxypropyl)-CGDPKHSPKSF and S-(2,3-bispalmitoyloxypropyl)-CGNNDESNISFKEK activated a monocytic cell line, THP-1 cells, to produce tumor necrosis factor alpha. The activity of the lipopeptides was augmented by ATP in a dose-dependent manner. In addition, the level of expression of mRNAs for tumor necrosis factor alpha and interleukin-1 beta, -6, and -8 was also upregulated by the lipopeptides and/or extracellular ATP, but that of interleukin-10 was not. The P2X purinergic receptor antagonists pyridoxal phosphate 6-azophenyl 2',4'-disulfonic acid and periodate-oxidized ATP suppressed the activity of ATP to augment the activation of THP-1 cells by the lipopeptides, suggesting that P2X receptors play important roles in the activity of ATP. The nuclear factor kappa B inhibitor dexamethasone also suppressed the activity, suggesting that the activity of ATP is dependent upon the nuclear factor kappa B. Thus, these results suggest that the interaction of extracellular ATP with the P2X receptors is attributed to the activity of ATP to augment the activation of THP-1 cells by mycoplasmal lipopeptides.

Gene cloning, recombinant expression, purification and characterization of l-methionine decarboxylase from Streptomyces sp. 590.

PubMed

Hayashi, Masaya; Okada, Akane; Yamamoto, Kumiko; Okugochi, Tomomi; Kusaka, Chika; Kudou, Daizou; Nemoto, Michiko; Inagaki, Junko; Hirose, Yuu; Okajima, Toshihide; Tamura, Takashi; Soda, Kenji; Inagaki, Kenji

2017-04-01

l-Methionine decarboxylase (MetDC) from Streptomyces sp. 590 depends on pyridoxal 5'-phosphate and catalyzes the non-oxidative decarboxylation of l-methionine to produce 3-methylthiopropylamine and carbon dioxide. MetDC gene (mdc) was determined to consist of 1,674 bp encoding 557 amino acids, and the amino acid sequence is similar to that of l-histidine decarboxylases and l-valine decarboxylases from Streptomyces sp. strains. The mdc gene was cloned and recombinant MetDC was heterologously expressed by Escherichia coli. The purification of recombinant MetDC was carried out by DEAE-Toyopearl and Ni-NTA agarose column chromatography. The recombinant enzyme was homodimeric with a molecular mass of 61,000 Da and showed optimal activity between 45 to 55 °C and at pH 6.6, and the stability below 30 °C and between pH 4.6 to 7.0. l-Methionine and l-norleucine were good substrates for MetDC. The Michaelis constants for l-methionine and l-norleucine were 30 and 73 mM, respectively. The recombinant MetDC (0.50 U/ml) severely inhibited growth of human tumour cells A431 (epidermoid ovarian carcinoma cell line) and MDA-MB-231 (breast cancer cell line), however showed relatively low cytotoxicity for human normal cell NHDF-Neo (dermal fibroblast cell line from neonatal foreskin). This study revealed the properties of the gene and the protein sequence of MetDC for the first time. © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Riboflavin Has Neuroprotective Potential: Focus on Parkinson’s Disease and Migraine

PubMed Central

Marashly, Eyad T.; Bohlega, Saeed A.

2017-01-01

With the huge negative impact of neurological disorders on patient’s life and society resources, the discovery of neuroprotective agents is critical and cost-effective. Neuroprotective agents can prevent and/or modify the course of neurological disorders. Despite being underestimated, riboflavin offers neuroprotective mechanisms. Significant pathogenesis-related mechanisms are shared by, but not restricted to, Parkinson’s disease (PD) and migraine headache. Those pathogenesis-related mechanisms can be tackled through riboflavin proposed neuroprotective mechanisms. In fact, it has been found that riboflavin ameliorates oxidative stress, mitochondrial dysfunction, neuroinflammation, and glutamate excitotoxicity; all of which take part in the pathogenesis of PD, migraine headache, and other neurological disorders. In addition, riboflavin-dependent enzymes have essential roles in pyridoxine activation, tryptophan-kynurenine pathway, and homocysteine metabolism. Indeed, pyridoxal phosphate, the active form of pyridoxine, has been found to have independent neuroprotective potential. Also, the produced kynurenines influence glutamate receptors and its consequent excitotoxicity. In addition, methylenetetrahydrofolate reductase requires riboflavin to ensure normal folate cycle influencing the methylation cycle and consequently homocysteine levels which have its own negative neurovascular consequences if accumulated. In conclusion, riboflavin is a potential neuroprotective agent affecting a wide range of neurological disorders exemplified by PD, a disorder of neurodegeneration, and migraine headache, a disorder of pain. In this article, we will emphasize the role of riboflavin in neuroprotection elaborating on its proposed neuroprotective mechanisms in opposite to the pathogenesis-related mechanisms involved in two common neurological disorders, PD and migraine headache, as well as, we encourage the clinical evaluation of riboflavin in PD and migraine headache patients in the future. PMID:28775706

Sources and Fates of Carbamyl Phosphate: A Labile Energy-Rich Molecule with Multiple Facets.

PubMed

Shi, Dashuang; Caldovic, Ljubica; Tuchman, Mendel

2018-06-12

Carbamyl phosphate (CP) is well-known as an essential intermediate of pyrimidine and arginine/urea biosynthesis. Chemically, CP can be easily synthesized from dihydrogen phosphate and cyanate. Enzymatically, CP can be synthesized using three different classes of enzymes: (1) ATP-grasp fold protein based carbamyl phosphate synthetase (CPS); (2) Amino-acid kinase fold carbamate kinase (CK)-like CPS (anabolic CK or aCK); and (3) Catabolic transcarbamylase. The first class of CPS can be further divided into three different types of CPS as CPS I, CPS II, and CPS III depending on the usage of ammonium or glutamine as its nitrogen source, and whether N -acetyl-glutamate is its essential co-factor. CP can donate its carbamyl group to the amino nitrogen of many important molecules including the most well-known ornithine and aspartate in the arginine/urea and pyrimidine biosynthetic pathways. CP can also donate its carbamyl group to the hydroxyl oxygen of a variety of molecules, particularly in many antibiotic biosynthetic pathways. Transfer of the carbamyl group to the nitrogen group is catalyzed by the anabolic transcarbamylase using a direct attack mechanism, while transfer of the carbamyl group to the oxygen group is catalyzed by a different class of enzymes, CmcH/NodU CTase, using a different mechanism involving a three-step reaction, decomposition of CP to carbamate and phosphate, transfer of the carbamyl group from carbamate to ATP to form carbamyladenylate and pyrophosphate, and transfer of the carbamyl group from carbamyladenylate to the oxygen group of the substrate. CP is also involved in transferring its phosphate group to ADP to generate ATP in the fermentation of many microorganisms. The reaction is catalyzed by carbamate kinase, which may be termed as catabolic CK (cCK) in order to distinguish it from CP generating CK. CP is a thermally labile molecule, easily decomposed into phosphate and cyanate, or phosphate and carbamate depending on the pH of the solution, or the presence of enzyme. Biological systems have developed several mechanisms including channeling between enzymes, increased affinity of CP to enzymes, and keeping CP in a specific conformation to protect CP from decomposition. CP is highly important for our health as both a lack of, or decreased, CP production and CP accumulation results in many disease conditions.

Molecular dynamics simulation unveils the conformational flexibility of the interdomain linker in the bacterial transcriptional regulator GabR from Bacillus subtilis bound to pyridoxal 5’-phosphate

PubMed Central

Narzi, Daniele; Guidoni, Leonardo

2017-01-01

GabR from Bacillus subtilis is a transcriptional regulator belonging to the MocR subfamily of the GntR regulators. The structure of the MocR regulators is characterized by the presence of two domains: i) a N-terminal domain, about 60 residue long, possessing the winged-Helix-Turn-Helix (wHTH) architecture with DNA recognition and binding capability; ii) a C-terminal domain (about 350 residue) folded as the pyridoxal 5’-phosphate (PLP) dependent aspartate aminotransferase (AAT) with dimerization and effector binding functions. The two domains are linked to each other by a peptide bridge. Although structural and functional characterization of MocRs is proceeding at a fast pace, virtually nothing is know about the molecular changes induced by the effector binding and on how these modifications influence the properties of the regulator. An extensive molecular dynamics simulation on the crystallographic structure of the homodimeric B. subtilis GabR has been undertaken with the aim to envisage the role and the importance of conformational flexibility in the action of GabR. Molecular dynamics has been calculated for the apo (without PLP) and holo (with PLP bound) forms of the GabR. A comparison between the molecular dynamics trajectories calculated for the two GabR forms suggested that one of the wHTH domain detaches from the AAT-like domain in the GabR PLP-bound form. The most evident conformational change in the holo PLP-bound form is represented by the rotation and the subsequent detachment from the subunit surface of one of the wHTH domains. The movement is mediated by a rearrangement of the linker connecting the AAT domain possibly triggered by the presence of the negative charge of the PLP cofactor. This is the second most significant conformational modification. The C-terminal section of the linker docks into the “active site” pocket and establish stabilizing contacts consisting of hydrogen-bonds, salt-bridges and hydrophobic interactions. PMID:29253008

Plasma homocysteine is associated with increased oxidative stress and antioxidant enzyme activity in welders.

PubMed

Liu, Hung-Hsin; Shih, Tung-Sheng; Huang, Hsin-Ru; Huang, Shih-Chien; Lee, Lien-Hsiung; Huang, Yi-Chia

2013-01-01

The purpose of this study was to examine the association of vitamin B6 status and plasma homocysteine with oxidative stress and antioxidant capacities in welders. Workers were divided into either the welding exposure group (n = 57) or the nonexposure controls (n = 42) based on whether they were employed as welders. There were no significant differences in vitamin B₆ status and plasma homocysteine concentration between the welding exposure group and the nonexposure controls. The welding exposure group had significantly higher levels of oxidized low-density lipoprotein cholesterol and lower erythrocyte glutathione concentration and superoxide dismutase (SOD) activities when compared to nonexposure controls. Plasma pyridoxal 5'-phosphate concentration did not correlate with oxidative stress indicators or antioxidant capacities in either group. However, plasma homocysteine significantly correlated with total antioxidant capacity (TAC) (partial r(s) = -0.34, P < 0.05) and erythrocyte SOD activities (partial r(s) = 0.29, P < 0.05) after adjusting for potential confounders in the welding exposure group. In the welding exposure group, adequate vitamin B₆ status was not associated with oxidative stress or antioxidant capacities. However, elevated plasma homocysteine seemed to be a major contributing factor to antioxidant capacities (TAC and erythrocyte SOD activities) in welders.

Structure and mechanism of a cysteine sulfinate desulfinase engineered on the aspartate aminotransferase scaffold.

PubMed

Fernandez, Francisco J; de Vries, Dominique; Peña-Soler, Esther; Coll, Miquel; Christen, Philipp; Gehring, Heinz; Vega, M Cristina

2012-02-01

The joint substitution of three active-site residues in Escherichia coli (L)-aspartate aminotransferase increases the ratio of l-cysteine sulfinate desulfinase to transaminase activity 10(5)-fold. This change in reaction specificity results from combining a tyrosine-shift double mutation (Y214Q/R280Y) with a non-conservative substitution of a substrate-binding residue (I33Q). Tyr214 hydrogen bonds with O3 of the cofactor and is close to Arg374 which binds the α-carboxylate group of the substrate; Arg280 interacts with the distal carboxylate group of the substrate; and Ile33 is part of the hydrophobic patch near the entrance to the active site, presumably participating in the domain closure essential for the transamination reaction. In the triple-mutant enzyme, k(cat)' for desulfination of l-cysteine sulfinate increased to 0.5s(-1) (from 0.05s(-1) in wild-type enzyme), whereas k(cat)' for transamination of the same substrate was reduced from 510s(-1) to 0.05s(-1). Similarly, k(cat)' for β-decarboxylation of l-aspartate increased from<0.0001s(-1) to 0.07s(-1), whereas k(cat)' for transamination was reduced from 530s(-1) to 0.13s(-1). l-Aspartate aminotransferase had thus been converted into an l-cysteine sulfinate desulfinase that catalyzes transamination and l-aspartate β-decarboxylation as side reactions. The X-ray structures of the engineered l-cysteine sulfinate desulfinase in its pyridoxal-5'-phosphate and pyridoxamine-5'-phosphate form or liganded with a covalent coenzyme-substrate adduct identified the subtle structural changes that suffice for generating desulfinase activity and concomitantly abolishing transaminase activity toward dicarboxylic amino acids. Apparently, the triple mutation impairs the domain closure thus favoring reprotonation of alternative acceptor sites in coenzyme-substrate intermediates by bulk water. Copyright © 2011 Elsevier B.V. All rights reserved.

Effects of B group vitamins on reactions of various alpha-hydroxyl-containing organic radicals.

PubMed

Lagutin, P Yu; Shadyro, O I

2005-08-15

Effects of vitamins B1, B2, B6, and pyridoxal phosphate (PPh) on final product formation in radiolysis of aqueous solutions of ethanol, ethylene glycol, alpha-methylglycoside, and maltose were studied. It has been found that vitamin B2 and PPh effectively oxidize R*CHOH species, while suppressing their recombination and fragmentation reactions, thereby increasing the yields of the respective oxidation products. Vitamins B1 and B2 are capable of reducing alcohol radicals to the respective initial molecules, decreasing the yields of the radical transformation products.

The effect of phosphomonoesterases on the oxygen isotope composition of phosphate

NASA Astrophysics Data System (ADS)

von Sperber, Christian; Kries, Hajo; Tamburini, Federica; Bernasconi, Stefano M.; Frossard, Emmanuel

2014-01-01

Plants and microorganisms under phosphorus (P) stress release extracellular phosphatases as a strategy to acquire inorganic phosphate (Pi). These enzymes catalyze the hydrolysis of phosphoesters leading to a release of Pi. During the enzymatic hydrolysis an isotopic fractionation (ε) occurs leaving an imprint on the oxygen isotope composition of the released Pi which might be used to trace phosphorus in the environment. Therefore, enzymatic assays with acid phosphatases from wheat germ and potato tuber and alkaline phosphatase from Escherichia coli were prepared in order to determine the oxygen isotope fractionation caused by these enzymes. Adenosine 5‧ monophosphate and glycerol phosphate were used as substrates. The oxygen isotope fractionation caused by acid phosphatases is 20-30‰ smaller than for alkaline phosphatases, resulting in a difference of 5-7.5‰ in δ18O of Pi depending on the enzyme. We attribute the enzyme dependence of the isotopic fractionation to distinct reaction mechanisms of the two types of phosphatases. The observed difference is large enough to distinguish between the two enzymatic processes in environmental samples. These findings show that the oxygen isotope composition of Pi can be used to trace different enzymatic processes, offering an analytical tool that might contribute to a better understanding of the P-cycle in the environment.

Identification and Characterization of Heptaprenylglyceryl Phosphate Processing Enzymes in Bacillus subtilis.

PubMed

Linde, Mona; Peterhoff, David; Sterner, Reinhard; Babinger, Patrick

2016-07-08

In Archaea, ether lipids play an essential role as the main building blocks of the cellular membrane. Recently, ether lipids have also been discovered in the domain of Bacteria, and the key enzymes that catalyze their synthesis, glycerol-1-phosphate dehydrogenase and heptaprenylglyceryl phosphate synthase, have been described. In Bacillales, heptaprenylglyceryl phosphate does not become linked to a second polyprenyl moiety like ether lipids in Archaea but is dephosphorylated and acetylated. Here, we report on the enzymes that catalyze these reactions. We enriched the phosphatase activity from a B. subtilis cell extract and suppose that dephosphorylation is catalyzed by the phosphatase PhoB or by any other phosphatase in an unspecific manner. By screening a B. subtilis knock-out library for deficiency in acetylation, the yvoF gene product was identified to be the acetyltransferase. The acetyl-CoA-dependent enzyme YvoF is a close relative of maltose O-acetyltransferase (MAT). Its catalytic properties were analyzed and compared with MAT. YvoF and MAT partially overlap in substrate and product range in vitro, but MAT is not able to complement the yvoF knock-out in vivo. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

S-adenosylmethionine decarboxylase from baker's yeast.

PubMed Central

Pösö, H; Sinervirta, R; Jänne, J

1975-01-01

1. S-Adenosyl-L-methionine decarboxylase (S-adenosyl-L-methionine carboxy-lyase, EC 4.1.1.50) was purified more than 1100-fold from extracts of Saccharomyces cerevisiae by affinity chromatography on columns of Sepharose containing covalently bound methylglyoxal bis(guanylhydrazone) (1,1'[(methylethanediylidene)dinitrilo]diguanidine) [Pegg, (1974) Biochem J. 141, 581-583]. The final preparation appeared to be homogeneous on polyacrylamide-gel electrophoresis at pH 8.4. 2. S-Adenosylmethionine decarboxylase activity was completely separated from spermidine synthase activity [5'-deoxyadenosyl-(5'),3-aminopropyl-(1),methylsulphonium-salt-putrescine 3-aminopropyltransferase, EC 2.5.1.16] during the purification procedure. 3. Adenosylmethionine decarboxylase activity from crude extracts of baker's yeast was stimulated by putrescine, 1,3-diamino-propane, cadaverine (1,5-diaminopentane) and spermidine; however, the purified enzyme, although still stimulated by the diamines, was completely insensitive to spermidine. 4. Adenosylmethionine decarboxylase has an apparent Km value of 0.09 mM for adenosylmethionine in the presence of saturating concentrations of putrescine. The omission of putrescine resulted in a five-fold increase in the apparent Km value for adenosylmethionine. 5. The apparent Ka value for putrescine, as the activator of the reaction, was 0.012 mM. 6. Methylglyoxal bis(guanylhydrazone) and S-methyladenosylhomocysteamine (decarboxylated adenosylmethionine) were powerful inhibitors of the enzyme. 7. Adenosylmethionine decarboxylase from baker's yeast was inhibited by a number of conventional carbonyl reagents, but in no case could the inhibition be reversed with exogenous pyridoxal 5'-phosphate. PMID:1108876

Characteristics of a ugp-encoded and phoB-dependent glycerophosphoryl diester phosphodiesterase which is physically dependent on the ugp transport system of Escherichia coli.

PubMed

Brzoska, P; Boos, W

1988-09-01

The ugp-encoded transport system of Escherichia coli accumulates sn-glycerol-3-phosphate with high affinity; it is binding protein mediated and part of the pho regulon. Here, we report that glycerophosphoryl diesters (deacylated phospholipids) are also high-affinity substrates for the ugp-encoded system. The diesters are not taken up in an unaltered form but are hydrolyzed during transport to sn-glycerol-3-phosphate plus the corresponding alcohols. The enzyme responsible for this reaction is not essential for the translocation of sn-glycerol-3-phosphate or for the glycerophosphoryl diesters but can only hydrolyze diesters that are in the process of being transported. Diesters in the periplasm or in the cytoplasm were not recognized, and no enzymatic activity could be detected in cellular extracts. The enzyme is encoded by the last gene in the ugp operon, termed ugpQ. The product of the ugpQ gene, expressed in minicells, has an apparent molecular weight of 17,500. We present evidence that only one major phoB-dependent promoter controls all ugp genes.

Cd²⁺-induced alteration of the global proteome of human skin fibroblast cells.

PubMed

Prins, John M; Fu, Lijuan; Guo, Lei; Wang, Yinsheng

2014-03-07

Cadmium (Cd(2+)) is a toxic heavy metal and a well-known human carcinogen. The toxic effects of Cd(2+) on biological systems are diverse and thought to be exerted through a complex array of mechanisms. Despite the large number of studies aimed to elucidate the toxic mechanisms of action of Cd(2+), few have been targeted toward investigating the ability of Cd(2+) to disrupt multiple cellular pathways simultaneously and the overall cellular responses toward Cd(2+) exposure. In this study, we employed a quantitative proteomic method, relying on stable isotope labeling by amino acids in cell culture (SILAC) and LC-MS/MS, to assess the Cd(2+)-induced simultaneous alterations of multiple cellular pathways in cultured human skin fibroblast cells. By using this approach, we were able to quantify 2931 proteins, and 400 of them displayed significantly changed expression following Cd(2+) exposure. Our results unveiled that Cd(2+) treatment led to the marked upregulation of several antioxidant enzymes (e.g., metallothionein-1G, superoxide dismutase, pyridoxal kinase, etc.), enzymes associated with glutathione biosynthesis and homeostasis (e.g., glutathione S-transferases, glutathione synthetase, glutathione peroxidase, etc.), and proteins involved in cellular energy metabolism (e.g., glycolysis, pentose phosphate pathway, and the citric acid cycle). Additionally, we found that Cd(2+) treatment resulted in the elevated expression of two isoforms of dimethylarginine dimethylaminohydrolase (DDAH I and II), enzymes known to play a key role in regulating nitric oxide biosynthesis. Consistent with these findings, we observed elevated formation of nitric oxide in human skin (GM00637) and lung (IMR-90) fibroblast cells following Cd(2+) exposure. The upregulation of DDAH I and II suggests a role of nitric oxide synthesis in Cd(2+)-induced toxicity in human cells.

Consequences of missense mutations for dimerization and turnover of alanine:glyoxylate aminotransferase: study of a spectrum of mutations.

PubMed

Coulter-Mackie, M B; Lian, Q

2006-12-01

Alanine:glyoxylate aminotransferase (AGT) is a liver peroxisomal enzyme, deficiency of which results in primary hyperoxaluria type 1 (PH1). More than 65 PH1-related mutations are now documented in the AGT gene (AGXT), of which about 50% are missense. We have generated a spectrum of 15 missense changes including the most common PH1 mutation, G170R, and expressed them on the appropriate background of the major or minor allele, in an Escherichia coli overexpression system and in a rabbit reticulocyte transcription/translation system. We have investigated their effects on enzyme activity, dimerization, aggregation, and turnover. The effect of pyridoxal phosphate (PLP) on dimerization and stability was also investigated. Although all 15 mutant AGTs were expressed as intact proteins in E. coli, only three: G41R and G41V on the major allele, and the common mutation G170R, resulted in significant amounts of enzymatic activity. Dimerization failure was a frequent observation (13/15) except for G41V and D183N. Dimerization was poor with S187F but was substantially improved with PLP. Proteasome-mediated protein degradation was observed for all the mutations except G41R on the major allele, G41V, D183N, G170R, and S218L. Increases in the stability of the mutant enzymes in the presence of PLP were small; however, G41R on the minor allele showed a direct relationship between its half life and the concentration of PLP. The minor allele AGT product and many of the mutants were subject to a limited non-proteasomal proteolytic cleavage when ATP was depleted.

S81L and G170R mutations causing Primary Hyperoxaluria type I in homozygosis and heterozygosis: an example of positive interallelic complementation

PubMed Central

Montioli, Riccardo; Roncador, Alessandro; Oppici, Elisa; Mandrile, Giorgia; Giachino, Daniela Francesca; Cellini, Barbara; Borri Voltattorni, Carla

2014-01-01

Primary Hyperoxaluria type I (PH1) is a rare disease due to the deficit of peroxisomal alanine:glyoxylate aminotransferase (AGT), a homodimeric pyridoxal-5′-phosphate (PLP) enzyme present in humans as major (Ma) and minor (Mi) allele. PH1-causing mutations are mostly missense identified in both homozygous and compound heterozygous patients. Until now, the pathogenesis of PH1 has been only studied by approaches mimicking homozygous patients, whereas the molecular aspects of the genotype-enzymatic-clinical phenotype relationship in compound heterozygous patients are completely unknown. Here, for the first time, we elucidate the enzymatic phenotype linked to the S81L mutation on AGT-Ma, relative to a PLP-binding residue, and how it changes when the most common mutation G170R on AGT-Mi, known to cause AGT mistargeting without affecting the enzyme functionality, is present in the second allele. By using a bicistronic eukaryotic expression vector, we demonstrate that (i) S81L-Ma is mainly in its apo-form and has a significant peroxisomal localization and (ii) S81L and G170R monomers interact giving rise to the G170R-Mi/S81L-Ma holo-form, which is imported into peroxisomes and exhibits an enhanced functionality with respect to the parental enzymes. These data, integrated with the biochemical features of the heterodimer and the homodimeric counterparts in their purified recombinant form, (i) highlight the molecular basis of the pathogenicity of S81L-Ma and (ii) provide evidence for a positive interallelic complementation between the S81L and G170R monomers. Our study represents a valid approach to investigate the molecular pathogenesis of PH1 in compound heterozygous patients. PMID:24990153

Cysteine S-conjugate β-lyases: Important roles in the metabolism of naturally occurring sulfur and selenium-containing compounds, xenobiotics and anticancer agents

PubMed Central

Cooper, Arthur J. L.; Krasnikov, Boris F.; Niatsetskaya, Zoya V.; Pinto, John T.; Callery, Patrick S.; Villar, Maria T.; Artigues, Antonio; Bruschi, Sam A.

2010-01-01

Summary Cysteine S-conjugate β-lyases are pyridoxal 5′-phosphate-containing enzymes that catalyze β-elimination reactions with cysteine S-conjugates that possess a good leaving group in the β-position. The end products are aminoacrylate and a sulfur-containing fragment. The aminoacrylate tautomerizes and hydrolyzes to pyruvate and ammonia. The mammalian cysteine S-conjugate β-lyases thus far identified are enzymes involved in amino acid metabolism that catalyze β-lyase reactions as non-physiological side reactions. Most are aminotransferases. In some cases the lyase is inactivated by reaction products. The cysteine S-conjugate β-lyases are of much interest to toxicologists because they play an important key role in the bioactivation (toxication) of halogenated alkenes, some of which are produced on an industrial scale and are environmental contaminants. The cysteine S-conjugate β-lyases have been reviewed in this journal previously [Cooper and Pinto, 2006]. Here we focus on more recent findings regarding: 1) the identification of enzymes associated with high-Mr cysteine S-conjugate β-lyases in the cytosolic and mitochondrial fractions of rat liver and kidney; 2) the mechanism of syncatalytic inactivation of rat liver mitochondrial aspartate aminotransferase by the nephrotoxic β-lyase substrate S-(1,1,2,2-tetrafluoroethyl)-L-cysteine (the cysteine S-conjugate of tetrafluoroethylene); 3) toxicant channeling of reactive fragments from the active site of mitochondrial aspartate aminotransferase to susceptible proteins in the mitochondria; 4) the involvement of cysteine S-conjugate β-lyases in the metabolism/bioactivation of drugs and natural products; and 5) the role of cysteine S-conjugate β-lyases in the metabolism of selenocysteine Se-conjugates. This review emphasizes the fact that the cysteine S-conjugate β-lyases are biologically more important than hitherto appreciated. PMID:20306345

NADP-dependent enzymes are involved in response to salt and hypoosmotic stress in cucumber plants.

PubMed

Hýsková, Veronika; Plisková, Veronika; Červený, Václav; Ryšlavá, Helena

2017-07-01

Salt stress is one of the most damaging plant stressors, whereas hypoosmotic stress is not considered to be a dangerous type of stress in plants and has been less extensively studied. This study was performed to compare the metabolism of cucumber plants grown in soil with plants transferred to distilled water and to a 100 mM NaCl solution. Even though hypoosmotic stress caused by distilled water did not cause such significant changes in the relative water content, Na+/K+ ratio and Rubisco content as those caused by salt stress, it was accompanied by more pronounced changes in the specific activities of NADP-dependent enzymes. After 3 days, the specific activities of NADP-isocitrate dehydrogenase, glucose-6-phosphate dehydrogenase, NADP-malic enzyme and non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase in leaves were highest under hypoosmotic stress, and lowest in plants grown in soil. In roots, salt stress caused a decrease in the specific activities of major NADP-enzymes. However, at the beginning of salt stress, NADP-galactose-1-dehydrogenase and ribose-1-dehydrogenase were involved in a plant defense response in both roots and leaves. Therefore, the enhanced demands of NADPH in stress can be replenished by a wide range of NADP-dependent enzymes.

Isolation and Compositional Analysis of a CP12-Associated Complex of Calvin Cycle Enzymes from Nicotiana tabacum

USDA-ARS?s Scientific Manuscript database

CP12 is a small intrinsically unstructured protein that forms a multiprotein complex with two Calvin Cycle enzymes, phosphoribulokinase (PRK) and NAD(P)-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The complex can be reconstituted in vitro from recombinant proteins under conditions t...

Micronutrient supplementation improves physical performance measures in Asian Indian school-age children.

PubMed

Vaz, Mario; Pauline, Maria; Unni, Uma S; Parikh, Panam; Thomas, Tinku; Bharathi, A V; Avadhany, Sandhya; Muthayya, Sumithra; Mehra, Ruchika; Kurpad, Anura V

2011-11-01

Micronutrients are important in physical work capacity and therefore performance. The impact of a multi-micronutrient-fortified nutritional beverage on physical performance measures among clinically healthy school-age children was assessed in a double-blind (for test and placebo groups), placebo-controlled, randomized trial in children aged between 7 and 10.5 y (n = 300). The participants with height- and weight-for-age Z-scores between 0 and ≥ -3 were randomized to 1 of 3 study arms: fortified choco-malt beverage powder (F), matched energy equivalent unfortified placebo (U), and untreated control (C). Participants in the F and C groups were given 40 g fortified (19 key vitamins and minerals) and unfortified choco-malt beverage, respectively, daily for 120 d. Primary efficacy outcomes included endurance and aerobic capacity using a 20-m shuttle test and step test. Other physical performance measures included speed (40-m sprint), visual reaction time, maximal hand grip, and forearm static endurance. Micronutrient status included thiamin, riboflavin, folate, niacin, iron, pyridoxal phosphate, and vitamins B-12 and C. All measurements were made at baseline and the end of the intervention. There was a within-subject increase in aerobic capacity and whole body endurance (P < 0.05) accompanied by a significant improvement in the status of iron thiamin, riboflavin, pyridoxal phosphate, folate, and vitamins C and B-12 in the F group compared to the within-subject changes in the other 2 groups (P < 0.05). The study suggests that multiple micronutrient supplementation in similar populations may be beneficial in improving micronutrient status and enhancing aerobic capacity and endurance in children.

An adaptation to life in acid through a novel mevalonate pathway

DOE PAGES

Vinokur, Jeffrey M.; Cummins, Matthew C.; Korman, Tyler P.; ...

2016-12-22

Here, extreme acidophiles are capable of growth at pH values near zero. Sustaining life in acidic environments requires extensive adaptations of membranes, proton pumps, and DNA repair mechanisms. Here we describe an adaptation of a core biochemical pathway, the mevalonate pathway, in extreme acidophiles. Two previously known mevalonate pathways involve ATP dependent decarboxylation of either mevalonate 5-phosphate or mevalonate 5-pyrophosphate, in which a single enzyme carries out two essential steps: (1) phosphorylation of the mevalonate moiety at the 3-OH position and (2) subsequent decarboxylation. We now demonstrate that in extreme acidophiles, decarboxylation is carried out by two separate steps: previouslymore » identified enzymes generate mevalonate 3,5-bisphosphate and a new decarboxylase we describe here, mevalonate 3,5-bisphosphate decarboxylase, produces isopentenyl phosphate. Why use two enzymes in acidophiles when one enzyme provides both functionalities in all other organisms examined to date? We find that at low pH, the dual function enzyme, mevalonate 5-phosphate decarboxylase is unable to carry out the first phosphorylation step, yet retains its ability to perform decarboxylation. We therefore propose that extreme acidophiles had to replace the dual-purpose enzyme with two specialized enzymes to efficiently produce isoprenoids in extremely acidic environments.« less

An adaptation to life in acid through a novel mevalonate pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vinokur, Jeffrey M.; Cummins, Matthew C.; Korman, Tyler P.

Here, extreme acidophiles are capable of growth at pH values near zero. Sustaining life in acidic environments requires extensive adaptations of membranes, proton pumps, and DNA repair mechanisms. Here we describe an adaptation of a core biochemical pathway, the mevalonate pathway, in extreme acidophiles. Two previously known mevalonate pathways involve ATP dependent decarboxylation of either mevalonate 5-phosphate or mevalonate 5-pyrophosphate, in which a single enzyme carries out two essential steps: (1) phosphorylation of the mevalonate moiety at the 3-OH position and (2) subsequent decarboxylation. We now demonstrate that in extreme acidophiles, decarboxylation is carried out by two separate steps: previouslymore » identified enzymes generate mevalonate 3,5-bisphosphate and a new decarboxylase we describe here, mevalonate 3,5-bisphosphate decarboxylase, produces isopentenyl phosphate. Why use two enzymes in acidophiles when one enzyme provides both functionalities in all other organisms examined to date? We find that at low pH, the dual function enzyme, mevalonate 5-phosphate decarboxylase is unable to carry out the first phosphorylation step, yet retains its ability to perform decarboxylation. We therefore propose that extreme acidophiles had to replace the dual-purpose enzyme with two specialized enzymes to efficiently produce isoprenoids in extremely acidic environments.« less

Enzyme characteristics of aminotransferase FumI of Sphingopyxis sp. MTA144 for deamination of hydrolyzed fumonisin B₁.

PubMed

Hartinger, Doris; Schwartz, Heidi; Hametner, Christian; Schatzmayr, Gerd; Haltrich, Dietmar; Moll, Wulf-Dieter

2011-08-01

Fumonisins are carcinogenic mycotoxins that are frequently found as natural contaminants in maize from warm climate regions around the world. The aminotransferase FumI is encoded as part of a gene cluster of Sphingopyxis sp. MTA144, which enables this bacterial strain to degrade fumonisin B(1) and related fumonisins. FumI catalyzes the deamination of the first intermediate of the catabolic pathway, hydrolyzed fumonisin B(1). We used a preparation of purified, His-tagged FumI, produced recombinantly in Escherichia coli in soluble form, for enzyme characterization. The structure of the reaction product was studied by NMR and identified as 2-keto hydrolyzed fumonisin B(1). Pyruvate was found to be the preferred co-substrate and amino group receptor (K (M) = 490 μM at 10 μM hydrolyzed fumonisin B(1)) of FumI, but other α-keto acids were also accepted as co-substrates. Addition of the co-enzyme pyridoxal phosphate to the enzyme preparation enhanced activity, and saturation was already reached at the lowest tested concentration of 10 μM. The enzyme showed activity in the range of pH 6 to 10 with an optimum at pH 8.5, and in the range of 6°C to 50°C with an optimum at 35°C. The aminotransferase worked best at low salt concentration. FumI activity could be recovered after preincubation at pH 4.0 or higher, but not lower. The aminotransferase was denatured after preincubation at 60°C for 1 h, and the residual activity was also reduced after preincubation at lower temperatures. At optimum conditions, the kinetic parameters K (M) = 1.1 μM and k (cat) = 104/min were determined with 5 mM pyruvate as co-substrate. Based on the enzyme characteristics, a technological application of FumI, in combination with the fumonisin carboxylesterase FumD for hydrolysis of fumonisins, for deamination and detoxification of hydrolyzed fumonisins seems possible, if the enzyme properties are considered.

Biochemical analyses are instrumental in identifying the impact of mutations on holo and/or apo-forms and on the region(s) of alanine:glyoxylate aminotransferase variants associated with Primary Hyperoxaluria Type I☆

PubMed Central

Oppici, Elisa; Montioli, Riccardo; Lorenzetto, Antonio; Bianconi, Silvia; Borri Voltattorni, Carla; Cellini, Barbara

2012-01-01

Primary Hyperoxaluria Type I (PH1) is a disorder of glyoxylate metabolism caused by mutations in the human AGXT gene encoding liver peroxisomal alanine:glyoxylate aminotransferase (AGT), a pyridoxal 5′-phosphate (PLP) dependent enzyme. Previous investigations highlighted that, although PH1 is characterized by a significant variability in terms of enzymatic phenotype, the majority of the pathogenic variants are believed to share both structural and functional defects, as mainly revealed by data on AGT activity and expression level in crude cellular extracts. However, the knowledge of the defects of the AGT variants at a protein level is still poor. We therefore performed a side-by-side comparison between normal AGT and nine purified recombinant pathogenic variants in terms of catalytic activity, coenzyme binding mode and affinity, spectroscopic features, oligomerization, and thermal stability of both the holo- and apo-forms. Notably, we chose four variants in which the mutated residues are located in the large domain of AGT either within the active site and interacting with the coenzyme or in its proximity, and five variants in which the mutated residues are distant from the active site either in the large or in the small domain. Overall, this integrated analysis of enzymatic activity, spectroscopic and stability information is used to (i) reassess previous data obtained with crude cellular extracts, (ii) establish which form(s) (i.e. holoenzyme and/or apoenzyme) and region(s) (i.e. active site microenvironment, large and/or small domain) of the protein are affected by each mutation, and (iii) suggest the possible therapeutic approach for patients bearing the examined mutations. PMID:22018727

Biochemical analyses are instrumental in identifying the impact of mutations on holo and/or apo-forms and on the region(s) of alanine:glyoxylate aminotransferase variants associated with primary hyperoxaluria type I.

PubMed

Oppici, Elisa; Montioli, Riccardo; Lorenzetto, Antonio; Bianconi, Silvia; Borri Voltattorni, Carla; Cellini, Barbara

2012-01-01

Primary Hyperoxaluria Type I (PH1) is a disorder of glyoxylate metabolism caused by mutations in the human AGXT gene encoding liver peroxisomal alanine:glyoxylate aminotransferase (AGT), a pyridoxal 5'-phosphate (PLP) dependent enzyme. Previous investigations highlighted that, although PH1 is characterized by a significant variability in terms of enzymatic phenotype, the majority of the pathogenic variants are believed to share both structural and functional defects, as mainly revealed by data on AGT activity and expression level in crude cellular extracts. However, the knowledge of the defects of the AGT variants at a protein level is still poor. We therefore performed a side-by-side comparison between normal AGT and nine purified recombinant pathogenic variants in terms of catalytic activity, coenzyme binding mode and affinity, spectroscopic features, oligomerization, and thermal stability of both the holo- and apo-forms. Notably, we chose four variants in which the mutated residues are located in the large domain of AGT either within the active site and interacting with the coenzyme or in its proximity, and five variants in which the mutated residues are distant from the active site either in the large or in the small domain. Overall, this integrated analysis of enzymatic activity, spectroscopic and stability information is used to (i) reassess previous data obtained with crude cellular extracts, (ii) establish which form(s) (i.e. holoenzyme and/or apoenzyme) and region(s) (i.e. active site microenvironment, large and/or small domain) of the protein are affected by each mutation, and (iii) suggest the possible therapeutic approach for patients bearing the examined mutations. Copyright © 2011 Elsevier Inc. All rights reserved.

Crystal Structures of Aedes Aegypt Alanine Glyoxylate Aminotransferase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Han,Q.; Robinson, H.; Gao, Y.

Mosquitoes are unique in having evolved two alanine glyoxylate aminotransferases (AGTs). One is 3-hydroxykynurenine transaminase (HKT), which is primarily responsible for catalyzing the transamination of 3-hydroxykynurenine (3-HK) to xanthurenic acid (XA). Interestingly, XA is used by malaria parasites as a chemical trigger for their development within the mosquito. This 3-HK to XA conversion is considered the major mechanism mosquitoes use to detoxify the chemically reactive and potentially toxic 3-HK. The other AGT is a typical dipteran insect AGT and is specific for converting glyoxylic acid to glycine. Here we report the 1.75{angstrom} high-resolution three-dimensional crystal structure of AGT from themore » mosquito Aedes aegypti (AeAGT) and structures of its complexes with reactants glyoxylic acid and alanine at 1.75 and 2.1{angstrom} resolution, respectively. This is the first time that the three-dimensional crystal structures of an AGT with its amino acceptor, glyoxylic acid, and amino donor, alanine, have been determined. The protein is dimeric and adopts the type I-fold of pyridoxal 5-phosphate (PLP)-dependent aminotransferases. The PLP co-factor is covalently bound to the active site in the crystal structure, and its binding site is similar to those of other AGTs. The comparison of the AeAGT-glyoxylic acid structure with other AGT structures revealed that these glyoxylic acid binding residues are conserved in most AGTs. Comparison of the AeAGT-alanine structure with that of the Anopheles HKT-inhibitor complex suggests that a Ser-Asn-Phe motif in the latter may be responsible for the substrate specificity of HKT enzymes for 3-HK.« less

The Human SepSecS-tRNASec Complex Reveals the Mechanism of Selenocysteine Formation

PubMed Central

Palioura, Sotiria; Sherrer, R. Lynn; Steitz, Thomas A.; Söll, Dieter; Simonović, Miljan

2010-01-01

Selenocysteine is the only genetically encoded amino acid in humans whose biosynthesis occurs on its cognate transfer RNA (tRNA). O-Phosphoseryl-tRNA:selenocysteinyl-tRNA synthase (SepSecS) catalyzes the final step of selenocysteine formation by a poorly understood tRNA-dependent mechanism. The crystal structure of human tRNASec in complex with SepSecS, phosphoserine, and thiophosphate, together with in vivo and in vitro enzyme assays, supports a pyridoxal phosphate–dependent mechanism of Sec-tRNASec formation. Two tRNASec molecules, with a fold distinct from other canonical tRNAs, bind to each SepSecS tetramer through their 13–base pair acceptor-TΨC arm (where Ψ indicates pseudouridine). The tRNA binding is likely to induce a conformational change in the enzyme’s active site that allows a phosphoserine covalently attached to tRNASec, but not free phosphoserine, to be oriented properly for the reaction to occur. PMID:19608919

A highly specific l-galactose-1-phosphate phosphatase on the path to ascorbate biosynthesis

PubMed Central

Laing, William A.; Bulley, Sean; Wright, Michele; Cooney, Janine; Jensen, Dwayne; Barraclough, Di; MacRae, Elspeth

2004-01-01

Ascorbate is a critical compound in plants and animals. Humans are unable to synthesize ascorbate, and their main source of this essential vitamin are plants. However, the pathway of synthesis in plants is yet to be established, and several unknown enzymes are only postulated to exist. We describe a specific l-galactose-1-phosphate (l-gal-1-P) phosphatase that we partially purified from young kiwifruit (Actinidia deliciosa) berries. The enzyme had a native molecular mass of ≈65 kDa, was completely dependent on Mg2+ for activity and was very specific in its ability to hydrolyze l-gal-1-P. The activity had a pH optimum of 7.0, a KM(l-gal-1-P) of 20–40 μM and a Ka(Mg2+) of 0.2 mM. The activity was inhibited by Mg2+ at concentrations >2 mM. The enzyme from Arabidopsis thaliana shoots showed similar properties to the kiwifruit enzyme. The Arabidopsis thaliana enzyme preparation was digested with trypsin, and proteins present were identified by using liquid chromatography–MS. One of 24 proteins present in our preparation was an Arabidopsis thaliana protein, At3g02870, annotated myo-inositol-1-phosphate phosphatase in GenBank, that matched the characteristics of the purified l-gal-1-phosphate phosphatase. We then expressed a kiwifruit homologue of this gene in Escherichia coli and found that it showed 14-fold higher maximum velocity for l-gal-1-P than myo-inositol-1-P. The expressed enzyme showed very similar properties to the enzyme purified from kiwifruit and Arabidopsis, except that its KM(l-gal-1-P) and Ka(Mg2+) were higher in the expressed enzyme. The data are discussed in terms of the pathway to ascorbate biosynthesis in plants PMID:15550539

An aqueous friendly chemosensor derived from vitamin B6 cofactor for colorimetric sensing of Cu2 + and fluorescent turn-off sensing of Fe3 +

NASA Astrophysics Data System (ADS)

Sharma, Darshna; Kuba, Aman; Thomas, Rini; Kumar, Rajender; Choi, Heung-Jin; Sahoo, Suban K.

2016-01-01

Chemosensor L derived from vitamin B6 cofactor pyridoxal-5-phosphate was investigated for the selective detection of Cu2 + and Fe3 + in aqueous medium. Sensor L formed a 1:1 complex with Cu2 + and displays a perceptible color change from colorless to yellow brown with the appearance of a new charge transfer band at 450 nm. In contrast, the fluorescence of L was quenched selectively in the presence of Fe3 + without any interference from other metal ions including Cu2 +.

Synergic Effects of Mycoplasmal Lipopeptides and Extracellular ATP on Activation of Macrophages

PubMed Central

Into, Takeshi; Fujita, Mari; Okusawa, Tsugumi; Hasebe, Akira; Morita, Manabu; Shibata, Ken-Ichiro

2002-01-01

Mycoplasmal lipopeptides S-(2,3-bispalmitoyloxypropyl)-CGDPKHSPKSF and S-(2,3-bispalmitoyloxypropyl)-CGNNDESNISFKEK activated a monocytic cell line, THP-1 cells, to produce tumor necrosis factor alpha. The activity of the lipopeptides was augmented by ATP in a dose-dependent manner. In addition, the level of expression of mRNAs for tumor necrosis factor alpha and interleukin-1β, -6, and -8 was also upregulated by the lipopeptides and/or extracellular ATP, but that of interleukin-10 was not. The P2X purinergic receptor antagonists pyridoxal phosphate 6-azophenyl 2′,4′-disulfonic acid and periodate-oxidized ATP suppressed the activity of ATP to augment the activation of THP-1 cells by the lipopeptides, suggesting that P2X receptors play important roles in the activity of ATP. The nuclear factor κB inhibitor dexamethasone also suppressed the activity, suggesting that the activity of ATP is dependent upon the nuclear factor κB. Thus, these results suggest that the interaction of extracellular ATP with the P2X receptors is attributed to the activity of ATP to augment the activation of THP-1 cells by mycoplasmal lipopeptides. PMID:12065499

Reductive trapping of substrate to bovine plasma amine oxidase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hartmann, C.; Klinman, J.P.

1987-01-25

Plasma amine oxidases catalyze the oxidative deamination of amines to aldehydes, followed by a 2e- reduction of O/sub 2/ to H/sub 2/O/sub 2/. Pyrroloquinoline quinone (PQQ), previously believed to be restricted to prokaryotes, has recently been proposed to be the cofactor undergoing reduction in the first half-reaction of bovine plasma amine oxidase (Ameyama, M., Hayashi, U., Matsushita, K., Shinagawa, E., and Adachi, O. (1984) Agric. Biol. Chem. 48, 561-565; Lobenstein-Verbeek, C. L., Jongejan, J. A., Frank, J., and Duine, J. A. (1984) FEBS Lett. 170, 305-309). This result is unexpected, since model studies with PQQ implicate Schiff's base formation betweenmore » a reactive carbonyl and substrates, whereas experiments with bovine plasma amine oxidase have failed to provide evidence for a carbonyl cofactor. We have, therefore, re-examined putative adducts between substrate and enzyme-bound cofactor, employing a combination of (/sup 14/C)benzylamine and (/sup 3/H)NaCNBH/sub 3/. The use of the relatively weak reductant, NaCNBH/sub 3/, affords Schiff's base specificity and permits the study of enzyme below pH 7.0. As we show, enzyme can only be inactivated by NaCNBH/sub 3/ in the presence of substrate, leading to the incorporation of 1 mol of (/sup 14/C)benzylamine/mol of enzyme subunit at complete inactivation. By contrast, we are unable to detect any labeling with (/sup 3/H)NaCNBH/sub 3/, analogous to an earlier study with (/sup 3/H)NaCNBH/sub 4/ (Suva, R. H., and Abeles, R. H. (1978) Biochemistry 17, 3538-3545). We conclude, first, that our inability to obtain adducts containing both carbon 14 and tritium rules out the reductive trapping either of amine substrate with pyridoxal phosphate or of aldehyde product with a lysyl side chain and, second, that the observed pattern of labeling is fully consistent with the presence of PQQ at the active site of bovine plasma amine oxidase.« less

Lysosomal enzymes and their receptors in invertebrates: an evolutionary perspective.

PubMed

Kumar, Nadimpalli Siva; Bhamidimarri, Poorna M

2015-01-01

Lysosomal biogenesis is an important process in eukaryotic cells to maintain cellular homeostasis. The key components that are involved in the biogenesis such as the lysosomal enzymes, their modifications and the mannose 6-phosphate receptors have been well studied and their evolutionary conservation across mammalian and non-mammalian vertebrates is clearly established. Invertebrate lysosomal biogenesis pathway on the other hand is not well studied. Although, details on mannose 6-phosphate receptors and enzymes involved in lysosomal enzyme modifications were reported earlier, a clear cut pathway has not been established. Recent research on the invertebrate species involving biogenesis of lysosomal enzymes suggests a possible conserved pathway in invertebrates. This review presents certain observations based on these processes that include biochemical, immunological and functional studies. Major conclusions include conservation of MPR-dependent pathway in higher invertebrates and recent evidence suggests that MPR-independent pathway might have been more prominent among lower invertebrates. The possible components of MPR-independent pathway that may play a role in lysosomal enzyme targeting are also discussed here.

Interactions between serine acetyltransferase and O-acetylserine (thiol) lyase in higher plants--structural and kinetic properties of the free and bound enzymes.

PubMed

Droux, M; Ruffet, M L; Douce, R; Job, D

1998-07-01

The last steps of cysteine synthesis in plants involve two consecutive enzymes. The first enzyme, serine acetyltransferase, catalyses the acetylation of L-serine in the presence of acetyl-CoA to form O-acetylserine. The second enzyme, O-acetylserine (thiol) lyase, converts O-acetylserine to L-cysteine in the presence of sulfide. We have, in the present work, over-produced in Escherichia coli harboring various type of plasmids, either a plant serine acetyltransferase or this enzyme with a plant O-acetylserine (thiol) lyase. The free recombinant serine acetyltransferase (subunit mass of 34 kDa) exhibited a high propensity to form high-molecular-mass aggregates and was found to be highly unstable in solution. However, these aggregates were prevented in the presence of O-acetylserine (thiol) lyase (subunit mass of 36 kDa). Under these conditions homotetrameric serine acetyltransferase associated with two molecules of homodimeric O-acetylserine (thiol) lyase to form a bienzyme complex (molecular mass approximately 300 kDa) called cysteine synthase containing 4 mol pyridoxal 5'-phosphate/mol complex. O-Acetylserine triggered the dissociation of the bienzyme complex, whereas sulfide counteracted the action of O-acetylserine. Protein-protein interactions within the bienzyme complex strongly modified the kinetic properties of plant serine acetyltransferase: there was a transition from a typical Michaelis-Menten model to a model displaying positive kinetic co-operativity with respect to serine and acetyl-CoA. On the other hand, the formation of the bienzyme complex resulted in a very dramatic decrease in the catalytic efficiency of bound O-acetylserine (thiol) lyase. The latter enzyme behaved as if it were a structural and/or regulatory subunit of serine acetyltransferase. Our results also indicated that bound serine acetyltransferase produces a build-up of O-acetylserine along the reaction path and that the full capacity for cysteine synthesis can only be achieved in the presence of a large excess of free O-acetylserine (thiol) lyase. These findings contradict the widely held belief that such a bienzyme complex is required to channel the metabolite intermediate O-acetylserine.

Vitamin B-6 and colorectal cancer risk: a prospective population-based study using 3 distinct plasma markers of vitamin B-6 status.

PubMed

Gylling, Björn; Myte, Robin; Schneede, Jörn; Hallmans, Göran; Häggström, Jenny; Johansson, Ingegerd; Ulvik, Arve; Ueland, Per M; Van Guelpen, Bethany; Palmqvist, Richard

2017-04-01

Background: Higher plasma concentrations of the vitamin B-6 marker pyridoxal 5'-phosphate (PLP) have been associated with reduced colorectal cancer (CRC) risk. Inflammatory processes, including vitamin B-6 catabolism, could explain such findings. Objective: We investigated 3 biomarkers of vitamin B-6 status in relation to CRC risk. Design: This was a prospective case-control study of 613 CRC cases and 1190 matched controls nested within the Northern Sweden Health and Disease Study ( n = 114,679). Participants were followed from 1985 to 2009, and the median follow-up from baseline to CRC diagnosis was 8.2 y. PLP, pyridoxal, pyridoxic acid (PA), 3-hydroxykynurenine, and xanthurenic acids (XAs) were measured in plasma with the use of liquid chromatography-tandem mass spectrometry. We calculated relative and absolute risks of CRC for PLP and the ratios 3-hydroxykynurenine:XA (HK:XA), an inverse marker of functional vitamin B-6 status, and PA:(PLP + pyridoxal) (PAr), a marker of inflammation and oxidative stress and an inverse marker of vitamin B-6 status. Results: Plasma PLP concentrations were associated with a reduced CRC risk for the third compared with the first quartile and for PLP sufficiency compared with deficiency [OR: 0.60 (95% CI: 0.44, 0.81) and OR: 0.55 (95% CI: 0.37, 0.81), respectively]. HK:XA and PAr were both associated with increased CRC risk [OR: 1.48 (95% CI: 1.08, 2.02) and OR: 1.50 (95% CI: 1.10, 2.04), respectively] for the fourth compared with the first quartile. For HK:XA and PAr, the findings were mainly observed in study participants with <10.5 y of follow-up between sampling and diagnosis. Conclusions: Vitamin B-6 deficiency as measured by plasma PLP is associated with a clear increase in CRC risk. Furthermore, our analyses of novel markers of functional vitamin B-6 status and vitamin B-6-associated oxidative stress and inflammation suggest a role in tumor progression rather than initiation. © 2017 American Society for Nutrition.

The Production and Utilization of GDP-glucose in the Biosynthesis of Trehalose 6-Phosphate by Streptomyces venezuelae*

PubMed Central

Asención Diez, Matías D.; Miah, Farzana; Stevenson, Clare E. M.; Lawson, David M.; Iglesias, Alberto A.; Bornemann, Stephen

2017-01-01

Trehalose-6-phosphate synthase OtsA from streptomycetes is unusual in that it uses GDP-glucose as the donor substrate rather than the more commonly used UDP-glucose. We now confirm that OtsA from Streptomyces venezuelae has such a preference for GDP-glucose and can utilize ADP-glucose to some extent too. A crystal structure of the enzyme shows that it shares twin Rossmann-like domains with the UDP-glucose-specific OtsA from Escherichia coli. However, it is structurally more similar to Streptomyces hygroscopicus VldE, a GDP-valienol-dependent pseudoglycosyltransferase enzyme. Comparison of the donor binding sites reveals that the amino acids associated with the binding of diphosphoribose are almost all identical in these three enzymes. By contrast, the amino acids associated with binding guanine in VldE (Asn, Thr, and Val) are similar in S. venezuelae OtsA (Asp, Ser, and Phe, respectively) but not conserved in E. coli OtsA (His, Leu, and Asp, respectively), providing a rationale for the purine base specificity of S. venezuelae OtsA. To establish which donor is used in vivo, we generated an otsA null mutant in S. venezuelae. The mutant had a cell density-dependent growth phenotype and accumulated galactose 1-phosphate, glucose 1-phosphate, and GDP-glucose when grown on galactose. To determine how the GDP-glucose is generated, we characterized three candidate GDP-glucose pyrophosphorylases. SVEN_3027 is a UDP-glucose pyrophosphorylase, SVEN_3972 is an unusual ITP-mannose pyrophosphorylase, and SVEN_2781 is a pyrophosphorylase that is capable of generating GDP-glucose as well as GDP-mannose. We have therefore established how S. venezuelae can make and utilize GDP-glucose in the biosynthesis of trehalose 6-phosphate. PMID:27903647

Markers of vitamin B6 status and metabolism as predictors of incident cancer: the Hordaland Health Study.

PubMed

Zuo, Hui; Ueland, Per M; Eussen, Simone J P M; Tell, Grethe S; Vollset, Stein E; Nygård, Ottar; Midttun, Øivind; Meyer, Klaus; Ulvik, Arve

2015-06-15

Dietary intake and/or circulating concentrations of vitamin B6 have been associated with risk of cancer, but results are inconsistent and mechanisms uncertain. Pyridoxal 5'-phosphate (PLP) is the most commonly used marker of B6 status. We recently proposed the ratio 3-hydroxykynurenine/xanthurenic acid (HK/XA) as an indicator of functional vitamin B6 status, and the 4-pyridoxic acid (PA) /(pyridoxal (PL) +PLP) ratio (PAr) as a marker of vitamin B6 catabolism during inflammation. We compared plasma PLP, HK/XA and PAr as predictors of cancer incidence in a prospective community-based cohort in Norway. This study included 6,539 adults without known cancer at baseline (1998-99) from the Hordaland Health Study (HUSK). HR and 95% CI were calculated for the risk of overall and site-specific cancers using multivariate Cox proportional hazards regression with adjustment for potential confounders. After a median follow-up time of 11.9 years, 963 cancer cases (501 men and 462 women) were identified. Multivariate-adjusted Cox-regression showed no significant relation of plasma PLP or HK/XA with risk of incident cancer. In contrast, PAr was significantly associated with risk of cancer with HR (95% CI) = 1.31 (1.12-1.52) per two standard deviation (SD) increment (p < 0.01). Further analysis showed that PAr was a particular strong predictor of lung cancer with HR (95% CI) = 2.46 (1.49-4.05) per two SD increment (p < 0.01). The present results indicate that associations of vitamin B6 with cancer may be related to increased catabolism of vitamin B6, in particular for lung cancer where inflammation may be largely involved in carcinogenesis. © 2014 The Authors. Published by Wiley Periodicals, Inc. on behalf of UICC.

Plasma vitamins B2, B6, B12, and related genetic variants as predictors of colorectal cancer risk

PubMed Central

Eussen, Simone JPM; Vollset, Stein Emil; Hustad, Steinar; Midttun, Øivind; Meyer, Klaus; Fredriksen, Åse; Ueland, Per Magne; Jenab, Mazda; Slimani, Nadia; Boffetta, Paolo; Overvad, Kim; Thorlacius-Ussing, Ole; Tjønneland, Anne; Olsen, Anja; Clavel-Chapelon, Françoise; Boutron-Ruault, Marie-Christine; Morois, Sophie; Weikert, Cornelia; Pischon, Tobias; Linseisen, Jakob; Kaaks, Rudolf; Trichopoulou, Antonia; Zilis, Demosthenes; Katsoulis, Michael; Palli, Domenico; Pala, Valeria; Vineis, Paolo; Tumino, Rosario; Panico, Salvatore; Peeters, Petra HM; Bueno-de-Mesquita, H Bas; van Duijnhoven, Fränzel JB; Skeie, Guri; Muñoz, Xavier; Martínez, Carmen; Dorronsoro, Miren; Ardanaz, Eva; Navarro, Carmen; Rodríguez, Laudina; Van Guelpen, Bethany; Palmqvist, Richard; Manjer, Jonas; Ericson, Ulrika; Bingham, Sheila; Khaw, Kay-Tee; Norat, Teresa; Riboli, Elio

2010-01-01

Background B-vitamins are essential for one-carbon metabolism and have been linked to colorectal cancer (CRC). Although associations with folate have frequently been studied, studies on other plasma vitamins B2, B6, and B12 and CRC are scarce or inconclusive. Methods Nested case-control study within the European Prospective Investigation into Cancer and Nutrition, including 1365 incident CRC cases and 2319 controls matched for study center, age, and sex. We measured the sum of B2 species riboflavin and flavin mononucleotide, and the sum of B6 species pyridoxal 5′-phosphate, pyridoxal, and 4-pyridoxic acid as indicators for vitamin B2 and B6 status, as well as vitamin B12 in plasma samples collected at baseline. In addition, we determined eight polymorphisms related to one-carbon metabolism. Relative risks (RRs) for CRC were estimated using conditional logistic regression, adjusted for smoking, education, physical activity, BMI, alcohol consumption, and intakes of fiber, red- and processed meat. Results RRs comparing highest to lowest quintile (95% confidence interval, Ptrend) were: 0.71 (0.56–0.91, 0.02) for vitamin B2, 0.68 (0.53–0.87, <0.001) for vitamin B6, and 1.02 (0.80–1.29, 0.19) for vitamin B12. The associations for vitamin B6 were stronger in males who consumed ≥ 30g alcohol/day. The polymorphisms were not associated with CRC. Conclusions Higher plasma concentrations of vitamins B2 and B6 are associated with a lower CRC risk. Impact This European population-based study is the first to indicate that vitamin B2 is inversely associated with CRC, and is in agreement to previously suggested inverse associations of vitamin B6 with CRC. PMID:20813848

Surface expression of ω-transaminase in Escherichia coli.

PubMed

Gustavsson, Martin; Muraleedharan, Madhu Nair; Larsson, Gen

2014-04-01

Chiral amines are important for the chemical and pharmaceutical industries, and there is rapidly growing interest to use transaminases for their synthesis. Since the cost of the enzyme is an important factor for process economy, the use of whole-cell biocatalysts is attractive, since expensive purification and immobilization steps can be avoided. Display of the protein on the cell surface provides a possible way to reduce the mass transfer limitations of such biocatalysts. However, transaminases need to dimerize in order to become active, and furthermore, they require the cofactor pyridoxal phosphate; consequently, successful transaminase surface expression has not been reported thus far. In this work, we produced an Arthrobacter citreus ω-transaminase in Escherichia coli using a surface display vector based on the autotransporter adhesin involved in diffuse adherence (AIDA-I), which has previously been used for display of dimeric proteins. The correct localization of the transaminase in the E. coli outer membrane and its orientation toward the cell exterior were verified. Furthermore, transaminase activity was detected exclusively in the outer membrane protein fraction, showing that successful dimerization had occurred. The transaminase was found to be present in both full-length and proteolytically degraded forms. The removal of this proteolysis is considered to be the main obstacle to achieving sufficient whole-cell transaminase activity.

Surface Expression of ω-Transaminase in Escherichia coli

PubMed Central

Gustavsson, Martin; Muraleedharan, Madhu Nair

2014-01-01

Chiral amines are important for the chemical and pharmaceutical industries, and there is rapidly growing interest to use transaminases for their synthesis. Since the cost of the enzyme is an important factor for process economy, the use of whole-cell biocatalysts is attractive, since expensive purification and immobilization steps can be avoided. Display of the protein on the cell surface provides a possible way to reduce the mass transfer limitations of such biocatalysts. However, transaminases need to dimerize in order to become active, and furthermore, they require the cofactor pyridoxal phosphate; consequently, successful transaminase surface expression has not been reported thus far. In this work, we produced an Arthrobacter citreus ω-transaminase in Escherichia coli using a surface display vector based on the autotransporter adhesin involved in diffuse adherence (AIDA-I), which has previously been used for display of dimeric proteins. The correct localization of the transaminase in the E. coli outer membrane and its orientation toward the cell exterior were verified. Furthermore, transaminase activity was detected exclusively in the outer membrane protein fraction, showing that successful dimerization had occurred. The transaminase was found to be present in both full-length and proteolytically degraded forms. The removal of this proteolysis is considered to be the main obstacle to achieving sufficient whole-cell transaminase activity. PMID:24487538

Plasma Homocysteine Is Associated with Increased Oxidative Stress and Antioxidant Enzyme Activity in Welders

PubMed Central

Liu, Hung-Hsin; Shih, Tung-Sheng; Huang, Hsin-Ru; Huang, Shih-Chien; Lee, Lien-Hsiung; Huang, Yi-Chia

2013-01-01

The purpose of this study was to examine the association of vitamin B6 status and plasma homocysteine with oxidative stress and antioxidant capacities in welders. Workers were divided into either the welding exposure group (n = 57) or the nonexposure controls (n = 42) based on whether they were employed as welders. There were no significant differences in vitamin B6 status and plasma homocysteine concentration between the welding exposure group and the nonexposure controls. The welding exposure group had significantly higher levels of oxidized low-density lipoprotein cholesterol and lower erythrocyte glutathione concentration and superoxide dismutase (SOD) activities when compared to nonexposure controls. Plasma pyridoxal 5′-phosphate concentration did not correlate with oxidative stress indicators or antioxidant capacities in either group. However, plasma homocysteine significantly correlated with total antioxidant capacity (TAC) (partial r s = −0.34, P < 0.05) and erythrocyte SOD activities (partial r s = 0.29, P < 0.05) after adjusting for potential confounders in the welding exposure group. In the welding exposure group, adequate vitamin B6 status was not associated with oxidative stress or antioxidant capacities. However, elevated plasma homocysteine seemed to be a major contributing factor to antioxidant capacities (TAC and erythrocyte SOD activities) in welders. PMID:24106453

Cloning and sequence analysis of the meso-diaminopimelate decarboxylase gene from Bacillus methanolicus MGA3 and comparison to other decarboxylase genes.

PubMed Central

Mills, D A; Flickinger, M C

1993-01-01

The lysA gene of Bacillus methanolicus MGA3 was cloned by complementation of an auxotrophic Escherichia coli lysA22 mutant with a genomic library of B. methanolicus MGA3 chromosomal DNA. Subcloning localized the B. methanolicus MGA3 lysA gene into a 2.3-kb SmaI-SstI fragment. Sequence analysis of the 2.3-kb fragment indicated an open reading frame encoding a protein of 48,223 Da, which was similar to the meso-diaminopimelate (DAP) decarboxylase amino acid sequences of Bacillus subtilis (62%) and Corynebacterium glutamicum (40%). Amino acid sequence analysis indicated several regions of conservation among bacterial DAP decarboxylases, eukaryotic ornithine decarboxylases, and arginine decarboxylases, suggesting a common structural arrangement for positioning of substrate and the cofactor pyridoxal 5'-phosphate. The B. methanolicus MGA3 DAP decarboxylase was shown to be a dimer (M(r) 86,000) with a subunit molecular mass of approximately 50,000 Da. This decarboxylase is inhibited by lysine (Ki = 0.93 mM) with a Km of 0.8 mM for DAP. The inhibition pattern suggests that the activity of this enzyme in lysine-overproducing strains of B. methanolicus MGA3 may limit lysine synthesis. Images PMID:8215365

Cloning and sequence analysis of the meso-diaminopimelate decarboxylase gene from Bacillus methanolicus MGA3 and comparison to other decarboxylase genes.

PubMed

Mills, D A; Flickinger, M C

1993-09-01

The lysA gene of Bacillus methanolicus MGA3 was cloned by complementation of an auxotrophic Escherichia coli lysA22 mutant with a genomic library of B. methanolicus MGA3 chromosomal DNA. Subcloning localized the B. methanolicus MGA3 lysA gene into a 2.3-kb SmaI-SstI fragment. Sequence analysis of the 2.3-kb fragment indicated an open reading frame encoding a protein of 48,223 Da, which was similar to the meso-diaminopimelate (DAP) decarboxylase amino acid sequences of Bacillus subtilis (62%) and Corynebacterium glutamicum (40%). Amino acid sequence analysis indicated several regions of conservation among bacterial DAP decarboxylases, eukaryotic ornithine decarboxylases, and arginine decarboxylases, suggesting a common structural arrangement for positioning of substrate and the cofactor pyridoxal 5'-phosphate. The B. methanolicus MGA3 DAP decarboxylase was shown to be a dimer (M(r) 86,000) with a subunit molecular mass of approximately 50,000 Da. This decarboxylase is inhibited by lysine (Ki = 0.93 mM) with a Km of 0.8 mM for DAP. The inhibition pattern suggests that the activity of this enzyme in lysine-overproducing strains of B. methanolicus MGA3 may limit lysine synthesis.

Effects of Cadmium and Mercury on the Upper Part of Skeletal Muscle Glycolysis in Mice

PubMed Central

Ortega, Fernando; Westerhoff, Hans V.; Gelpí, Josep Lluis; Centelles, Josep J.; Cascante, Marta

2014-01-01

The effects of pre-incubation with mercury (Hg2+) and cadmium (Cd2+) on the activities of individual glycolytic enzymes, on the flux and on internal metabolite concentrations of the upper part of glycolysis were investigated in mouse muscle extracts. In the range of metal concentrations analysed we found that only hexokinase and phosphofructokinase, the enzymes that shared the control of the flux, were inhibited by Hg2+ and Cd2+. The concentrations of the internal metabolites glucose-6-phosphate and fructose-6-phosphate did not change significantly when Hg2+ and Cd2+ were added. A mathematical model was constructed to explore the mechanisms of inhibition of Hg2+ and Cd2+ on hexokinase and phosphofructokinase. Equations derived from detailed mechanistic models for each inhibition were fitted to the experimental data. In a concentration-dependent manner these equations describe the observed inhibition of enzyme activity. Under the conditions analysed, the integral model showed that the simultaneous inhibition of hexokinase and phosphofructokinase explains the observation that the concentrations of glucose-6-phosphate and fructose-6-phosphate did not change as the heavy metals decreased the glycolytic flux. PMID:24489641

Replacing Escherichia coli NAD-dependent glyceraldehyde 3-phosphate dehydrogenase (GAPDH) with a NADP-dependent enzyme from Clostridium acetobutylicum facilitates NADPH dependent pathways.

PubMed

Martínez, Irene; Zhu, Jiangfeng; Lin, Henry; Bennett, George N; San, Ka-Yiu

2008-11-01

Reactions requiring reducing equivalents, NAD(P)H, are of enormous importance for the synthesis of industrially valuable compounds such as carotenoids, polymers, antibiotics and chiral alcohols among others. The use of whole-cell biocatalysis can reduce process cost by acting as catalyst and cofactor regenerator at the same time; however, product yields might be limited by cofactor availability within the cell. Thus, our study focussed on the genetic manipulation of a whole-cell system by modifying metabolic pathways and enzymes to improve the overall production process. In the present work, we genetically engineered an Escherichia coli strain to increase NADPH availability to improve the productivity of products that require NADPH in its biosynthesis. The approach involved an alteration of the glycolysis step where glyceraldehyde-3-phosphate (GAP) is oxidized to 1,3 bisphophoglycerate (1,3-BPG). This reaction is catalyzed by NAD-dependent endogenous glyceraldehyde-3-phosphate dehydrogenase (GAPDH) encoded by the gapA gene. We constructed a recombinant E. coli strain by replacing the native NAD-dependent gapA gene with a NADP-dependent GAPDH from Clostridium acetobutylicum, encoded by the gene gapC. The beauty of this approach is that the recombinant E. coli strain produces 2 mol of NADPH, instead of NADH, per mole of glucose consumed. Metabolic flux analysis showed that the flux through the pentose phosphate (PP) pathway, one of the main pathways that produce NADPH, was reduced significantly in the recombinant strain when compared to that of the parent strain. The effectiveness of the NADPH enhancing system was tested using the production of lycopene and epsilon-caprolactone as model systems using two different background strains. The recombinant strains, with increased NADPH availability, consistently showed significant higher productivity than the parent strains.

An efficient protocol for the production of tRNA-free recombinant Selenocysteine Synthase (SELA) from Escherichia coli and its biophysical characterization.

PubMed

Manzine, Livia Regina; Cassago, Alexandre; da Silva, Marco Túlio Alves; Thiemann, Otavio Henrique

2013-03-01

Selenocysteine Synthase (SELA, E.C. 2.9.1.1) from Escherichia coli is a homodecamer pyridoxal-5'-phosphate containing enzyme responsible for the conversion of seryl-tRNA(sec) into selenocysteyl-tRNA(sec) in the biosynthesis of the 21th amino acid, selenocysteine (Sec or U). This paper describes the cloning of the E. coli selA gene into a modified pET29a(+) vector and its expression in E. coli strain WL81460, a crucial modification allowing SELA expression without bound endogenous tRNA(sec). This expression strategy enabled the purification and additional biochemical and biophysical characterization of the SELA decamer. The homogeneous SELA protein was obtained using three chromatographic steps. Size Exclusion Chromatography and Native Gel Electrophoresis showed that SELA maintains a decameric state with molecular mass of approximately 500 kDa with an isoelectric point of 6,03. A predominance of α-helix structures was detected by circular dichroism with thermal stability up to 45 °C. The oligomeric assemblage of SELA was investigated by glutaraldehyde crosslinking experiments indicate that SELA homodecameric structure is the result of a stepwise addition of intermediate oligomeric states and not a direct monomer to homodecamer transition. Our results have contributed to the establishment of a robust expression model for the enzyme free of bound RNA and are of general interest to be taken into consideration in all cases of heterologous/homologous expressions of RNA-binding proteins avoiding the carryover of endogenous RNAs, which may interfere with further biochemical characterizations. Copyright © 2012 Elsevier Inc. All rights reserved.

21 CFR 862.1720 - Triose phosphate isomerase test system.

Code of Federal Regulations, 2011 CFR

2011-04-01

... isomerase test system is a device intended to measure the activity of the enzyme triose phosphate isomerase in erythrocytes (red blood cells). Triose phosphate isomerase is an enzyme important in glycolysis... this device are used in the diagnosis and treatment of congenital triose phosphate isomerase enzyme...

21 CFR 862.1720 - Triose phosphate isomerase test system.

Code of Federal Regulations, 2010 CFR

2010-04-01

... isomerase test system is a device intended to measure the activity of the enzyme triose phosphate isomerase in erythrocytes (red blood cells). Triose phosphate isomerase is an enzyme important in glycolysis... this device are used in the diagnosis and treatment of congenital triose phosphate isomerase enzyme...

Effects of protonation on the hydrolysis of triphosphate in vacuum and the implications for catalysis by nucleotide hydrolyzing enzymes.

PubMed

Kiani, Farooq Ahmad; Fischer, Stefan

2016-06-29

Nucleoside triphosphate (NTP) hydrolysis is a key reaction in biology. It involves breaking two very stable bonds (one P-O bond and one O-H bond of water), in either a concurrent or a sequential way. Here, we systematically examine how protonation of the triphosphate affects the mechanism of hydrolysis. The hydrolysis reaction of methyl triphosphate in vacuum is computed with protons in various numbers and position on the three phosphate groups. Protonation is seen to have a strong catalytic effect, with the reaction mechanism depending highly on the protonation pattern. This dependence is apparently complicated, but is shown to obey a well-defined set of rules: Protonation of the α- and β-phosphate groups favors a sequential hydrolysis mechanism, whereas γ-protonation favors a concurrent mechanism, the two effects competing with each other in cases of simultaneous protonation. The rate-limiting step is always the breakup of the water molecule while it attacks the γ-phosphorus, and its barrier is lowered by γ-protonation. This step has significantly lower barriers in the sequential reactions, because the dissociated γ-metaphosphate intermediate (P γ O 3 - ) is a much better target for water attack than the un-dissociated γ-phosphate (-P γ O 4 2- ). The simple chemical logic behind these rules helps to better understand the catalytic strategy used by NTPase enzymes, as illustrated here for the catalytic pocket of myosin. A set of rules was determined that describes how protonating the phosphate groups affects the hydrolysis mechanism of methyl triphosphate: Protonation of the α- and/or β- phosphate groups promotes a sequential mechanism in which P-O bond breaking precedes the breakup of the attacking water, whereas protonation of the γ-phosphate promotes a concurrent mechanism and lowers the rate-limiting barrier of water breakup. The role played by individual protein residues in the catalytic pocket of triphosphate hydrolysing enzymes can be assigned accordingly.

Probing the origins of catalytic discrimination between phosphate and sulfate monoester hydrolysis: comparative analysis of alkaline phosphatase and protein tyrosine phosphatases.

PubMed

Andrews, Logan D; Zalatan, Jesse G; Herschlag, Daniel

2014-11-04

Catalytic promiscuity, the ability of enzymes to catalyze multiple reactions, provides an opportunity to gain a deeper understanding of the origins of catalysis and substrate specificity. Alkaline phosphatase (AP) catalyzes both phosphate and sulfate monoester hydrolysis reactions with a ∼10(10)-fold preference for phosphate monoester hydrolysis, despite the similarity between these reactions. The preponderance of formal positive charge in the AP active site, particularly from three divalent metal ions, was proposed to be responsible for this preference by providing stronger electrostatic interactions with the more negatively charged phosphoryl group versus the sulfuryl group. To test whether positively charged metal ions are required to achieve a high preference for the phosphate monoester hydrolysis reaction, the catalytic preference of three protein tyrosine phosphatases (PTPs), which do not contain metal ions, were measured. Their preferences ranged from 5 × 10(6) to 7 × 10(7), lower than that for AP but still substantial, indicating that metal ions and a high preponderance of formal positive charge within the active site are not required to achieve a strong catalytic preference for phosphate monoester over sulfate monoester hydrolysis. The observed ionic strength dependences of kcat/KM values for phosphate and sulfate monoester hydrolysis are steeper for the more highly charged phosphate ester with both AP and the PTP Stp1, following the dependence expected based on the charge difference of these two substrates. However, the dependences for AP were not greater than those of Stp1 and were rather shallow for both enzymes. These results suggest that overall electrostatics from formal positive charge within the active site is not the major driving force in distinguishing between these reactions and that substantial discrimination can be attained without metal ions. Thus, local properties of the active site, presumably including multiple positioned dipolar hydrogen bond donors within the active site, dominate in defining this reaction specificity.

Mechanism of Substrate Recognition And PLP-Induced Conformational Changes in II-Diaminopimelate Aminotransferase From Arabidopsis Thaliana

DOE Office of Scientific and Technical Information (OSTI.GOV)

Watanabe, N.; Clay, M.D.; Belkum, M.J.van

2009-05-26

LL-Diaminopimelate aminotransferase (LL-DAP-AT), a pyridoxal phosphate (PLP)-dependent enzyme in the lysine biosynthetic pathways of plants and Chlamydia, is a potential target for the development of herbicides or antibiotics. This homodimeric enzyme converts L-tetrahydrodipicolinic acid (THDP) directly to LL-DAP using L-glutamate as the source of the amino group. Earlier, we described the 3D structures of native and malate-bound LL-DAP-AT from Arabidopsis thaliana (AtDAP-AT). Seven additional crystal structures of AtDAP-AT and its variants are reported here as part of an investigation into the mechanism of substrate recognition and catalysis. Two structures are of AtDAP-AT with reduced external aldimine analogues: N-(5'-phosphopyridoxyl)-L-glutamate (PLP-Glu) andmore » N-(5'-phosphopyridoxyl)- LL-Diaminopimelate (PLP-DAP) bound in the active site. Surprisingly, they reveal that both L-glutamate and LL-DAP are recognized in a very similar fashion by the same sets of amino acid residues; both molecules adopt twisted V-shaped conformations. With both substrates, the {alpha}-carboxylates are bound in a salt bridge with Arg404, whereas the distal carboxylates are recognized via hydrogen bonds to the well-conserved side chains of Tyr37, Tyr125 and Lys129. The distal C{sup {var_epsilon}} amino group of LL-DAP is specifically recognized by several non-covalent interactions with residues from the other subunit (Asn309*, Tyr94*, Gly95*, and Glu97* (Amino acid designators followed by an asterisk (*) indicate that the residues originate in the other subunit of the dimer)) and by three bound water molecules. Two catalytically inactive variants of AtDAP-AT were created via site-directed mutagenesis of the active site lysine (K270N and K270Q). The structures of these variants permitted the observation of the unreduced external aldimines of PLP with L-glutamate and with LL-DAP in the active site, and revealed differences in the torsion angle about the PLP-substrate bond. Lastly, an apo-AtDAP-AT structure missing PLP revealed details of conformational changes induced by PLP binding and substrate entry into the active site.« less

A Dopa Decarboxylase Modulating the Immune Response of Scallop Chlamys farreri

PubMed Central

Zhou, Zhi; Yang, Jialong; Wang, Lingling; Zhang, Huan; Gao, Yang; Shi, Xiaowei; Wang, Mengqiang; Kong, Pengfei; Qiu, Limei; Song, Linsheng

2011-01-01

Background Dopa decarboxylase (DDC) is a pyridoxal 5-phosphate (PLP)-dependent enzyme that catalyzes the decarboxylation of L-Dopa to dopamine, and involved in complex neuroendocrine-immune regulatory network. The function for DDC in the immunomodulation remains unclear in invertebrate. Methodology The full-length cDNA encoding DDC (designated CfDDC) was cloned from mollusc scallop Chlamys farreri. It contained an open reading frame encoding a polypeptide of 560 amino acids. The CfDDC mRNA transcripts could be detected in all the tested tissues, including the immune tissues haemocytes and hepatopancreas. After scallops were treated with LPS stimulation, the mRNA expression level of CfDDC in haemocytes increased significantly (5.5-fold, P<0.05) at 3 h and reached the peak at 12 h (9.8-fold, P<0.05), and then recovered to the baseline level. The recombinant protein of CfDDC (rCfDDC) was expressed in Escherichia coli BL21 (DE3)-Transetta, and 1 mg rCfDDC could catalyze the production of 1.651±0.22 ng dopamine within 1 h in vitro. When the haemocytes were incubated with rCfDDC-coated agarose beads, the haemocyte encapsulation to the beads was increased significantly from 70% at 6 h to 93% at 24 h in vitro in comparison with that in the control (23% at 6 h to 25% at 24 h), and the increased haemocyte encapsulation was repressed by the addition of rCfDDC antibody (which is acquired via immunization 6-week old rats with rCfDDC). After the injection of DDC inhibitor methyldopa, the ROS level in haemocytes of scallops was decreased significantly to 0.41-fold (P<0.05) of blank group at 12 h and 0.47-fold (P<0.05) at 24 h, respectively. Conclusions These results collectively suggested that CfDDC, as a homologue of DDC in scallop, modulated the immune responses such as haemocytes encapsulation as well as the ROS level through its catalytic activity, functioning as an indispensable immunomodulating enzyme in the neuroendocrine-immune regulatory network of mollusc. PMID:21533240

Glycosylation differences contribute to distinct catalytic properties among bone alkaline phosphatase isoforms

PubMed Central

Linder, Cecilia Halling; Narisawa, Sonoko; Millán, José Luis; Magnusson, Per

2009-01-01

Three circulating human bone alkaline phosphatase (BALP) isoforms (B1, B2, and B/I) can be distinguished in healthy individuals and a fourth isoform (B1x) has been discovered in patients with chronic kidney disease and in bone tissue. The present study was designed to correlate differing glycosylation patterns of each BALP isoform with their catalytic activity towards presumptive physiological substrates and to compare those properties with two recombinant isoforms of the tissue-nonspecific ALP (TNALP) isozyme, i.e., TNALP-flag, used extensively for mutation analysis of hypophosphatasia mutations and sALP-FcD10, a chimeric enzyme recently used as therapeutic drug in a mouse model of infantile hypophosphatasia. The BALP isoforms were prepared from human osteosarcoma (SaOS-2) cells and the kinetic properties were evaluated using the synthetic substrate p-nitrophenylphosphate (pNPP) at pH 7.4 and 9.8, and the three suggested endogenous physiological substrates, i.e., inorganic pyrophosphate (PPi), pyridoxal 5′-phosphate (PLP), and phosphoethanolamine (PEA) at pH 7.4. Qualitative glycosylation differences were also assessed by lectin binding and precipitation. The kcat/KM was higher for B2 for all the investigated substrates. The catalytic activity towards PEA was essentially undetectable. The kinetic activity for TNALP-flag and sALP-FcD10 was similar to the activity of the human BALP isoforms. The BALP isoforms differed in their lectin-binding properties and dose-dependent lectin precipitation, which also demonstrated differences between native and denatured BALP isoforms. The observed differences in lectin specificity were attributed to N-linked carbohydrates. In conclusion, we demonstrate significantly different catalytic properties among the BALP isoforms due to structural differences in posttranslational glycosylation. Our data also suggests that PEA is not an endogenous substrate for the BALP isoforms or for the recombinant TNALP isoforms. The TNALP-flag and the sALP-FcD10 isoforms faithfully mimic the biological properties of the human BALP isoforms in vivo validating the use of these recombinant enzymes in studies aimed at dissecting the pathophysiology and treating hypophosphatasia. PMID:19631305

Glycosylation differences contribute to distinct catalytic properties among bone alkaline phosphatase isoforms.

PubMed

Halling Linder, Cecilia; Narisawa, Sonoko; Millán, José Luis; Magnusson, Per

2009-11-01

Three circulating human bone alkaline phosphatase (BALP) isoforms (B1, B2, and B/I) can be distinguished in healthy individuals and a fourth isoform (B1x) has been discovered in patients with chronic kidney disease and in bone tissue. The present study was designed to correlate differing glycosylation patterns of each BALP isoform with their catalytic activity towards presumptive physiological substrates and to compare those properties with two recombinant isoforms of the tissue-nonspecific ALP (TNALP) isozyme, i.e., TNALP-flag, used extensively for mutation analysis of hypophosphatasia mutations and sALP-FcD(10), a chimeric enzyme recently used as therapeutic drug in a mouse model of infantile hypophosphatasia. The BALP isoforms were prepared from human osteosarcoma (SaOS-2) cells and the kinetic properties were evaluated using the synthetic substrate p-nitrophenylphosphate (pNPP) at pH 7.4 and 9.8, and the three suggested endogenous physiological substrates, i.e., inorganic pyrophosphate (PP(i)), pyridoxal 5'-phosphate (PLP), and phosphoethanolamine (PEA) at pH 7.4. Qualitative glycosylation differences were also assessed by lectin binding and precipitation. The k(cat)/K(M) was higher for B2 for all the investigated substrates. The catalytic activity towards PEA was essentially undetectable. The kinetic activity for TNALP-flag and sALP-FcD(10) was similar to the activity of the human BALP isoforms. The BALP isoforms differed in their lectin binding properties and dose-dependent lectin precipitation, which also demonstrated differences between native and denatured BALP isoforms. The observed differences in lectin specificity were attributed to N-linked carbohydrates. In conclusion, we demonstrate significantly different catalytic properties among the BALP isoforms due to structural differences in posttranslational glycosylation. Our data also suggests that PEA is not an endogenous substrate for the BALP isoforms or for the recombinant TNALP isoforms. The TNALP-flag and the sALP-FcD(10) isoforms faithfully mimic the biological properties of the human BALP isoforms in vivo validating the use of these recombinant enzymes in studies aimed at dissecting the pathophysiology and treating hypophosphatasia.

Transaminases for the synthesis of enantiopure beta-amino acids

PubMed Central

2012-01-01

Optically pure β-amino acids constitute interesting building blocks for peptidomimetics and a great variety of pharmaceutically important compounds. Their efficient synthesis still poses a major challenge. Transaminases (also known as aminotransferases) possess a great potential for the synthesis of optically pure β-amino acids. These pyridoxal 5'-dependent enzymes catalyze the transfer of an amino group from a donor substrate to an acceptor, thus enabling the synthesis of a wide variety of chiral amines and amino acids. Transaminases can be applied either for the kinetic resolution of racemic compounds or the asymmetric synthesis starting from a prochiral substrate. This review gives an overview over microbial transaminases with activity towards β-amino acids and their substrate spectra. It also outlines current strategies for the screening of new biocatalysts. Particular emphasis is placed on activity assays which are applicable to high-throughput screening. PMID:22293122

Submerged fermentation of Lactobacillus rhamnosus YS9 for γ-aminobutyric acid (GABA) production

PubMed Central

Lin, Qian

2013-01-01

γ-Aminobutyric acid (GABA) is a major inhibitory neurotransmitter in central nervous system, and its application in drugs and functional foods has attracted great attention. To enhance production of γ-aminobutyric acid, Lactobacillus rhamnosus YS9, a strain isolated from Chinese traditional fermented food pickled vegetable, was grown under submerged fermentation. Its cultivation conditions were investigated. When culture pH condition was adjusted to the optimal pH of glutamate decarboxylase activity, culture of Lb. rhamnosus YS9 in medium supplemented with 200 mM of monosodium glutamate and 200 μM of pyridoxal phosphate (PLP), produced 187 mM of GABA. PMID:24159304

Structure of L-Xylulose-5-Phosphate 3-Epimerase (UlaE) from the Anaerobic L-Ascorbate Utilization Pathway of Escherichia coli: Identification of a Novel Phosphate Binding Motif within a TIM Barrel Fold

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shi, Rong; Pineda, Marco; Ajamian, Eunice

2009-01-15

Three catabolic enzymes, UlaD, UlaE, and UlaF, are involved in a pathway leading to fermentation of L-ascorbate under anaerobic conditions. UlaD catalyzes a {beta}-keto acid decarboxylation reaction to produce L-xylulose-5-phosphate, which undergoes successive epimerization reactions with UlaE (L-xylulose-5-phosphate 3-epimerase) and UlaF (L-ribulose-5-phosphate 4-epimerase), yielding D-xylulose-5-phosphate, an intermediate in the pentose phosphate pathway. We describe here crystallographic studies of UlaE from Escherichia coli O157:H7 that complete the structural characterization of this pathway. UlaE has a triosephosphate isomerase (TIM) barrel fold and forms dimers. The active site is located at the C-terminal ends of the parallel {beta}-strands. The enzyme binds Zn{sup 2+},more » which is coordinated by Glu155, Asp185, His211, and Glu251. We identified a phosphate-binding site formed by residues from the {beta}1/{alpha}1 loop and {alpha}3' helix in the N-terminal region. This site differs from the well-characterized phosphate-binding motif found in several TIM barrel superfamilies that is located at strands {beta}7 and {beta}8. The intrinsic flexibility of the active site region is reflected by two different conformations of loops forming part of the substrate-binding site. Based on computational docking of the L-xylulose 5-phosphate substrate to UlaE and structural similarities of the active site of this enzyme to the active sites of other epimerases, a metal-dependent epimerization mechanism for UlaE is proposed, and Glu155 and Glu251 are implicated as catalytic residues. Mutation and activity measurements for structurally equivalent residues in related epimerases supported this mechanistic proposal.« less

Lactose metabolism by Staphylococcus aureus: characterization of lacABCD, the structural genes of the tagatose 6-phosphate pathway.

PubMed Central

Rosey, E L; Oskouian, B; Stewart, G C

1991-01-01

The nucleotide and deduced amino acid sequences of the lacA and lacB genes of the Staphylococcus aureus lactose operon (lacABCDFEG) are presented. The primary translation products are polypeptides of 142 (Mr = 15,425) and 171 (Mr = 18,953) amino acids, respectively. The lacABCD loci were shown to encode enzymes of the tagatose 6-phosphate pathway through both in vitro studies and complementation analysis in Escherichia coli. A serum aldolase assay, modified to allow detection of the tagatose 6-phosphate pathway enzymes utilizing galactose 6-phosphate or fructose phosphate analogs as substrate, is described. Expression of both lacA and lacB was required for galactose 6-phosphate isomerase activity. LacC (34 kDa) demonstrated tagatose 6-phosphate kinase activity and was found to share significant homology with LacC from Lactococcus lactis and with both the minor 6-phosphofructokinase (PfkB) and 1-phosphofructokinase (FruK) from E. coli. Detection of tagatose 1,6-bisphosphate aldolase activity was dependent on expression of the 36-kDa protein specified by lacD. The LacD protein is highly homologous with LacD of L. lactis. Thus, the lacABCD genes comprise the tagatose 6-phosphate pathway and are cotranscribed with genes lacFEG, which specify proteins for transport and cleavage of lactose in S. aureus. PMID:1655695

Mutation of cysteine 111 in Dopa decarboxylase leads to active site perturbation.

PubMed Central

Dominici, P.; Moore, P. S.; Castellani, S.; Bertoldi, M.; Voltattorni, C. B.

1997-01-01

Cysteine 111 in Dopa decarboxylase (DDC) has been replaced by alanine or serine by site-directed mutagenesis. Compared to the wild-type enzyme, the resultant C111A and C111S mutant enzymes exhibit Kcat values of about 50% and 15%, respectively, at pH 6.8, while the K(m) values remain relatively unaltered for L-3,4-dihydroxyphenylalanine (L-Dopa) and L-5-hydroxytryptophan (L-5-HTP). While a significant decrease of the 280 nm optically active band present in the wild type is observed in mutant DDCs, their visible co-enzyme absorption and CD spectra are similar to those of the wild type. With respect to the wild type, the Cys-111-->Ala mutant displays a reduced affinity for pyridoxal 5'-phosphate (PLP), slower kinetics of reconstitution to holoenzyme, a decreased ability to anchor the external aldimine formed between D-Dopa and the bound co-enzyme, and a decreased efficiency of energy transfer between tryptophan residue(s) and reduced PLP. Values of pKa and pKb for the groups involved in catalysis were determined for the wild-type and the C111A mutant enzymes. The mutant showed a decrease in both pK values by about 1 pH unit, resulting in a shift of the pH of the maximum velocity from 7.2 (wild-type) to 6.2 (mutant). This change in maximum velocity is mirrored by a similar shift in the spectrophotometrically determined pK value of the 420-->390 nm transition of the external aldimine. These results demonstrate that the sulfhydryl group of Cys-111 is catalytically nonessential and provide strong support for previous suggestion that this residue is located at or near the PLP binding site (Dominici P, Maras B, Mei G, Borri Voltattorni C. 1991. Eur J Biochem 201:393-397). Moreover, our findings provide evidence that Cys-111 has a structural role in PLP binding and suggest that this residue is required for maintenance of proper active-site conformation. PMID:9300500

Glutamine Hydrolysis by Imidazole Glycerol Phosphate Synthase Displays Temperature Dependent Allosteric Activation

PubMed Central

Lisi, George P.; Currier, Allen A.; Loria, J. Patrick

2018-01-01

The enzyme imidazole glycerol phosphate synthase (IGPS) is a model for studies of long-range allosteric regulation in enzymes. Binding of the allosteric effector ligand N'-[5'-phosphoribulosyl)formimino]-5-aminoimidazole-4-carboxamide-ribonucleotide (PRFAR) stimulates millisecond (ms) timescale motions in IGPS that enhance its catalytic function. We studied the effect of temperature on these critical conformational motions and the catalytic mechanism of IGPS from the hyperthermophile Thermatoga maritima in an effort to understand temperature-dependent allostery. Enzyme kinetic and NMR dynamics measurements show that apo and PRFAR-activated IGPS respond differently to changes in temperature. Multiple-quantum Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments performed at 303, 323, and 343 K (30, 50, and 70°C) reveal that millisecond flexibility is enhanced to a higher degree in apo IGPS than in the PRFAR-bound enzyme as the sample temperature is raised. We find that the flexibility of the apo enzyme is nearly identical to that of its PRFAR activated state at 343 K, whereas conformational motions are considerably different between these two forms of the enzyme at room temperature. Arrhenius analyses of these flexible sites show a varied range of activation energies that loosely correlate to allosteric communities identified by computational methods and reflect local changes in dynamics that may facilitate conformational sampling of the active conformation. In addition, kinetic assays indicate that allosteric activation by PRFAR decreases to 65-fold at 343 K, compared to 4,200-fold at 303 K, which mirrors the decreased effect of PRFAR on ms motions relative to the unactivated enzyme. These studies indicate that at the growth temperature of T. maritima, PFRAR is a weaker allosteric activator than it is at room temperature and illustrate that the allosteric mechanism of IGPS is temperature dependent. PMID:29468164

Functional and molecular characterization of a glycosomal PPi-dependent enzyme in trypanosomatids: Pyruvate, phosphate dikinase

PubMed Central

Bringaud, Frederic; Baltz, Dominique; Baltz, Theo

1998-01-01

Trypanosomatids are parasitic protists that have an ATP-dependent glycolysis with no indication of PPi-dependent metabolism. Most of the glycolysis takes place in peroxisome-like organelles, the glycosomes. We characterized in Trypanosoma brucei a single-copy gene encoding a PPi-dependent enzyme, pyruvate, phosphate dikinase (PPDK), which was expressed functionally in Escherichia coli. Specific antibodies detected a 100-kDa protein in procyclic forms but not in mammalian forms of T. brucei, indicating a differential expression. Glycosomal localization of PPDK was determined by immunofluorescence analysis and was confirmed by Western blot analysis on glycosomal fractions by using anti-PPDK antibodies. Expression and localization of recombinant PPDKs in procyclic forms of T. brucei showed that the AKL motif at the C-terminal extremity of PPDK is necessary for glycosomal targeting. PPDK was detected in every trypanosomatid tested—Trypanosoma congolense, Trypanosoma vivax, Trypanosoma cruzi, Phytomonas, Crithidia and Leishmania—with a good correlation between amount of protein and enzymatic activity. The precise role of PPDK in trypanosomatid carbohydrate metabolism remains to be clarified. PMID:9653123

Partial reactions of d-glucose 6-phosphate–1 l-myoinositol 1-phosphate cyclase

PubMed Central

Barnett, J. E. G.; Rasheed, A.; Corina, D. L.

1973-01-01

After removal of tightly bound NAD+ by using charcoal, a preparation of d-glucose 6-phosphate–1 l-myoinositol 1-phosphate cyclase catalysed the reduction of 5-keto-d-glucitol 6-phosphate and 5-keto-d-glucose 6-phosphate by [4-3H]NADH to give [5-3H]-glucitol 6-phosphate and [5-3H]glucose 6-phosphate respectively. The position of the tritium atom in the latter was shown by degradation. Both enzyme-catalysed reductions were strongly inhibited by 2-deoxy-d-glucose 6-phosphate, a powerful competitive inhibitor of inositol cyclase. The charcoal-treated enzyme preparation also converted 5-keto-d-glucose 6-phosphate into [3H]myoinositol 1-phosphate in the presence of [4-3H]NADH, but less effectively. These partial reactions of inositol cyclase are interpreted as providing strong evidence for the formation of 5-keto-d-glucose 6-phosphate as an enzyme-bound intermediate in the conversion of d-glucose 6-phosphate into 1 l-myoinositol 1-phosphate. The enzyme was partially inactivated by NaBH4 in the presence of NAD+. Glucose 6-phosphate did not increase the inactivation, and there was no inactivation in the absence of NAD+. There was no evidence for Schiff base formation during the cyclization. d-Glucitol 6-phosphate (l-sorbitol 1-phosphate) was a good inhibitor of the overall reaction. It did not inactivate the enzyme. The apparent molecular weight of inositol cyclase as determined by Sephadex chromatography was 2.15×105. PMID:4352864

The Putative Mevalonate Diphosphate Decarboxylase from Picrophilus torridus Is in Reality a Mevalonate-3-Kinase with High Potential for Bioproduction of Isobutene

PubMed Central

Hall, Stephen J.; Eastham, Graham; Licence, Peter; Stephens, Gill

2015-01-01

Mevalonate diphosphate decarboxylase (MVD) is an ATP-dependent enzyme that catalyzes the phosphorylation/decarboxylation of (R)-mevalonate-5-diphosphate to isopentenyl pyrophosphate in the mevalonate (MVA) pathway. MVD is a key enzyme in engineered metabolic pathways for bioproduction of isobutene, since it catalyzes the conversion of 3-hydroxyisovalerate (3-HIV) to isobutene, an important platform chemical. The putative homologue from Picrophilus torridus has been identified as a highly efficient variant in a number of patents, but its detailed characterization has not been reported. In this study, we have successfully purified and characterized the putative MVD from P. torridus. We discovered that it is not a decarboxylase per se but an ATP-dependent enzyme, mevalonate-3-kinase (M3K), which catalyzes the phosphorylation of MVA to mevalonate-3-phosphate. The enzyme's potential in isobutene formation is due to the conversion of 3-HIV to an unstable 3-phosphate intermediate that undergoes consequent spontaneous decarboxylation to form isobutene. Isobutene production rates were as high as 507 pmol min−1 g cells−1 using Escherichia coli cells expressing the enzyme and 2,880 pmol min−1 mg protein−1 with the purified histidine-tagged enzyme, significantly higher than reported previously. M3K is a key enzyme of the novel MVA pathway discovered very recently in Thermoplasma acidophilum. We suggest that P. torridus metabolizes MVA by the same pathway. PMID:25636853

Glucose-6-phosphate dehydrogenase status and risk of hemolysis in Plasmodium falciparum-infected African children receiving single-dose primaquine.

PubMed

Eziefula, Alice C; Pett, Helmi; Grignard, Lynn; Opus, Salome; Kiggundu, Moses; Kamya, Moses R; Yeung, Shunmay; Staedke, Sarah G; Bousema, Teun; Drakeley, Chris

2014-08-01

Glucose-6-phosphate dehydrogenase (G6PD) enzyme function and genotype were determined in Ugandan children with uncomplicated falciparum malaria enrolled in a primaquine trial after exclusion of severe G6PD deficiency by fluorescent spot test. G6PD A- heterozygotes and hemizygotes/homozygotes experienced dose-dependent lower hemoglobin concentrations after treatment. No severe anemia was observed. Copyright © 2014, Eziefula et al.

Glucose-6-phosphate metabolism in Plasmodium falciparum.

PubMed

Preuss, Janina; Jortzik, Esther; Becker, Katja

2012-07-01

Malaria is still one of the most threatening diseases worldwide. The high drug resistance rates of malarial parasites make its eradication difficult and furthermore necessitate the development of new antimalarial drugs. Plasmodium falciparum is responsible for severe malaria and therefore of special interest with regard to drug development. Plasmodium parasites are highly dependent on glucose and very sensitive to oxidative stress; two observations that drew interest to the pentose phosphate pathway (PPP) with its key enzyme glucose-6-phosphate dehydrogenase (G6PD). A central position of the PPP for malaria parasites is supported by the fact that human G6PD deficiency protects to a certain degree from malaria infections. Plasmodium parasites and the human host possess a complete PPP, both of which seem to be important for the parasites. Interestingly, there are major differences between parasite and human G6PD, making the enzyme of Plasmodium a promising target for antimalarial drug design. This review gives an overview of the current state of research on glucose-6-phosphate metabolism in P. falciparum and its impact on malaria infections. Moreover, the unique characteristics of the enzyme G6PD in P. falciparum are discussed, upon which its current status as promising target for drug development is based. Copyright © 2012 Wiley Periodicals, Inc.

Kinetics and mechanism of the condensation of pyridoxal hydrochloride with L-tryptophan and D-tryptophan, and the chemical transformation of their products

NASA Astrophysics Data System (ADS)

Pishchugin, F. V.; Tuleberdiev, I. T.

2017-10-01

The kinetics and mechanism of interaction between pyridoxal and L-tryptophan, D-tryptophan, and their derivatives are studied. It is found that condensation reactions proceed via three kinetically distinguishable stages: (1) the rapid intraplanar addition of the NH2 groups of the amino acids to pyridoxal with the formation of amino alcohols; (2) the rotational isomerism of amino alcohol fragments with their subsequent dehydration and the formation of a Schiff base with a specific configuration; (3) the abstraction of α-hydrogen in the product of condensation of pyridoxal with L-tryptophan, or the abstraction of CO2 in the product of condensation of pyridoxal with D-tryptophan with the formation of quinoid structures, hydrolysis of which results in the preparation of pyridoxamine and keto acid or pyridoxal and tryptamine, respectively. Schiff bases resistant to further chemical transformations are formed in the reaction with tryptophan methyl ester.

Structural and Functional Adaptation of Vancomycin Resistance VanT Serine Racemases.

PubMed

Meziane-Cherif, Djalal; Stogios, Peter J; Evdokimova, Elena; Egorova, Olga; Savchenko, Alexei; Courvalin, Patrice

2015-08-11

Vancomycin resistance in Gram-positive bacteria results from the replacement of the D-alanyl-D-alanine target of peptidoglycan precursors with D-alanyl-D-lactate or D-alanyl-D-serine (D-Ala-D-Ser), to which vancomycin has low binding affinity. VanT is one of the proteins required for the production of D-Ala-D-Ser-terminating precursors by converting L-Ser to D-Ser. VanT is composed of two domains, an N-terminal membrane-bound domain, likely involved in L-Ser uptake, and a C-terminal cytoplasmic catalytic domain which is related to bacterial alanine racemases. To gain insight into the molecular function of VanT, the crystal structure of the catalytic domain of VanTG from VanG-type resistant Enterococcus faecalis BM4518 was determined. The structure showed significant similarity to type III pyridoxal 5'-phosphate (PLP)-dependent alanine racemases, which are essential for peptidoglycan synthesis. Comparative structural analysis between VanTG and alanine racemases as well as site-directed mutagenesis identified three specific active site positions centered around Asn696 which are responsible for the L-amino acid specificity. This analysis also suggested that VanT racemases evolved from regular alanine racemases by acquiring additional selectivity toward serine while preserving that for alanine. The 4-fold-lower relative catalytic efficiency of VanTG against L-Ser versus L-Ala implied that this enzyme relies on its membrane-bound domain for L-Ser transport to increase the overall rate of d-Ser production. These findings illustrate how vancomycin pressure selected for molecular adaptation of a housekeeping enzyme to a bifunctional enzyme to allow for peptidoglycan remodeling, a strategy increasingly observed in antibiotic-resistant bacteria. Vancomycin is one of the drugs of last resort against Gram-positive antibiotic-resistant pathogens. However, bacteria have evolved a sophisticated mechanism which remodels the drug target, the D-alanine ending precursors in cell wall synthesis, into precursors terminating with D-lactate or D-serine, to which vancomycin has less affinity. D-Ser is synthesized by VanT serine racemase, which has two unusual characteristics: (i) it is one of the few serine racemases identified in bacteria and (ii) it contains a membrane-bound domain involved in L-Ser uptake. The structure of the catalytic domain of VanTG showed high similarity to alanine racemases, and we identified three specific active site substitutions responsible for L-Ser specificity. The data provide the molecular basis for VanT evolution to a bifunctional enzyme coordinating both transport and racemization. Our findings also illustrate the evolution of the essential alanine racemase into a vancomycin resistance enzyme in response to antibiotic pressure. Copyright © 2015 Meziane-Cherif et al.

Degradation of Phytate by the 6-Phytase from Hafnia alvei: A Combined Structural and Solution Study

PubMed Central

Blagova, Elena V.; Turkenburg, Johan P.; Waterman, Jitka; Roberts, Shirley M.; Vind, Jesper; Sjøholm, Carsten; Lassen, Søren F.; De Maria, Leonardo; Glitsoe, Vibe; Skov, Lars K.; Wilson, Keith S.

2013-01-01

Phytases hydrolyse phytate (myo-inositol hexakisphosphate), the principal form of phosphate stored in plant seeds to produce phosphate and lower phosphorylated myo-inositols. They are used extensively in the feed industry, and have been characterised biochemically and structurally with a number of structures in the PDB. They are divided into four distinct families: histidine acid phosphatases (HAP), β-propeller phytases, cysteine phosphatases and purple acid phosphatases and also split into three enzyme classes, the 3-, 5- and 6-phytases, depending on the position of the first phosphate in the inositol ring to be removed. We report identification, cloning, purification and 3D structures of 6-phytases from two bacteria, Hafnia alvei and Yersinia kristensenii, together with their pH optima, thermal stability, and degradation profiles for phytate. An important result is the structure of the H. alvei enzyme in complex with the substrate analogue myo-inositol hexakissulphate. In contrast to the only previous structure of a ligand-bound 6-phytase, where the 3-phosphate was unexpectedly in the catalytic site, in the H. alvei complex the expected scissile 6-phosphate (sulphate in the inhibitor) is placed in the catalytic site. PMID:23741456

Degradation of phytate by the 6-phytase from Hafnia alvei: a combined structural and solution study.

PubMed

Ariza, Antonio; Moroz, Olga V; Blagova, Elena V; Turkenburg, Johan P; Waterman, Jitka; Roberts, Shirley M; Vind, Jesper; Sjøholm, Carsten; Lassen, Søren F; De Maria, Leonardo; Glitsoe, Vibe; Skov, Lars K; Wilson, Keith S

2013-01-01

Phytases hydrolyse phytate (myo-inositol hexakisphosphate), the principal form of phosphate stored in plant seeds to produce phosphate and lower phosphorylated myo-inositols. They are used extensively in the feed industry, and have been characterised biochemically and structurally with a number of structures in the PDB. They are divided into four distinct families: histidine acid phosphatases (HAP), β-propeller phytases, cysteine phosphatases and purple acid phosphatases and also split into three enzyme classes, the 3-, 5- and 6-phytases, depending on the position of the first phosphate in the inositol ring to be removed. We report identification, cloning, purification and 3D structures of 6-phytases from two bacteria, Hafnia alvei and Yersinia kristensenii, together with their pH optima, thermal stability, and degradation profiles for phytate. An important result is the structure of the H. alvei enzyme in complex with the substrate analogue myo-inositol hexakissulphate. In contrast to the only previous structure of a ligand-bound 6-phytase, where the 3-phosphate was unexpectedly in the catalytic site, in the H. alvei complex the expected scissile 6-phosphate (sulphate in the inhibitor) is placed in the catalytic site.

Enzyme activity in dialkyl phosphate ionic liquids

DOE Office of Scientific and Technical Information (OSTI.GOV)

Thomas, M.F.; Dunn, J.; Li, L.-L.

2011-12-01

The activity of four metagenomic enzymes and an enzyme cloned from the straw mushroom, Volvariellavolvacea were studied in the following ionic liquids, 1,3-dimethylimidazolium dimethyl phosphate, [mmim][dmp], 1-ethyl-3-methylimidazolium dimethyl phosphate, [emim][dmp], 1-ethyl-3-methylimidazolium diethyl phosphate, [emim][dep] and 1-ethyl-3-methylimidazolium acetate, [emim][OAc]. Activity was determined by analyzing the hydrolysis of para-nitrobenzene carbohydrate derivatives. In general, the enzymes were most active in the dimethyl phosphate ionic liquids, followed by acetate. Generally speaking, activity decreased sharply for concentrations of [emim][dep] above 10% v/v, while the other ionic liquids showed less impact on activity up to 20% v/v.

Structure of RNA 3′-phosphate cyclase bound to substrate RNA

PubMed Central

Desai, Kevin K.; Bingman, Craig A.; Cheng, Chin L.; Phillips, George N.

2014-01-01

RNA 3′-phosphate cyclase (RtcA) catalyzes the ATP-dependent cyclization of a 3′-phosphate to form a 2′,3′-cyclic phosphate at RNA termini. Cyclization proceeds through RtcA–AMP and RNA(3′)pp(5′)A covalent intermediates, which are analogous to intermediates formed during catalysis by the tRNA ligase RtcB. Here we present a crystal structure of Pyrococcus horikoshii RtcA in complex with a 3′-phosphate terminated RNA and adenosine in the AMP-binding pocket. Our data reveal that RtcA recognizes substrate RNA by ensuring that the terminal 3′-phosphate makes a large contribution to RNA binding. Furthermore, the RNA 3′-phosphate is poised for in-line attack on the P–N bond that links the phosphorous atom of AMP to Nε of His307. Thus, we provide the first insights into RNA 3′-phosphate termini recognition and the mechanism of 3′-phosphate activation by an Rtc enzyme. PMID:25161314

A B12-dependent radical SAM enzyme involved in Oxetanocin-A biosynthesis

PubMed Central

Bridwell-Rabb, Jennifer; Zhong, Aoshu; Sun, He G.; Drennan, Catherine L.; Liu, Hung-wen

2017-01-01

Summary Oxetanocin-A (OXT-A, 1) is a potent antitumor, antiviral, and antibacterial compound. Biosynthesis of OXT-A has been linked to a plasmid-borne, Bacillus megaterium gene cluster that contains four genes, oxsA, oxsB, oxrA, and oxrB. Here, we show that the oxsA and oxsB genes are both required for the production of OXT-A. Biochemical analysis of the encoded proteins, a cobalamin (Cbl)-dependent S-adenosylmethionine (AdoMet) radical enzyme, OxsB, and an HD-domain phosphohydrolase, OxsA, revealed that OXT-A is derived from 2′-deoxyadenosine phosphate in an OxsB-catalyzed ring contraction reaction initiated by H-atom abstraction from C2′. Hence, OxsB represents the first biochemically characterized non-methylating Cbl-dependent AdoMet radical enzyme. X-ray analysis of OxsB reveals the fold of a Cbl-dependent AdoMet radical enzyme for which there are an estimated 7000 members. Overall, this work provides a framework for understanding the interplay of AdoMet and Cbl cofactors and expands the catalytic repertoire of Cbl-dependent AdoMet radical enzymes. PMID:28346939

Plasmid linkage of the D-tagatose 6-phosphate pathway in Streptococcus lactis: effect on lactose and galactose metabolism.

PubMed Central

Crow, V L; Davey, G P; Pearce, L E; Thomas, T D

1983-01-01

The three enzymes of the D-tagatose 6-phosphate pathway (galactose 6-phosphate isomerase, D-tagatose 6-phosphate kinase, and tagatose 1,6-diphosphate aldolase) were absent in lactose-negative (Lac-) derivatives of Streptococcus lactis C10, H1, and 133 grown on galactose. The lactose phosphoenolpyruvate-dependent phosphotransferase system and phospho-beta-galactosidase activities were also absent in Lac- derivatives of strains H1 and 133 and were low (possibly absent) in C10 Lac-. In all three Lac- derivatives, low galactose phosphotransferase system activity was found. On galactose, Lac- derivatives grew more slowly (presumably using the Leloir pathway) than the wild-type strains and accumulated high intracellular concentrations of galactose 6-phosphate (up to 49 mM); no intracellular tagatose 1,6-diphosphate was detected. The data suggest that the Lac phenotype is plasmid linked in the three strains studied, with the evidence being more substantial for strain H1. A Lac- derivative of H1 contained a single plasmid (33 megadaltons) which was absent from the Lac- mutant. We suggest that the genes linked to the lactose plasmid in S. lactis are more numerous than previously envisaged, coding for all of the enzymes involved in lactose metabolism from initial transport to the formation of triose phosphates via the D-tagatose 6-phosphate pathway. Images PMID:6294064

Physiological role of glucose-6-phosphate dehydrogenase in cold acclimation of strawberry (Fragaria × ananassa)

NASA Astrophysics Data System (ADS)

Zhang, Yong; Yu, Dingqun; Luo, Ya; Wang, Xiaorong; Chen, Qing; Sun, Bo; Wang, Yan; Liu, Zejing; Tang, Haoru

2018-04-01

In recent years, there has been an increasing interest in study of new resistance mechanism in fruit trees. All these regard the climate change and subsequent fruit production. Glucose-6-phosphate dehydrogenase (G6PDH) catalyzes the first and rate-limiting step of the oxidative pentose phosphate pathway (OPPP), and the expression of this enzyme is related to different biotic and abiotic stresses. Under accumulation of low temperature stress, the significant increase in G6PDH activity was found to be closely correlated to the levels of antioxidant enzymes, malondialdehyde (MDA) contents, sugar contents as well as changes of superoxide (O2•-). It is suggested that the enhancement of cold resistance of strawberry, which induced by cold acclimation, related to the significant increase in G6PDH activity. On one hand, G6PDH activates NADPH oxidase to generate reactive oxygen species (ROS); on the other hand, it may be involved in the activation of antioxidant enzymes, and accelerates many other important NADPH-dependent enzymatic reactions. Then further result in the elevation of membrane stability and cold resistance of strawberry. Interestingly, even though the plants were placed again under a temperature of 25°C for 1 d, the higher cold resistance, enzyme activities and soluble sugar content acquired.

Consequences of a deficit in vitamin B6 biosynthesis de novo for hormone homeostasis and root development in Arabidopsis.

PubMed

Boycheva, Svetlana; Dominguez, Ana; Rolcik, Jakub; Boller, Thomas; Fitzpatrick, Teresa B

2015-01-01

Vitamin B(6) (pyridoxal 5'-phosphate) is an essential cofactor of many metabolic enzymes. Plants biosynthesize the vitamin de novo employing two enzymes, pyridoxine synthase1 (PDX1) and PDX2. In Arabidopsis (Arabidopsis thaliana), there are two catalytically active paralogs of PDX1 (PDX1.1 and PDX1.3) producing the vitamin at comparable rates. Since single mutants are viable but the pdx1.1 pdx1.3 double mutant is lethal, the corresponding enzymes seem redundant. However, the single mutants exhibit substantial phenotypic differences, particularly at the level of root development, with pdx1.3 being more impaired than pdx1.1. Here, we investigate the differential regulation of PDX1.1 and PDX1.3 by identifying factors involved in their disparate phenotypes. Swapped-promoter experiments clarify the presence of distinct regulatory elements in the upstream regions of both genes. Exogenous sucrose (Suc) triggers impaired ethylene production in both mutants but is more severe in pdx1.3 than in pdx1.1. Interestingly, Suc specifically represses PDX1.1 expression, accounting for the stronger vitamin B6 deficit in pdx1.3 compared with pdx1.1. Surprisingly, Suc enhances auxin levels in pdx1.1, whereas the levels are diminished in pdx1.3. In the case of pdx1.3, the previously reported reduced meristem activity combined with the impaired ethylene and auxin levels manifest the specific root developmental defects. Moreover, it is the deficit in ethylene production and/or signaling that triggers this outcome. On the other hand, we hypothesize that it is the increased auxin content of pdx1.1 that is responsible for the root developmental defects observed therein. We conclude that PDX1.1 and PDX1.3 play partially nonredundant roles and are differentially regulated as manifested in disparate root growth impairment morphologies. © 2015 American Society of Plant Biologists. All Rights Reserved.

Consequences of a Deficit in Vitamin B6 Biosynthesis de Novo for Hormone Homeostasis and Root Development in Arabidopsis1[OPEN

PubMed Central

Boycheva, Svetlana; Dominguez, Ana; Rolcik, Jakub; Boller, Thomas; Fitzpatrick, Teresa B.

2015-01-01

Vitamin B6 (pyridoxal 5′-phosphate) is an essential cofactor of many metabolic enzymes. Plants biosynthesize the vitamin de novo employing two enzymes, pyridoxine synthase1 (PDX1) and PDX2. In Arabidopsis (Arabidopsis thaliana), there are two catalytically active paralogs of PDX1 (PDX1.1 and PDX1.3) producing the vitamin at comparable rates. Since single mutants are viable but the pdx1.1 pdx1.3 double mutant is lethal, the corresponding enzymes seem redundant. However, the single mutants exhibit substantial phenotypic differences, particularly at the level of root development, with pdx1.3 being more impaired than pdx1.1. Here, we investigate the differential regulation of PDX1.1 and PDX1.3 by identifying factors involved in their disparate phenotypes. Swapped-promoter experiments clarify the presence of distinct regulatory elements in the upstream regions of both genes. Exogenous sucrose (Suc) triggers impaired ethylene production in both mutants but is more severe in pdx1.3 than in pdx1.1. Interestingly, Suc specifically represses PDX1.1 expression, accounting for the stronger vitamin B6 deficit in pdx1.3 compared with pdx1.1. Surprisingly, Suc enhances auxin levels in pdx1.1, whereas the levels are diminished in pdx1.3. In the case of pdx1.3, the previously reported reduced meristem activity combined with the impaired ethylene and auxin levels manifest the specific root developmental defects. Moreover, it is the deficit in ethylene production and/or signaling that triggers this outcome. On the other hand, we hypothesize that it is the increased auxin content of pdx1.1 that is responsible for the root developmental defects observed therein. We conclude that PDX1.1 and PDX1.3 play partially nonredundant roles and are differentially regulated as manifested in disparate root growth impairment morphologies. PMID:25475669

Enzyme replacement therapy on hypophosphatasia mouse model

PubMed Central

Montaño, Adriana M.; Shimada, Tsutomu; Sly, William S.

2013-01-01

Hypophosphatasia (HPP) is an inborn error of metabolism caused by deficiency of the tissue-nonspecific alkaline phosphatase (TNSALP), resulting in a defect of bone mineralization. Natural substrates for this ectoenzyme accumulate extracellulary including inorganic pyrophosphate (PPi), an inhibitor of mineralization, and pyridoxal 5-phosphate (PLP), a co-factor form of vitamin B6. Enzyme replacement therapy (ERT) for HPP by functional TNSALP is one of the therapeutic options. The C-terminal-anchorless human recombinant TNSALP derived from Chinese hamster ovary cell lines was purified. TNSALP-null mice (Akp2−/−), an infantile model of HPP, were treated from birth using TNSALP and vitamin B6 diet. Long-term efficacy studies of ERT consisted of every 3 days subcutaneous or intravenous injections till 28 days old (dose 20 U/g) and subsequently every 3 days intravenous injections for 6 months (dose 10 U/g). We assessed therapeutic effect by growth and survival rates, fertility, skeletal manifestations, and radiographic and pathological finding. Treated Akp2−/− mice grew normally till 4 weeks and appeared well with a minimum skeletal abnormality as well as absence of epilepsy, compared with untreated mice which died by 3 weeks old. The prognosis of TNSALP-treated Akp2−/− mice was improved substantially: 1) prolonged life span over 6 months, 2) improvement of the growth, and 3) normal fertility. After 6 months of treatment, we found moderate hypomineralization with abnormal proliferative chondrocytes in growth plate and articular cartilage. In conclusion, ERT with human native TNSALP improves substantial clinical manifestations in Akp2−/− mice, suggesting that ERT with anchorless TNSALP is also a potential therapy for HPP. PMID:23978959

Recyclable Thermoresponsive Polymer-Cellulase Bioconjugates for Biomass Depolymerization

PubMed Central

Mackenzie, Katherine J.; Francis, Matthew B.

2013-01-01

Here we report the construction and characterization of a recoverable, thermoresponsive polymer-endoglucanase bioconjugate that matches the activity of unmodified enzymes on insoluble cellulose substrates. Two copolymers exhibiting a thermoresponsive lower critical solution temperature (LCST) were created through the copolymerization of an aminooxy-bearing methacrylamide with N-isopropylacrylamide (NIPAm) or N-isopropylmethacrylamide (NIPMa). The aminooxy group provided a handle through which the LCST was adjusted through small-molecule quenching. This allowed materials with LCSTs ranging from 20.9 °C to 60.5 °C to be readily obtained after polymerization. The thermostable endoglucanase EGPh from the hypothermophilic Pyrococcus horikoshii was transaminated with pyridoxal-5’-phosphate to produce a ketone-bearing protein, which was then site-selectively modified through oxime linkage with benzylalkoxyamine or 5 kDa-poly(ethylene glycol)-alkoxyamine. These modified proteins showed activity comparable to the controls when assayed on an insoluble cellulosic substrate. Two polymer bioconjugates were then constructed using transaminated EGPh and the aminooxy-bearing copolymers. After twelve hours, both bioconjugates produced an equivalent amount of free reducing sugars as the unmodified control using insoluble cellulose as a substrate. The recycling ability of the NIPAm copolymer-EGPh conjugate was determined through three rounds of activity, maintaining over 60% activity after two cycles of reuse and affording significantly more soluble carbohydrates than unmodified enzyme alone. When assayed on acid-pretreated Miscanthus, this bioconjugate increased the amount of reducing sugars by 2.8-fold over three rounds of activity. The synthetic strategy of this bioconjugate allows the LCST of the material to be changed readily from a common stock of copolymer and the method of attachment is applicable to a variety of proteins, enabling the same approach to be amenable to thermophile-derived cellulases or to the separation of multiple species using polymers with different recovery temperatures. PMID:23270527

The Production and Utilization of GDP-glucose in the Biosynthesis of Trehalose 6-Phosphate by Streptomyces venezuelae.

PubMed

Asención Diez, Matías D; Miah, Farzana; Stevenson, Clare E M; Lawson, David M; Iglesias, Alberto A; Bornemann, Stephen

2017-01-20

Trehalose-6-phosphate synthase OtsA from streptomycetes is unusual in that it uses GDP-glucose as the donor substrate rather than the more commonly used UDP-glucose. We now confirm that OtsA from Streptomyces venezuelae has such a preference for GDP-glucose and can utilize ADP-glucose to some extent too. A crystal structure of the enzyme shows that it shares twin Rossmann-like domains with the UDP-glucose-specific OtsA from Escherichia coli However, it is structurally more similar to Streptomyces hygroscopicus VldE, a GDP-valienol-dependent pseudoglycosyltransferase enzyme. Comparison of the donor binding sites reveals that the amino acids associated with the binding of diphosphoribose are almost all identical in these three enzymes. By contrast, the amino acids associated with binding guanine in VldE (Asn, Thr, and Val) are similar in S. venezuelae OtsA (Asp, Ser, and Phe, respectively) but not conserved in E. coli OtsA (His, Leu, and Asp, respectively), providing a rationale for the purine base specificity of S. venezuelae OtsA. To establish which donor is used in vivo, we generated an otsA null mutant in S. venezuelae The mutant had a cell density-dependent growth phenotype and accumulated galactose 1-phosphate, glucose 1-phosphate, and GDP-glucose when grown on galactose. To determine how the GDP-glucose is generated, we characterized three candidate GDP-glucose pyrophosphorylases. SVEN_3027 is a UDP-glucose pyrophosphorylase, SVEN_3972 is an unusual ITP-mannose pyrophosphorylase, and SVEN_2781 is a pyrophosphorylase that is capable of generating GDP-glucose as well as GDP-mannose. We have therefore established how S. venezuelae can make and utilize GDP-glucose in the biosynthesis of trehalose 6-phosphate. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Pyridoxine-Dependent Epilepsy: An Expanding Clinical Spectrum.

PubMed

van Karnebeek, Clara D M; Tiebout, Sylvia A; Niermeijer, Jikkemien; Poll-The, Bwee Tien; Ghani, Aisha; Coughlin, Curtis R; Van Hove, Johan L K; Richter, Jost Wigand; Christen, Hans Juergen; Gallagher, Renata; Hartmann, Hans; Stockler-Ipsiroglu, Sylvia

2016-06-01

Pyridoxine-dependent epilepsy is a rare autosomal recessive epileptic encephalopathy caused by antiquitin (ALDH7A1) deficiency. In spite of adequate seizure control, 75% of patients suffer intellectual developmental disability. Antiquitin deficiency affects lysine catabolism resulting in accumulation of α-aminoadipic semialdehyde/pyrroline 6' carboxylate and pipecolic acid. Beside neonatal refractory epileptic encephalopathy, numerous neurological manifestations and metabolic/biochemical findings have been reported. We present a phenotypic spectrum of antiquitin deficiency based on a literature review (2006 to 2015) of reports (n = 49) describing the clinical presentation of confirmed patients (n > 200) and a further six patient vignettes. Possible presentations include perinatal asphyxia; neonatal withdrawal syndrome; sepsis; enterocolitis; hypoglycemia; neuroimaging abnormalities (corpus callosum and cerebellar abnormalities, hemorrhage, white matter lesions); biochemical abnormalities (lactic acidosis, electrolyte disturbances, neurotransmitter abnormalities); and seizure response to pyridoxine, pyridoxal-phosphate, and folinic acid dietary interventions. The phenotypic spectrum of pyridoxine-dependent epilepsy is wide, including a myriad of neurological and systemic symptoms. Its hallmark feature is refractory seizures during the first year of life. Given its amenability to treatment with lysine-lowering strategies in addition to pyridoxine supplementation for optimal seizure control and developmental outcomes, early diagnosis of pyridoxine-dependent epilepsy is essential. All infants presenting with unexplained seizures should be screened for antiquitin deficiency by determination of α-aminoadipic semialdehyde/pyrroline 6' carboxylate (in urine, plasma or cerebrospinal fluid) and ALDH7A1 molecular analysis. Copyright © 2016 Elsevier Inc. All rights reserved.

Fructose Degradation in the Haloarchaeon Haloferax volcanii Involves a Bacterial Type Phosphoenolpyruvate-Dependent Phosphotransferase System, Fructose-1-Phosphate Kinase, and Class II Fructose-1,6-Bisphosphate Aldolase

PubMed Central

Pickl, Andreas; Johnsen, Ulrike

2012-01-01

The halophilic archaeon Haloferax volcanii utilizes fructose as a sole carbon and energy source. Genes and enzymes involved in fructose uptake and degradation were identified by transcriptional analyses, deletion mutant experiments, and enzyme characterization. During growth on fructose, the gene cluster HVO_1495 to HVO_1499, encoding homologs of the five bacterial phosphotransferase system (PTS) components enzyme IIB (EIIB), enzyme I (EI), histidine protein (HPr), EIIA, and EIIC, was highly upregulated as a cotranscript. The in-frame deletion of HVO_1499, designated ptfC (ptf stands for phosphotransferase system for fructose) and encoding the putative fructose-specific membrane component EIIC, resulted in a loss of growth on fructose, which could be recovered by complementation in trans. Transcripts of HVO_1500 (pfkB) and HVO_1494 (fba), encoding putative fructose-1-phosphate kinase (1-PFK) and fructose-1,6-bisphosphate aldolase (FBA), respectively, as well as 1-PFK and FBA activities were specifically upregulated in fructose-grown cells. pfkB and fba knockout mutants did not grow on fructose, whereas growth on glucose was not inhibited, indicating the functional involvement of both enzymes in fructose catabolism. Recombinant 1-PFK and FBA obtained after homologous overexpression were characterized as having kinetic properties indicative of functional 1-PFK and a class II type FBA. From these data, we conclude that fructose uptake in H. volcanii involves a fructose-specific PTS generating fructose-1-phosphate, which is further converted via fructose-1,6-bisphosphate to triose phosphates by 1-PFK and FBA. This is the first report of the functional involvement of a bacterial-like PTS and of class II FBA in the sugar metabolism of archaea. PMID:22493022

Multiple regulatory elements for the glpA operon encoding anaerobic glycerol-3-phosphate dehydrogenase and the glpD operon encoding aerobic glycerol-3-phosphate dehydrogenase in Escherichia coli: further characterization of respiratory control.

PubMed

Iuchi, S; Cole, S T; Lin, E C

1990-01-01

In Escherichia coli, sn-glycerol-3-phosphate can be oxidized by two different flavo-dehydrogenases, an anaerobic enzyme encoded by the glpACB operon and an aerobic enzyme encoded by the glpD operon. These two operons belong to the glp regulon specifying the utilization of glycerol, sn-glycerol-3-phosphate, and glycerophosphodiesters. In glpR mutant cells grown under conditions of low catabolite repression, the glpA operon is best expressed anaerobically with fumarate as the exogenous electron acceptor, whereas the glpD operon is best expressed aerobically. Increased anaerobic expression of glpA is dependent on the fnr product, a pleiotropic activator of genes involved in anaerobic respiration. In this study we found that the expression of a glpA1(Oxr) (oxygen-resistant) mutant operon, selected for increased aerobic expression, became less dependent on the FNR protein but more dependent on the cyclic AMP-catabolite gene activator protein complex mediating catabolite repression. Despite the increased aerobic expression of glpA1(Oxr), a twofold aerobic repressibility persisted. Moreover, anaerobic repression by nitrate respiration remained normal. Thus, there seems to exist a redox control apart from the FNR-mediated one. We also showed that the anaerobic repression of the glpD operon was fully relieved by mutations in either arcA (encoding a presumptive DNA recognition protein) or arcB (encoding a presumptive redox sensor protein). The arc system is known to mediate pleiotropic control of genes of aerobic function.

Two continuous coupled assays for ornithine-δ-aminotransferase.

PubMed

Juncosa, Jose I; Lee, Hyunbeom; Silverman, Richard B

2013-09-15

We have developed two new continuous coupled assays for ornithine-δ-aminotransferase (OAT) that are more sensitive than previous methods, measure activity in real time, and can be carried out in multiwell plates for convenience and high throughput. The first assay is based on the reduction of Δ(1)-pyrroline-5-carboxylate (P5C), generated from ornithine by OAT, using human pyrroline 5-carboxylate reductase 1 (PYCR1), which results in the concomitant oxidation of NADH (nicotinamide adenine dinucleotide, reduced form) to NAD⁺ (nicotinamide adenine dinucleotide, oxidized form). This procedure was found to be three times more sensitive than previous methods and is suitable for the study of small molecules as inhibitors or inactivators of OAT or as a method to determine OAT activity in unknown samples. The second method involves the detection of L-glutamate, produced during the regeneration of the cofactor pyridoxal 5'-phosphate (PLP) of OAT by an unamplified modification of the commercially available Amplex Red L-glutamate detection kit (Life Technologies). This assay is recommended for the determination of the substrate activity of small molecules against OAT; measuring the transformation of L-ornithine at high concentrations by this assay is complicated by the fact that it also acts as a substrate for the L-glutamate oxidase (GluOx) reporter enzyme. Copyright © 2013 Elsevier Inc. All rights reserved.

Decreased serum pyridoxal levels in schizophrenia: meta-analysis and Mendelian randomization analysis

PubMed Central

Tomioka, Yukiko; Kinoshita, Makoto; Umehara, Hidehiro; Watanabe, Shin-ya; Nakataki, Masahito; Iwayama, Yoshimi; Toyota, Tomoko; Ikeda, Masashi; Yamamori, Hidenaga; Shimodera, Shinji; Tajima, Atsushi; Hashimoto, Ryota; Iwata, Nakao; Yoshikawa, Takeo; Ohmori, Tetsuro

2018-01-01

Background Alterations in one-carbon metabolism have been associated with schizophrenia, and vitamin B6 is one of the key components in this pathway. Methods We first conducted a case–control study of serum pyridoxal levels and schizophrenia in a large Japanese cohort (n = 1276). Subsequently, we conducted a meta-analysis of association studies (n = 2125). Second, we investigated whether rs4654748, which was identified in a genome-wide association study as a vitamin B6-related single nucleotide polymorphism, was genetically implicated in patients with schizophrenia in the Japanese population (n = 10 689). Finally, we assessed the effect of serum pyridoxal levels on schizophrenia risk using a Mendelian randomization (MR) approach. Results Serum pyridoxal levels were significantly lower in patients with schizophrenia than in controls, not only in our cohort, but also in the pooled data set of the meta-analysis of association studies (standardized mean difference −0.48, 95% confidence interval [CI] −0.57 to −0.39, p = 9.8 × 10−24). We failed to find a significant association between rs4654748 and schizophrenia. Furthermore, an MR analysis failed to find a causal relationship between pyridoxal levels and schizophrenia risk (odds ratio 0.99, 95% CI 0.65–1.51, p = 0.96). Limitations Food consumption and medications may have affected serum pyridoxal levels in our cross-sectional study. Sample size, number of instrumental variables and substantial heterogeneity among patients with schizophrenia are limitations of an MR analysis. Conclusion We found decreased serum pyridoxal levels in patients with schizophrenia in this observational study. However, we failed to obtain data supporting a causal relationship between pyridoxal levels and schizophrenia risk using the MR approach. PMID:29688875

Glucose-6-phosphate dehydrogenase Buenos Aires: a novel de novo missense mutation associated with severe enzyme deficiency.

PubMed

Minucci, Angelo; Concolino, Paola; Vendittelli, Francesca; Giardina, Bruno; Zuppi, Cecilia; Capoluongo, Ettore

2008-06-01

: Glucose 6-phosphate dehydrogenase (G6PD) catalyzes the first committed steps in the pentose phosphate pathway: the generation of NADPH by this enzyme is essential for protection against oxidative stress. The human enzyme is in a dimer<-->tetramer equilibrium and its stability depends on NADP(+) concentration. Herein, we report a case of a symptomatic baby affected by severe deficiency of G6PD activity due to a novel de novo genetic mutation (g1465C>T) in the thirteenth exon of its gene. : Clinical, biochemical and genetic evaluations of the affected baby and his mother were performed. : We found the g1465C>T novel mutation, in the thirteenth exon of G6PD gene (named "G6PD Buenos Aires variant"). This g1465C>T mutation produce a P489S substitution at protein level. The P489S mutation was absent in his mother, suggesting that G6PD Buenos Aires resulted from a de novo mutation. : The absence of mosaicism in the baby's DNA (from saliva and blood samples) suggests that a de novo mutation event may occur in the very early stages in embryogenesis or in the mother's germ cell lines.

Comparison of blood aminotransferase methods for assessment of myopathy and hepatopathy in Florida manatees (Trichechus manatus latirostris)

USGS Publications Warehouse

Harr, K.E.; Allison, K.; Bonde, R.K.; Murphy, D.; Harvey, J.W.

2008-01-01

Muscle injury is common in Florida manatees (Trichechus manatus latirostris). Plasma aspartate amino-transferase (AST) is frequently used to assess muscular damage in capture myopathy and traumatic injury. Therefore, accurate measurement of AST and alanine aminotransferase (ALT) is important in managed, free-ranging animals, as well as in those rehabilitating from injury. Activities of these enzymes, however, are usually not increased in manatees with either acute or chronic muscle damage, despite marked increases in plasma creatine kinase activity. It is hypothesized that this absence of response is due to apoenzymes in the blood not detected by commonly used veterinary assays. Addition of coenzyme pyridoxal-5-phosphate (P5P or vitamin B6) should, therefore, result in higher measured enzyme activities. The objective of this study was to determine the most accurate, precise, and diagnostically useful method for aminotransferase measurement in manatees that can be used in veterinary practices and diagnostic laboratories. Additionally, appropriate collection and storage techniques were assessed. The use of an optimized commercial wet chemical assay with 100 ??mol P5P resulted in a positive bias of measured enzyme activities in a healthy population of animals. However, AST and ALT were still much lower than that typically observed in domestic animals and should not be used alone in the assessment of capture myopathy and muscular trauma. Additionally, the dry chemistry analyzer, typically used in clinics, reported significantly higher and less precise AST and ALT activities with poor correlation to those measured with wet chemical methods found in diagnostic laboratories. Therefore, these results cannot be clinically compared. Overall, the optimized wet chemical method was the most precise and diagnostically useful measurement of aminotransferase in samples. Additionally, there was a statistically significant difference between paired serum and plasma measurement, indicating that separate reference intervals should be established for serum and plasma. Finally, storage of these enzymes at -70??C for 1 mo resulted in up to a 25% decrease in enzymatic activity in manatee plasma. Copyright 2008 by American Association of Zoo Veterinarians.

Comparison of blood aminotransferase methods for assessment of myopathy and hepatopathy in Florida manatees (Trichechus manatus latirostris).

PubMed

Harr, Kendal E; Allison, Kathryn; Bonde, Robert K; Murphy, David; Harvey, John W

2008-06-01

Muscle injury is common in Florida manatees (Trichechus manatus latirostris). Plasma aspartate aminotransferase (AST) is frequently used to assess muscular damage in capture myopathy and traumatic injury. Therefore, accurate measurement of AST and alanine aminotransferase (ALT) is important in managed, free-ranging animals, as well as in those rehabilitating from injury. Activities of these enzymes, however, are usually not increased in manatees with either acute or chronic muscle damage, despite marked increases in plasma creatine kinase activity. It is hypothesized that this absence of response is due to apoenzymes in the blood not detected by commonly used veterinary assays. Addition of coenzyme pyridoxal-5-phosphate (P5P or vitamin B6) should, therefore, result in higher measured enzyme activities. The objective of this study was to determine the most accurate, precise, and diagnostically useful method for aminotransferase measurement in manatees that can be used in veterinary practices and diagnostic laboratories. Additionally, appropriate collection and storage techniques were assessed. The use of an optimized commercial wet chemical assay with 100 micromol P5P resulted in a positive bias of measured enzyme activities in a healthy population of animals. However, AST and ALT were still much lower than that typically observed in domestic animals and should not be used alone in the assessment of capture myopathy and muscular trauma. Additionally, the dry chemistry analyzer, typically used in clinics, reported significantly higher and less precise AST and ALT activities with poor correlation to those measured with wet chemical methods found in diagnostic laboratories. Therefore, these results cannot be clinically compared. Overall, the optimized wet chemical method was the most precise and diagnostically useful measurement of aminotransferase in samples. Additionally, there was a statistically significant difference between paired serum and plasma measurement, indicating that separate reference intervals should be established for serum and plasma. Finally, storage of these enzymes at -70 degrees C for 1 mo resulted in up to a 25% decrease in enzymatic activity in manatee plasma.

Adenine phosphoribosyltransferase from Sulfolobus solfataricus is an enzyme with unusual kinetic properties and a crystal structure that suggests it evolved from a 6-oxopurine phosphoribosyltransferase.

PubMed

Jensen, Kaj Frank; Hansen, Michael Riis; Jensen, Kristine Steen; Christoffersen, Stig; Poulsen, Jens-Christian Navarro; Mølgaard, Anne; Kadziola, Anders

2015-04-14

The adenine phosphoribosyltransferase (APRTase) encoded by the open reading frame SSO2342 of Sulfolobus solfataricus P2 was subjected to crystallographic, kinetic, and ligand binding analyses. The enzyme forms dimers in solution and in the crystals, and binds one molecule of the reactants 5-phosphoribosyl-α-1-pyrophosphate (PRPP) and adenine or the product adenosine monophosphate (AMP) or the inhibitor adenosine diphosphate (ADP) in each active site. The individual subunit adopts an overall structure that resembles a 6-oxopurine phosphoribosyltransferase (PRTase) more than known APRTases implying that APRT functionality in Crenarchaeotae has its evolutionary origin in this family of PRTases. Only the N-terminal two-thirds of the polypeptide chain folds as a traditional type I PRTase with a five-stranded β-sheet surrounded by helices. The C-terminal third adopts an unusual three-helix bundle structure that together with the nucleobase-binding loop undergoes a conformational change upon binding of adenine and phosphate resulting in a slight contraction of the active site. The inhibitor ADP binds like the product AMP with both the α- and β-phosphates occupying the 5'-phosphoribosyl binding site. The enzyme shows activity over a wide pH range, and the kinetic and ligand binding properties depend on both pH and the presence/absence of phosphate in the buffers. A slow hydrolysis of PRPP to ribose 5-phosphate and pyrophosphate, catalyzed by the enzyme, may be facilitated by elements in the C-terminal three-helix bundle part of the protein.

Effects of folic acid supplementation to rations differing in the concentrate to roughage ratio on ruminal fermentation, nutrient flow at the duodenum, and on serum and milk variables of dairy cows.

PubMed

Ragaller, Veronika; Lebzien, Peter; Bigalke, Wiebke; Südekum, Karl-Heinz; Hüthera, Liane; Flachowsky, Gerhard

2010-12-01

The present study was undertaken to determine the effects of dietary folic acid (FOL) supplementation on ruminal fermentation, duodenal nutrient flow, serum and milk variables, and on B-vitamin concentration in serum. The study was divided into two experiments: in Exp. 1 the forage to concentrate (F:C) ratio of the diet (DM basis) was 34:66 (high concentrate, HC), while in Exp. 2 the F:C ratio was 66:34 (high forage, HF). In addition, the cows received 0 or 1 g FOL/d. In Exp. 1, two German Holstein cows equipped with cannulas in the dorsal sac of the rumen and in the proximal duodenum were dry and five were lactating (186 +/- 144 days in milk); in Exp. 2 four cows were dry and four were lactating (165 +/- 57 days in milk). In cows fed the HC diet, FOL supplementation decreased the ruminally-fermented organic matter. Thus, less energy was available for ruminal microorganisms, which resulted in a reduced microbial crude protein flow at the duodenum. Feeding the HF diet, FOL supplementation only increased the apparent ruminal digestibility of acid detergent fibre (ADF). With the HF diet, FOL had no influence on the serum levels of glucose, non-esterified fatty acids, beta-hydroxybutyrate, urea, thiamine, riboflavin, pyridoxal-5'-phosphate, pyridoxic acid, pyridoxal, pyridoxine, pyridoxamine, pantothenic acid, nicotinamide or nicotinic acid, whereas supplementing FOL to the HC diet lowered the serum glucose and riboflavin levels. In both experiments, the supplementation of FOL had no effects on milk composition. Folic acid supplementation to both diets increased the concentrations of serum 5-methyl-tetrahydrofolate. However, no beneficial effects to dairy cows were obvious. Therefore, to achieve certain results, studies with a higher number of non-fistulated cows would be necessary.

Activation of pollen tube callose synthase by detergents. Evidence for different mechanisms of action.

PubMed Central

Li, H; Bacic, A; Read, S M

1997-01-01

In pollen tubes of Nicotiana alata, a membrane-bound, Ca(2+)-independent callose synthase (CalS) is responsible for the biosynthesis of the (1,3)-beta-glucan backbone of callose, the main cell wall component. Digitonin increases CalS activity 3- to 4-fold over a wide range of concentrations, increasing the maximum initial velocity without altering the Michaelis constant for UDP-glucose. The CalS activity that requires digitonin for assay (the latent CalS activity) is not inhibited by the membrane-impermeant, active site-directed reagent UDP-pyridoxal when the reaction is conducted in the absence of digitonin. This is consistent with digitonin increasing CalS activity by the permeabilization of membrane vesicles. A second group of detergents, including 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate (CHAPS), Zwittergent 3-16, and 1-alpha-lysolecithin, activate pollen tube CalS 10- to 15-fold, but only over a narrow range of concentrations just below their respective critical micellar concentrations. This activation could not be attributed to any particular chemical feature of these detergents. CHAPS increases maximum initial velocity and decreases the Michaelis constant for UDP-glucose and activates CalS even in the presence of permeabilizing concentrations of digitonin. Inhibition studies with UDP-pyridoxal indicate that activation by CHAPS occurs by recruitment of previously inactive CalS molecules to the pool of active enzyme. The activation of pollen tube CalS by these detergents therefore resembles activation of the enzyme by trypsin. PMID:9276948

An ensemble of structures of Burkholderia pseudomallei 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Davies, Douglas R.; Staker, Bart L.; Abendroth, Jan A.

2011-12-07

Burkholderia pseudomallei is a soil-dwelling bacterium endemic to Southeast Asia and Northern Australia. Burkholderia is responsible for melioidosis, a serious infection of the skin. The enzyme 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase (PGAM) catalyzes the interconversion of 3-phosphoglycerate and 2-phosphoglycerate, a key step in the glycolytic pathway. As such it is an extensively studied enzyme and X-ray crystal structures of PGAM enzymes from multiple species have been elucidated. Vanadate is a phosphate mimic that is a powerful tool for studying enzymatic mechanisms in phosphoryl-transfer enzymes such as phosphoglycerate mutase. However, to date no X-ray crystal structures of phosphoglycerate mutase have been solved withmore » vanadate acting as a substrate mimic. Here, two vanadate complexes together with an ensemble of substrate and fragment-bound structures that provide a comprehensive picture of the function of the Burkholderia enzyme are reported.« less

Biogenesis and Metabolic Maintenance of Rubisco.

PubMed

Bracher, Andreas; Whitney, Spencer M; Hartl, F Ulrich; Hayer-Hartl, Manajit

2017-04-28

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) mediates the fixation of atmospheric CO 2 in photosynthesis by catalyzing the carboxylation of the 5-carbon sugar ribulose-1,5-bisphosphate (RuBP). Rubisco is a remarkably inefficient enzyme, fixing only 2-10 CO 2 molecules per second. Efforts to increase crop yields by bioengineering Rubisco remain unsuccessful, owing in part to the complex cellular machinery required for Rubisco biogenesis and metabolic maintenance. The large subunit of Rubisco requires the chaperonin system for folding, and recent studies have shown that assembly of hexadecameric Rubisco is mediated by specific assembly chaperones. Moreover, Rubisco function can be inhibited by a range of sugar-phosphate ligands, including RuBP. Metabolic repair depends on remodeling of Rubisco by the ATP-dependent Rubisco activase and hydrolysis of inhibitory sugar phosphates by specific phosphatases. Here, we review our present understanding of the structure and function of these auxiliary factors and their utilization in efforts to engineer more catalytically efficient Rubisco enzymes.

The characteristics of pyrophosphate: D-fructose-6-phosphate 1-phosphotransferases from Sansevieria trifasciata leaves and Phaseolus coccineus stems.

PubMed

Kowalczyk, S

1987-01-01

Three different molecular forms of pyrophosphate-dependent phosphofructokinase have been isolated: one from Sansevieria trifasciata leaves and two from Phaseolus coccineus stems. The form isolated from S. trifasciata has the molecular weight of about 115,000. The apparent molecular weights for the two forms from mung bean were approximately 220,000 and 450,000. All three forms have the same pH optima, an absolute requirement for Mg2+ ions both in the forward and reverse reaction, but differ in their sensitivity toward fructose 2,6-bisphosphate. Kinetic properties of the partially purified enzymes have been investigated in the presence and absence of fructose 2,6-bisphosphate. Pyrophosphate-dependent phosphofructokinase from S. trifasciata exhibited hyperbolic kinetics with all substrates tested. The saturation curves of the enzyme (form A) from mung bean for pyrophosphate, fructose 6-phosphate and fructose 1,6-bisphosphate were sigmoidal in the absence of fructose 2,6-bisphosphate. In the presence of fructose 2,6-bisphosphate these kinetics became hyperbolic.

Use of Chimeras, Point Mutants, and Molecular Modeling to Map the Antagonist-binding Site of 4,4′,4″,4‴-(Carbonylbis-(imino-5,1,3-benzenetriylbis(carbonylimino)))tetrakisbenzene-1,3-disulfonic Acid (NF449) at P2X1 Receptors for ATP*

PubMed Central

Farmer, Louise K.; Schmid, Ralf; Evans, Richard J.

2015-01-01

P2X receptor subtype-selective antagonists are promising candidates for treatment of a range of pathophysiological conditions. However, in contrast to high resolution structural understanding of agonist action in the receptors, comparatively little is known about the molecular basis of antagonist binding. We have generated chimeras and point mutations in the extracellular ligand-binding loop of the human P2X1 receptor, which is inhibited by NF449, suramin, and pyridoxal-phosphate-6-azophenyl-2,4-disulfonate, with residues from the rat P2X4 receptor, which is insensitive to these antagonists. There was little or no effect on sensitivity to suramin and pyridoxal-phosphate-6-azophenyl-2,4-disulfonate in chimeric P2X1/4 receptors, indicating that a significant number of residues required for binding of these antagonists are present in the P2X4 receptor. Sensitivity to the P2X1 receptor-selective antagonist NF449 was reduced by ∼60- and ∼135-fold in chimeras replacing the cysteine-rich head, and the dorsal fin region below it in the adjacent subunit, respectively. Point mutants identified the importance of four positively charged residues at the base of the cysteine-rich head and two variant residues in the dorsal fin for high affinity NF449 binding. These six residues were used as the starting area for molecular docking. The four best potential NF449-binding poses were then discriminated by correspondence with the mutagenesis data and an additional mutant to validate the binding of one lobe of NF449 within the core conserved ATP-binding pocket and the other lobes coordinated by positive charge on the cysteine-rich head region and residues in the adjacent dorsal fin. PMID:25425641

Use of chimeras, point mutants, and molecular modeling to map the antagonist-binding site of 4,4',4″,4‴-(carbonylbis-(imino-5,1,3-benzenetriylbis(carbonylimino)))tetrakisbenzene-1,3-disulfonic acid (NF449) at P2X1 receptors for ATP.

PubMed

Farmer, Louise K; Schmid, Ralf; Evans, Richard J

2015-01-16

P2X receptor subtype-selective antagonists are promising candidates for treatment of a range of pathophysiological conditions. However, in contrast to high resolution structural understanding of agonist action in the receptors, comparatively little is known about the molecular basis of antagonist binding. We have generated chimeras and point mutations in the extracellular ligand-binding loop of the human P2X1 receptor, which is inhibited by NF449, suramin, and pyridoxal-phosphate-6-azophenyl-2,4-disulfonate, with residues from the rat P2X4 receptor, which is insensitive to these antagonists. There was little or no effect on sensitivity to suramin and pyridoxal-phosphate-6-azophenyl-2,4-disulfonate in chimeric P2X1/4 receptors, indicating that a significant number of residues required for binding of these antagonists are present in the P2X4 receptor. Sensitivity to the P2X1 receptor-selective antagonist NF449 was reduced by ∼60- and ∼135-fold in chimeras replacing the cysteine-rich head, and the dorsal fin region below it in the adjacent subunit, respectively. Point mutants identified the importance of four positively charged residues at the base of the cysteine-rich head and two variant residues in the dorsal fin for high affinity NF449 binding. These six residues were used as the starting area for molecular docking. The four best potential NF449-binding poses were then discriminated by correspondence with the mutagenesis data and an additional mutant to validate the binding of one lobe of NF449 within the core conserved ATP-binding pocket and the other lobes coordinated by positive charge on the cysteine-rich head region and residues in the adjacent dorsal fin. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Biochemical characterization of rice trehalose-6-phosphate phosphatases supports distinctive functions of these plant enzymes.

PubMed

Shima, Shuhei; Matsui, Hirokazu; Tahara, Satoshi; Imai, Ryozo

2007-03-01

Substantial levels of trehalose accumulate in bacteria, fungi, and invertebrates, where it serves as a storage carbohydrate or as a protectant against environmental stresses. In higher plants, trehalose is detected at fairly low levels; therefore, a regulatory or signaling function has been proposed for this molecule. In many organisms, trehalose-6-phosphate phosphatase is the enzyme governing the final step of trehalose biosynthesis. Here we report that OsTPP1 and OsTPP2 are the two major trehalose-6-phosphate phosphatase genes expressed in vegetative tissues of rice. Similar to results obtained from our previous OsTPP1 study, complementation analysis of a yeast trehalose-6-phosphate phosphatase mutant and activity measurement of the recombinant protein demonstrated that OsTPP2 encodes a functional trehalose-6-phosphate phosphatase enzyme. OsTPP2 expression is transiently induced in response to chilling and other abiotic stresses. Enzymatic characterization of recombinant OsTPP1 and OsTPP2 revealed stringent substrate specificity for trehalose 6-phosphate and about 10 times lower K(m) values for trehalose 6-phosphate as compared with trehalose-6-phosphate phosphatase enzymes from microorganisms. OsTPP1 and OsTPP2 also clearly contrasted with microbial enzymes, in that they are generally unstable, almost completely losing activity when subjected to heat treatment at 50 degrees C for 4 min. These characteristics of rice trehalose-6-phosphate phosphatase enzymes are consistent with very low cellular substrate concentration and tightly regulated gene expression. These data also support a plant-specific function of trehalose biosynthesis in response to environmental stresses.

Molecular Clone and Expression of a NAD+-Dependent Glycerol-3-Phosphate Dehydrogenase Isozyme Gene from the Halotolerant alga Dunaliella salina

PubMed Central

Cai, Ma; He, Li-Hong; Yu, Tu-Yuan

2013-01-01

Glycerol is an important osmotically compatible solute in Dunaliella. Glycerol-3-phosphate dehydrogenase (G3PDH) is a key enzyme in the pathway of glycerol synthesis, which converts dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate. Generally, the glycerol-DHAP cycle pathway, which is driven by G3PDH, is considered as the rate-limiting enzyme to regulate the glycerol level under osmotic shocks. Considering the peculiarity in osmoregulation, the cDNA of a NAD+-dependent G3PDH was isolated from D. salina using RACE and RT-PCR approaches in this study. Results indicated that the length of the cDNA sequence of G3PDH was 2,100 bp encoding a 699 amino acid deduced polypeptide whose computational molecular weight was 76.6 kDa. Conserved domain analysis revealed that the G3PDH protein has two independent functional domains, SerB and G3PDH domains. It was predicted that the G3PDH was a nonsecretory protein and may be located in the chloroplast of D. salina. Phylogenetic analysis demonstrated that the D. salina G3PDH had a closer relationship with the G3PDHs from the Dunaliella genus than with those from other species. In addition, the cDNA was subsequently subcloned in the pET-32a(+) vector and was transformed into E. coli strain BL21 (DE3), a expression protein with 100 kDa was identified, which was consistent with the theoretical value. PMID:23626797

Adaptation of red cell enzymes and intermediates in metabolic disorders.

PubMed

Goebel, K M; Goebel, F D; Neitzert, A; Hausmann, L; Schneider, J

1975-01-01

The metabolic activity of the red cell glycolytic pathway hexose monophosphate shunt (HMP) with dependent glutathione system was studied in patients with hyperthyroidism (n = 10), hyperlipoproteinemia (n = 16), hypoglycemia (n = 25) and hyperglycemia (n = 23). In uncontrolled diabetics and patients with hyperthyroidism the mean value of glucose phosphate isomerase (GPI), glucose-6-phosphate dehydrogenase (G-6-PD), glutathione reductase (GR) was increased, whereas these enzyme activities were reduced in patients with hypoglycemia. Apart from a few values of hexokinase (HK) which were lower than normal the results in hyperlipoproteinemia patients remained essentially unchanged, including the intermediates such as 2,3-diphosphoglycerate (2,3-DPG), adenosine triphosphate (ATP) and reduced glutathione (GSH). While increased rates of 2,3-DPG and ATP in hypoglycemia patients were obtained, these substrates were markedly reduced in diabetics.

Structural insights into the catalytic mechanism of cysteine (hydroxyl) lyase from the hydrogen sulfide-producing oral pathogen, Fusobacterium nucleatum.

PubMed

Kezuka, Yuichiro; Ishida, Tetsuo; Yoshida, Yasuo; Nonaka, Takamasa

2018-02-16

Hydrogen sulfide (H 2 S) plays important roles in the pathogenesis of periodontitis. Oral pathogens typically produce H 2 S from l-cysteine in addition to pyruvate and [Formula: see text] However, fn1055 from Fusobacterium nucleatum subsp. nucleatum ATCC 25586 encodes a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the production of H 2 S and l-serine from l-cysteine and H 2 O, an unusual cysteine (hydroxyl) lyase reaction (β-replacement reaction). To reveal the reaction mechanism, the crystal structure of substrate-free Fn1055 was determined. Based on this structure, a model of the l-cysteine-PLP Schiff base suggested that the thiol group forms hydrogen bonds with Asp 232 and Ser 74 , and the substrate α-carboxylate interacts with Thr 73 and Gln 147 Asp 232 is a unique residue to Fn1055 and its substitution to asparagine (D232N) resulted in almost complete loss of β-replacement activity. The D232N structure obtained in the presence of l-cysteine contained the α-aminoacrylate-PLP Schiff base in the active site, indicating that Asp 232 is essential for the addition of water to the α-aminoacrylate to produce the l-serine-PLP Schiff base. Rapid-scan stopped-flow kinetic analyses showed an accumulation of the α-aminoacrylate intermediate during the reaction cycle, suggesting that water addition mediated by Asp 232 is the rate-limiting step. In contrast, mutants containing substitutions of other active-site residues (Ser 74 , Thr 73 , and Gln 147 ) exhibited reduced β-replacement activity by more than 100-fold. Finally, based on the structural and biochemical analyses, we propose a mechanism of the cysteine (hydroxyl) lyase reaction by Fn1055. The present study leads to elucidation of the H 2 S-producing mechanism in F. nucleatum . © 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

The galactose-induced decrease in phosphate levels leads to toxicity in yeast models of galactosemia.

PubMed

Machado, Caio M; De-Souza, Evandro A; De-Queiroz, Ana Luiza F V; Pimentel, Felipe S A; Silva, Guilherme F S; Gomes, Fabio M; Montero-Lomelí, Mónica; Masuda, Claudio A

2017-06-01

Classic galactosemia is an inborn error of metabolism caused by deleterious mutations in the GALT gene. A number of evidences indicate that the galactose-1-phosphate accumulation observed in patient cells is a cause of toxicity in this disease. Nevertheless, the consequent molecular events caused by the galactose-1-phosphate accumulation remain elusive. Here we show that intracellular inorganic phosphate levels decreased when yeast models of classic galactosemia were exposed to galactose. The decrease in phosphate levels is probably due to the trapping of phosphate in the accumulated galactose-1-phosphate since the deletion of the galactokinase encoding gene GAL1 suppressed this phenotype. Galactose-induced phosphate depletion caused an increase in glycogen content, an expected result since glycogen breakdown by the enzyme glycogen phosphorylase is dependent on inorganic phosphate. Accordingly, an increase in intracellular phosphate levels suppressed the galactose effect on glycogen content and conferred galactose tolerance to yeast models of galactosemia. These results support the hypothesis that the galactose-induced decrease in phosphate levels leads to toxicity in galactosemia and opens new possibilities for the development of better treatments for this disease. Copyright © 2017 Elsevier B.V. All rights reserved.

Behavior of soluble and immobilized acid phosphatase in hydro-organic media.

PubMed

Wan, H; Horvath, C

1975-11-20

The hydrolysis of p-nitrophenyl phosphate by wheat germ acid phosphatase (orthophosphoric monoester phosphohydrolase, EC 3.1.3.2) has been investigated in mixtures of aqueous buffers with acetone, dioxane and acetonitrile. The enzyme was either in free solution or immobilized on a pellicular support which consisted of a porous carbonaceous layer on solid glass beads. The highest enzyme activity was obtained in acetone and acetonitrile mixed with citrate buffer over a wide range of organic solvent concentration. In 50% (v/v) acetone both V and Km of the immobilized enzyme were about half of the values in the neat aqueous buffer, but the Ki for inorganic phosphate was unchanged. In 50% (v/v) mixtures of various solvents and citrate buffers of different pH, the enzymic activity was found to depend on the pH of the aqueous buffer component rather than the pH of the hydro-organic mixture as measured with the glass-calomel electrode. The relatively high rates of p-nitrophenol liberation in the presence of glucose even at high organic solvent concentrations suggest that transphosphorylation is facilitated at low water activity.

Overexpression, purification, and characterization of SHPTP1, a Src homology 2-containing protein-tyrosine-phosphatase.

PubMed Central

Pei, D; Neel, B G; Walsh, C T

1993-01-01

A protein-tyrosine-phosphatase (PTPase; EC 3.1.3.48) containing two Src homology 2 (SH2) domains, SHPTP1, was previously identified in hematopoietic and epithelial cells. By placing the coding sequence of the PTPase behind a bacteriophage T7 promoter, we have overexpressed both the full-length enzyme and a truncated PTPase domain in Escherichia coli. In each case, the soluble enzyme was expressed at levels of 3-4% of total soluble E. coli protein. The recombinant proteins had molecular weights of 63,000 and 45,000 for the full-length protein and the truncated PTPase domain, respectively, as determined by SDS/PAGE. The recombinant enzymes dephosphorylated p-nitrophenyl phosphate, phosphotyrosine, and phosphotyrosyl peptides but not phosphoserine, phosphothreonine, or phosphoseryl peptides. The enzymes showed a strong dependence on pH and ionic strength for their activity, with pH optima of 5.5 and 6.3 for the full-length enzyme and the catalytic domain, respectively, and an optimal NaCl concentration of 250-300 mM. The recombinant PTPases had high Km values for p-nitrophenyl phosphate and exhibited non-Michaelis-Menten kinetics for phosphotyrosyl peptides. Images PMID:8430079

Involvement of Vitamin B6 Biosynthesis Pathways in the Insecticidal Activity of Photorhabdus luminescens.

PubMed

Sato, Kazuki; Yoshiga, Toyoshi; Hasegawa, Koichi

2016-06-15

Photorhabdus luminescens is a Gram-negative entomopathogenic bacterium which symbiotically associates with the entomopathogenic nematode Heterorhabditis bacteriophora P. luminescens is highly virulent to many insects and nonsymbiotic nematodes, including Caenorhabditis elegans To understand the virulence mechanisms of P. luminescens, we obtained virulence-deficient and -attenuated mutants against C. elegans through a transposon-mutagenized library. From the genetic screening, we identified the pdxB gene, encoding erythronate-4-phosphate dehydrogenase, as required for de novo vitamin B6 biosynthesis. Mutation in pdxB caused growth deficiency of P. luminescens in nutrient-poor medium, which was restored under nutrient-rich conditions or by supplementation with pyridoxal 5'-phosphate (PLP), an active form of vitamin B6 Supplementation with three other B6 vitamers (pyridoxal, pyridoxine, and pyridoxamine) also restored the growth of the pdxB mutant, suggesting the existence of a salvage pathway for vitamin B6 biosynthesis in P. luminescens Moreover, supplementation with PLP restored the virulence-deficient phenotype against C. elegans Combining these results with the fact that pdxB mutation also caused attenuation of insecticidal activity, we concluded that the production of appropriate amounts of vitamin B6 is critical for P. luminescens pathogenicity. The Gram-negative entomopathogenic bacterium Photorhabdus luminescens symbiotically associates with the entomopathogenic nematode Heterorhabditis bacteriophora P. luminescens is highly virulent to many insects and nonsymbiotic nematodes, including Caenorhabditis elegans We have obtained several virulence-deficient and -attenuated P. luminescens mutants against C. elegans through genetic screening. From the genetic analysis, we present the vitamin B6 biosynthetic pathways in P. luminescens that are important for its insecticidal activity. Mutation in pdxB, encoding erythronate-4-phosphate dehydrogenase and required for the de novo vitamin B6 biosynthesis pathway, caused virulence deficiency against C. elegans and growth deficiency of P. luminescens in nutrient-poor medium. Because such phenotypes were restored under nutrient-rich conditions or by supplementation with B6 vitamers, we showed the presence of the two vitamin B6 synthetic pathways (de novo and salvage) in P. luminescens and also showed that the ability to produce an appropriate amount of vitamin B6 is critical for P. luminescens pathogenicity. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Time-dependent 31P saturation transfer in the phosphoglucomutase reaction. Characterization of the spin system for the Cd(II) enzyme and evaluation of rate constants for the transfer process.

PubMed

Post, C B; Ray, W J; Gorenstein, D G

1989-01-24

Time-dependent 31P saturation-transfer studies were conducted with the Cd2+-activated form of muscle phosphoglucomutase to probe the origin of the 100-fold difference between its catalytic efficiency (in terms of kcat) and that of the more efficient Mg2+-activated enzyme. The present paper describes the equilibrium mixture of phosphoglucomutase and its substrate/product pair when the concentration of the Cd2+ enzyme approaches that of the substrate and how the nine-spin 31P NMR system provided by this mixture was treated. It shows that the presence of abortive complexes is not a significant factor in the reduced activity of the Cd2+ enzyme since the complex of the dephosphoenzyme and glucose 1,6-bisphosphate, which accounts for a large majority of the enzyme present at equilibrium, is catalytically competent. It also shows that rate constants for saturation transfer obtained at three different ratios of enzyme to free substrate are mutually compatible. These constants, which were measured at chemical equilibrium, can be used to provide a quantitative kinetic rationale for the reduced steady-state activity elicited by Cd2+ relative to Mg2+ [cf. Ray, W.J., Post, C.B., & Puvathingal, J.M. (1989) Biochemistry (following paper in this issue)]. They also provide minimal estimates of 350 and 150 s-1 for the rate constants describing (PO3-) transfer from the Cd2+ phosphoenzyme to the 6-position of bound glucose 1-phosphate and to the 1-position of bound glucose 6-phosphate, respectively. These minimal estimates are compared with analogous estimates for the Mg2+ and Li+ forms of the enzyme in the accompanying paper.

Vitamin B6 deficiency: a potential cause of refractory seizures in adults.

PubMed

Gerlach, Anthony T; Thomas, Sheela; Stawicki, Stanislaw P; Whitmill, Melissa L; Steinberg, Steven M; Cook, Charles H

2011-03-01

In children, vitamin B(6) (pyridoxine) deficiency has been described as a cause of seizures that are refractory to conventional antiepileptic medications. We describe the clinical presentation of 3 adults with refractory seizures (later diagnosed with vitamin B(6) deficiency) that resolved after pyridoxine treatment. Case series. Tertiary care surgical intensive care unit. In the first case, a 54-year-old male with history of alcoholic cirrhosis developed new-onset seizures refractory to phenytoin and levetiracetam 8 days after liver transplantation. In the second case, a 59-year-old male with hepatitis C infection developed intracranial hemorrhage and new-onset seizures refractory to phenytoin, levetiracetam, and pentobarbital. The third patient is a 78-year-old male with a history of alcohol dependence who was admitted for an intraventricular bleed and developed new onset of refractory seizures. Intravenous pyridoxine followed by oral pyridoxine. In all 3 cases, seizures persisted despite escalation of conventional antiepileptic medications but resolved within 2 days of pyridoxine supplementation. In each case, low serum pyridoxal 5'-phosphate concentrations normalized with pyroxidine administration. Although refractory seizures caused by vitamin B(6) deficiency are rare in adults, it should be considered in critically ill adult patients with refractory seizures.

Redox self-sufficient whole cell biotransformation for amination of alcohols.

PubMed

Klatte, Stephanie; Wendisch, Volker F

2014-10-15

Whole cell biotransformation is an upcoming tool to replace common chemical routes for functionalization and modification of desired molecules. In the approach presented here the production of various non-natural (di)amines was realized using the designed whole cell biocatalyst Escherichia coli W3110/pTrc99A-ald-adh-ta with plasmid-borne overexpression of genes for an l-alanine dehydrogenase, an alcohol dehydrogenase and a transaminase. Cascading alcohol oxidation with l-alanine dependent transamination and l-alanine dehydrogenase allowed for redox self-sufficient conversion of alcohols to the corresponding amines. The supplementation of the corresponding (di)alcohol precursors as well as amino group donor l-alanine and ammonium chloride were sufficient for amination and redox cofactor recycling in a resting buffer system. The addition of the transaminase cofactor pyridoxal-phosphate and the alcohol dehydrogenase cofactor NAD(+) was not necessary to obtain complete conversion. Secondary and cyclic alcohols, for example, 2-hexanol and cyclohexanol were not aminated. However, efficient redox self-sufficient amination of aliphatic and aromatic (di)alcohols in vivo was achieved with 1-hexanol, 1,10-decanediol and benzylalcohol being aminated best. Copyright © 2014 Elsevier Ltd. All rights reserved.

Arsenic Toxicity: The Effects on Plant Metabolism

PubMed Central

Finnegan, Patrick M.; Chen, Weihua

2012-01-01

The two forms of inorganic arsenic, arsenate (AsV) and arsenite (AsIII), are easily taken up by the cells of the plant root. Once in the cell, AsV can be readily converted to AsIII, the more toxic of the two forms. AsV and AsIII both disrupt plant metabolism, but through distinct mechanisms. AsV is a chemical analog of phosphate that can disrupt at least some phosphate-dependent aspects of metabolism. AsV can be translocated across cellular membranes by phosphate transport proteins, leading to imbalances in phosphate supply. It can compete with phosphate during phosphorylation reactions, leading to the formation of AsV adducts that are often unstable and short-lived. As an example, the formation and rapid autohydrolysis of AsV-ADP sets in place a futile cycle that uncouples photophosphorylation and oxidative phosphorylation, decreasing the ability of cells to produce ATP and carry out normal metabolism. AsIII is a dithiol reactive compound that binds to and potentially inactivates enzymes containing closely spaced cysteine residues or dithiol co-factors. Arsenic exposure generally induces the production of reactive oxygen species that can lead to the production of antioxidant metabolites and numerous enzymes involved in antioxidant defense. Oxidative carbon metabolism, amino acid and protein relationships, and nitrogen and sulfur assimilation pathways are also impacted by As exposure. Readjustment of several metabolic pathways, such as glutathione production, has been shown to lead to increased arsenic tolerance in plants. Species- and cultivar-dependent variation in arsenic sensitivity and the remodeling of metabolite pools that occurs in response to As exposure gives hope that additional metabolic pathways associated with As tolerance will be identified. PMID:22685440

Synthesis and Physicochemical Characterization of D-Tagatose-1-Phosphate: The Substrate of the Tagatose-1-Phosphate Kinase in the Phosphotransferase System-Mediated D-Tagatose Catabolic Pathway of Bacillus licheniformis.

PubMed

Van der Heiden, Edwige; Delmarcelle, Michaël; Simon, Patricia; Counson, Melody; Galleni, Moreno; Freedberg, Darón I; Thompson, John; Joris, Bernard; Battistel, Marcos D

2015-01-01

We report the first enzymatic synthesis of D-tagatose-1-phosphate (Tag-1P) by the multicomponent phosphoenolpyruvate:sugar phosphotransferase system (PEP-PTS) present in tagatose-grown cells of Klebsiella pneumoniae. Physicochemical characterization by (31)P and (1)H nuclear magnetic resonance spectroscopy reveals that, in solution, this derivative is primarily in the pyranose form. Tag-1P was used to characterize the putative tagatose-1-phosphate kinase (TagK) of the Bacillus licheniformis PTS-mediated D-tagatose catabolic pathway (Bli-TagP). For this purpose, a soluble protein fusion was obtained with the 6 His-tagged trigger factor (TF(His6)) of Escherichia coli. The active fusion enzyme was named TagK-TF(His6). Tag-1P and D-fructose-1-phosphate are substrates for the TagK-TF(His6) enzyme, whereas the isomeric derivatives D-tagatose-6-phosphate and D-fructose-6-phosphate are inhibitors. Studies of catalytic efficiency (kcat/Km) reveal that the enzyme specificity is markedly in favor of Tag-1P as the substrate. Importantly, we show in vivo that the transfer of the phosphate moiety from PEP to the B. licheniformis tagatose-specific Enzyme II in E. coli is inefficient. The capability of the PTS general cytoplasmic components of B. subtilis, HPr and Enzyme I to restore the phosphate transfer is demonstrated. © 2015 S. Karger AG, Basel.

Balancing of B6 Vitamers Is Essential for Plant Development and Metabolism in Arabidopsis

PubMed Central

Tohge, Takayuki; Fernie, Alisdair R.

2016-01-01

Vitamin B6 comprises a family of compounds that is essential for all organisms, most notable among which is the cofactor pyridoxal 5′-phosphate (PLP). Other forms of vitamin B6 include pyridoxamine 5′-phosphate (PMP), pyridoxine 5′-phosphate (PNP), and the corresponding nonphosphorylated derivatives. While plants can biosynthesize PLP de novo, they also have salvage pathways that serve to interconvert the different vitamers. The selective contribution of these various pathways to cellular vitamin B6 homeostasis in plants is not fully understood. Although biosynthesis de novo has been extensively characterized, the salvage pathways have received comparatively little attention in plants. Here, we show that the PMP/PNP oxidase PDX3 is essential for balancing B6 vitamer levels in Arabidopsis thaliana. In the absence of PDX3, growth and development are impaired and the metabolite profile is altered. Surprisingly, RNA sequencing reveals strong induction of stress-related genes in pdx3, particularly those associated with biotic stress that coincides with an increase in salicylic acid levels. Intriguingly, exogenous ammonium rescues the growth and developmental phenotype in line with a severe reduction in nitrate reductase activity that may be due to the overaccumulation of PMP in pdx3. Our analyses demonstrate an important link between vitamin B6 homeostasis and nitrogen metabolism. PMID:26858304

3-phosphoglycerate kinase from Hydrogenomonas facilis.

NASA Technical Reports Server (NTRS)

Mcfadden, B. A.; Schuster, E.

1972-01-01

Description of studies of the kinetics of heat inactivation of phosphoglycerate kinase in the soluble fraction from Hydrogenomonas facilis, its extensive purification, and inhibition by adenosine monophosphate (AMP). No evidence was found for an enzyme which catalyzes adenosine-triphosphate-dependent conversion of 3-phosphoglycerate to 1,3-diphosphoglycerate, AMP, and phosphate.

Structural basis for glucose-6-phosphate activation of glycogen synthase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Baskaran, Sulochanadevi; Roach, Peter J.; DePaoli-Roach, Anna A.

2010-11-22

Regulation of the storage of glycogen, one of the major energy reserves, is of utmost metabolic importance. In eukaryotes, this regulation is accomplished through glucose-6-phosphate levels and protein phosphorylation. Glycogen synthase homologs in bacteria and archaea lack regulation, while the eukaryotic enzymes are inhibited by protein kinase mediated phosphorylation and activated by protein phosphatases and glucose-6-phosphate binding. We determined the crystal structures corresponding to the basal activity state and glucose-6-phosphate activated state of yeast glycogen synthase-2. The enzyme is assembled into an unusual tetramer by an insertion unique to the eukaryotic enzymes, and this subunit interface is rearranged by themore » binding of glucose-6-phosphate, which frees the active site cleft and facilitates catalysis. Using both mutagenesis and intein-mediated phospho-peptide ligation experiments, we demonstrate that the enzyme's response to glucose-6-phosphate is controlled by Arg583 and Arg587, while four additional arginine residues present within the same regulatory helix regulate the response to phosphorylation.« less

The route of non-enzymic and enzymic breakdown of 5-phosphoribosyl 1-pyrophosphate to ribose 1-phosphate.

PubMed Central

Trembacz, H; Jezewska, M M

1990-01-01

Spontaneous decomposition of 5-phosphoribosyl 1-pyrophosphate at pH 5.5 was established to occur as follows: 5-Phosphoribosyl 1-pyrophosphate----5-phosphoribosyl 1,2-(cyclic)phosphate----ribose 1-phosphate----ribose Enzymic degradation of 5-phosphoribosyl 1-pyrophosphate by alkaline phosphatase from calf intestine and by acid phosphatases from potato and Aspergillus niger was found to proceed according to this pathway within the pH range 2.5-7.4 with accumulation of ribose 1-phosphate. In the case of alkaline phosphatase, Mg2+ ions inhibit the pyrophosphorolysis of 5-phosphoribosyl 1-pyrophosphate and stimulate the hydrolysis of ribose 1-phosphate. PMID:1700897

Phosphate starvation promoted the accumulation of phenolic acids by inducing the key enzyme genes in Salvia miltiorrhiza hairy roots.

PubMed

Liu, Lin; Yang, DongFeng; Liang, TongYao; Zhang, HaiHua; He, ZhiGui; Liang, ZongSuo

2016-09-01

Phosphate starvation increased the production of phenolic acids by inducing the key enzyme genes in a positive feedback pathway in Saliva miltiorrhiza hairy roots. SPX may be involved in this process. Salvia miltiorrhiza is a wildly popular traditional Chinese medicine used for the treatment of coronary heart diseases and inflammation. Phosphate is an essential plant macronutrient that is often deficient, thereby limiting crop yield. In this study, we investigated the effects of phosphate concentration on the biomass and accumulation of phenolic acid in S. miltiorrhiza. Results show that 0.124 mM phosphate was favorable for plant growth. Moreover, 0.0124 mM phosphate was beneficial for the accumulation of phenolic acids, wherein the contents of danshensu, caffeic acid, rosmarinic acid, and salvianolic acid B were, respectively, 2.33-, 1.02-, 1.68-, and 2.17-fold higher than that of the control. By contrast, 12.4 mM phosphate inhibited the accumulation of phenolic acids. The key enzyme genes in the phenolic acid biosynthesis pathway were investigated to elucidate the mechanism of phosphate starvation-induced increase of phenolic acids. The results suggest that phosphate starvation induced the gene expression from the downstream pathway to the upstream pathway, i.e., a feedback phenomenon. In addition, phosphate starvation response gene SPX (SYG1, Pho81, and XPR1) was promoted by phosphate deficiency (0.0124 mM). We inferred that SPX responded to phosphate starvation, which then affected the expression of later responsive key enzyme genes in phenolic acid biosynthesis, resulting in the accumulation of phenolic acids. Our findings provide a resource-saving and environmental protection strategy to increase the yield of active substance in herbal preparations. The relationship between SPX and key enzyme genes and the role they play in phenolic acid biosynthesis during phosphate deficiency need further studies.

The role of phosphate in a multistep enzymatic reaction: reactions of the substrate and intermediate in pieces.

PubMed

Kholodar, Svetlana A; Allen, C Leigh; Gulick, Andrew M; Murkin, Andrew S

2015-02-25

Several mechanistically unrelated enzymes utilize the binding energy of their substrate's nonreacting phosphoryl group to accelerate catalysis. Evidence for the involvement of the phosphodianion in transition state formation has come from reactions of the substrate in pieces, in which reaction of a truncated substrate lacking its phosphorylmethyl group is activated by inorganic phosphite. What has remained unknown until now is how the phosphodianion group influences the reaction energetics at different points along the reaction coordinate. 1-Deoxy-D-xylulose-5-phosphate (DXP) reductoisomerase (DXR), which catalyzes the isomerization of DXP to 2-C-methyl-D-erythrose 4-phosphate (MEsP) and subsequent NADPH-dependent reduction, presents a unique opportunity to address this concern. Previously, we have reported the effect of covalently linked phosphate on the energetics of DXP turnover. Through the use of chemically synthesized MEsP and its phosphate-truncated analogue, 2-C-methyl-D-glyceraldehyde, the current study revealed a loss of 6.1 kcal/mol of kinetic barrier stabilization upon truncation, of which 4.4 kcal/mol was regained in the presence of phosphite dianion. The activating effect of phosphite was accompanied by apparent tightening of its interactions within the active site at the intermediate stage of the reaction, suggesting a role of the phosphodianion in disfavoring intermediate release and in modulation of the on-enzyme isomerization equilibrium. The results of kinetic isotope effect and structural studies indicate rate limitation by physical steps when the covalent linkage is severed. These striking differences in the energetics of the natural reaction and the reactions in pieces provide a deeper insight into the contribution of enzyme-phosphodianion interactions to the reaction coordinate.

Light-regulation of enzyme activity in anacystis nidulans (Richt.).

PubMed

Duggan, J X; Anderson, L E

1975-01-01

The effect of light on the levels of activity of six enzymes which are light-modulated in higher plants was examined in the photosynthetic procaryot Anacystis nidulans. Ribulose-5-phosphate kinase (EC 2.7.1.19) was found to be light-activated in vivo and dithiothreitol-activated in vitro while glucose-6-phosphate dehydrogenase (EC 1.1.1.49) was light-inactivated and dithiothreitol-inactivated. The enzymes fructose-1,6-diphosphate phosphatase (EC 3.1.3.11), sedoheptulose-1,7-diphosphate phosphatase, NAD- and NADP-linked glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12; EC 1.2.1.13) were not affected by light treatment of the intact algae, but sedoheptulose-diphosphate phosphatase and the glyceraldehyde-3-phosphate dehydrogenases were dithiothreitol-activated in crude extracts. Light apparently controls the activity of the reductive and oxidative pentose phosphate pathway in this photosynthetic procaryot as in higher plants, through a process which probably involves reductive modulation of enzyme activity.

Production of 2-deoxyribose 5-phosphate from fructose to demonstrate a potential of artificial bio-synthetic pathway using thermophilic enzymes.

PubMed

Honda, Kohsuke; Maya, Shohei; Omasa, Takeshi; Hirota, Ryuichi; Kuroda, Akio; Ohtake, Hisao

2010-08-02

Six thermophilic enzymes from Thermus thermophilus were used to construct an 'artificial bio-synthetic pathway' for the production of 2-deoxyribose 5-phosphate from fructose. By a simple operation using six recombinant Escherichia coli strains producing the thermophilic enzymes, respectively, fructose was converted to 2-deoxyribose 5-phosphate with a molar yield of 55%. Copyright 2010 Elsevier B.V. All rights reserved.

Crystal structure of 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase from the ESKAPE pathogen Acinetobacter baumannii

PubMed Central

Sutton, Kristin A.; Breen, Jennifer; Russo, Thomas A.; Schultz, L. Wayne; Umland, Timothy C.

2016-01-01

The enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase catalyzes the sixth step of the seven-step shikimate pathway. Chorismate, the product of the pathway, is a precursor for the biosynthesis of aromatic amino acids, siderophores and metabolites such as folate, ubiquinone and vitamin K. The shikimate pathway is present in bacteria, fungi, algae, plants and apicomplexan parasites, but is absent in humans. The EPSP synthase enzyme produces 5-enolpyruvylshikimate 3-phosphate and phosphate from phosphoenolpyruvate and shikimate 3-phosphate via a transferase reaction, and is the target of the herbicide glyphosate. The Acinetobacter baumannii gene encoding EPSP synthase, aroA, has previously been demonstrated to be essential during host infection for the growth and survival of this clinically important drug-resistant ESKAPE pathogen. Prephenate dehydrogenase is also encoded by the bifunctional A. baumannii aroA gene, but its activity is dependent upon EPSP synthase since it operates downstream of the shikimate pathway. As part of an effort to evaluate new antimicrobial targets, recombinant A. baumannii EPSP (AbEPSP) synthase, comprising residues Ala301–Gln756 of the aroA gene product, was overexpressed in Escherichia coli, purified and crystallized. The crystal structure, determined to 2.37 Å resolution, is described in the context of a potential antimicrobial target and in comparison to EPSP synthases that are resistant or sensitive to the herbicide glyphosate. PMID:26919521

Crystal structure of 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase from the ESKAPE pathogen Acinetobacter baumannii.

PubMed

Sutton, Kristin A; Breen, Jennifer; Russo, Thomas A; Schultz, L Wayne; Umland, Timothy C

2016-03-01

The enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase catalyzes the sixth step of the seven-step shikimate pathway. Chorismate, the product of the pathway, is a precursor for the biosynthesis of aromatic amino acids, siderophores and metabolites such as folate, ubiquinone and vitamin K. The shikimate pathway is present in bacteria, fungi, algae, plants and apicomplexan parasites, but is absent in humans. The EPSP synthase enzyme produces 5-enolpyruvylshikimate 3-phosphate and phosphate from phosphoenolpyruvate and shikimate 3-phosphate via a transferase reaction, and is the target of the herbicide glyphosate. The Acinetobacter baumannii gene encoding EPSP synthase, aroA, has previously been demonstrated to be essential during host infection for the growth and survival of this clinically important drug-resistant ESKAPE pathogen. Prephenate dehydrogenase is also encoded by the bifunctional A. baumannii aroA gene, but its activity is dependent upon EPSP synthase since it operates downstream of the shikimate pathway. As part of an effort to evaluate new antimicrobial targets, recombinant A. baumannii EPSP (AbEPSP) synthase, comprising residues Ala301-Gln756 of the aroA gene product, was overexpressed in Escherichia coli, purified and crystallized. The crystal structure, determined to 2.37 Å resolution, is described in the context of a potential antimicrobial target and in comparison to EPSP synthases that are resistant or sensitive to the herbicide glyphosate.

21 CFR 862.1720 - Triose phosphate isomerase test system.

Code of Federal Regulations, 2014 CFR

2014-04-01

... isomerase test system is a device intended to measure the activity of the enzyme triose phosphate isomerase in erythrocytes (red blood cells). Triose phosphate isomerase is an enzyme important in glycolysis... device is exempt from the premarket notification procedures in subpart E of part 807 subject to the...

21 CFR 862.1720 - Triose phosphate isomerase test system.

Code of Federal Regulations, 2013 CFR

2013-04-01

... isomerase test system is a device intended to measure the activity of the enzyme triose phosphate isomerase in erythrocytes (red blood cells). Triose phosphate isomerase is an enzyme important in glycolysis... device is exempt from the premarket notification procedures in subpart E of part 807 subject to the...

21 CFR 862.1720 - Triose phosphate isomerase test system.

Code of Federal Regulations, 2012 CFR

2012-04-01

... isomerase test system is a device intended to measure the activity of the enzyme triose phosphate isomerase in erythrocytes (red blood cells). Triose phosphate isomerase is an enzyme important in glycolysis... device is exempt from the premarket notification procedures in subpart E of part 807 subject to the...

Discovery of a novel amino acid racemase through exploration of natural variation in Arabidopsis thaliana

PubMed Central

Strauch, Renee C.; Svedin, Elisabeth; Dilkes, Brian; Chapple, Clint; Li, Xu

2015-01-01

Plants produce diverse low-molecular-weight compounds via specialized metabolism. Discovery of the pathways underlying production of these metabolites is an important challenge for harnessing the huge chemical diversity and catalytic potential in the plant kingdom for human uses, but this effort is often encumbered by the necessity to initially identify compounds of interest or purify a catalyst involved in their synthesis. As an alternative approach, we have performed untargeted metabolite profiling and genome-wide association analysis on 440 natural accessions of Arabidopsis thaliana. This approach allowed us to establish genetic linkages between metabolites and genes. Investigation of one of the metabolite–gene associations led to the identification of N-malonyl-d-allo-isoleucine, and the discovery of a novel amino acid racemase involved in its biosynthesis. This finding provides, to our knowledge, the first functional characterization of a eukaryotic member of a large and widely conserved phenazine biosynthesis protein PhzF-like protein family. Unlike most of known eukaryotic amino acid racemases, the newly discovered enzyme does not require pyridoxal 5′-phosphate for its activity. This study thus identifies a new d-amino acid racemase gene family and advances our knowledge of plant d-amino acid metabolism that is currently largely unexplored. It also demonstrates that exploitation of natural metabolic variation by integrating metabolomics with genome-wide association is a powerful approach for functional genomics study of specialized metabolism. PMID:26324904

Discovery of a novel amino acid racemase through exploration of natural variation in Arabidopsis thaliana

DOE PAGES

Strauch, Renee C.; Svedin, Elisabeth; Dilkes, Brian; ...

2015-08-31

Plants produce diverse low-molecular-weight compounds via specialized metabolism. Discovery of the pathways underlying production of these metabolites is an important challenge for harnessing the huge chemical diversity and catalytic potential in the plant kingdom for human uses, but this effort is often encumbered by the necessity to initially identify compounds of interest or purify a catalyst involved in their synthesis. Here, as an alternative approach, we have performed untargeted metabolite profiling and genome-wide association analysis on 440 natural accessions of Arabidopsis thaliana. This approach allowed us to establish genetic linkages between metabolites and genes. Investigation of one of the metabolite-genemore » associations led to the identification of N-malonyl-D-allo-isoleucine, and the discovery of a novel amino acid racemase involved in its biosynthesis. This finding provides, to our knowledge, the first functional characterization of a eukaryotic member of a large and widely conserved phenazine biosynthesis protein PhzF-like protein family. Unlike most of known eukaryotic amino acid racemases, the newly discovered enzyme does not require pyridoxal 5'-phosphate for its activity. In conclusion, this study thus identifies a new d-amino acid racemase gene family and advances our knowledge of plant d-amino acid metabolism that is currently largely unexplored. As a result, it also demonstrates that exploitation of natural metabolic variation by integrating metabolomics with genome-wide association is a powerful approach for functional genomics study of specialized metabolism.« less

Discovery of a novel amino acid racemase through exploration of natural variation in Arabidopsis thaliana

DOE Office of Scientific and Technical Information (OSTI.GOV)

Strauch, Renee C.; Svedin, Elisabeth; Dilkes, Brian

Plants produce diverse low-molecular-weight compounds via specialized metabolism. Discovery of the pathways underlying production of these metabolites is an important challenge for harnessing the huge chemical diversity and catalytic potential in the plant kingdom for human uses, but this effort is often encumbered by the necessity to initially identify compounds of interest or purify a catalyst involved in their synthesis. Here, as an alternative approach, we have performed untargeted metabolite profiling and genome-wide association analysis on 440 natural accessions of Arabidopsis thaliana. This approach allowed us to establish genetic linkages between metabolites and genes. Investigation of one of the metabolite-genemore » associations led to the identification of N-malonyl-D-allo-isoleucine, and the discovery of a novel amino acid racemase involved in its biosynthesis. This finding provides, to our knowledge, the first functional characterization of a eukaryotic member of a large and widely conserved phenazine biosynthesis protein PhzF-like protein family. Unlike most of known eukaryotic amino acid racemases, the newly discovered enzyme does not require pyridoxal 5'-phosphate for its activity. In conclusion, this study thus identifies a new d-amino acid racemase gene family and advances our knowledge of plant d-amino acid metabolism that is currently largely unexplored. As a result, it also demonstrates that exploitation of natural metabolic variation by integrating metabolomics with genome-wide association is a powerful approach for functional genomics study of specialized metabolism.« less

Energizing Eukaryotic Cell-Free Protein Synthesis With Glucose Metabolism

PubMed Central

Hodgman, C. Eric; Jewett, Michael C.

2015-01-01

Eukaryotic cell-free protein synthesis (CFPS) is limited by the dependence on costly high-energy phosphate compounds and exogenous enzymes to power protein synthesis (e.g., creatine phosphate and creatine kinase, CrP/CrK). Here, we report the ability to use glucose as a secondary energy substrate to regenerate ATP in a Saccharomyces cerevisiae crude extract CFPS platform. We observed synthesis of 3.64±0.35 μg mL−1 active luciferase in batch reactions with 16mM glucose and 25mM phosphate, resulting in a 16% increase in relative protein yield (μg protein/$ reagents) compared to the CrP/CrK system. Our demonstration provides the foundation for development of cost-effective eukaryotic CFPS platforms. PMID:26054976

Structural intermediates and directionality of the swiveling motion of Pyruvate Phosphate Dikinase

NASA Astrophysics Data System (ADS)

Minges, Alexander; Ciupka, Daniel; Winkler, Christian; Höppner, Astrid; Gohlke, Holger; Groth, Georg

2017-03-01

Pyruvate phosphate dikinase (PPDK) is a vital enzyme in cellular energy metabolism catalyzing the ATP- and Pi-dependent formation of phosphoenolpyruvate from pyruvate in C4 -plants, but the reverse reaction forming ATP in bacteria and protozoa. The multi-domain enzyme is considered an efficient molecular machine that performs one of the largest single domain movements in proteins. However, a comprehensive understanding of the proposed swiveling domain motion has been limited by not knowing structural intermediates or molecular dynamics of the catalytic process. Here, we present crystal structures of PPDKs from Flaveria, a model genus for studying the evolution of C4 -enzymes from phylogenetic ancestors. These structures resolve yet unknown conformational intermediates and provide the first detailed view on the large conformational transitions of the protein in the catalytic cycle. Independently performed unrestrained MD simulations and configurational free energy calculations also identified these intermediates. In all, our experimental and computational data reveal strict coupling of the CD swiveling motion to the conformational state of the NBD. Moreover, structural asymmetries and nucleotide binding states in the PPDK dimer support an alternate binding change mechanism for this intriguing bioenergetic enzyme.

Generation of reactive oxygen species (ROS) is a key factor for stimulation of macrophage proliferation by ceramide 1-phosphate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Arana, Lide; Gangoiti, Patricia; Ouro, Alberto

2012-02-15

We previously demonstrated that ceramide 1-phosphate (C1P) is mitogenic for fibroblasts and macrophages. However, the mechanisms involved in this action were only partially described. Here, we demonstrate that C1P stimulates reactive oxygen species (ROS) formation in primary bone marrow-derived macrophages, and that ROS are required for the mitogenic effect of C1P. ROS production was dependent upon prior activation of NADPH oxidase by C1P, which was determined by measuring phosphorylation of the p40phox subunit and translocation of p47phox from the cytosol to the plasma membrane. In addition, C1P activated cytosolic calcium-dependent phospholipase A{sub 2} and protein kinase C-{alpha}, and NADPH oxidasemore » activation was blocked by selective inhibitors of these enzymes. These inhibitors, and inhibitors of ROS production, blocked the mitogenic effect of C1P. By using BHNB-C1P (a photolabile caged-C1P analog), we demonstrate that all of these C1P actions are caused by intracellular C1P. It can be concluded that the enzyme responsible for C1P-stimulated ROS generation in bone marrow-derived macrophages is NADPH oxidase, and that this enzyme is downstream of PKC-{alpha} and cPLA{sub 2}-{alpha} in this pathway. -- Highlights: Black-Right-Pointing-Pointer Ceramide 1-phosphate (C1P) stimulates reactive oxygen species (ROS) formation. Black-Right-Pointing-Pointer The enzyme responsible for ROS generation by C1P in macrophages is NADPH oxidase. Black-Right-Pointing-Pointer NADPH oxidase lies downstream of cPLA{sub 2}-{alpha} and PKC-{alpha} in this pathway. Black-Right-Pointing-Pointer ROS generation is essential for the stimulation of macrophage proliferation by C1P.« less

[Intensity of pentose phosphate metabolism of carbohydrates in various brain areas in normal and starved animals].

PubMed

Kerimov, B F

2002-01-01

The activities of key enzymes of pentose phosphate pathway, glucose-6-phosphate dehydrogenase (G-6 PD) and 6-phosphogluconate dehydrogenase (6-PGD), were studied in cytoplasmatic fractions of brain cortical (limbic, orbital, sensorimotor cortex) and subcortical (myelencefalon, mesencefalon, hypothalamus) structures of rats subjected to starvation for 1, 2, 3, 5 and 7 days. Short-term starvation (1-3 days) caused activation of 6-GPD and 6-PGD both in cortical and subcortical structures. Long-term starvation for 5-7 days caused a decrease of activities of the pentose phosphate pathway enzymes in all studied structures. It is suggested that enzymes of pentose phosphate pathway in nervous tissues are functionally and metabolically related to glutathione system and during starvation they indirectly participate in the regulation lipid peroxidation processes.

Structural Characterizations of Glycerol Kinase: Unraveling Phosphorylation-Induced Long-Range Activation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yeh, Joanne I.; Kettering, Regina; Saxl, Ruth

2009-09-11

Glycerol metabolism provides a central link between sugar and fatty acid catabolism. In most bacteria, glycerol kinase plays a crucial role in regulating channel/facilitator-dependent uptake of glycerol into the cell. In the firmicute Enterococcus casseliflavus, this enzyme's activity is enhanced by phosphorylation of the histidine residue (His232) located in its activation loop, approximately 25 A from its catalytic cleft. We reported earlier that some mutations of His232 altered enzyme activities; we present here the crystal structures of these mutant GlpK enzymes. The structure of a mutant enzyme with enhanced enzymatic activity, His232Arg, reveals that residues at the catalytic cleft aremore » more optimally aligned to bind ATP and mediate phosphoryl transfer. Specifically, the position of Arg18 in His232Arg shifts by approximately 1 A when compared to its position in wild-type (WT), His232Ala, and His232Glu enzymes. This new conformation of Arg18 is more optimally positioned at the presumed gamma-phosphate location of ATP, close to the glycerol substrate. In addition to structural changes exhibited at the active site, the conformational stability of the activation loop is decreased, as reflected by an approximately 35% increase in B factors ('thermal factors') in a mutant enzyme displaying diminished activity, His232Glu. Correlating conformational changes to alteration of enzymatic activities in the mutant enzymes identifies distinct localized regions that can have profound effects on intramolecular signal transduction. Alterations in pairwise interactions across the dimer interface can communicate phosphorylation states over 25 A from the activation loop to the catalytic cleft, positioning Arg18 to form favorable interactions at the beta,gamma-bridging position with ATP. This would offset loss of the hydrogen bonds at the gamma-phosphate of ATP during phosphoryl transfer to glycerol, suggesting that appropriate alignment of the second substrate of glycerol kinase, the ATP molecule, may largely determine the rate of glycerol 3-phosphate production.« less

Cloning and Characterization of the Pseudomonas aeruginosa zwf Gene Encoding Glucose-6-Phosphate Dehydrogenase, an Enzyme Important in Resistance to Methyl Viologen (Paraquat)

PubMed Central

Ma, Ju-Fang; Hager, Paul W.; Howell, Michael L.; Phibbs, Paul V.; Hassett, Daniel J.

1998-01-01

In this study, we cloned the Pseudomonas aeruginosa zwf gene, encoding glucose-6-phosphate dehydrogenase (G6PDH), an enzyme that catalyzes the NAD+- or NADP+-dependent conversion of glucose-6-phosphate to 6-phosphogluconate. The predicted zwf gene product is 490 residues, which could form a tetramer with a molecular mass of ∼220 kDa. G6PDH activity and zwf transcription were maximal in early logarithmic phase when inducing substrates such as glycerol, glucose, or gluconate were abundant. In contrast, both G6PDH activity and zwf transcription plummeted dramatically when bacteria approached stationary phase, when inducing substrate was limiting, or when the organisms were grown in a citrate-, succinate-, or acetate-containing basal salts medium. G6PDH was purified to homogeneity, and its molecular mass was estimated to be ∼220 kDa by size exclusion chromatography. Estimated Km values of purified G6PDH acting on glucose-6-phosphate, NADP+, and NAD+ were 530, 57, and 333 μM, respectively. The specific activities with NAD+ and NADP+ were calculated to be 176 and 69 μmol/min/mg. An isogenic zwf mutant was unable to grow on minimal medium supplemented with mannitol. The mutant also demonstrated increased sensitivity to the redox-active superoxide-generating agent methyl viologen (paraquat). Since one by-product of G6PDH activity is NADPH, the latter data suggest that this cofactor is essential for the activity of enzymes critical in defense against paraquat toxicity. PMID:9537370

Glucose-6-phosphate dehydrogenase (G-6-PD) deficiency in Switzerland. Demonstration of a new variant (G-6-PD Aarau) with chronic nonsphaerocytic haemolytic anaemia.

PubMed

Gahr, M; Schröter, W; Sturzenegger, M; Bornhalm, D; Marti, H R

1976-08-01

A new variant of erythrocytic glucose-6-phosphate dehydrogenase has been found in a family of Swiss origin. It is associated with chronic nonsphaerocytic haemolytic anaemia. The enzyme from the erythrocytes of a young boy of this family was partially purified 110-fold and characterized. It revealed reduced catalytic activity, increased thermolability and two maxima of the pH activity curve at pH 7.0 and 8.5. The Km value for glucose-6-phosphate was reduced, that for NADP was normal. The enzyme showed an increased inhibitor constant for NADPH with respect to NADP. Electrophoretic mobility was normal (B+). 2-Desoxyglucose-6-phosphate and galactose-6-phosphate were utilized at normal rates, whereas the analogue deamino-NADP gave an increased utilization rate. The mother of the propositus could be identified as heterozygous for this enzyme deficiency. Chronic haemolysis is possibly due to the increased thermolability of the variant enzyme.

Optimization of γ-amino butyric acid production in a newly isolated Lactobacillus brevis.

PubMed

Binh, Tran Thi Thanh; Ju, Wan-Taek; Jung, Woo-Jin; Park, Ro-Dong

2014-01-01

An isolate from kimchi, identified as Lactobacillus brevis, accumulated γ-aminobutyric acid (GABA), a major inhibitory neurotransmitter, in the culture medium. Optimal culture conditions for growth of L. brevis and production of GABA were 6 % (w/v) l-glutamic acid, 4 % (w/v) maltose, 2 % (w/v) yeast extract, 1 % (w/v) NaCl, 1 % (w/v) CaCl2, 2 g Tween 80/l, and 0.02 mM pyridoxal 5′-phosphate at initial pH 5.25 and 37 °C. GABA reached 44.4 g/l after 72 h cultivation with a conversion rate 99.7 %, based on the amount (6 %) of l-glutamic acid added. GABA was purified using ion exchange column chromatography with 70 % recovery and 97 % purity.

An unexpected phosphate binding site in Glyceraldehyde 3-Phosphate Dehydrogenase: Crystal structures of apo, holo and ternary complex of Cryptosporidium parvum enzyme

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cook, William J; Senkovich, Olga; Chattopadhyay, Debasish

2009-06-08

The structure, function and reaction mechanism of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) have been extensively studied. Based on these studies, three anion binding sites have been identified, one 'Ps' site (for binding the C-3 phosphate of the substrate) and two sites, 'Pi' and 'new Pi', for inorganic phosphate. According to the original flip-flop model, the substrate phosphate group switches from the 'Pi' to the 'Ps' site during the multistep reaction. In light of the discovery of the 'new Pi' site, a modified flip-flop mechanism, in which the C-3 phosphate of the substrate binds to the 'new Pi' site and flips tomore » the 'Ps' site before the hydride transfer, was proposed. An alternative model based on a number of structures of B. stearothermophilus GAPDH ternary complexes (non-covalent and thioacyl intermediate) proposes that in the ternary Michaelis complex the C-3 phosphate binds to the 'Ps' site and flips from the 'Ps' to the 'new Pi' site during or after the redox step. We determined the crystal structure of Cryptosporidium parvum GAPDH in the apo and holo (enzyme + NAD) state and the structure of the ternary enzyme-cofactor-substrate complex using an active site mutant enzyme. The C. parvum GAPDH complex was prepared by pre-incubating the enzyme with substrate and cofactor, thereby allowing free movement of the protein structure and substrate molecules during their initial encounter. Sulfate and phosphate ions were excluded from purification and crystallization steps. The quality of the electron density map at 2{angstrom} resolution allowed unambiguous positioning of the substrate. In three subunits of the homotetramer the C-3 phosphate group of the non-covalently bound substrate is in the 'new Pi' site. A concomitant movement of the phosphate binding loop is observed in these three subunits. In the fourth subunit the C-3 phosphate occupies an unexpected site not seen before and the phosphate binding loop remains in the substrate-free conformation. Orientation of the substrate with respect to the active site histidine and serine (in the mutant enzyme) also varies in different subunits. The structures of the C. parvum GAPDH ternary complex and other GAPDH complexes demonstrate the plasticity of the substrate binding site. We propose that the active site of GAPDH can accommodate the substrate in multiple conformations at multiple locations during the initial encounter. However, the C-3 phosphate group clearly prefers the 'new Pi' site for initial binding in the active site.« less

O-acetylserine(thiol)lyase from spinach (Spinacia oleracea L.) leaf: cDNA cloning, characterization, and overexpression in Escherichia coli of the chloroplast isoform.

PubMed

Rolland, N; Droux, M; Lebrun, M; Douce, R

1993-01-01

The last enzymatic step for L-cysteine biosynthesis is catalyzed by O-acetylserine(thiol)lyase (OASTL, EC 4.2.99.8) which synthesizes L-cysteine from O-acetylserine and "sulfide." We have isolated and characterized a full-length cDNA (1432 bp) from a lambda gt11 library of spinach leaf encoding the complete precursor of the chloroplast isoform. The 1149-nucleotide open reading frame coding for O-acetylserine(thiol)lyase was in the direction opposite that of the lambda gt11 beta-galactosidase gene. The derived amino acid sequence indicates that the protein precursor consists of 383 amino acid residues including a N-terminal presequence peptide of 52 residues. The amino acid sequence of mature spinach chloroplast O-acetylserine(thiol)lyase shows 40 and 57% homology with its bacterial counterparts. Sequence comparison with several pyridoxal 5'-phosphate-containing proteins reveals the presence of a lysine residue assumed to be involved in cofactor binding. A synthetic cDNA was constructed, coding for the entire 331-amino-acid mature O-acetylserine(thiol)lyase and for an initiating methionine. A high level of expression of the active mature chloroplast isoform was achieved in an Escherichia coli strain carrying the T7 RNA polymerase system (F. W. Studier, A. H. Rosenberg, J. J. Dunn, and J. W. Dubendorff, 1990, in Methods in Enzymology, D. V. Goeddel, Ed., Vol. 185, pp. 60-89, Academic Press, San Diego, CA). Addition of pyridoxine to the bacterial growth medium enhanced the enzyme activity due to the recombinant protein. The extent of production is 25-fold higher than in chloroplast from spinach leaves and the recombinant protein presents the relative molecular mass and immunological properties of the natural enzyme from spinach leaf chloroplast. This work, together with our previous biochemical studies, are in accordance with a prokaryotic type enzyme for L-cysteine biosynthesis in higher plant chloroplasts. Southern blot analysis indicated that O-acetylserine(thiol)lyase is encoded by multiple genes in the spinach leaf genomic DNA.

The PAr index, an indicator reflecting altered vitamin B-6 homeostasis, is associated with long-term risk of stroke in the general population: the Hordaland Health Study (HUSK).

PubMed

Zuo, Hui; Tell, Grethe S; Ueland, Per M; Nygård, Ottar; Vollset, Stein E; Midttun, Øivind; Meyer, Klaus; Ulvik, Arve

2018-01-01

Vitamin B-6 homeostasis is altered during inflammation and immune activation. It is unknown whether altered vitamin B-6 homeostasis is associated with the risk of stroke. We investigated the relation between the ratio plasma 4-pyridoxic acid: (pyridoxal + pyridoxal-5'-phosphate) (PAr) as an indicator of altered vitamin B-6 homeostasis and the risk of stroke in the general population. We conducted a prospective analysis of the community-based Hordaland Health Study (HUSK) in 6891 adults (born during 1925-1927 and 1950-1951) without known stroke at baseline (1998-1999). Participants were followed via linkage to the CVDNOR (Cardiovascular Disease in Norway) project and the Cause of Death Registry. HRs and 95% CIs were calculated using Cox proportional hazards analyses. A total of 390 participants (193 men and 197 women) developed stroke over a median follow-up period of 11 y. Study participants with elevated PAr experienced a higher risk of incident stroke in an essentially linear dose-response fashion. The HR (95% CI) for the highest compared with the lowest quartile of PAr was 1.97 (1.42, 2.73; P-trend <0.001) for total stroke and 2.09 (1.42, 3.09; P-trend <0.001) for ischemic stroke after adjustment for age, sex, body mass index (BMI), smoking, education, physical activity, estimated glomerular filtration rate, hypertension, diabetes, total cholesterol, and statin use. PAr had greater predictive strength than did C-reactive protein, current smoking, diabetes, hypertension, estimated glomerular filtration rate, and physical activity. The associations were similar in subgroups stratified by age group, sex, BMI, current smoking, hypertension, diabetes, and statin use at baseline. Higher plasma PAr was independently associated with increased risk of incident stroke in all participants and across all subgroups stratified by conventional risk predictors. Our novel findings point to and expand the range of inflammation and immune activation processes that may be relevant for the pathogenesis and prevention of stroke. This trial was registered at clinicaltrials.gov as NCT03013725. © 2018 American Society for Nutrition.

Most blood biomarkers related to vitamin status, one-carbon metabolism, and the kynurenine pathway show adequate preanalytical stability and within-person reproducibility to allow assessment of exposure or nutritional status in healthy women and cardiovascular patients.

PubMed

Midttun, Oivind; Townsend, Mary K; Nygård, Ottar; Tworoger, Shelley S; Brennan, Paul; Johansson, Mattias; Ueland, Per Magne

2014-05-01

Knowledge of stability during sample transportation and changes in biomarker concentrations within person over time are paramount for proper design and interpretation of epidemiologic studies based on a single measurement of biomarker status. Therefore, we investigated stability and intraindividual vs. interindividual variation in blood concentrations of biomarkers related to vitamin status, one-carbon metabolism, and the kynurenine pathway. Whole blood (EDTA and heparin, n = 12) was stored with an icepack for 24 or 48 h, and plasma concentrations of 38 biomarkers were determined. Stability was calculated as change per hour, intraclass correlation coefficient (ICC), and simple Spearman correlation. Within-person reproducibility of biomarkers was expressed as ICC in samples collected 1-2 y apart from 40 postmenopausal women and in samples collected up to 3 y apart from 551 patients with stable angina pectoris. Biomarker stability was similar in EDTA and heparin blood. Most biomarkers were essentially stable, except for choline and total homocysteine (tHcy), which increased markedly. Within-person reproducibility in postmenopausal women was excellent (ICC > 0.75) for cotinine, all-trans retinol, cobalamin, riboflavin, α-tocopherol, Gly, pyridoxal, methylmalonic acid, creatinine, pyridoxal 5'-phosphate, and Ser; was good to fair (ICC of 0.74-0.40) for pyridoxic acid, kynurenine, tHcy, cholecalciferol, flavin mononucleotide, kynurenic acid, xanthurenic acid, 3-hydroxykynurenine, sarcosine, anthranilic acid, cystathionine, homoarginine, 3-hydroxyanthranilic acid, betaine, Arg, folate, total cysteine, dimethylglycine, asymmetric dimethylarginine, neopterin, symmetric dimethylarginine, and Trp; and poor (ICC of 0.39-0.15) for methionine sulfoxide, Met, choline, and trimethyllysine. Similar reproducibilities were observed in patients with coronary heart disease. Thus, most biomarkers investigated were essentially stable in cooled whole blood for up to 48 h and had a sufficient within-person reproducibility to allow one-exposure assessment of biomarker status in epidemiologic studies. The Western Norway B Vitamin Intervention Trial was registered at clinicaltrials.gov as NTC00354081.

Turning tryptophanase into odor-generating biosensors.

PubMed

Xu, Yaqin; Zhang, Zhuyuan; Ali, M Monsur; Sauder, Joanna; Deng, Xudong; Giang, Karen; Aguirre, Sergio D; Pelton, Robert; Li, Yingfu; Filipe, Carlos D M

2014-03-03

An odor-based sensor system that exploits the metabolic enzyme tryptophanase (TPase) as the key component is reported. This enzyme is able to convert an odorless substrate like S-methyl-L-cysteine or L-tryptophan into the odorous products methyl mercaptan or indole. To make a biosensor, TPase was biotinylated so that it could be coupled with a molecular recognition element, such as an antibody, to develop an ELISA-like assay. This method was used for the detection of an antibody present in nM concentrations by the human nose. TPase can also be combined with the enzyme pyridoxal kinase (PKase) for use in a coupled assay to detect adenosine 5'-triphosphate (ATP). When ATP is present in the low μM concentration range, the coupled enzymatic system generates an odor that is easily detectable by the human nose. Biotinylated TPase can be combined with various biotin-labeled molecular recognition elements, thereby enabling a broad range of applications for this odor-based reporting system. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Glucose-1-phosphate uridylyltransferase from Erwinia amylovora: Activity, structure and substrate specificity.

PubMed

Benini, Stefano; Toccafondi, Mirco; Rejzek, Martin; Musiani, Francesco; Wagstaff, Ben A; Wuerges, Jochen; Cianci, Michele; Field, Robert A

2017-11-01

Erwinia amylovora, a Gram-negative plant pathogen, is the causal agent of Fire Blight, a contagious necrotic disease affecting plants belonging to the Rosaceae family, including apple and pear. E. amylovora is highly virulent and capable of rapid dissemination in orchards; effective control methods are still lacking. One of its most important pathogenicity factors is the exopolysaccharide amylovoran. Amylovoran is a branched polymer made by the repetition of units mainly composed of galactose, with some residues of glucose, glucuronic acid and pyruvate. E. amylovora glucose-1-phosphate uridylyltransferase (UDP-glucose pyrophosphorylase, EC 2.7.7.9) has a key role in amylovoran biosynthesis. This enzyme catalyses the production of UDP-glucose from glucose-1-phosphate and UTP, which the epimerase GalE converts into UDP-galactose, the main building block of amylovoran. We determined EaGalU kinetic parameters and substrate specificity with a range of sugar 1-phosphates. At time point 120min the enzyme catalysed conversion of the sugar 1-phosphate into the corresponding UDP-sugar reached 74% for N-acetyl-α-d-glucosamine 1-phosphate, 28% for α-d-galactose 1-phosphate, 0% for α-d-galactosamine 1-phosphate, 100% for α-d-xylose 1-phosphate, 100% for α-d-glucosamine 1-phosphate, 70% for α-d-mannose 1-phosphate, and 0% for α-d-galacturonic acid 1-phosphate. To explain our results we obtained the crystal structure of EaGalU and augmented our study by docking the different sugar 1-phosphates into EaGalU active site, providing both reliable models for substrate binding and enzyme specificity, and a rationale that explains the different activity of EaGalU on the sugar 1-phosphates used. These data demonstrate EaGalU potential as a biocatalyst for biotechnological purposes, as an alternative to the enzyme from Escherichia coli, besides playing an important role in E. amylovora pathogenicity. Copyright © 2017 Elsevier B.V. All rights reserved.

Consensus structures of the Mo(v) sites of sulfite-oxidizing enzymes derived from variable frequency pulsed EPR spectroscopy, isotopic labelling and DFT calculations.

PubMed

Enemark, John H

2017-10-10

Sulfite-oxidizing enzymes from eukaryotes and prokaryotes have five-coordinate distorted square-pyramidal coordination about the molybdenum atom. The paramagnetic Mo(v) state is easily generated, and over the years four distinct CW EPR spectra have been identified, depending upon enzyme source and the reaction conditions, namely high and low pH (hpH and lpH), phosphate inhibited (P i ) and sulfite (or blocked). Extensive studies of these paramagnetic forms of sulfite-oxidizing enzymes using variable frequency pulsed electron spin echo (ESE) spectroscopy, isotopic labeling and density functional theory (DFT) calculations have led to the consensus structures that are described here. Errors in some of the previously proposed structures are corrected.

Copper stress and filamentous fungus Humicola lutea 103 - ultrastructural changes and activities of key metabolic enzymes.

PubMed

Krumova, Ekaterina Ts; Stoitsova, Stoyanka R; Paunova-Krasteva, Tsvetelina S; Pashova, Svetlana B; Angelova, Maria B

2012-12-01

Humicola lutea 103 is a copper-tolerant fungal strain able to grow in the presence of 300 μg·mL(-1) Cu(2+) under submerged cultivation. To prevent the consequences of copper overload, microorganisms have evolved molecular mechanisms that regulate its uptake, intracellular traffic, storage, and efflux. In spite of this avoidance strategy, high heavy-metal concentrations caused distinct and widespread ultrastructural alterations in H. lutea. The mitochondria were the first and main target of the toxic action. The effect of copper on activities of the key enzymes (hexokinase, glucose-6-phosphate dehydrogenase, malate dehydrogenase, and isocitrate dehydrogenase) included in the 3 main metabolic pathways, glycolysis, pentose phosphate pathway, and tricarboxylic acid cycle, was investigated. High metal concentrations exhibited a dramatic negative effect on hexokinase, while the other 3 enzymes showed a significant and dose-dependent stimulation. On the basis of the present and previous results we concluded that the copper-induced oxidative stress plays an important role in the fungal tolerance to high Cu (2+) concentrations.

Inhibition of Glucuronokinase by Substrate Analogs 1

PubMed Central

Gillard, Douglas F.; Dickinson, David B.

1978-01-01

Glucuronokinase from Lilium longiflorum pollen was purified 30- to 40- fold on a blue dextran-Sepharose column. Substrate analogs were tested for inhibitory effects, and nucleotide substrate specificity of the enzyme was determined. Nine nucleotides were tested, and all were inhibitory when the substrate was ATP. ADP was competitive with ATP and had a Ki value of 0.23 mm. None of the other nucleotide triphosphates could effectively substitute for ATP as a nucleotide substrate. Ten mm dATP and ITP reacted only 3% as rapidly as 10 mm ATP, while the rates for 10 mm GTP, CTP, UTP, and TTP were less than 1%. The glucuronic acid analogs, methyl α-glucuronoside, methyl β-glucuronoside, β-glucuronic acid-1-phosphate, and 4-O-methylglucuronic acid were tested as possible enzyme inhibitors. The three methyl derivatives showed little or no inhibition. The β-glucuronic acid-1-phosphate was inhibitory, with 50% inhibition obtained at 1 to 3 mm depending on the concentration of the glucuronic acid. It is concluded that the glucuronic acid-binding site on the enzyme is highly selective. PMID:16660589

Molecular Recognition at Purine and Pyrimidine Nucleotide (P2) Receptors

PubMed Central

Jacobson, Kenneth A.; Constanzi, Stefano; Ohno, Michihiro; Joshi, Bhalchandra V.; Besada, Pedro; Xu, Bin; Tchilibon, Susanna

2015-01-01

In comparison to other classes of cell surface receptors, the medicinal chemistry at P2X (ligand-gated ion channels) and P2Y (G protein-coupled) nucleotide receptors has been relatively slow to develop. Recent effort to design selective agonists and antagonists based on a combination of library screening, empirical modification of known ligands, and rational design have led to the introduction of potent antagonists of the P2X1 (derivatives of pyridoxal phosphates and suramin), P2X3 (A-317491), P2X7 (derivatives of the isoquinoline KN-62), P2Y1 (nucleotide analogues MRS 2179 and MRS 2279), P2Y2 (thiouracil derivatives such as AR-C126313), and P2Y12 (nucleotide/nucleoside analogues AR-C69931X and AZD6140) receptors. A variety of native agonist ligands (ATP, ADP, UTP, UDP, and UDP-glucose) are currently the subject of structural modification efforts to improve selectivity. MRS2365 is a selective agonist for P2Y1 receptors. The dinucleotide INS 37217 potently activates the P2Y2 receptor. UTP-γ-S and UDP-β-S are selective agonists for P2Y2/P2Y4 and P2Y6 receptors, respectively. The current knowledge of the structures of P2X and P2Y receptors, is derived mainly from mutagenesis studies. Site-directed mutagenesis has shown that ligand recognition in the human P2Y1 receptor involves individual residues of both the TMs (3, 5, 6, and 7), as well as EL 2 and 3. The binding of the negatively-charged phosphate moiety is dependent on positively charged lysine and arginine residues near the exofacial side of TMs 3 and 7. PMID:15078212

Cytosolic NADPH Homeostasis in Glucose-starved Procyclic Trypanosoma brucei Relies on Malic Enzyme and the Pentose Phosphate Pathway Fed by Gluconeogenic Flux*

PubMed Central

Allmann, Stefan; Morand, Pauline; Ebikeme, Charles; Gales, Lara; Biran, Marc; Hubert, Jane; Brennand, Ana; Mazet, Muriel; Franconi, Jean-Michel; Michels, Paul A. M.; Portais, Jean-Charles; Boshart, Michael; Bringaud, Frédéric

2013-01-01

All living organisms depend on NADPH production to feed essential biosyntheses and for oxidative stress defense. Protozoan parasites such as the sleeping sickness pathogen Trypanosoma brucei adapt to different host environments, carbon sources, and oxidative stresses during their infectious life cycle. The procyclic stage develops in the midgut of the tsetse insect vector, where they rely on proline as carbon source, although they prefer glucose when grown in rich media. Here, we investigate the flexible and carbon source-dependent use of NADPH synthesis pathways in the cytosol of the procyclic stage. The T. brucei genome encodes two cytosolic NADPH-producing pathways, the pentose phosphate pathway (PPP) and the NADP-dependent malic enzyme (MEc). Reverse genetic blocking of those pathways and a specific inhibitor (dehydroepiandrosterone) of glucose-6-phosphate dehydrogenase together established redundancy with respect to H2O2 stress management and parasite growth. Blocking both pathways resulted in ∼10-fold increase of susceptibility to H2O2 stress and cell death. Unexpectedly, the same pathway redundancy was observed in glucose-rich and glucose-depleted conditions, suggesting that gluconeogenesis can feed the PPP to provide NADPH. This was confirmed by (i) a lethal phenotype of RNAi-mediated depletion of glucose-6-phosphate isomerase (PGI) in the glucose-depleted Δmec/Δmec null background, (ii) an ∼10-fold increase of susceptibility to H2O2 stress observed for the Δmec/Δmec/RNAiPGI double mutant when compared with the single mutants, and (iii) the 13C enrichment of glycolytic and PPP intermediates from cells incubated with [U-13C]proline, in the absence of glucose. Gluconeogenesis-supported NADPH supply may also be important for nucleotide and glycoconjugate syntheses in the insect host. PMID:23665470

Identification by Virtual Screening and In Vitro Testing of Human DOPA Decarboxylase Inhibitors

PubMed Central

Cellini, Barbara; Macchiarulo, Antonio; Giardina, Giorgio; Bossa, Francesco; Borri Voltattorni, Carla

2012-01-01

Dopa decarboxylase (DDC), a pyridoxal 5′-phosphate (PLP) enzyme responsible for the biosynthesis of dopamine and serotonin, is involved in Parkinson's disease (PD). PD is a neurodegenerative disease mainly due to a progressive loss of dopamine-producing cells in the midbrain. Co-administration of L-Dopa with peripheral DDC inhibitors (carbidopa or benserazide) is the most effective symptomatic treatment for PD. Although carbidopa and trihydroxybenzylhydrazine (the in vivo hydrolysis product of benserazide) are both powerful irreversible DDC inhibitors, they are not selective because they irreversibly bind to free PLP and PLP-enzymes, thus inducing diverse side effects. Therefore, the main goals of this study were (a) to use virtual screening to identify potential human DDC inhibitors and (b) to evaluate the reliability of our virtual-screening (VS) protocol by experimentally testing the “in vitro” activity of selected molecules. Starting from the crystal structure of the DDC-carbidopa complex, a new VS protocol, integrating pharmacophore searches and molecular docking, was developed. Analysis of 15 selected compounds, obtained by filtering the public ZINC database, yielded two molecules that bind to the active site of human DDC and behave as competitive inhibitors with Ki values ≥10 µM. By performing in silico similarity search on the latter compounds followed by a substructure search using the core of the most active compound we identified several competitive inhibitors of human DDC with Ki values in the low micromolar range, unable to bind free PLP, and predicted to not cross the blood-brain barrier. The most potent inhibitor with a Ki value of 500 nM represents a new lead compound, targeting human DDC, that may be the basis for lead optimization in the development of new DDC inhibitors. To our knowledge, a similar approach has not been reported yet in the field of DDC inhibitors discovery. PMID:22384042

Identification by virtual screening and in vitro testing of human DOPA decarboxylase inhibitors.

PubMed

Daidone, Frederick; Montioli, Riccardo; Paiardini, Alessandro; Cellini, Barbara; Macchiarulo, Antonio; Giardina, Giorgio; Bossa, Francesco; Borri Voltattorni, Carla

2012-01-01

Dopa decarboxylase (DDC), a pyridoxal 5'-phosphate (PLP) enzyme responsible for the biosynthesis of dopamine and serotonin, is involved in Parkinson's disease (PD). PD is a neurodegenerative disease mainly due to a progressive loss of dopamine-producing cells in the midbrain. Co-administration of L-Dopa with peripheral DDC inhibitors (carbidopa or benserazide) is the most effective symptomatic treatment for PD. Although carbidopa and trihydroxybenzylhydrazine (the in vivo hydrolysis product of benserazide) are both powerful irreversible DDC inhibitors, they are not selective because they irreversibly bind to free PLP and PLP-enzymes, thus inducing diverse side effects. Therefore, the main goals of this study were (a) to use virtual screening to identify potential human DDC inhibitors and (b) to evaluate the reliability of our virtual-screening (VS) protocol by experimentally testing the "in vitro" activity of selected molecules. Starting from the crystal structure of the DDC-carbidopa complex, a new VS protocol, integrating pharmacophore searches and molecular docking, was developed. Analysis of 15 selected compounds, obtained by filtering the public ZINC database, yielded two molecules that bind to the active site of human DDC and behave as competitive inhibitors with K(i) values ≥10 µM. By performing in silico similarity search on the latter compounds followed by a substructure search using the core of the most active compound we identified several competitive inhibitors of human DDC with K(i) values in the low micromolar range, unable to bind free PLP, and predicted to not cross the blood-brain barrier. The most potent inhibitor with a K(i) value of 500 nM represents a new lead compound, targeting human DDC, that may be the basis for lead optimization in the development of new DDC inhibitors. To our knowledge, a similar approach has not been reported yet in the field of DDC inhibitors discovery.

Functional role of a distal (3'-phosphate) group of CoA in the recombinant human liver medium-chain acyl-CoA dehydrogenase-catalysed reaction.

PubMed Central

Peterson, K L; Srivastava, D K

1997-01-01

The X-ray crystallographic structure of medium-chain acyl-CoA dehydrogenase (MCAD)-octenoyl-CoA complex reveals that the 3'-phosphate group of CoA is confined to the exterior of the protein structure [approx. 15 A (1.5 nm) away from the enzyme active site], and is fully exposed to the outside solvent environment. To ascertain whether such a distal (3'-phosphate) fragment of CoA plays any significant role in the enzyme catalysis, we investigated the recombinant human liver MCAD (HMCAD)-catalysed reaction by using normal (phospho) and 3'-phosphate-truncated (dephospho) forms of octanoyl-CoA and butyryl-CoA substrates. The steady-state kinetic data revealed that deletion of the 3'-phosphate group from octanoyl-CoA substrate increased the turnover rate of the enzyme to about one-quarter, whereas that from butyryl-CoA substrate decreased the turnover rate of the enzyme to about one-fifth; the Km values of both these substrates were increased by 5-10-fold on deletion of the 3'-phosphate group from the corresponding acyl-CoA substrates. The transient kinetics for the reductive half-reaction, oxidative half-reaction and the dissociation 'off-rate' (of the reaction product from the oxidized enzyme site) were all found to be affected by deletions of the 3'-phosphate group from octanoyl-CoA and butyryl-CoA substrates. A cumulative account of these results reveals that, although the 3'-phosphate group of acyl-CoA substrates might seem 'useless' on the basis of the structural data, it has an essential functional role during HMCAD catalysis. PMID:9271097

Spontaneous interfacial reaction between metallic copper and PBS to form cupric phosphate nanoflower and its enzyme hybrid with enhanced activity.

PubMed

He, Guangli; Hu, Weihua; Li, Chang Ming

2015-11-01

We herein report the spontaneous interfacial reaction between copper foil with 0.01 M phosphate buffered saline (PBS) to form free-standing cupric phosphate (Cu3(PO4)2) nanoflowers at ambient temperature. The underlying chemistry was thoroughly investigated and it is found that the formation of nanoflower is synergistically caused by dissolved oxygen, chlorine ions and phosphate ions. Enzyme-Cu3(PO4)2 hybrid nanoflower was further prepared successfully by using an enzyme-dissolving PBS solution and the enzymes in the hybrid exhibit enhanced biological activity. This work provides a facile route for large-scale synthesis of hierarchical inorganic and functional protein-inorganic hybrid architectures via a simple one-step solution-immersion reaction without using either template or surfactant, thus offering great potential for biosensing application among others. Copyright © 2015 Elsevier B.V. All rights reserved.

Systemic bioavailability and pharmacokinetics of the doxylamine-pyridoxine delayed-release combination (Diclectin).

PubMed

Gill, Simerpal Kaur; Garcia-Bournissen, Facundo; Koren, Gideon

2011-02-01

Diclectin, composed of 10 mg doxylamine succinate (DOX) and 10 mg pyridoxine hydrochloride, is the drug combination of choice for the management of nausea and vomiting during pregnancy in Canada. However, there is large variability in its onset and duration of action among women. To understand and improve its effectiveness, the variability in the pharmacokinetics of the ingredients in this doxylamine succinate/pyridoxine hydrochloride combination must be studied. To determine the pharmacokinetics of DOX and pyridoxine after oral administration of two tablets of this drug combination in the form of Diclectin and to calculate their respective relative bioavailability by comparison with intravenous administration in another population. Eighteen nonpregnant, nonlactating, healthy females between 18 and 45 years of age were administered two tablets of Diclectin with 240 mL of water under empty stomach conditions. Blood samples were analyzed for DOX and pyridoxine along with its four active metabolites: pyridoxal, pyridoxal-5'-phosphate (PLP), pyridoxamine, and pyridoxamine-5'-phosphate using tandem mass spectrometry. For the purpose of this study, pharmacokinetic values for DOX and PLP were adjusted for body weight. The mean DOX-AUC0→∞ was calculated to be 3137.22 ± 633.57 ng·hr/mL (range, 2056.59-4376.06 ng·hr/mL). The mean PLP-AUC0→∞ was calculated to be 5513.10 ± 2362.35 ng·hr/mL (range, 1572.56-10,153.77 ng·hr/mL). Based on literature values of the PLP-AUC0→∞ after intravenous administration and data from the current study, the relative bioavailability of pyridoxine in Diclectin was calculated at 100%. There was a 2.1-fold variability in the DOX-AUC0→∞ and 6.5-fold variability in the PLP-AUC0→∞ after oral administration of 20 mg Diclectin. Using literature values and data from the current study, we estimated the oral bioavailability of pyridoxine to be 100%. Therefore, interindividual differences in metabolism, and not in bioavailability, may be important sources of variability that need to be addressed in dosing guidelines.

Vitamin B6 nutritional status and cellular availability of pyridoxal 5'-phosphate govern the function of the transsulfuration pathway's canonical reactions and hydrogen sulfide production via side reactions.

PubMed

Gregory, Jesse F; DeRatt, Barbara N; Rios-Avila, Luisa; Ralat, Maria; Stacpoole, Peter W

2016-07-01

The transsulfuration pathway (TS) acts in sulfur amino acid metabolism by contributing to the regulation of cellular homocysteine, cysteine production, and the generation of H2S for signaling functions. Regulation of TS pathway kinetics involves stimulation of cystathionine β-synthase (CBS) by S-adenosylmethionine (SAM) and oxidants such as H2O2, and by Michaelis-Menten principles whereby substrate concentrations affect reaction rates. Although pyridoxal phosphate (PLP) serves as coenzyme for both CBS and cystathionine γ-lyase (CSE), CSE exhibits much greater loss of activity than CBS during PLP insufficiency. Thus, cellular and plasma cystathionine concentrations increase in vitamin B6 deficiency mainly due to the bottleneck caused by reduced CSE activity. Because of the increase in cystathionine, the canonical production of cysteine (homocysteine → cystathionine → cysteine) is largely maintained even during vitamin B6 deficiency. Typical whole body transsulfuration flux in humans is 3-7 μmol/h per kg body weight. The in vivo kinetics of H2S production via side reactions of CBS and CSE in humans are unknown but they have been reported for cultured HepG2 cells. In these studies, cells exhibit a pronounced reduction in H2S production capacity and rates of lanthionine and homolanthionine synthesis in deficiency. In humans, plasma concentrations of lanthionine and homolanthionine exhibit little or no mean change due to 4-wk vitamin B6 restriction, nor do they respond to pyridoxine supplementation of subjects in chronically low-vitamin B6 status. Wide individual variation in responses of the H2S biomarkers to such perturbations of human vitamin B6 status suggests that the resulting modulation of H2S production may have physiological consequences in a subset of people. Supported by NIH grant DK072398. This paper refers to data from studies registered at clinicaltrials.gov as NCT01128244 and NCT00877812. Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Purification and characterization of rat liver nuclear thyroid hormone receptors.

PubMed Central

Ichikawa, K; DeGroot, L J

1987-01-01

Nuclear thyroid hormone receptor was purified to 904 pmol of L-3,5,3'-triiodothyronine (T3) binding capacity per mg of protein with 2.5-5.2% recovery by sequentially using hydroxylapatite column chromatography, ammonium sulfate precipitation, Sephadex G-150 gel filtration, DNA-cellulose column chromatography, DEAE-Sephadex column chromatography, and heparin-Sepharose column chromatography. Assuming that one T3 molecule binds to the 49,000-Da unit of the receptor, we reproducibly obtained 6.4-14.7 micrograms of receptor protein with 4.2-4.9% purity from 4-5 kg of rat liver. Elution of receptor from the heparin-Sepharose column was performed using 10 mM pyridoxal 5'-phosphate, which was observed to diminish binding of receptor to heparin-Sepharose or DNA-cellulose. This effect was specific for pyridoxal 5'-phosphate, since related compounds were not effective. Purified receptor bound T3 with high affinity (6.0 X 10(9) liter/mol), and the order of affinity of iodothyronine analogues to purified receptor was identical to that observed with crude receptor preparations [3,5,3'-triiodothyroacetic acid greater than L-T3 greater than D-3,5,3'-triiodothyronine (D-T3) greater than L-thyroxine greater than D-thyroxine]. Purified receptor had a sedimentation coefficient of 3.4 S, Stokes radius of 34 A, and calculated molecular mass of 49,000. Among several bands identified by silver staining after electrophoresis in NaDodSO4/polyacrylamide gels, one 49,000-Da protein showed photoaffinity labeling with [125I]thyroxine that was displaceable with excess unlabeled T3. The tryptic fragment and endogenous proteinase-digested fragment of the affinity-labeled receptor showed saturable binding in 27,000-Da and 36,000-Da peptides, respectively. These molecular masses are in agreement with estimates from gel filtration and gradient sedimentation, indicating that affinity labeling occurred at the hormone binding domain of nuclear thyroid hormone receptor. This procedure reproducibly provides classical native rat liver T3 nuclear receptor in useful quantity and purity and of the highest specific activity so far reported. Images PMID:3472213

Simultaneous determination of water-soluble vitamins in selected food matrices by liquid chromatography/electrospray ionization tandem mass spectrometry.

PubMed

Gentili, Alessandra; Caretti, Fulvia; D'Ascenzo, Giuseppe; Marchese, Stefano; Perret, Daniela; Di Corcia, Daniele; Rocca, Lucia Mainero

2008-07-01

A rapid, simple and sensitive method based on liquid chromatography/tandem mass spectrometry (LC/MS/MS) with an electrospray ionization (ESI) source for the simultaneous analysis of fourteen water-soluble vitamins (B1, B2, two B3 vitamers, B5, five B6 vitamers, B8, B9, B12 and C) in various food matrices, i.e. maize flour, green and golden kiwi and tomato pulp, is presented here. Analytes were separated by ion-suppression reversed-phase liquid chromatography in less than 10 min and detected in positive ion mode. Sensitivity and specificity of this method allowed two important results to be achieved: (i) limits of detection of the analytes at ng g(-1) levels (except for vitamin C); (ii) development of a rapid sample treatment that minimizes analyte exposition to light, air and heat, eliminating any step of extract concentration. Analyte recovery depended on the type of matrix. In particular, recovery of the analytes in maize flour was > or =70%, with the exception of vitamin C, pyridoxal-5'-phosphate and vitamin B9 (ca 40%); with tomato pulp, recovery was > or =64%, except for vitamin C (41%); with kiwi, recovery was > or =73%, except for nicotinamide (ca. 30%).

Production of monospecific antibodies to rat liver ornithine decarboxylase and their use in turnover studies.

PubMed

Obenrader, M F; Prouty, W F

1977-05-10

Two forms of ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) were purified from the livers of rats which had been treated with thioacetamide for 16 h (for details, see miniprint to Obenrader, M.F., and Prouty, W. F. (1977) J. Biol. Chem. 252, 2860-2865). The enzyme was purified over 7,000-fold from liver cytosol with an overall yield of 8%. Enzyme activity was eluted finally in two distinct fractions by chromatography on activated thiol-Sepharose 4B. Both forms appear to be dimeric proteins having molecular weights of approximately 100,000 by equilibrium sedimentation and analysis on a calibrated Sephadex G-200 column. The apparent subunits are approximately 50,000 daltons as determined by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate. Since electrophoresis in the presence of detergent is the only method used here to indicate subunits, the possibility that conditions of sample preparation resulted in splitting of a labile protein cannot be excluded from consideration. Ornithine decarboxylase has a very broad pH-activity curve with an optimum that shifts from pH 7.0 to pH 7.8 as the enzyme is purified. The apparent Km values for a highly purified mixture of the two forms of enzyme for L-ornithine and pyridoxal 5'-phosphate were determined to be 0.13 mM and 0.25 micronM, respectively. Both sodium and potassium chloride were shown to inhibit enzymatic activity; 50% inhibition occurred at 270 mM for each when Km amounts or ornithine were used. Rat liver ornithine decarboxylase antiserum was prepared in rabbits using Form I of the enzyme as the antigen. The antibody was shown to precipitate quantitatively the ornithine decarboxylase activity isolated from induced rat liver and rat ventral prostate. The specificity of the antiserum was demonstrated by rocket immunoelectrophoresis and by gel electrophoresis in the presence of sodium dodecyl sulfate using immunoprecipitates obtained from enzyme preparations labeled either in vivo, with [3H]leucine, or in vitro, by reductive methylation using formaldehyde and sodium [3H]borohydride. The antibody preparation has been used in a titration method to assess the half-life of antigen in livers of rats induced for ornithine decarboxylase by injection of thioacetamide. In two experiments, the t1/2 of activity at the height of induction, following injection of cycloheximide, was 19 and 24 min, while the t1/2 of disappearance of antigen was 28 and 33 min, respectively. In each experiment the t1/2 for antigen was significantly longer than the t1/2 for loss of enzyme activity. Enzyme levels appear to be modulated primarily by synthesis and degradation of antigen. Furthermore, the observation that enzyme activity is lost with a shorter t1/2 than antigen is consistent with the idea that denaturation is an initial step in the degradation of this enzyme...

Differentiating phosphate-dependent and phosphate-independent systemic phosphate-starvation response networks in Arabidopsis thaliana through the application of phosphite

PubMed Central

Jost, Ricarda; Pharmawati, Made; Lapis-Gaza, Hazel R.; Rossig, Claudia; Berkowitz, Oliver; Lambers, Hans; Finnegan, Patrick M.

2015-01-01

Phosphite is a less oxidized form of phosphorus than phosphate. Phosphite is considered to be taken up by the plant through phosphate transporters. It can mimic phosphate to some extent, but it is not metabolized into organophosphates. Phosphite could therefore interfere with phosphorus signalling networks. Typical physiological and transcriptional responses to low phosphate availability were investigated and the short-term kinetics of their reversion by phosphite, compared with phosphate, were determined in both roots and shoots of Arabidopsis thaliana. Phosphite treatment resulted in a strong growth arrest. It mimicked phosphate in causing a reduction in leaf anthocyanins and in the expression of a subset of the phosphate-starvation-responsive genes. However, the kinetics of the response were slower than for phosphate, which may be due to discrimination against phosphite by phosphate transporters PHT1;8 and PHT1;9 causing delayed shoot accumulation of phosphite. Transcripts encoding PHT1;7, lipid-remodelling enzymes such as SQD2, and phosphocholine-producing NMT3 were highly responsive to phosphite, suggesting their regulation by a direct phosphate-sensing network. Genes encoding components associated with the ‘PHO regulon’ in plants, such as At4, IPS1, and PHO1;H1, generally responded more slowly to phosphite than to phosphate, except for SPX1 in roots and MIR399d in shoots. Two uncharacterized phosphate-responsive E3 ligase genes, PUB35 and C3HC4, were also highly phosphite responsive. These results show that phosphite is a valuable tool to identify network components directly responsive to phosphate. PMID:25697796

Leishmania UDP-sugar pyrophosphorylase: the missing link in galactose salvage?

PubMed

Damerow, Sebastian; Lamerz, Anne-Christin; Haselhorst, Thomas; Führing, Jana; Zarnovican, Patricia; von Itzstein, Mark; Routier, Françoise H

2010-01-08

The Leishmania parasite glycocalyx is rich in galactose-containing glycoconjugates that are synthesized by specific glycosyltransferases that use UDP-galactose as a glycosyl donor. UDP-galactose biosynthesis is thought to be predominantly a de novo process involving epimerization of the abundant nucleotide sugar UDP-glucose by the UDP-glucose 4-epimerase, although galactose salvage from the environment has been demonstrated for Leishmania major. Here, we present the characterization of an L. major UDP-sugar pyrophosphorylase able to reversibly activate galactose 1-phosphate into UDP-galactose thus proving the existence of the Isselbacher salvage pathway in this parasite. The ordered bisubstrate mechanism and high affinity of the enzyme for UTP seem to favor the synthesis of nucleotide sugar rather than their pyrophosphorolysis. Although L. major UDP-sugar pyrophosphorylase preferentially activates galactose 1-phosphate and glucose 1-phosphate, the enzyme is able to act on a variety of hexose 1-phosphates as well as pentose 1-phosphates but not hexosamine 1-phosphates and hence presents a broad in vitro specificity. The newly identified enzyme exhibits a low but significant homology with UDP-glucose pyrophosphorylases and conserved in particular is the pyrophosphorylase consensus sequence and residues involved in nucleotide and phosphate binding. Saturation transfer difference NMR spectroscopy experiments confirm the importance of these moieties for substrate binding. The described leishmanial enzyme is closely related to plant UDP-sugar pyrophosphorylases and presents a similar substrate specificity suggesting their common origin.

Oxidoreductases Involved in Cell Carbon Synthesis of Methanobacterium thermoautotrophicum

PubMed Central

Zeikus, J. G.; Fuchs, G.; Kenealy, W.; Thauer, R. K.

1977-01-01

Cell-free extracts of Methanobacterium thermoautotrophicum were found to contain high activities of the following oxidoreductases (at 60°C): pyruvate dehydrogenase (coenzyme A acetylating), 275 nmol/min per mg of protein; α-ketoglutarate dehydrogenase (coenzyme A acylating), 100 nmol/min per mg; fumarate reductase, 360 nmol/min per mg; malate dehydrogenase, 240 nmol/min per mg; and glyceraldehyde-3-phosphate dehydrogenase, 100 nmol/min per mg. The kinetic properties (apparent Vmax and KM values), pH optimum, temperature dependence of the rate, and specificity for electron acceptors/donors of the different oxidoreductases were examined. Pyruvate dehydrogenase and α-ketoglutarate dehydrogenase were shown to be two separate enzymes specific for factor 420 rather than for nicotinamide adenine dinucleotide (NAD), NADP, or ferredoxin as the electron acceptor. Both activities catalyzed the reduction of methyl viologen with the respective α-ketoacid and a coenzyme A-dependent exchange between the carboxyl group of the α-ketoacid and CO2. The data indicate that the two enzymes are similar to pyruvate synthase and α-ketoglutarate synthase, respectively. Fumarate reductase was found in the soluble cell fraction. This enzyme activity coupled with reduced benzyl viologen as the electron donor, but reduced factor 420, NADH, or NADPH was not effective. The cells did not contain menaquinone, thus excluding this compound as the physiological electron donor for fumarate reduction. NAD was the preferred coenzyme for malate dehydrogenase, whereas NADP was preferred for glyceraldehyde-3-phosphate dehydrogenase. The organism also possessed a factor 420-dependent hydrogenase and a factor 420-linked NADP reductase. The involvement of the described oxidoreductases in cell carbon synthesis is discussed. PMID:914779

Spectrophotometric determination of ternary mixtures of thiamin, riboflavin and pyridoxal in pharmaceutical and human plasma by least-squares support vector machines.

PubMed

Niazi, Ali; Zolgharnein, Javad; Afiuni-Zadeh, Somaie

2007-11-01

Ternary mixtures of thiamin, riboflavin and pyridoxal have been simultaneously determined in synthetic and real samples by applications of spectrophotometric and least-squares support vector machines. The calibration graphs were linear in the ranges of 1.0 - 20.0, 1.0 - 10.0 and 1.0 - 20.0 microg ml(-1) with detection limits of 0.6, 0.5 and 0.7 microg ml(-1) for thiamin, riboflavin and pyridoxal, respectively. The experimental calibration matrix was designed with 21 mixtures of these chemicals. The concentrations were varied between calibration graph concentrations of vitamins. The simultaneous determination of these vitamin mixtures by using spectrophotometric methods is a difficult problem, due to spectral interferences. The partial least squares (PLS) modeling and least-squares support vector machines were used for the multivariate calibration of the spectrophotometric data. An excellent model was built using LS-SVM, with low prediction errors and superior performance in relation to PLS. The root mean square errors of prediction (RMSEP) for thiamin, riboflavin and pyridoxal with PLS and LS-SVM were 0.6926, 0.3755, 0.4322 and 0.0421, 0.0318, 0.0457, respectively. The proposed method was satisfactorily applied to the rapid simultaneous determination of thiamin, riboflavin and pyridoxal in commercial pharmaceutical preparations and human plasma samples.

Inorganic phosphate blocks binding of pre-miRNA to Dicer-2 via its PAZ domain

PubMed Central

Fukunaga, Ryuya; Colpan, Cansu; Han, Bo W; Zamore, Phillip D

2014-01-01

In Drosophila, Dicer-1 produces microRNAs (miRNAs) from pre-miRNAs, whereas Dicer-2 generates small interfering RNAs from long double-stranded RNA (dsRNA), a process that requires ATP hydrolysis. We previously showed that inorganic phosphate inhibits Dicer-2 cleavage of pre-miRNAs, but not long dsRNAs. Here, we report that phosphate-dependent substrate discrimination by Dicer-2 reflects dsRNA substrate length. Efficient processing by Dicer-2 of short dsRNA requires a 5′ terminal phosphate and a two-nucleotide, 3′ overhang, but does not require ATP. Phosphate inhibits cleavage of such short substrates. In contrast, cleavage of longer dsRNA requires ATP but no specific end structure: phosphate does not inhibit cleavage of these substrates. Mutation of a pair of conserved arginine residues in the Dicer-2 PAZ domain blocked cleavage of short, but not long, dsRNA. We propose that inorganic phosphate occupies a PAZ domain pocket required to bind the 5′ terminal phosphate of short substrates, blocking their use and restricting pre-miRNA processing in flies to Dicer-1. Our study helps explain how a small molecule can alter the substrate specificity of a nucleic acid processing enzyme. PMID:24488111

Phosphate forms an unusual tripodal complex with the Fe–Mn center of sweet potato purple acid phosphatase

PubMed Central

Schenk, Gerhard; Gahan, Lawrence R.; Carrington, Lyle E.; Mitić, Nataša; Valizadeh, Mohsen; Hamilton, Susan E.; de Jersey, John; Guddat, Luke W.

2005-01-01

Purple acid phosphatases (PAPs) are a family of binuclear metalloenzymes that catalyze the hydrolysis of phosphoric acid esters and anhydrides. A PAP in sweet potato has a unique, strongly antiferromagnetically coupled Fe(III)–Mn(II) center and is distinguished from other PAPs by its increased catalytic efficiency for a range of activated and unactivated phosphate esters, its strict requirement for Mn(II), and the presence of a μ-oxo bridge at pH 4.90. This enzyme displays maximum catalytic efficiency (kcat/Km) at pH 4.5, whereas its catalytic rate constant (kcat) is maximal at near-neutral pH, and, in contrast to other PAPs, its catalytic parameters are not dependent on the pKa of the leaving group. The crystal structure of the phosphate-bound Fe(III)–Mn(II) PAP has been determined to 2.5-Å resolution (final Rfree value of 0.256). Structural comparisons of the active site of sweet potato, red kidney bean, and mammalian PAPs show several amino acid substitutions in the sweet potato enzyme that can account for its increased catalytic efficiency. The phosphate molecule binds in an unusual tripodal mode to the two metal ions, with two of the phosphate oxygen atoms binding to Fe(III) and Mn(II), a third oxygen atom bridging the two metal ions, and the fourth oxygen pointing toward the substrate binding pocket. This binding mode is unique among the known structures in this family but is reminiscent of phosphate binding to urease and of sulfate binding to λ protein phosphatase. The structure and kinetics support the hypothesis that the bridging oxygen atom initiates hydrolysis. PMID:15625111

Comparative molecular analysis of evolutionarily distant glyceraldehyde-3-phosphate dehydrogenase from Sardina pilchardus and Octopus vulgaris.

PubMed

Baibai, Tarik; Oukhattar, Laila; Mountassif, Driss; Assobhei, Omar; Serrano, Aurelio; Soukri, Abdelaziz

2010-12-01

The NAD(+)-dependent cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12), which is recognized as a key to central carbon metabolism in glycolysis and gluconeogenesis and as an important allozymic polymorphic biomarker, was purified from muscles of two marine species: the skeletal muscle of Sardina pilchardus Walbaum (Teleost, Clupeida) and the incompressible arm muscle of Octopus vulgaris (Mollusca, Cephalopoda). Comparative biochemical studies have revealed that they differ in their subunit molecular masses and in pI values. Partial cDNA sequences corresponding to an internal region of the GapC genes from Sardina and Octopus were obtained by polymerase chain reaction using degenerate primers designed from highly conserved protein motifs. Alignments of the deduced amino acid sequences were used to establish the 3D structures of the active site of two enzymes as well as the phylogenetic relationships of the sardine and octopus enzymes. These two enzymes are the first two GAPDHs characterized so far from teleost fish and cephalopod, respectively. Interestingly, phylogenetic analyses indicated that the sardina GAPDH is in a cluster with the archetypical enzymes from other vertebrates, while the octopus GAPDH comes together with other molluscan sequences in a distant basal assembly closer to bacterial and fungal orthologs, thus suggesting their different evolutionary scenarios.

Revisiting the Nucleotide and Aminoglycoside Substrate Specificity of the Bifunctional Aminoglycoside Acetyltransferase(6′)-Ie/Aminoglycoside Phosphotransferase(2″)-Ia Enzyme*

PubMed Central

Frase, Hilary; Toth, Marta; Vakulenko, Sergei B.

2012-01-01

The bifunctional aminoglycoside-modifying enzyme aminoglycoside acetyltransferase(6′)-Ie/aminoglycoside phosphotransferase(2″)-Ia, or AAC(6′)-Ie/APH(2″)-Ia, is the major source of aminoglycoside resistance in Gram-positive bacterial pathogens. In previous studies, using ATP as the cosubstrate, it was reported that the APH(2″)-Ia domain of this enzyme is unique among aminoglycoside phosphotransferases, having the ability to inactivate an unusually broad spectrum of aminoglycosides, including 4,6- and 4,5-disubstituted and atypical. We recently demonstrated that GTP, and not ATP, is the preferred cosubstrate of this enzyme. We now show, using competition assays between ATP and GTP, that GTP is the exclusive phosphate donor at intracellular nucleotide levels. In light of these findings, we reevaluated the substrate profile of the phosphotransferase domain of this clinically important enzyme. Steady-state kinetic characterization using the phosphate donor GTP demonstrates that AAC(6′)-Ie/APH(2″)-Ia phosphorylates 4,6-disubstituted aminoglycosides with high efficiency (kcat/Km = 105-107 m−1 s−1). Despite this proficiency, no resistance is conferred to some of these antibiotics by the enzyme in vivo. We now show that phosphorylation of 4,5-disubstituted and atypical aminoglycosides are negligible and thus these antibiotics are not substrates. Instead, these aminoglycosides tend to stimulate an intrinsic GTPase activity of the enzyme. Taken together, our data show that the bifunctional enzyme efficiently phosphorylates only 4,6-disubstituted antibiotics; however, phosphorylation does not necessarily result in bacterial resistance. Hence, the APH(2″)-Ia domain of the bifunctional AAC(6′)-Ie/APH(2″)-Ia enzyme is a bona fide GTP-dependent kinase with a narrow substrate profile, including only 4,6-disubstituted aminoglycosides. PMID:23115238

The inhibitory effect of metals and other ions on acid phosphatase activity from Vigna aconitifolia seeds.

PubMed

Srivastava, Pramod Kumar; Anand, Asha

2015-01-01

Sensitivity of acid phosphatase from Vigna aconitifolia seeds to metal ions, fluoride, and phosphate was examined. All the effectors had different degree of inhibitory effect on the enzyme. Among metal ions, molybdate and ferric ion were observed to be most potent inhibitors and both exhibited mixed type of inhibition. Acid phosphatase activity was inhibited by Cu2+ in a noncompetitive manner. Zn and Mn showed mild inhibition on the enzyme activity. Inhibition kinetics analysis explored molybdate as a potent inhibitor for acid phosphatase in comparison with other effectors used in this study. Fluoride was the next most strong inhibitor for the enzyme activity, and caused a mixed type of inhibition. Phosphate inhibited the enzyme competitively, which demonstrates that inhibition due to phosphate is one of the regulatory factors for enzyme activity.

6-phospho-alpha-D-glucosidase from Fusobacterium mortiferum: cloning, expression, and assignment to family 4 of the glycosylhydrolases.

PubMed Central

Bouma, C L; Reizer, J; Reizer, A; Robrish, S A; Thompson, J

1997-01-01

The Fusobacterium mortiferum malH gene, encoding 6-phospho-alpha-glucosidase (maltose 6-phosphate hydrolase; EC 3.2.1.122), has been isolated, characterized, and expressed in Escherichia coli. The relative molecular weight of the polypeptide encoded by malH (441 residues; Mr of 49,718) was in agreement with the estimated value (approximately 49,000) obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the enzyme purified from F. mortiferum. The N-terminal sequence of the MalH protein obtained by Edman degradation corresponded to the first 32 amino acids deduced from the malH sequence. The enzyme produced by the strain carrying the cloned malH gene cleaved [U-14C]maltose 6-phosphate to glucose 6-phosphate (Glc6P) and glucose. The substrate analogs p-nitrophenyl-alpha-D-glucopyranoside 6-phosphate (pNP alphaGlc6P) and 4-methylumbelliferyl-alpha-D-glucopyranoside 6-phosphate (4MU alphaGlc6P) were hydrolyzed to yield Glc6P and the yellow p-nitrophenolate and fluorescent 4-methylumbelliferyl aglycons, respectively. The 6-phospho-alpha-glucosidase expressed in E. coli (like the enzyme purified from F. mortiferum) required Fe2+, Mn2+, Co2+, or Ni2+ for activity and was inhibited in air. Synthesis of maltose 6-phosphate hydrolase from the cloned malH gene in E. coli was modulated by addition of various sugars to the growth medium. Computer-based analyses of MalH and its homologs revealed that the phospho-alpha-glucosidase from F. mortiferum belongs to the seven-member family 4 of the glycosylhydrolase superfamily. The cloned 2.2-kb Sau3AI DNA fragment from F. mortiferum contained a second partial open reading frame of 83 residues (designated malB) that was located immediately upstream of malH. The high degree of sequence identity of MalB with IIB(Glc)-like proteins of the phosphoenol pyruvate dependent:sugar phosphotransferase system suggests participation of MalB in translocation of maltose and related alpha-glucosides in F. mortiferum. PMID:9209025

Deletion Mapping of zwf, the Gene for a Constitutive Enzyme, Glucose 6-Phosphate Dehydrogenase in ESCHERICHIA COLI

PubMed Central

Fraenkel, D. G.; Banerjee, Santimoy

1972-01-01

Genes for three enzymes of intermediary sugar metabolism in E. coli, zwf (glucose 6-phosphate dehydrogenase, constitutive), edd (gluconate 6-phosphate dehydrase, inducible), and eda (2-keto-3-deoxygluconate 6-phosphate aldolase, differently inducible) are closely linked on the E. coli genetic map, the overall gene order being man... old... eda. edd. zwf... cheB... uvrC... his. One class of apparent revertants of an eda mutant strain contains a secondary mutation in edd, and some of these mutations are deletions extending into zwf. We have used a series of spontaneous edd-zwf deletions to map a series of point mutants in zwf and thus report the first fine structure map of a gene for a constitutive enzyme (zwf). PMID:4560065

Agonists and antagonists acting at P2X receptors: selectivity profiles and functional implications.

PubMed

Lambrecht, G

2000-11-01

P2X receptors are nucleotide-gated cation channels composed of homomeric or heteromeric assemblies of three subunits. In the past 7 years, an extended series (P2X1-7) of P2X subunits has been cloned from vertebrate tissues. In this rapidly expanding field, one of the main current challenges is to relate the cloned P2X receptor subtypes to the diverse physiological responses mediated by the native P2X receptors. However, the paucity of useful ligands, especially subtype-selective agonists and antagonists as well as radioligands, acts as a considerable impediment to progress. Most of the ligands available are highly limited in terms of their kinetics of action, receptor-affinity, subtype-selectivity and P2X receptor-specificity. Their suspected ability to be a substrate for ecto-nucleotidases or to inhibit these enzymes also complicates their use. A number of new antagonists at P2X receptors have recently been described which to some degree are more potent and more selective than earlier antagonists like suramin or pyridoxal-5'-phosphate-6-azophenyl 2',4'-disulfonate (PPADS). This work moves us closer to the ideal goal of classifying the recombinant and native P2X receptor subtypes on the basis of antagonist profiles. This review begins with a brief account of the current status of P2X receptors. It then focuses on the pharmacological properties of a series of key P2 receptor agonists and antagonists and will finish with the discussion of some related therapeutic possibilities.

Functional Characterization of Alanine Racemase from Schizosaccharomyces pombe: a Eucaryotic Counterpart to Bacterial Alanine Racemase

PubMed Central

Uo, Takuma; Yoshimura, Tohru; Tanaka, Naotaka; Takegawa, Kaoru; Esaki, Nobuyoshi

2001-01-01

Schizosaccharomyces pombe has an open reading frame, which we named alr1+, encoding a putative protein similar to bacterial alanine racemase. We cloned the alr1+ gene in Escherichia coli and purified the gene product (Alr1p), with an Mr of 41,590, to homogeneity. Alr1p contains pyridoxal 5′-phosphate as a coenzyme and catalyzes the racemization of alanine with apparent Km and Vmax values as follows: for l-alanine, 5.0 mM and 670 μmol/min/mg, respectively, and for d-alanine, 2.4 mM and 350 μmol/min/mg, respectively. The enzyme is almost specific to alanine, but l-serine and l-2-aminobutyrate are racemized slowly at rates 3.7 and 0.37% of that of l-alanine, respectively. S. pombe uses d-alanine as a sole nitrogen source, but deletion of the alr1+ gene resulted in retarded growth on the same medium. This indicates that S. pombe has catabolic pathways for both enantiomers of alanine and that the pathway for l-alanine coupled with racemization plays a major role in the catabolism of d-alanine. Saccharomyces cerevisiae differs markedly from S. pombe: S. cerevisiae uses l-alanine but not d-alanine as a sole nitrogen source. Moreover, d-alanine is toxic to S. cerevisiae. However, heterologous expression of the alr1+ gene enabled S. cerevisiae to grow efficiently on d-alanine as a sole nitrogen source. The recombinant yeast was relieved from the toxicity of d-alanine. PMID:11244061

Glutathione-related enzymes and the eye.

PubMed

Ganea, Elena; Harding, John J

2006-01-01

Glutathione and the related enzymes belong to the defence system protecting the eye against chemical and oxidative stress. This review focuses on GSH and two key enzymes, glutathione reductase and glucose-6-phosphate dehydrogenase in lens, cornea, and retina. Lens contains a high concentration of reduced glutathione, which maintains the thiol groups in the reduced form. These contribute to lens complete transparency as well as to the transparent and refractive properties of the mammalian cornea, which are essential for proper image formation on the retina. In cornea, gluthatione also plays an important role in maintaining normal hydration level, and in protecting cellular membrane integrity. In retina, glutathione is distributed in the different types of retinal cells. Intracellular enzyme, glutathione reductase, involved in reducing the oxidized glutathione has been found at highest activity in human and primate lenses, as compared to other species. Besides the enzymes directly involved in maintaining the normal redox status of the cell, glucose-6-phosphate dehydrogenase which catalyzes the first reaction of the pentose phosphate pathway, plays a key role in protection of the eye against reactive oxygen species. Cornea has a high activity of the pentose phosphate pathway and glucose-6-phosphate dehydrogenase activity. Glycation, the non-enzymic reaction between a free amino group in proteins and a reducing sugar, slowly inactivates gluthathione-related and other enzymes. In addition, glutathione can be also glycated. The presence of glutathione, and of the related enzymes has been also reported in other parts of the eye, such as ciliary body and trabecular meshwork, suggesting that the same enzyme systems are present in all tissues of the eye to generate NADPH and to maintain gluthatione in the reduced form. Changes of glutathione and related enzymes activity in lens, cornea, retina and other eye tissues, occur with ageing, cataract, diabetes, irradiation and administration of some drugs.

Energizing eukaryotic cell-free protein synthesis with glucose metabolism.

PubMed

Anderson, Mark J; Stark, Jessica C; Hodgman, C Eric; Jewett, Michael C

2015-07-08

Eukaryotic cell-free protein synthesis (CFPS) is limited by the dependence on costly high-energy phosphate compounds and exogenous enzymes to power protein synthesis (e.g., creatine phosphate and creatine kinase, CrP/CrK). Here, we report the ability to use glucose as a secondary energy substrate to regenerate ATP in a Saccharomyces cerevisiae crude extract CFPS platform. We observed synthesis of 3.64±0.35 μg mL(-1) active luciferase in batch reactions with 16 mM glucose and 25 mM phosphate, resulting in a 16% increase in relative protein yield (μg protein/$ reagents) compared to the CrP/CrK system. Our demonstration provides the foundation for development of cost-effective eukaryotic CFPS platforms. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Cellular localization of Na(+), K(+)-ATPase in the mammalian vestibular system

NASA Technical Reports Server (NTRS)

Kerr, T. P.

1984-01-01

Two different, but complementary, procedures for cellular localization of Na+, K+-ATPase in the guinea pig vestibular system were employed. One of these techniques, devised by Stirling, depends upon the well documented ability of the specific inhibitor ouabain to bind selectively to Na+,K+-ATPase, blocking catalytic activity. Microdisected vestibular tissues are incubated with tritium-labelled (3H-) ouabain, and regions with a high concentration of Na+,K+-ATPase are subsequently identified by light microscope autoradiography. A second method, originated by Ernst, detects inorganic phosphate released from an artificial substrate (nitrophenyl phosphate) by catalytic activity of the enzyme. In the presence of strontium ion, phosphate is precipitated near regions of high activity, then converted to a product which may finally be visualized in the electron microscope. This cytochemical enzymatic reaction is inhibited by ouabain.

The pentose phosphate pathway of glucose metabolism. Hormonal and dietary control of the oxidative and non-oxidative reactions of the cycle in liver

PubMed Central

Novello, F.; Gumaa, J. A.; McLean, Patricia

1969-01-01

1. Measurements were made of the non-oxidative reactions of the pentose phosphate cycle in liver (transketolase, transaldolase, ribulose 5-phosphate epimerase and ribose 5-phosphate isomerase activities) in a variety of hormonal and nutritional conditions. In addition, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities were measured for comparison with the oxidative reactions of the cycle; hexokinase, glucokinase and phosphoglucose isomerase activities were also included. Starvation for 2 days caused significant lowering of activity of all the enzymes of the pentose phosphate cycle based on activity in the whole liver. Re-feeding with a high-carbohydrate diet restored all the enzyme activities to the range of the control values with the exception of that of glucose 6-phosphate dehydrogenase, which showed the well-known `overshoot' effect. Re-feeding with a high-fat diet also restored the activities of all the enzymes of the pentose phosphate cycle and of hexokinase; glucokinase activity alone remained unchanged. Expressed as units/g. of liver or units/mg. of protein hexokinase, glucose 6-phosphate dehydrogenase, transketolase and pentose phosphate isomerase activities were unchanged by starvation; both 6-phosphogluconate dehydrogenase and ribulose 5-phosphate epimerase activities decreased faster than the liver weight or protein content. 2. Alloxan-diabetes resulted in a decrease of approx. 30–40% in the activities of 6-phosphogluconate dehydrogenase, ribose 5-phosphate isomerase, ribulose 5-phosphate epimerase and transketolase; in contrast with this glucose 6-phosphate dehydrogenase, transaldolase and phosphoglucose isomerase activities were unchanged. Treatment of alloxan-diabetic rats with protamine–zinc–insulin for 3 days caused a very marked increase to above normal levels of activity in all the enzymes of the pentose phosphate pathway except ribulose 5-phosphate epimerase, which was restored to the control value. Hexokinase activity was also raised by this treatment. After 7 days treatment of alloxan-diabetic rats with protamine–zinc–insulin the enzyme activities returned towards the control values. 3. In adrenalectomized rats the two most important changes were the rise in hexokinase activity and the fall in transketolase activity; in addition, ribulose 5-phosphate epimerase activity was also decreased. These effects were reversed by cortisone treatment. In addition, in cortisone-treated adrenalectomized rats glucokinase activity was significantly lower than the control value. 4. In thyroidectomized rats both ribose 5-phosphate isomerase and transketolase activities were decreased; in contrast with this transaldolase activity did not change significantly. Hypophysectomy caused a 50% fall in transketolase activity that was partially reversed by treatment with thyroxine and almost fully reversed by treatment with growth hormone for 8 days. 5. The results are discussed in relation to the hormonal control of the non-oxidative reactions of the pentose phosphate cycle, the marked changes in transketolase activity being particularly outstanding. PMID:5791534

Kinetic and molecular characterization of the pyruvate phosphate dikinase from Trypanosoma cruzi.

PubMed

González-Marcano, Eglys; Acosta, Héctor; Mijares, Alfredo; Concepción, Juan Luis

2016-06-01

Trypanosoma cruzi, like other trypanosomatids analyzed so far, can use both glucose and amino acids as carbon and energy source. In these parasites, glycolysis is compartmentalized in glycosomes, authentic but specialized peroxisomes. The major part of this pathway, as well as a two-branched glycolytic auxiliary system, are present in these organelles. The first enzyme of one branch of this auxiliary system is the PPi-dependent pyruvate phosphate dikinase (PPDK) that converts phosphoenolpyruvate (PEP), inorganic pyrophosphate (PPi) and AMP into pyruvate, inorganic phosphate (Pi) and ATP, thus contributing to the ATP/ADP balance within the glycosomes. In this work we cloned, expressed and purified the T. cruzi PPDK. It kinetic parameters were determined, finding KM values for PEP, PPi and AMP of 320, 70 and 17 μM, respectively. Using molecular exclusion chromatography, two native forms of the enzyme were found with estimated molecular weights of 200 and 100 kDa, corresponding to a homodimer and monomer, respectively. It was established that T. cruzi PPDK's specific activity can be enhanced up to 2.6 times by the presence of ammonium in the assay mixture. During growth of epimastigotes in batch culture an apparent decrease in the specific activity of PPDK was observed. However, when its activity is normalized for the presence of ammonium in the medium, no significant modification of the enzyme activity per cell in time was found. Copyright © 2016 Elsevier Inc. All rights reserved.

Human glucose-6-phosphate dehydrogenase: the crystal structure reveals a structural NADP(+) molecule and provides insights into enzyme deficiency.

PubMed

Au, S W; Gover, S; Lam, V M; Adams, M J

2000-03-15

Glucose-6-phosphate dehydrogenase (G6PD) catalyses the first committed step in the pentose phosphate pathway; the generation of NADPH by this enzyme is essential for protection against oxidative stress. The human enzyme is in a dimer<-->tetramer equilibrium and its stability is dependent on NADP(+) concentration. G6PD deficiency results from many different point mutations in the X-linked gene encoding G6PD and is the most common human enzymopathy. Severe deficiency causes chronic non-spherocytic haemolytic anaemia; the usual symptoms are neonatal jaundice, favism and haemolytic anaemia. We have determined the first crystal structure of a human G6PD (the mutant Canton, Arg459-->Leu) at 3 A resolution. The tetramer is a dimer of dimers. Despite very similar dimer topology, there are two major differences from G6PD of Leuconostoc mesenteroides: a structural NADP(+) molecule, close to the dimer interface but integral to the subunit, is visible in all subunits of the human enzyme; and an intrasubunit disulphide bond tethers the otherwise disordered N-terminal segment. The few dimer-dimer contacts making the tetramer are charge-charge interactions. The importance of NADP(+) for stability is explained by the structural NADP(+) site, which is not conserved in prokaryotes. The structure shows that point mutations causing severe deficiency predominate close to the structural NADP(+) and the dimer interface, primarily affecting the stability of the molecule. They also indicate that a stable dimer is essential to retain activity in vivo. As there is an absolute requirement for some G6PD activity, residues essential for coenzyme or substrate binding are rarely modified.

Renal cysteine conjugate C-S lyase mediated toxicity of halogenated alkenes in primary cultures of human and rat proximal tubular cells.

PubMed

McGoldrick, Trevor A; Lock, Edward A; Rodilla, Vicente; Hawksworth, Gabrielle M

2003-07-01

Proximal tubular cells from human (HPT) and rat (RPT) kidneys were isolated, grown to confluence and incubated with S-(1,2-dichlorovinyl)- l-cysteine (DCVC), S-(1,2,2-trichlorovinyl)- l-cysteine (TCVC), S-(1,1,2,2-tetrafluoroethyl)- l-cysteine (TFEC) and S-(2-chloro-1,1-difluorethyl)- l-cysteine (CDFEC), the cysteine conjugates of nephrotoxicants. The cultures were exposed to the conjugates for 12, 24 and 48 h and the toxicity determined using the MTT assay. All four conjugates caused dose-dependent toxicity to RPT cells over the range 50-1,000 microM, the order of toxicity being DCVC>TCVC>TFEC=CDFEC. The inclusion of aminooxyacetic acid (AOAA; 250 microM), an inhibitor of pyridoxal phosphate-dependent enzymes such as C-S lyase, afforded protection, indicating that C-S lyase has a role in the bioactivation of these conjugates. In HPT cultures only DCVC caused significant time- and dose-dependent toxicity. Exposure to DCVC (500 microM) for 48 h decreased cell viability to 7% of control cell values, whereas co-incubation of DCVC (500 microM) with AOAA (250 microM) resulted in cell viability of 71%. Human cultures were also exposed to S-(1,2-dichlorovinyl)-glutathione (DCVG). DCVG was toxic to HPT cells, but the onset of toxicity was delayed compared with the corresponding cysteine conjugate. AOAA afforded almost complete protection from DCVG toxicity. Acivicin (250 microM), an inhibitor of gamma-glutamyl transferase (gamma-GT), partially protected against DCVG (500 microM)-induced toxicity at 48 h (5% viability and 53% viability in the absence and presence of acivicin, respectively). These results suggest that DCVG requires processing by gamma-GT prior to bioactivation by C-S lyase in HPT cells. The activity of C-S lyase, using TFEC as a substrate, and glutamine transaminase K (GTK) was measured in rat and human cells with time in culture. C-S lyase activity in RPT and HPT cells decreased to approximately 30% of fresh cell values by the time the cells reached confluence (120 h), whereas the decline in GTK activity was less marked (50% of the fresh cell values at confluence). Rat cells had threefold higher activity than human cells at each time point. This higher activity may partly explain the differences in toxicity between rat and human proximal tubular cells in culture.

Properties of thymidylate synthetase from Ehrlich ascites carcinoma cells. Effect of Mg2/ and MgATP2-.

PubMed

Jastreboff, M; Kedzierska, B; Rode, W

1982-01-15

Ehrlich ascites carcinoma thymidylate synthetase was purified to electrophoretic homogeneity by affinity chromatography on 10-formyl-5,8-dideazofolate-ethyl-Sepharose. Electrophoretic analysis of the formation of the enzyme-5-fluorodeoxyuridylate-5,10-methylenetetrahydrofolate complexes showed the presence of two binding sites for 5-fluorodeoxyuridylate on the enzyme molecule. Molecular weight of the native enzyme was found to be 78,5000, whereas that of its monomer was 38, 500. The apparent Michaelis constants for dUMP and (+/-)-L-5,10-methylenetetrahydrofolate were 1.3 +/- 0.4 and 32.2 +/- 0.7 micrometers respectively. Phosphate acted as a weak inhibitor, competitive toward dUMP. The enzyme reaction exhibited a temperature-dependent change of activation energy, reflected in the binding affinity of dUMP, with a transitional temperature of 35.8 degrees. Both Mg2+ and MgATP2- were strong activators of the enzyme, MgATP2- being more effective.

Broad-spectrum non-nucleoside inhibitors for caliciviruses.

PubMed

Netzler, Natalie E; Enosi Tuipulotu, Daniel; Eltahla, Auda A; Lun, Jennifer H; Ferla, Salvatore; Brancale, Andrea; Urakova, Nadya; Frese, Michael; Strive, Tanja; Mackenzie, Jason M; White, Peter A

2017-10-01

Viruses of the Caliciviridae cause significant and sometimes lethal diseases, however despite substantial research efforts, specific antivirals are lacking. Broad-spectrum antivirals could combat multiple viral pathogens, offering a rapid solution when no therapies exist. The RNA-dependent RNA polymerase (RdRp) is an attractive antiviral target as it is essential for viral replication and lacks mammalian homologs. To focus the search for pan-Caliciviridae antivirals, the RdRp was probed with non-nucleoside inhibitors (NNIs) developed against hepatitis C virus (HCV) to reveal both allosteric ligands for structure-activity relationship enhancement, and highly-conserved RdRp pockets for antiviral targeting. The ability of HCV NNIs to inhibit calicivirus RdRp activities was assessed using in vitro enzyme and murine norovirus cell culture assays. Results revealed that three NNIs which bound the HCV RdRp Thumb I (TI) site also inhibited transcriptional activities of six RdRps spanning the Norovirus, Sapovirus and Lagovirus genera of the Caliciviridae. These NNIs included JTK-109 (RdRp inhibition range: IC 50 4.3-16.6 μM), TMC-647055 (IC 50 range: 18.8-45.4 μM) and Beclabuvir (IC 50 range: 23.8->100 μM). In silico studies and site-directed mutagenesis indicated the JTK-109 binding site was within the calicivirus RdRp thumb domain, in a pocket termed Site-B, which is highly-conserved within all calicivirus RdRps. Additionally, RdRp inhibition assays revealed that JTK-109 was antagonistic with the previously reported RdRp inhibitor pyridoxal-5'-phosphate-6-(2'-naphthylazo-6'-nitro-4',8'-disulfonate) tetrasodium salt (PPNDS), that also binds to Site-B. Moreover, like JTK-109, PPNDS was also a potent inhibitor of polymerases from six viruses spanning the three Caliciviridae genera tested (IC 50 range: 0.1-2.3 μM). Together, this study demonstrates the potential for de novo development of broad-spectrum antivirals that target the highly-conserved RdRp thumb pocket, Site-B. We also revealed three broad-spectrum HCV NNIs that could be used as antiviral scaffolds for further development against caliciviruses and other viruses. Copyright © 2017 Elsevier B.V. All rights reserved.

Analysis of class II (hydrolytic) and class I (beta-lyase) apurinic/apyrimidinic endonucleases with a synthetic DNA substrate.

PubMed Central

Levin, J D; Demple, B

1990-01-01

We have developed simple and sensitive assays that distinguish the main classes of apurinic/apyrimidinic (AP) endonucleases: Class I enzymes that cleave on the 3' side of AP sites by beta-elimination, and Class II enzymes that cleave by hydrolysis on the 5' side. The distinction of the two types depends on the use of a synthetic DNA polymer that contains AP sites with 5'-[32P]phosphate residues. Using this approach, we now show directly that Escherichia coli endonuclease IV and human AP endonuclease are Class II enzymes, as inferred previously on the basis of indirect assays. The assay method does not exhibit significant interference by nonspecific nucleases or primary amines, which allows the ready determination of different AP endonuclease activities in crude cell extracts. In this way, we show that virtually all of the Class II AP endonuclease activity in E. coli can be accounted for by two enzymes: exonuclease III and endonuclease IV. In the yeast Saccharomyces cerevisiae, the Class II AP endonuclease activity is totally dependent on a single enzyme, the Apn1 protein, but there are probably multiple Class I enzymes. The versatility and ease of our approach should be useful for characterizing this important class of DNA repair enzymes in diverse systems. PMID:1698278

Patterns of soil calcium and aluminum across the conterminous United States (Chapter 9)

Treesearch

Charles H. Perry; Michael C. Amacher

2012-01-01

The health and growth of forests depends upon soil nutrients. Calcium (Ca) is a cation - positive ion - used by plants to build cell walls (Marschner 1986). It is also involved in root and leaf development and the activation of plant enzymes (Potash and Phosphate Institute 1995). Soils that develop in areas with low rainfall tend to have greater Ca supplies than soils...

Characterization of human triosephosphate isomerase S-nitrosylation.

PubMed

Romero, Jorge Miguel; Carrizo, María Elena; Curtino, Juan Agustín

2018-07-01

Triosephosphate isomerase (TPI), the glycolytic enzyme that catalyzes the isomerization of dihydroxyacetone phosphate (DHAP) to glyceraldehyde-3-phosphate (G3P), has been frequently identified as a target of S-nitrosylation by proteomic studies. However, the effect of S-nitrosylation on its activity has only been explored in plants and algae. Here, we describe the in vitro S-nitrosylation of human TPI (hTPI), and the effect of the modification on its enzymatic parameters. NO-incorporation into the enzyme cysteine residues occurred by a time-dependent S-transnitrosylation from both, S-nitrosocysteine (CySNO) and S-nitrosoglutathione (GSNO), with CySNO being the more efficient NO-donor. Both X-ray crystal structure and mass spectrometry analyses showed that only Cys217 was S-nitrosylated. hTPI S-nitrosylation produced a 30% inhibition of the Vmax of the DHAP conversion to G3P, without affecting the Km for DHAP. This is the first study describing features of human TPI S-nitrosylation. Copyright © 2018 Elsevier Inc. All rights reserved.

Immobilization Increases the Stability and Reusability of Pigeon Pea NADP+ Linked Glucose-6-Phosphate Dehydrogenase.

PubMed

Singh, Siddhartha; Singh, Amit Kumar; Singh, M Chandrakumar; Pandey, Pramod Kumar

2017-02-01

Immobilization of enzymes is valuably important as it improves the stability and hence increases the reusability of enzymes. The present investigation is an attempt for immobilization of purified glucose-6-phosphate dehydrogenase from pigeon pea on different matrix. Maximum immobilization was achieved when alginate was used as immobilization matrix. As compared to soluble enzyme the alginate immobilized enzyme exhibited enhanced optimum pH and temperature. The alginate immobilized enzyme displayed more than 80% activity up to 7 continuous reactions and more than 50% activity up to 11 continuous reactions.

Systematic engineering of pentose phosphate pathway improves Escherichia coli succinate production.

PubMed

Tan, Zaigao; Chen, Jing; Zhang, Xueli

2016-01-01

Succinate biosynthesis of Escherichia coli is reducing equivalent-dependent and the EMP pathway serves as the primary reducing equivalent source under anaerobic condition. Compared with EMP, pentose phosphate pathway (PPP) is reducing equivalent-conserving but suffers from low efficacy. In this study, the ribosome binding site library and modified multivariate modular metabolic engineering (MMME) approaches are employed to overcome the low efficacy of PPP and thus increase succinate production. Altering expression levels of different PPP enzymes have distinct effects on succinate production. Specifically, increased expression of five enzymes, i.e., Zwf, Pgl, Gnd, Tkt, and Tal, contributes to increased succinate production, while the increased expression of two enzymes, i.e., Rpe and Rpi, significantly decreases succinate production. Modular engineering strategy is employed to decompose PPP into three modules according to position and function. Engineering of Zwf/Pgl/Gnd and Tkt/Tal modules effectively increases succinate yield and production, while engineering of Rpe/Rpi module decreases. Imbalance of enzymatic reactions in PPP is alleviated using MMME approach. Finally, combinational utilization of engineered PPP and SthA transhydrogenase enables succinate yield up to 1.61 mol/mol glucose, which is 94% of theoretical maximum yield (1.71 mol/mol) and also the highest succinate yield in minimal medium to our knowledge. In summary, we systematically engineered the PPP for improving the supply of reducing equivalents and thus succinate production. Besides succinate, these PPP engineering strategies and conclusions can also be applicable to the production of other reducing equivalent-dependent biorenewables.

The source of phosphate in the oxidation zone of ore deposits: Evidence from oxygen isotope compositions of pyromorphite

NASA Astrophysics Data System (ADS)

Burmann, Fabian; Keim, Maximilian F.; Oelmann, Yvonne; Teiber, Holger; Marks, Michael A. W.; Markl, Gregor

2013-12-01

Pyromorphite (Pb5[PO4]3Cl) is an abundant mineral in oxidized zones of lead-bearing ore deposits and due to its very low solubility product effectively binds Pb during supergene alteration of galena (PbS). The capacity of a soil or near-surface fluid to immobilize dissolved Pb depends critically on the availability of phosphate in this soil or fluid. Potential phosphorus sources in soil include (i) release during biological processes, i.e. leaching from litter/lysis of microbial cells (after intracellular enzyme activity) in soil and hydrolysis from soil organic matter by extracellular enzymes and (ii) inorganic phosphate from the dissolution of apatite in the adjacent basement rocks. Intracellular enzyme activity in plants/microorganisms associated with kinetic fractionation produces an oxygen isotope composition distinctly different from inorganic processes in soil. This study presents the first oxygen isotope data for phosphate (δ18OP) in pyromorphite and a comprehensive data set for apatite from crystalline rocks. We investigated 38 pyromorphites from 26 localities in the Schwarzwald (Southwest Germany) and five samples from localities outside the Schwarzwald in addition to 12 apatite separates from gneissic and granitic host rocks. Pyromorphites had δ18OP values between +10‰ and +19‰, comparable to literature data on δ18OP in the readily available P fraction in soil (resin-extractable P) from which minerals potentially precipitate in soils. δ18OP values below the range of equilibrium isotope fractionation can be attributed either to apatites that formed geochemically (δ18OP of apatites:+6‰ to +9‰) or less likely to biological processes (extracellular enzyme activity). However, for most of our samples isotopic equilibrium with ambient water was indicated, which suggests biological activity. Therefore, we conclude that the majority of pyromorphites in oxidized zones of ore bodies formed from biologically cycled phosphate. This study highlights that biological activity and Pb mobilization are intimately connected: in humid regions with high biological activity in soil, Pb might be precipitated rapidly due to biologically-released phosphate, whereas Pb will be released to the environment from ore deposits or mine dumps much more easily in arid regions with low biological activity, because pyromorphite cannot form due to limited supply of phosphorus. Phosphate from magmatic, metamorphic or sedimentary rocks: The most important phosphate-bearing mineral in such rocks is apatite (Ca5[(PO4)3(F,Cl,OH)]). In magmatic and metamorphic rocks it generally occurs as fluorapatite (Piccoli and Candela, 2002; Filippelli, 2008), whereas sedimentary rocks may also contain considerable amounts of carbonate-fluorapatite. Phosphorites are present in the geological record since the Lower Proterozoic (Cook and McElhinny, 1979; Shemesh et al., 1983). Alteration with low-pH fluids can dissolve apatite and thereby release geochemical phosphate (Filippelli, 2008). Low pH values may be attained by dissolution of atmospheric CO2 or by reaction with sulfides present in the rocks or in adjacent ore deposits. Phosphate of organic origin, such as from plants, animals or microorganisms: Phosphorus is required in most biological systems, as it is an essential element in major organic molecules such as adenosine triphosphate in the energy cycle, or in phospholipids, which form cell walls (Bucher, 2007; Filippelli, 2008). Organisms take up phosphorus as dissolved inorganic phosphate and cycle it through metabolic processes (intracellular enzyme activity). Once entering the soil, the organic material is decomposed by extracellular enzyme activity (hydrolysis of ester bonds) and phosphate is being released (Bünemann et al., 2011). Phosphate of anthropogenic origin: Since phosphate is a limiting factor in organism growth, it is an important ingredient of fertilizers in the agricultural industry. Also, phosphate can be found as ingredients in detergents, toothpaste and as a release of waste water treatment plants (Young et al., 2009). Anthropogenic effects will not be discussed further in the following. On this basis, we consider three different cases of pyromorphite formation as illustrated on the conceptual scheme of Fig. 1. Case 1: Pyromorphite grown recently (within the last hundreds of years) on rock surfaces in former mines. Both, phosphate released geochemically from igneous rocks and phosphate released biologically during leaching from litter/lysis of microbial cells and soil organic matter decomposition are possible sources. Case 2: Pyromorphite formation on mine dumps, below vegetation (recent, during tens to hundreds of years). Based on the specific setting of these samples investigated here (they were found exclusively below a large fern; see more details in the section on sample description), biologically-mediated P release provides the phosphate for pyromorphite growth. Case 3: Pyromorphite growth in the oxidized zones of ore bodies prior to human interference. Most samples of our study belong to this case.Phosphorus generally forms very strong covalent bonds (Huminicki and Hawthorne, 2002) and there is only negligible exchange of oxygen isotopes between phosphate and ambient water under most near-surface conditions without biological activity (Winter et al., 1940; Longinelli, 1965). The only important exchange of oxygen isotopes between phosphate and ambient water involves biological activity and the oxygen isotope composition of phosphate (δ18OP) may be modified by different enzymatic/cellular processes. Once phosphate is taken up by organisms, intracellular pyrophosphatase mediates internal P cycling. This is associated with a temperature-dependent equilibrium isotope fractionation due to the reversible exchange of O atoms between the phosphate molecule and cell water. As a result the δ18OP is equilibrated with the ambient water, and the equilibrium temperature can be calculated following the revised empirical equation from Longinelli and Nuti (1973) presented by Puceat et al. (2010): T(°C)=118.7-4.22[(δ18OP+(22.6-δ18ONBS120c))-δ18OW] where T is the temperature of the ambient water, δ18OP is the oxygen isotope composition of the phosphate at equilibrium conditions, δ18ONBS120c is the oxygen isotope composition of reference material NBS120c according to Vennemann et al. (2002) and δ18OW is the oxygen isotope composition of the ambient water. Knowledge of the δ18OP of ambient water and its temperature renders it possible to calculate a theoretical equilibrium value for δ18OP. If phosphate is again released from organisms into the soil, it will reflect the δ18OP of the cell-internal P cycling. In addition, extracellular enzymes are released in soil if the demand for P requires the hydrolysis of organic P in soil (McGill and Cole, 1981). Extracellular enzymes also transfer O atoms from water to phosphate and thus, change δ18OP. The associated isotopic fractionation factors vary between -10‰ (enzyme: 5‧-nucleotidase) and -30‰ (enzyme: alkaline phosphatase; Liang and Blake, 2006, 2009). All recent publications on δ18OP of phosphate in the readily available P fraction in soil (resin P) showed δ18OP values in the range calculated for isotopic equilibrium fractionation irrespective of environmental conditions (parent material, climate, biome). At most 20% down to 0% of the measured δ18OP fell outside the calculated isotopic equilibrium range (Angert et al., 2011, 2012; Tamburini et al., 2012). We therefore infer a dominant role of intracellular enzyme activity for δ18OP values in resin P in soil.Theoretical calculations by Lecuyer et al. (1999) imply that oxygen isotope exchange between phosphate and water can also occur in the absence of biological activity. An extrapolation of their equations to temperatures of 10 °C shows, however, that it takes more than 6000 years to exchange 10% of the phosphate oxygen (Colman et al., 2005). Traditionally, the oxygen isotope composition of phosphate has been used as a tool for determining paleotemperatures (e.g., Longinelli, 1984), but recent studies suggested to test its suitability for tracing phosphate sources in aquatic systems (Gruau et al., 2005; Elsbury et al., 2009; Young et al., 2009). Most of these studies deal with short-term ecological cycles and therefore the inorganic exchange of oxygen is negligible. However, this effect has to be considered for processes that happen in geological timescales.Due to the low phosphate concentrations in natural waters (Blake et al., 2005) and the low solubility product of pyromorphite, it is reasonable to assume almost all phosphate to precipitate as pyromorphite without any fractionation. Accordingly, the δ18OP of pyromorphite reflects the oxygen isotope composition of the dissolved phosphate in the water from which it precipitated and records the source, if this phosphate was not modified during fluid transport.Different phosphate reservoirs differ in their oxygen-isotope composition and with more and more data available it is possible to discriminate between different sources. Data for phosphates in aquatic systems are provided by Young et al. (2009): Phosphates of anthropogenic origin (fertilizers and the corresponding processing stages, detergents and toothpaste) show δ18OP values between +13.3‰ and +22.3‰, for phosphates from organic sources (vegetation leachate and animal waste) values between +14.2‰ and +23.1‰ are reported and a range between +8.4‰ and +14.2‰ is covered by phosphates of waste water treatment plants. For terrestrial ecosystems, Tamburini et al. (2012) reported δ18OP values between +4.5‰ and +31.4‰ with most data falling in the range of +12.4‰ to +31.4‰ for phosphate in plants (N = 11). Microbial phosphate in soil covered a range of +11‰ to +19‰. Resin-extractable P in soil as the readily available P fraction in soil from which P-containing minerals would precipitate, showed a range of 14.5-20.0‰ (Angert et al., 2011, 2012; Weiner et al., 2011; Tamburini et al., 2012). Additionally, Tamburini et al., 2012 reported values for apatite, most likely from the metamorphosed granitic bedrock, to be about +7‰. This is consistent with theoretical considerations by Shemesh et al. (1983) and with data from a gabbro (+4.1‰) and a tonalite (+6.7‰) reported by Taylor and Epstein (1962). Mizota et al. (1992) analyzed δ18OP of apatites from carbonatites, volcanic ashes and hydrothermal vugs covering a range of +0.2 to +12.2‰ (N = 10), whereas phosphate from phosphorites have higher values of up to +20‰ (e.g., Shemesh et al. (1983).This study investigates the oxygen isotope composition of phosphate in pyromorphite and in apatite from crystalline rocks. To evaluate possible phosphate sources, the results will be checked for isotopic equilibrium with different ambient waters and possible phosphate sources will be discussed.

A Single Amino Acid Substitution in the Active Site of Escherichia coli Aspartate Transcarbamoylase Prevents the Allosteric Transition

PubMed Central

Stieglitz, Kimberly A.; Pastra-Landis, Styliani C.; Xia, Jiarong; Tsuruta, Hiro; Kantrowitz, Evan R.

2005-01-01

Modeling of the tetrahedral intermediate within the active site of Escherichia coli aspartate transcarbamoylase revealed a specific interaction with the side chain of Gln137, an interaction not previously observed in the structure of the X-ray enzyme in the presence of N-phosphonacetyl-L-aspartate (PALA). Previous site-specific mutagenesis experiments showed that when Gln137 was replaced by alanine, the resulting mutant enzyme (Q137A) exhibited approximately 50-fold less activity than the wild-type enzyme, exhibited no homotropic cooperativity, and the binding of both carbamoyl phosphate and aspartate were extremely compromised. To elucidate the structural alterations in the mutant enzyme that might lead to such pronounced changes in kinetic and binding properties, the Q137A enzyme was studied by time-resolved small-angle X-ray scattering and its structure was determined in the presence of PALA to 2.7Å resolution. Time-resolved small-angle X-ray scattering established that the natural substrates, carbamoyl phosphate and L-aspartate, do not induce in the Q137A enzyme the same conformational changes as observed for the wild-type enzyme, although the scattering pattern of the Q137A and wild-type enzymes in the presence of PALA were identical. The overall structure of the Q137A enzyme is similar to that of the R-state structure of wild-type enzyme with PALA bound. However, there are differences in the manner by which the Q137A enzyme coordinates PALA, especially in the side chain positions of Arg105 and His134. The replacement of Gln137 by Ala also has a dramatic effect on the electrostatics of the active site. These data taken together suggest that the side chain of Gln137 in the wild-type enzyme is required for the binding of carbamoyl phosphate in the proper orientation so as to induce conformational changes required for the creation of the high-affinity aspartate binding site. The inability of carbamoyl phosphate to create the high-affinity binding site in the Q137A enzyme results in an enzyme locked in the low activity low affinity T state. These results emphasize the absolute requirement of the binding of carbamoyl phosphate for the creation of the high-affinity aspartate binding site and for inducing the homotropic cooperativity in aspartate transcarbamoylase. PMID:15890205

Crystal Structures of An F420-Dependent Glucose-6-Phosphate Dehydrogenase Fgd1 Involved in the Activation of the Anti-Tb Drug Candidate Pa-824 Reveal the Basis of Coenzyme And Substrate Binding

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bashiri, G.; Squire, C.J.; Moreland, N.J.

2009-05-11

The modified flavin coenzyme F{sub 420} is found in a restricted number of microorganisms. It is widely distributed in mycobacteria, however, where it is important in energy metabolism, and in Mycobacterium tuberculosis (Mtb) is implicated in redox processes related to non-replicating persistence. In Mtb, the F{sub 420}-dependent glucose-6-phosphate dehydrogenase FGD1 provides reduced F{sub 420} for the in vivo activation of the nitroimidazopyran prodrug PA-824, currently being developed for anti-tuberculosis therapy against both replicating and persistent bacteria. The structure of M. tuberculosis FGD1 has been determined by x-ray crystallography both in its apo state and in complex with F{sub 420} andmore » citrate at resolutions of 1.90 and 1.95{angstrom}, respectively. The structure reveals a highly specific F{sub 420} binding mode, which is shared with several other F{sub 420}-dependent enzymes. Citrate occupies the substrate binding pocket adjacent to F{sub 420} and is shown to be a competitive inhibitor (IC{sub 50} 43 {micro}m). Modeling of the binding of the glucose 6-phosphate (G6P) substrate identifies a positively charged phosphate binding pocket and shows that G6P, like citrate, packs against the isoalloxazine moiety of F{sub 420} and helps promote a butterfly bend conformation that facilitates F{sub 420} reduction and catalysis.« less

Chloroplast Phosphofructokinase

PubMed Central

Kelly, Grahame J.; Latzko, Erwin

1977-01-01

Chloroplast phosphofructokinase from spinach (Spinacia oleracea L.) was purified approximately 40-fold by a combination of fractionations with ammonium sulfate and acetone followed by chromatography on DEAE-Sephadex A-50. Positive cooperative kinetics was observed for the interaction between the enzyme and the substrate fructose 6-phosphate. The optimum pH shifted from 7.7 toward 7.0 as the fructose 6-phosphate concentration was taken below 0.5 mm. The second substrate was MgATP2− (Michaelis constant 30 μm). Free ATP inhibited the enzyme. Chloroplast phosphofructokinase was sensitive to inhibition by low concentration of phosphoenolpyruvate and glycolate 2-phosphate (especially at higher pH); these compounds inhibited in a positively cooperative fashion. Inhibitions by glycerate 2-phosphate (and probably glycerate 3-phosphate), citrate, and inorganic phosphate were also recorded; however, inorganic phosphate effectively relieved the inhibitions by phosphoenolpyruvate and glycolate 2-phosphate. These regulatory properties are considered to complement those of ADP-glucose pyrophosphorylase and fructosebisphosphatase in the regulation of chloroplast starch metabolism. PMID:16660079

Effect of N-benzoyl-D-phenylalanine and metformin on carbohydrate metabolic enzymes in neonatal streptozotocin diabetic rats.

PubMed

Ashokkumar, Natarajan; Pari, Leelavinothan

2005-01-01

The effect of N-benzoyl-D-phenylalanine (NBDP) and metformin was studied on the activities of carbohydrate metabolic enzymes in neonatal streptozotocin (nSTZ) non-insulin-dependent diabetic rats. To induce non-insulin-dependent diabetes mellitus (NIDDM), single dose injection of streptozotocin (STZ; 100 mg/kg body weight; i.p.) was given to 2-day old rats. After 10-12 weeks, rats weighing >150 g were selected for screening in NIDDM model, they were checked for fasting blood glucose concentrations to conform the status of NIDDM. NBDP (50,100 and 200 mg/kg body weight) was administered orally for 6 weeks into the confirmed diabetic rats. The activities of gluconeogenic enzymes were significantly increased, whereas the activities of hexokinase and glucose-6-phosphate dehydrogenase were significantly decreased in nSTZ diabetic rats. Both NBDP and metformin were able to restore the altered enzyme activities to almost control concentrations. Combination treatment was more effective than either drug alone. The administration of NBDP along with metformin to nSTZ diabetic rats normalizes blood glucose and causes marked improvement of altered carbohydrate metabolic enzymes during diabetes.

Action of nucleotide phosphotransferase of Escherichia coli on nicotinamide riboside and nicotinamide mononucleotide.

PubMed Central

Brunngraber, E F; Chargaff, E

1977-01-01

The action of the nucleotide phosphotransferase of Escherichia coli on nicotinamide riboside and on its 5'-phosphate results in the addition of one phosphate moiety to each of the substrates. Although the proof is not conclusive, it is likely that the phosphate group is transferred to the 3'-hydroxyl of the ribose. This is in contrast to the behavior of the enzyme toward NAD in which only the adenylic acid portion is phosphorylated enzymically. PMID:144913

Characterization of 9H-(1,3-dichlor-9, 9-dimethylacridin-2-ona-7-yl)-phosphate (DDAO) as substrate of PP-2A in a fluorimetric microplate assay for diarrhetic shellfish toxins (DSP).

PubMed

Leira, F; Vieites, J M; Vieytes, M R; Botana, L M

2000-12-01

Specific inhibition of protein-phosphatases by diarrhetic shellfish toxins (DSP) of the okadaic acid group, has led to the development of a fluorescent enzyme inhibition assay for these toxins using protein-phosphatase 2A (PP-2A) and fluorogenic substrates of the enzyme. Two different substrates of PP-2A have been previously used in this microplate assay: 4-methylumbelliferyl phosphate and fluorescein diphosphate (FDP). In this report, we present the results obtained using a new fluorogenic substrate of PP-2A, the compound dimethylacridinone phosphate (DDAO). A linear relationship between PP-2A concentration and DDAO-induced fluorescence was observed. Okadaic acid (0.0157-9.43 nM)-dependent inhibition of phosphatase activity showed similar results using FDP and DDAO. Recovery percentages obtained with FDP and DDAO in spiked mussel samples (both raw and canned) were very similar and reproducible. Comparative analysis of DSP-contaminated mussel samples by HPLC and FDP/DDAO-PP-2A showed a good correlation among all methods, thus demonstrating that DDAO can be used as a fluorogenic substrate to quantify okadaic acid and related toxins in bivalve molluscs with optimum reliability.

Metabolic switches and adaptations deduced from the proteomes of Streptomyces coelicolor wild type and phoP mutant grown in batch culture.

PubMed

Thomas, Louise; Hodgson, David A; Wentzel, Alexander; Nieselt, Kay; Ellingsen, Trond E; Moore, Jonathan; Morrissey, Edward R; Legaie, Roxane; Wohlleben, Wolfgang; Rodríguez-García, Antonio; Martín, Juan F; Burroughs, Nigel J; Wellington, Elizabeth M H; Smith, Margaret C M

2012-02-01

Bacteria in the genus Streptomyces are soil-dwelling oligotrophs and important producers of secondary metabolites. Previously, we showed that global messenger RNA expression was subject to a series of metabolic and regulatory switches during the lifetime of a fermentor batch culture of Streptomyces coelicolor M145. Here we analyze the proteome from eight time points from the same fermentor culture and, because phosphate availability is an important regulator of secondary metabolite production, compare this to the proteome of a similar time course from an S. coelicolor mutant, INB201 (ΔphoP), defective in the control of phosphate utilization. The proteomes provide a detailed view of enzymes involved in central carbon and nitrogen metabolism. Trends in protein expression over the time courses were deduced from a protein abundance index, which also revealed the importance of stress pathway proteins in both cultures. As expected, the ΔphoP mutant was deficient in expression of PhoP-dependent genes, and several putatively compensatory metabolic and regulatory pathways for phosphate scavenging were detected. Notably there is a succession of switches that coordinately induce the production of enzymes for five different secondary metabolite biosynthesis pathways over the course of the batch cultures.

Pyridoxic acid excretion during low vitamin B-6 intake, total fasting, and bed rest

NASA Technical Reports Server (NTRS)

Coburn, S. P.; Thampy, K. G.; Lane, H. W.; Conn, P. S.; Ziegler, P. J.; Costill, D. L.; Mahuren, J. D.; Fink, W. J.; Pearson, D. R.; Schaltenbrand, W. E.

1995-01-01

Vitamin B-6 metabolism in 10 volunteers during 21 d of total fasting was compared with results from 10 men consuming a diet low only in vitamin B-6 (1.76 mumol/d) and with men consuming a normal diet during bed rest. At the end of the fast mean plasma concentrations of vitamin B-6 metabolites and urinary excretion of 4-pyridoxic acid tended to be higher in the fasting subjects than in the low-vitamin B-6 group. The fasting subjects lost approximately 10% of their total vitamin B-6 pool and approximately 13% of their body weight. The low-vitamin B-6 group lost only approximately 4% of their vitamin B-6 pool. Compared with baseline, urinary excretion of pyridoxic acid was significantly increased during 17 wk of bed rest. There was no increase in pyridoxic acid excretion during a second 15-d bed rest study. These data suggest the possibility of complex interactions between diet and muscle metabolism that may influence indexes that are frequently used to assess vitamin B-6 status.

The hydrophobic substituent in aminophospholipids affects the formation kinetics of their Schiff bases.

PubMed

Caldés, Catalina; Vilanova, Bartolomé; Adrover, Miquel; Donoso, Josefa; Muñoz, Francisco

2013-04-01

Schiff bases (SBs) are the initial products of non-enzymatic glycation reactions, which are associated to some diabetes-related diseases. In this work, we used physiological pH and temperature conditions to study the formation kinetics of the SBs of 1,2-dihexanoyl-sn-glycero-3-phosphoethanolamine (DPHE) and 1,2-dihexanoyl-sn-glycero-3-phospho-l-serine (DHPS) with various glycating compounds and with pyridoxal 5'-phosphate (an effective glycation inhibitor). Based on the obtained results, the hydrophobic environment simultaneously decreases the nucleophilic character of the amino group (k1) and increases its pKa, thereby increasing the formation rate of SB (kobs). Therefore, the presence of hydrophobic chains in aminophospholipids facilitates the formation and stabilization of SBs, and also, in a biological environment, their glycation. Additionally, the results confirm the inhibitory action of B6 vitamers on aminophospholipid glycation. Copyright © 2013 Elsevier Ltd. All rights reserved.

Ribose 5-Phosphate Isomerase Investigations for the Undergraduate Biochemistry Laboratory

ERIC Educational Resources Information Center

Jewett, Kathy; Sandwick, Roger K.

2011-01-01

The enzyme ribose 5-phosphate isomerase (RpiA) has many features that make it attractive as a focal point of a semester-long, advanced biochemistry laboratory for undergraduate students. The protein can easily and inexpensively be isolated from spinach using traditional purification techniques. Characterization of RpiA enzyme activity can be…

Silencing trehalose-6-phosphate synthase incapacitates adult mosquitoes by interfering with the biosynthetic pathway for flight fuel

USDA-ARS?s Scientific Manuscript database

Trehalose is a disaccharide comprised of two glucose molecules. It is the main blood sugar of insects and is essential for flight. Trehalose is synthesized by two enzymes: trehalose-6-phosphate synthase (T6PS) converts glucose-6-phosphate to trehalose-6-phosphate, and trehalose-6-phosphate phosphata...

Stereospecificity of myo-inositol hexakisphosphate dephosphorylation by a phytate-degrading enzyme of baker's yeast.

PubMed

Greiner, R; Alminger, M L; Carlsson, N G

2001-05-01

During food processing such as baking, phytate is dephosphorylated to produce degradation products, such as myo-inositol pentakis-, tetrakis-, tris-, bis-, and monophosphates. Certain myo-inositol phosphates have been proposed to have positive effects on human health. The position of the phosphate groups on the myo-inositol ring is thereby of great significance for their physiological functions. Using a combination of high-performance ion chromatography analysis and kinetic studies the stereospecificity of myo-inositol hexakisphosphate dephosphorylation by a phytate-degrading enzyme from baker's yeast (Saccharomyces cerevisiae) was established. The data demonstrate that the phytate-degrading enzyme from baker's yeast dephosphorylates myo-inositol hexakisphosphate in a stereospecific way by sequential removal of phosphate groups via D-Ins(1,2,4,5,6)P(5), D-Ins(1,2,5,6)P(4), D-Ins(1,2,6)P(3), D-Ins(1,2)P(2), to finally Ins(2)P (notation 3/4/5/6/1). Knowledge of the absolute stereochemical specificity of the baker's yeast phytase allows use of the enzyme to produce defined myo-inositol phosphates for kinetic and physiological studies.

Purification and characterization of chromatin-bound DNA-dependent RNA polymerase I from parsley (Petroselinum crispum). Influence of nucleoside triphosphates.

PubMed Central

Grossmann, K; Friedrich, H; Seitz, U

1980-01-01

The isolation and purification of DNA-dependent RNA polymerase I (EC 2.7.7.6) from parsley (Petroselinum crispum) callus cells grown in suspension culture is described. The enzyme was solubilized from isolated chromatin. Purification was achieved by using DEAE- and phospho-cellulose in batches, followed by column chromatography on DEAE- and phospho-cellulose (two columns) and density-gradient centrifugation. The highly purified enzyme was stable over several months. The properties of purified parsley RNA polymerase I were investigated. Optimum concentration for Mn2+ was 1 mM, and for Mg2+ 4-6 mM, Mn2+ was slightly more stimulatory than Mg2+. The enzyme was most active at low ionic strengths [10-20 mM-(NH4)SO4]. The influence of various phosphates was tested: pyrophosphate inhibited RNA polymerase at low concentrations, whereas orthophosphate had no effect on the enzyme activity. ADP was slightly inhibitory, and AMP had no effect on the enzyme reaction. Nucleoside triphosphates and bivalent cations in equimolar concentrations in the range 4-11 mM did not influence the RNA synthesis in vitro. Free nucleoside triphosphates in excess of this 1:1 ratio inhibited the enzyme activity, unlike free bivalent cations, which stimulated RNA polymerase I. PMID:7470092

Characterization of Runella slithyformis HD-Pnk, a bifunctional DNA/RNA end-healing enzyme composed of an N-terminal 2',3' -phosphoesterase HD domain and a C-terminal 5' -OH polynucleotide kinase domain.

PubMed

Munir, Annum; Shuman, Stewart

2016-11-28

5' and 3' end healing are key steps in nucleic acid break repair in which 5' -OH ends are phosphorylated by a polynucleotide kinase and 3' -PO 4 or 2',3' -cyclic-PO 4 ends are hydrolyzed by a phosphoesterase to generate the 5' -PO 4 and 3' -OH termini required for sealing by classic polynucleotide ligases. End healing and sealing enzymes are present in diverse bacterial taxa, often organized as modular units within a single multifunctional polypeptide or as subunits of a repair complex. Here we identify and characterize Runella slithyformis HD-Pnk as a novel bifunctional end-healing enzyme composed of an N-terminal 2',3' -phosphoesterase HD domain and a C-terminal 5' -OH polynucleotide kinase P-loop domain. HD-Pnk phosphorylates 5' -OH polynucleotides (9-mers or longer) in the presence of magnesium and any NTP donor. HD-Pnk dephosphorylates RNA 2',3' -cyclic phosphate, RNA 3' -phosphate, RNA 2' -phosphate, and DNA 3' -phosphate ends in the presence of a transition metal cofactor, which can be nickel, copper or cobalt. HD-Pnkp homologs are present in genera from eleven bacterial phyla and are often encoded in an operon with a putative ATP-dependent polynucleotide ligase. The present study provides insights to the diversity of nucleic acid repair strategies via the characterization of Runella slithyformis HD-Pnkp as the exemplar of a novel clade of dual 5' and 3' end-healing enzymes that phosphorylate 5' -OH termini and dephosphorylate 2',3' -cyclic-PO 4 , 3' -PO 4 , and 2' -PO 4 ends. The distinctive feature of HD-Pnk is its domain composition: a fusion of an N-terminal HD phosphohydrolase module to a C-terminal P-loop polynucleotide kinase module. Homologs of Runella HD-Pnk with the same domain composition, domain order, and similar polypeptide size are distributed widely among genera from eleven bacterial phyla. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Mutations affecting transport of the hexitols D-mannitol, D-glucitol, and galactitol in Escherichia coli K-12: isolation and mapping.

PubMed Central

Lengeler, J

1975-01-01

Mutants of Escherichia coli K-12 unable to grow on any of the three naturally occurring hexitols D-manitol, D-glucitol, and galactitol and, among these specifically, mutants with altered transport and phosphorylating activity have been isolated. Different isolation procedures have been utilized, including suicide by D-[3H]mannitol, chemotaxis, and resistance to the toxic hexitol analogue 2-deoxy-arabino-hexitol. Mutations thus obtained have been mapped in four distinct operons. (i) Mutations affecting an enzyme II-complexmt1 activity of the phosphoenolpyruvate-dependent phosphotransferase system all map in gene mtlA. This gene has previously been shown (Solomon and Lin, 1972) to be part of an operon, mtl, located at 71 min on the E. coli linkage map containing, in addition to mtlA, the cis-dominant regulatory gene mtlC and mtlD, the structural gene for the enzyme D-mannitol-1-phosphate dehydrogenase. The gene order in this operon, induced by D-mannitol, is mtlC A D. (ii) Mutations in gene gutA affecting a second enzyme II-complexgut of the phosphotransferase system map at 51 min, clustered in operon gutC A D together with the cis-dominant regulatory gene gutC and the structural gene gutD for the enzyme D-glucitol-6-phosphate dehydrogenase. The gut operon, previously called sbl or srl, is induced by D-glucitol. (iii) Mutations affecting the transport and catabolism of galactitol are clustered in a third operon, gatC A D, located at 40.5 min. This operon again contains a cis-dominant regulatory gene, gatC, the structural gene gatD for galactitol-1-phosphate dehydrogenase, and gene gatA coding for a thrid hexitol-specific enzyme II-complexgat. Other genes coding for two additional enzymes involved in galactitol catabolism apparently are not linked to gatC A D. (iv) A fourth class of mutants pleiotropically negative for hexitol growth and transport maps in the pts operon. Triple-negative mutants (mtlA gutA gatA) do not have further transport or phosphorylating activity for any of the three hexitols. PMID:1100602

Mechanism of the 2,3-diphosphoglycerate-dependent phosphoglycerate mutase from rabbit muscle.

PubMed

Britton, H G; Clarke, J B

1972-11-01

1. The properties and kinetics of the 2,3-diphosphoglycerate-dependent phosphoglycerate mutases are discussed. There are at least three possible mechanisms for the reaction: (i) a phosphoenzyme (Ping Pong) mechanism; (ii) an intermolecular transfer of phosphate from 2,3-diphosphoglycerate to the substrates (sequential mechanism); (iii) an intramolecular transfer of phosphate. It is concluded that these mechanisms cannot be distinguished by conventional kinetic measurements. 2. The fluxes for the different mechanisms are calculated and it is shown that it should be possible to distinguish between the mechanisms by appropriate induced-transport tests and by comparing the fluxes of (32)P- and (14)C-labelled substrates at chemical equilibrium. 3. With (14)C-labelled substrates no induced transport was found over a wide concentration range, and with (32)P-labelled substrates co-transport occurred that was independent of concentration over a twofold range. (14)C-labelled substrates exchange at twice the rate of (32)P-labelled substrates at chemical equilibrium. The results were completely in accord with a phosphoenzyme mechanism and indicated a rate constant for the isomerization of the phosphoenzyme of not less than 4x10(6)s(-1). The intramolecular transfer of phosphate (and intermolecular transfer between two or more molecules of substrate) were completely excluded. The intermolecular transfer of phosphate from 2,3-diphosphoglycerate would have been compatible with the results only if the K(m) for 2-phosphoglycerate had been over 7.5-fold smaller than the observed value and if an isomerization of the enzyme-2,3-diphosphoglycerate complex had been the major rate-limiting step in the reaction. 4. The very rapid isomerization of the phosphoenzyme that the experiments demonstrate suggests a mechanism that does not involve a formal isomerization. According to this new scheme the enzyme is closely related mechanistically and perhaps evolutionarily to a 2,3-diphosphoglycerate diphosphatase.

Mechanism of the 2,3-diphosphoglycerate-dependent phosphoglycerate mutase from rabbit muscle

PubMed Central

Britton, H. G.; Clarke, J. B.

1972-01-01

1. The properties and kinetics of the 2,3-diphosphoglycerate-dependent phosphoglycerate mutases are discussed. There are at least three possible mechanisms for the reaction: (i) a phosphoenzyme (Ping Pong) mechanism; (ii) an intermolecular transfer of phosphate from 2,3-diphosphoglycerate to the substrates (sequential mechanism); (iii) an intramolecular transfer of phosphate. It is concluded that these mechanisms cannot be distinguished by conventional kinetic measurements. 2. The fluxes for the different mechanisms are calculated and it is shown that it should be possible to distinguish between the mechanisms by appropriate induced-transport tests and by comparing the fluxes of 32P- and 14C-labelled substrates at chemical equilibrium. 3. With 14C-labelled substrates no induced transport was found over a wide concentration range, and with 32P-labelled substrates co-transport occurred that was independent of concentration over a twofold range. 14C-labelled substrates exchange at twice the rate of 32P-labelled substrates at chemical equilibrium. The results were completely in accord with a phosphoenzyme mechanism and indicated a rate constant for the isomerization of the phosphoenzyme of not less than 4×106s−1. The intramolecular transfer of phosphate (and intermolecular transfer between two or more molecules of substrate) were completely excluded. The intermolecular transfer of phosphate from 2,3-diphosphoglycerate would have been compatible with the results only if the Km for 2-phosphoglycerate had been over 7.5-fold smaller than the observed value and if an isomerization of the enzyme-2,3-diphosphoglycerate complex had been the major rate-limiting step in the reaction. 4. The very rapid isomerization of the phosphoenzyme that the experiments demonstrate suggests a mechanism that does not involve a formal isomerization. According to this new scheme the enzyme is closely related mechanistically and perhaps evolutionarily to a 2,3-diphosphoglycerate diphosphatase. PMID:4677138

Detection of phosphohydrolytic enzyme activity through the oxygen isotope composition of dissolved phosphate

NASA Astrophysics Data System (ADS)

Colman, A. S.

2016-02-01

Phosphohydrolytic enzymes play an important role in phosphorus remineralization. As they release phosphate (Pi) from various organophosphorus compounds, these enzymes facilitate the transfer of oxygen atoms from water to the phosphoryl moieties. Most such enzymatic reactions impart a significant isotopic fractionation to the oxygen transferred. If this reaction occurs within a cell, then the resultant oxygen isotope signal is overprinted by continued recycling of the Pi. However, if this reaction occurs extracellularly, then the isotopic signal will be preserved until the Pi is transported back into a cell. Thus, the oxygen isotope composition of Pi (δ18Op) in an aquatic ecosystem can serve as a useful indicator of the mechanisms by which P is remineralized. We develop a time-dependent model illustrating the sensitivity of the δ18O of dissolved phosphate to various modes of P remineralization. The model is informed by cell lysis experiments that reveal the relative proportions of P­i that are directly liberated from cytosol vs. regenerated from co-liberated dissolved organic phosphorus compounds via extracellular hydrolysis. By incorporating both cellular uptake and release fluxes of P, we show that the degree of isotopic disequilibrium in an aquatic ecosystem can be a strong indicator of P remineralization mode. Apparent oxygen isotope equilibrium between Pi and water arises in this model as a steady-state scenario in which fractionation upon cellular uptake of Pi counterbalances the hydrolytic source flux of disequilibrated Pi. Low and high rates of extracellular phosphohydrolase activity are shown to produce steady-state δ18Op values that are respectively above or below thermodynamic equilibrium compositions.

Characterization of a Glutamate Decarboxylase (GAD) from Enterococcus avium M5 Isolated from Jeotgal, a Korean Fermented Seafood.

PubMed

Lee, Kang Wook; Shim, Jae Min; Yao, Zhuang; Kim, Jeong A; Kim, Hyun-Jin; Kim, Jeong Hwan

2017-07-28

To develop starters for the production of functional foods or materials, lactic acid bacteria producing γ-aminobutyric acid (GABA) were screened from jeotgals, Korean fermented seafoods. One isolate producing a high amount of GABA from monosodium L-glutamate (MSG) was identified as Enterococcus avium by 16S rRNA gene sequencing. E. avium M5 produced 18.47 ± 1.26 mg/ml GABA when incubated for 48 h at 37°C in MRS broth with MSG (3% (w/v)). A gadB gene encoding glutamate decarboxylase (GAD) was cloned and overexpressed in E. coli BL21 (DE3) using the pET26b (+) expression vector. Recombinant GAD was purified through a Ni-NTA column and the size was estimated to be 53 kDa by SDS-PAGE. Maximum GAD activity was observed at pH 4.5 and 55°C and the activity was dependent on pyridoxal 5'-phosphate. The K m and V max values of GAD were 3.26 ± 0.21 mM and 0.0120 ± 0.0001 mM/min, respectively, when MSG was used as a substrate. Enterococcus avium M5 secretes a lot of GABA when grown on MRS with MSG, and the strain is useful for the production of fermented foods containing a high amount of GABA.

Genetic basis of stage-specific melanism: a putative role for a cysteine sulfinic acid decarboxylase in insect pigmentation.

PubMed

Saenko, S V; Jerónimo, M A; Beldade, P

2012-06-01

Melanism, the overall darkening of the body, is a widespread form of animal adaptation to particular environments, and includes bookcase examples of evolution by natural selection, such as industrial melanism in the peppered moth. The major components of the melanin biosynthesis pathway have been characterized in model insects, but little is known about the genetic basis of life-stage specific melanism such as cases described in some lepidopteran species. Here, we investigate two melanic mutations of Bicyclus anynana butterflies, called Chocolate and melanine, that exclusively affect pigmentation of the larval and adult stages, respectively. Our analysis of Mendelian segregation patterns reveals that the larval and adult melanic phenotypes are due to alleles at different, independently segregating loci. Our linkage mapping analysis excludes the pigmentation candidate gene black as the melanine locus, and implicates a gene encoding a putative pyridoxal phosphate-dependant cysteine sulfinic acid decarboxylase as the Chocolate locus. We show variation in coding sequence and in expression levels for this candidate larval melanism locus. This is the first study that suggests a biological function for this gene in insects. Our findings open up exciting opportunities to study the role of this locus in the evolution of adaptive variation in pigmentation, and the uncoupling of regulation of pigment biosynthesis across developmental stages with different ecologies and pressures on body coloration.

Genetic basis of stage-specific melanism: a putative role for a cysteine sulfinic acid decarboxylase in insect pigmentation

PubMed Central

Saenko, S V; Jerónimo, M A; Beldade, P

2012-01-01

Melanism, the overall darkening of the body, is a widespread form of animal adaptation to particular environments, and includes bookcase examples of evolution by natural selection, such as industrial melanism in the peppered moth. The major components of the melanin biosynthesis pathway have been characterized in model insects, but little is known about the genetic basis of life-stage specific melanism such as cases described in some lepidopteran species. Here, we investigate two melanic mutations of Bicyclus anynana butterflies, called Chocolate and melanine, that exclusively affect pigmentation of the larval and adult stages, respectively. Our analysis of Mendelian segregation patterns reveals that the larval and adult melanic phenotypes are due to alleles at different, independently segregating loci. Our linkage mapping analysis excludes the pigmentation candidate gene black as the melanine locus, and implicates a gene encoding a putative pyridoxal phosphate-dependant cysteine sulfinic acid decarboxylase as the Chocolate locus. We show variation in coding sequence and in expression levels for this candidate larval melanism locus. This is the first study that suggests a biological function for this gene in insects. Our findings open up exciting opportunities to study the role of this locus in the evolution of adaptive variation in pigmentation, and the uncoupling of regulation of pigment biosynthesis across developmental stages with different ecologies and pressures on body coloration. PMID:22234245

Streptococcus mutans serotype c tagatose 6-phosphate pathway gene cluster.

PubMed Central

Jagusztyn-Krynicka, E K; Hansen, J B; Crow, V L; Thomas, T D; Honeyman, A L; Curtiss, R

1992-01-01

DNA cloned into Escherichia coli K-12 from a serotype c strain of Streptococcus mutans encodes three enzyme activities for galactose utilization via the tagatose 6-phosphate pathway: galactose 6-phosphate isomerase, tagatose 6-phosphate kinase, and tagatose-1,6-bisphosphate aldolase. The genes coding for the tagatose 6-phosphate pathway were located on a 3.28-kb HindIII DNA fragment. Analysis of the tagatose proteins expressed by recombinant plasmids in minicells was used to determine the sizes of the various gene products. Mutagenesis of these plasmids with transposon Tn5 was used to determine the order of the tagatose genes. Tagatose 6-phosphate isomerase appears to be composed of 14- and 19-kDa subunits. The sizes of the kinase and aldolase were found to be 34 and 36 kDa, respectively. These values correspond to those reported previously for the tagatose pathway enzymes in Staphylococcus aureus and Lactococcus lactis. Images PMID:1328153

Dextransucrase production using cashew apple juice as substrate: effect of phosphate and yeast extract addition.

PubMed

Chagas, Clarice M A; Honorato, Talita L; Pinto, Gustavo A S; Maia, Geraldo A; Rodrigues, Sueli

2007-05-01

Cashew apples are considered agriculture excess in the Brazilian Northeast because cashew trees are cultivated primarily with the aim of cashew nut production. In this work, the use of cashew apple juice as a substrate for Leuconostoc mesenteroides cultivation was investigated. The effect of yeast extract and phosphate addition was evaluated using factorial planning tools. Both phosphate and yeast extract addition were significant factors for biomass growth, but had no significant effect on maximum enzyme activity. The enzyme activities found in cashew apple juice assays were at least 3.5 times higher than the activity found in the synthetic medium. Assays with pH control (pH = 6.5) were also carried out. The pH-controlled fermentation enhanced biomass growth, but decreased the enzyme activity. Crude enzyme free of cells produced using cashew apple juice was stable for 16 h at 30 degrees C at a pH of 5.0.

Crystal structure of fuculose aldolase from the Antarctic psychrophilic yeast Glaciozyma antarctica PI12.

PubMed

Jaafar, Nardiah Rizwana; Littler, Dene; Beddoe, Travis; Rossjohn, Jamie; Illias, Rosli Md; Mahadi, Nor Muhammad; Mackeen, Mukram Mohamed; Murad, Abdul Munir Abdul; Abu Bakar, Farah Diba

2016-11-01

Fuculose-1-phosphate aldolase (FucA) catalyses the reversible cleavage of L-fuculose 1-phosphate to dihydroxyacetone phosphate (DHAP) and L-lactaldehyde. This enzyme from mesophiles and thermophiles has been extensively studied; however, there is no report on this enzyme from a psychrophile. In this study, the gene encoding FucA from Glaciozyma antarctica PI12 (GaFucA) was cloned and the enzyme was overexpressed in Escherichia coli, purified and crystallized. The tetrameric structure of GaFucA was determined to 1.34 Å resolution. The overall architecture of GaFucA and its catalytically essential histidine triad are highly conserved among other fuculose aldolases. Comparisons of structural features between GaFucA and its mesophilic and thermophilic homologues revealed that the enzyme has typical psychrophilic attributes, indicated by the presence of a high number of nonpolar residues at the surface and a lower number of arginine residues.

Metal cation controls phosphate release in the myosin ATPase.

PubMed

Ge, Jinghua; Huang, Furong; Nesmelov, Yuri E

2017-11-01

Myosin is an enzyme that utilizes ATP to produce a conformational change generating a force. The kinetics of the myosin reverse recovery stroke depends on the metal cation complexed with ATP. The reverse recovery stroke is slow for MgATP and fast for MnATP. The metal ion coordinates the γ phosphate of ATP in the myosin active site. It is accepted that the reverse recovery stroke is correlated with the phosphate release; therefore, magnesium "holds" phosphate tighter than manganese. Magnesium and manganese are similar ions in terms of their chemical properties and the shell complexation; hence, we propose to use these ions to study the mechanism of the phosphate release. Analysis of octahedral complexes of magnesium and manganese show that the partial charge of magnesium is higher than that of manganese and the slightly larger size of manganese ion makes its ionic potential smaller. We hypothesize that electrostatics play a role in keeping and releasing the abstracted γ phosphate in the active site, and the stronger electric charge of magnesium ion holds γ phosphate tighter. We used stable myosin-nucleotide analog complex and Raman spectroscopy to examine the effect of the metal cation on the relative position of γ phosphate analog in the active site. We found that in the manganese complex, the γ phosphate analog is 0.01 nm further away from ADP than in the magnesium complex. We conclude that the ionic potential of the metal cation plays a role in the retention of the abstracted phosphate. © 2017 The Protein Society.

Remobilization of Phytol from Chlorophyll Degradation Is Essential for Tocopherol Synthesis and Growth of Arabidopsis

PubMed Central

vom Dorp, Katharina; Hölzl, Georg; Plohmann, Christian; Eisenhut, Marion; Abraham, Marion

2015-01-01

Phytol from chlorophyll degradation can be phosphorylated to phytyl-phosphate and phytyl-diphosphate, the substrate for tocopherol (vitamin E) synthesis. A candidate for the phytyl-phosphate kinase from Arabidopsis thaliana (At1g78620) was identified via a phylogeny-based approach. This gene was designated VITAMIN E DEFICIENT6 (VTE6) because the leaves of the Arabidopsis vte6 mutants are tocopherol deficient. The vte6 mutant plants are incapable of photoautotrophic growth. Phytol and phytyl-phosphate accumulate, and the phytyl-diphosphate content is strongly decreased in vte6 leaves. Phytol feeding and enzyme assays with Arabidopsis and recombinant Escherichia coli cells demonstrated that VTE6 has phytyl-P kinase activity. Overexpression of VTE6 resulted in increased phytyl-diphosphate and tocopherol contents in seeds, indicating that VTE6 encodes phytyl-phosphate kinase. The severe growth retardation of vte6 mutants was partially rescued by introducing the phytol kinase mutation vte5. Double mutant plants (vte5 vte6) are tocopherol deficient and contain more chlorophyll, but reduced amounts of phytol and phytyl-phosphate compared with vte6 mutants, suggesting that phytol or phytyl-phosphate are detrimental to plant growth. Therefore, VTE6 represents the missing phytyl-phosphate kinase, linking phytol release from chlorophyll with tocopherol synthesis. Moreover, tocopherol synthesis in leaves depends on phytol derived from chlorophyll, not on de novo synthesis of phytyl-diphosphate from geranylgeranyl-diphosphate. PMID:26452599

The expression and regulation of enzymes mediating the biosynthesis of triglycerides and phospholipids in keratinocytes/epidermis

PubMed Central

Feingold, Kenneth R

2011-01-01

Triglycerides and phospholipids play an important role in epidermal permability barrier formation and function. They are synthesized de novo in the epidermis via the glycerol-3-phosphate pathway, catalyzed sequentially by a group of enzymes that have multiple isoforms including glycerol-3-phosphate acyltransferase (GPAT), 1-acylglycerol-3-phosphate acyltransferase (AGPAT), Lipin and diacylglycerol acyltransferase (DGAT). Here we review the current knowledge of GPAT, AGPAT, Lipin and DGAT enzymes in keratinocytes/epidermis focusing on the expression levels of the various isoforms and their localization in mouse epidermis. Additionally, the factors regulating their gene expression, including calcium induced differentiation, PPAR and LXR activators, and the effect of acute permeability barrier disruption will be discussed. PMID:21695015

Environmentally relevant organophosphate triesters in herring gulls: In vitro biotransformation and kinetics and diester metabolite formation using a hepatic microsomal assay.

PubMed

Greaves, Alana K; Su, Guanyong; Letcher, Robert J

2016-10-01

The in vitro biotransformation and kinetics of six organophosphate triester (OPE) flame retardants were investigated in herring gulls (Larus argentatus) from the Great Lakes using a hepatic microsomal metabolism assay. Administration of each individual OPE (tri-n-butyl phosphate (TNBP), tris(2-butoxyethyl) phosphate (TBOEP), triphenyl phosphate (TPHP), triethyl phosphate (TEP), tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) and tris(2-chloroisopropyl) phosphate (TCIPP)) to the in vitro assay (concentration range 0.01 to 10μM) resulted in rapid depletion with the exception of TEP. Following the Michaelis-Menten enzyme kinetics model, a preliminary 2-minute incubation period was used to estimate the Vmax (±SE) values (i.e., the maximal rate of reaction for a saturated enzyme system), which ranged from 5.0±0.4 (TPHP) to 29±18pmol/min/mg protein (TBOEP), as well as the KM (±SE) values (i.e., the OPE concentration corresponding to one half of the Vmax), which ranged from 9.8±1 (TPHP) to 189±135nM (TBOEP). Biotransformation assays over a 100-minute incubation period revealed that TNBP was metabolized most rapidly (with a depletion rate of 73±4pmol/min/mg protein), followed by TBOEP (53±8pmol/min/mg), TCIPP (27±1pmol/min/mg), TPHP (22±2pmol/min/mg) and TDCIPP (8±1pmol/min/mg). In vitro biotransformation of OP triesters was clearly structure-dependent where non-halogenated alkyl OP triesters were metabolized more rapidly than halogenated alkyl triesters. Halogenated OP triesters were transformed to their respective diesters more efficiently relative to non-halogenated OP triesters. To our knowledge, this is the first study to investigate OP triester metabolism and OP diester formation in an avian or wildlife model system, which is important to understand the fate and biological activity of OPEs in an exposed organism. Copyright © 2016 Elsevier Inc. All rights reserved.

[Activity and thermal stability of acid phosphatase in homogenates of Amoeba proteus, acclimated to various temperatures].

PubMed

Sopina, V A

2001-01-01

Activity and thermoresistance of acid phosphatase were determined in supernatant of Amoeba proteus homogenates using 1-naphthyl phosphate (pH 4.0) and p-nitrophenyl phosphate (pH 5.5). Although tartrate-resistant and tartrate-sensitive acid phosphatases hydrolyse both substrates, the former mainly hydrolyses p-nitrophenyl phosphate and the latter 1-naphthyl phosphate. A decrease in the activity of the total and tartrate-sensitive acid phosphatases, when using 1-naphthyl phosphate, and of the total and tartrate-resistant acid phosphatases, when using p-nitrophenyl phosphate, was found in amoebae acclimated to 10 degrees C (10 degrees-amoebae) compared to those acclimated to 25 degrees C (25 degrees-amoebae). Using 1-naphthyl phosphate, the thermoresistance of the total acid phosphatase was lower in 10 degrees-amoebae than in 25 degrees-amoebae, but the thermostability of tartrate-resistant enzyme was the same in both groups of amoebae. Using p-nitrophenyl phosphate, the thermoresistance of the total and tartrate-resistant acid phosphatases was lower (the latter only slightly) in 10 degrees-amoebae than in 25 degrees-amoebae. It is suggested that at least with the use of 1-naphthyl phosphate a decrease in thermostability of the total acid phosphatase may be due to a decrease in thermoresistance of tartrate-sensitive enzyme. The results obtained confirm the author's previous data on the activity and thermostability of electrophoretic forms of acid phosphatase using 2-naphthyl phosphate in 10- and 25 degrees-amoebae (Sopina, 2001). It is the first case of discovering a correlation between changes in primary cell thermoresistance of amoebae cultured at different temperatures and changes in the activity and thermostability of acid phosphatase in their homogenates, with the number of electrophoretic forms of this enzyme and their mobility being permanent.

Light-dependent activation of phosphoenolpyruvate carboxylase by reversible phosphorylation in cluster roots of white lupin plants: diurnal control in response to photosynthate supply

PubMed Central

Feil, Regina; Lunn, John E.; Plaxton, William C.

2016-01-01

Background and Aims Phosphoenolpyruvate carboxylase (PEPC) is a tightly regulated enzyme that controls carbohydrate partitioning to organic acid anions (malate, citrate) excreted in copious amounts by cluster roots of inorganic phosphate (Pi)-deprived white lupin plants. Excreted malate and citrate solubilize otherwise inaccessible sources of mineralized soil Pi for plant uptake. The aim of this study was to test the hypotheses that (1) PEPC is post-translationally activated by reversible phosphorylation in cluster roots of illuminated white lupin plants, and (2) light-dependent phosphorylation of cluster root PEPC is associated with elevated intracellular levels of sucrose and its signalling metabolite, trehalose-6-phosphate. Methods White lupin plants were cultivated hydroponically at low Pi levels (≤1 µm) and subjected to various light/dark pretreatments. Cluster root PEPC activity and in vivo phosphorylation status were analysed to assess the enzyme’s diurnal, post-translational control in response to light and dark. Levels of various metabolites, including sucrose and trehalose-6-phosphate, were also quantified in cluster root extracts using enzymatic and spectrometric methods. Key Results During the daytime the cluster root PEPC was activated by phosphorylation at its conserved N-terminal seryl residue. Darkness triggered a progressive reduction in PEPC phosphorylation to undetectable levels, and this was correlated with 75–80 % decreases in concentrations of sucrose and trehalose-6- phosphate. Conclusions Reversible, light-dependent regulatory PEPC phosphorylation occurs in cluster roots of Pi-deprived white lupin plants. This likely facilitates the well-documented light- and sucrose-dependent exudation of Pi-solubilizing organic acid anions by the cluster roots. PEPC’s in vivo phosphorylation status appears to be modulated by sucrose translocated from CO2-fixing leaves into the non-photosynthetic cluster roots. PMID:27063365

Formation of adenosine 5'-tetraphosphate from the acyl phosphate intermediate: a difference between the MurC and MurD synthetases of Escherichia coli.

PubMed

Bouhss, A; Dementin, S; van Heijenoort, J; Parquet, C; Blanot, D

1999-06-18

The mechanism of the Mur synthetases of peptidoglycan biosynthesis is thought to involve in each case the successive formation of an acyl phosphate and a tetrahedral intermediate. The existence of the acyl phosphates for the MurC and MurD enzymes from Escherichia coli was firmly established by their in situ reduction by sodium borohydride followed by acid hydrolysis, yielding the corresponding amino alcohols. Furthermore, it was found that MurD, but not MurC, catalyses the synthesis of adenosine 5'-tetraphosphate from the acyl phosphate, thereby substantiating its existence and pointing out a difference between the two enzymes.

Thermolabile triose phosphate isomerase in a psychrophilic Clostridium.

NASA Technical Reports Server (NTRS)

Shing, Y. W.; Akagi, J. M.; Himes, R. H.

1972-01-01

It was found that a psychrophilic Clostridium contains a triose phosphate isomerase which is very labile at moderate temperatures. An investigation showed that the optimal growth temperature of the psychrophile was between 15 and 20 deg C. No growth occurred at 25 deg C. The thermostability of the glycolytic enzymes in the cell-free extracts of Clostridium sp. strain 69 was studied. The data obtained show that the triose phosphate isomerase is quite labile at moderate temperatures. The instability of the enzyme is sufficient to explain the low maximum growth temperature of the psychrophile.

Different patterns of nuclear and mitochondrial penetration by the G3 PAMAM dendrimer and its biotin–pyridoxal bioconjugate BC-PAMAM in normal and cancer cells in vitro

PubMed Central

Uram, Łukasz; Szuster, Magdalena; Filipowicz, Aleksandra; Gargasz, Krzysztof; Wołowiec, Stanisław; Wałajtys-Rode, Elżbieta

2015-01-01

The intracellular localization and colocalization of a fluorescently labeled G3 amine-terminated cationic polyamidoamine (PAMAM) dendrimer and its biotin–pyridoxal (BC-PAMAM) bioconjugate were investigated in a concentration-dependent manner in normal human fibroblast (BJ) and squamous epithelial carcinoma (SCC-15) cell lines. After 24 hours treatment, both cell lines revealed different patterns of intracellular dendrimer accumulation depending on their cytotoxic effects. Cancer cells exhibited much higher (20-fold) tolerance for native PAMAM treatment than fibroblasts, whereas BC-PAMAM was significantly toxic only for fibroblasts at 50 µM concentration. Fibroblasts accumulated the native and bioconjugated dendrimers in a concentration-dependent manner at nontoxic range of concentration, with significantly lower bioconjugate loading. After reaching the cytotoxicity level, fluorescein isothiocyanate-PAMAM accumulation remains at high, comparable level. In cancer cells, native PAMAM loading at higher, but not cytotoxic concentrations, was kept at constant level with a sharp increase at toxic concentration. Mander’s coefficient calculated for fibroblasts and cancer cells confirmed more efficient native PAMAM penetration as compared to BC-PAMAM. Significant differences in nuclear dendrimer penetration were observed for both cell lines. In cancer cells, PAMAM signals amounted to ~25%–35% of the total nuclei area at all investigated concentrations, with lower level (15%–25%) observed for BC-PAMAM. In fibroblasts, the dendrimer nuclear signal amounted to 15% at nontoxic and up to 70% at toxic concentrations, whereas BC-PAMAM remained at a lower concentration-dependent level (0.3%–20%). Mitochondrial localization of PAMAM and BC-PAMAM revealed similar patterns in both cell lines, depending on the extracellular dendrimer concentration, and presented significantly lower signals from BC-PAMAM, which correlated well with the cytotoxicity. PMID:26379435

Allosteric Regulation of Lactobacillus plantarum Xylulose 5-Phosphate/Fructose 6-Phosphate Phosphoketolase (Xfp)

PubMed Central

Glenn, Katie

2015-01-01

ABSTRACT Xylulose 5-phosphate/fructose 6-phosphate phosphoketolase (Xfp), which catalyzes the conversion of xylulose 5-phosphate (X5P) or fructose 6-phosphate (F6P) to acetyl phosphate, plays a key role in carbohydrate metabolism in a number of bacteria. Recently, we demonstrated that the fungal Cryptococcus neoformans Xfp2 exhibits both substrate cooperativity for all substrates (X5P, F6P, and Pi) and allosteric regulation in the forms of inhibition by phosphoenolpyruvate (PEP), oxaloacetic acid (OAA), and ATP and activation by AMP (K. Glenn, C. Ingram-Smith, and K. S. Smith. Eukaryot Cell 13:657–663, 2014). Allosteric regulation has not been reported previously for the characterized bacterial Xfps. Here, we report the discovery of substrate cooperativity and allosteric regulation among bacterial Xfps, specifically the Lactobacillus plantarum Xfp. L. plantarum Xfp is an allosteric enzyme inhibited by PEP, OAA, and glyoxylate but unaffected by the presence of ATP or AMP. Glyoxylate is an additional inhibitor to those previously reported for C. neoformans Xfp2. As with C. neoformans Xfp2, PEP and OAA share the same or possess overlapping sites on L. plantarum Xfp. Glyoxylate, which had the lowest half-maximal inhibitory concentration of the three inhibitors, binds at a separate site. This study demonstrates that substrate cooperativity and allosteric regulation may be common properties among bacterial and eukaryotic Xfp enzymes, yet important differences exist between the enzymes in these two domains. IMPORTANCE Xylulose 5-phosphate/fructose 6-phosphate phosphoketolase (Xfp) plays a key role in carbohydrate metabolism in a number of bacteria. Although we recently demonstrated that the fungal Cryptococcus Xfp is subject to substrate cooperativity and allosteric regulation, neither phenomenon has been reported for a bacterial Xfp. Here, we report that the Lactobacillus plantarum Xfp displays substrate cooperativity and is allosterically inhibited by phosphoenolpyruvate and oxaloacetate, as is the case for Cryptococcus Xfp. The bacterial enzyme is unaffected by the presence of AMP or ATP, which act as a potent activator and inhibitor of the fungal Xfp, respectively. Our results demonstrate that substrate cooperativity and allosteric regulation may be common properties among bacterial and eukaryotic Xfps, yet important differences exist between the enzymes in these two domains. PMID:25605308

Allosteric regulation of Lactobacillus plantarum xylulose 5-phosphate/fructose 6-phosphate phosphoketolase (Xfp).

PubMed

Glenn, Katie; Smith, Kerry S

2015-04-01

Xylulose 5-phosphate/fructose 6-phosphate phosphoketolase (Xfp), which catalyzes the conversion of xylulose 5-phosphate (X5P) or fructose 6-phosphate (F6P) to acetyl phosphate, plays a key role in carbohydrate metabolism in a number of bacteria. Recently, we demonstrated that the fungal Cryptococcus neoformans Xfp2 exhibits both substrate cooperativity for all substrates (X5P, F6P, and Pi) and allosteric regulation in the forms of inhibition by phosphoenolpyruvate (PEP), oxaloacetic acid (OAA), and ATP and activation by AMP (K. Glenn, C. Ingram-Smith, and K. S. Smith. Eukaryot Cell 13: 657-663, 2014). Allosteric regulation has not been reported previously for the characterized bacterial Xfps. Here, we report the discovery of substrate cooperativity and allosteric regulation among bacterial Xfps, specifically the Lactobacillus plantarum Xfp. L. plantarum Xfp is an allosteric enzyme inhibited by PEP, OAA, and glyoxylate but unaffected by the presence of ATP or AMP. Glyoxylate is an additional inhibitor to those previously reported for C. neoformans Xfp2. As with C. neoformans Xfp2, PEP and OAA share the same or possess overlapping sites on L. plantarum Xfp. Glyoxylate, which had the lowest half-maximal inhibitory concentration of the three inhibitors, binds at a separate site. This study demonstrates that substrate cooperativity and allosteric regulation may be common properties among bacterial and eukaryotic Xfp enzymes, yet important differences exist between the enzymes in these two domains. Xylulose 5-phosphate/fructose 6-phosphate phosphoketolase (Xfp) plays a key role in carbohydrate metabolism in a number of bacteria. Although we recently demonstrated that the fungal Cryptococcus Xfp is subject to substrate cooperativity and allosteric regulation, neither phenomenon has been reported for a bacterial Xfp. Here, we report that the Lactobacillus plantarum Xfp displays substrate cooperativity and is allosterically inhibited by phosphoenolpyruvate and oxaloacetate, as is the case for Cryptococcus Xfp. The bacterial enzyme is unaffected by the presence of AMP or ATP, which act as a potent activator and inhibitor of the fungal Xfp, respectively. Our results demonstrate that substrate cooperativity and allosteric regulation may be common properties among bacterial and eukaryotic Xfps, yet important differences exist between the enzymes in these two domains. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

How Native and Alien Metal Cations Bind ATP: Implications for Lithium as a Therapeutic Agent

NASA Astrophysics Data System (ADS)

Dudev, Todor; Grauffel, Cédric; Lim, Carmay

2017-02-01

Adenosine triphosphate (ATP), the major energy currency of the cell, exists in solution mostly as ATP-Mg. Recent experiments suggest that Mg2+ interacts with the highly charged ATP triphosphate group and Li+ can co-bind with the native Mg2+ to form ATP-Mg-Li and modulate the neuronal purine receptor response. However, it is unclear how the negatively charged ATP triphosphate group binds Mg2+ and Li+ (i.e. which phosphate group(s) bind Mg2+/Li+) and how the ATP solution conformation depends on the type of metal cation and the metal-binding mode. Here, we reveal the preferred ATP-binding mode of Mg2+/Li+ alone and combined: Mg2+ prefers to bind ATP tridentately to each of the three phosphate groups, but Li+ prefers to bind bidentately to the terminal two phosphates. We show that the solution ATP conformation depends on the cation and its binding site/mode, but it does not change significantly when Li+ binds to Mg2+-loaded ATP. Hence, ATP-Mg-Li, like Mg2+-ATP, can fit in the ATP-binding site of the host enzyme/receptor, activating specific signaling pathways.

Quantitative Proteome Analysis of Mouse Liver Lysosomes Provides Evidence for Mannose 6-phosphate-independent Targeting Mechanisms of Acid Hydrolases in Mucolipidosis II.

PubMed

Markmann, Sandra; Krambeck, Svenja; Hughes, Christopher J; Mirzaian, Mina; Aerts, Johannes M F G; Saftig, Paul; Schweizer, Michaela; Vissers, Johannes P C; Braulke, Thomas; Damme, Markus

2017-03-01

The efficient receptor-mediated targeting of soluble lysosomal proteins to lysosomes requires the modification with mannose 6-phosphate (M6P) residues. Although the absence of M6P results in misrouting and hypersecretion of lysosomal enzymes in many cells, normal levels of lysosomal enzymes have been reported in liver of patients lacking the M6P-generating phosphotransferase (PT). The identity of lysosomal proteins depending on M6P has not yet been comprehensively analyzed. In this study we purified lysosomes from liver of PT-defective mice and 67 known soluble lysosomal proteins were identified that illustrated quantitative changes using an ion mobility-assisted data-independent label-free LC-MS approach. After validation of various differentially expressed lysosomal components by Western blotting and enzyme activity assays, the data revealed a small number of lysosomal proteins depending on M6P, including neuraminidase 1, cathepsin F, Npc2, and cathepsin L, whereas the majority reach lysosomes by alternative pathways. These data were compared with findings on cultured hepatocytes and liver sinusoid endothelial cells isolated from the liver of wild-type and PT-defective mice. Our findings show that the relative expression, targeting efficiency and lysosomal localization of lysosomal proteins tested in cultured hepatic cells resemble their proportion in isolated liver lysosomes. Hypersecretion of newly synthesized nonphosphorylated lysosomal proteins suggest that secretion-recapture mechanisms contribute to maintain major lysosomal functions in liver. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Ratios of One-Carbon Metabolites Are Functional Markers of B-Vitamin Status in a Norwegian Coronary Angiography Screening Cohort.

PubMed

Ulvik, Arve; Hustad, Steinar; McCann, Adrian; Midttun, Øivind; Nygård, Ottar K; Ueland, Per M

2017-06-01

Background: Functional (metabolic) markers of B-vitamin status, including plasma total homocysteine (tHcy) for folate and plasma methylmalonic acid (MMA) for vitamin B-12, suffer from moderate sensitivity and poor specificity. Ratios of metabolites belonging to the same pathway may have better performance characteristics. Objective: We evaluated the ratios of tHcy to total cysteine (tCys; Hcy:Cys), tHcy to creatinine (Hcy:Cre), and tHcy to tCys to creatinine (Hcy:Cys:Cre) as functional markers of B-vitamin status represented by a summary score composed of folate, cobalamin, betaine, pyridoxal 5'-phosphate (PLP), and riboflavin concentrations measured in plasma. Methods: Cross-sectional data were obtained from a cohort of patients with stable angina pectoris (2994 men and 1167 women) aged 21-88 y. The relative contribution of the B-vitamin score, age, sex, smoking, body mass index, and markers of renal function and inflammation to the variance of the functional B-vitamin markers was calculated by using multiple linear regression. Results: Compared with tHcy alone, Hcy:Cys, Hcy:Cre, and Hcy:Cys:Cre all showed improved sensitivity and specificity for detecting plasma B-vitamin status. Improvements in overall performance ranged from 4-fold for Hcy:Cys to ∼8-fold for Hcy:Cys:Cre and were particularly strong in subjects with the common 5,10-methylenetetrahydrofolate reductase (MTHFR) 677CC genotype. Conclusions: Ratios of tHcy to tCys and/or creatinine showed a severalfold improvement over tHcy alone as functional markers of B-vitamin status in Norwegian coronary angiography screenees. The biological rationale for these ratios is discussed in terms of known properties of enzymes involved in the catabolism of homocysteine and synthesis of creatine and creatinine. © 2017 American Society for Nutrition.

Vitamin B6 Generated by Obligate Symbionts Is Critical for Maintaining Proline Homeostasis and Fecundity in Tsetse Flies

PubMed Central

Michalkova, Veronika; Weiss, Brian L.; Attardo, Geoffrey M.; Aksoy, Serap

2014-01-01

The viviparous tsetse fly utilizes proline as a hemolymph-borne energy source. In tsetse, biosynthesis of proline from alanine involves the enzyme alanine-glyoxylate aminotransferase (AGAT), which requires pyridoxal phosphate (vitamin B6) as a cofactor. This vitamin can be synthesized by tsetse's obligate symbiont, Wigglesworthia glossinidia. In this study, we examined the role of Wigglesworthia-produced vitamin B6 for maintenance of proline homeostasis, specifically during the energetically expensive lactation period of the tsetse's reproductive cycle. We found that expression of agat, as well as genes involved in vitamin B6 metabolism in both host and symbiont, increases in lactating flies. Removal of symbionts via antibiotic treatment of flies (aposymbiotic) led to hypoprolinemia, reduced levels of vitamin B6 in lactating females, and decreased fecundity. Proline homeostasis and fecundity recovered partially when aposymbiotic tsetse were fed a diet supplemented with either yeast or Wigglesworthia extracts. RNA interference-mediated knockdown of agat in wild-type flies reduced hemolymph proline levels to that of aposymbiotic females. Aposymbiotic flies treated with agat short interfering RNA (siRNA) remained hypoprolinemic even upon dietary supplementation with microbial extracts or B vitamins. Flies infected with parasitic African trypanosomes display lower hemolymph proline levels, suggesting that the reduced fecundity observed in parasitized flies could result from parasite interference with proline homeostasis. This interference could be manifested by competition between tsetse and trypanosomes for vitamins, proline, or other factors involved in their synthesis. Collectively, these results indicate that the presence of Wigglesworthia in tsetse is critical for the maintenance of proline homeostasis through vitamin B6 production. PMID:25038091

Enzymatic and biochemical properties of a novel human serine dehydratase isoform.

PubMed

Ogawa, Hirofumi; Gomi, Tomoharu; Nishizawa, Mikio; Hayakawa, Yumiko; Endo, Shunro; Hayashi, Kyoko; Ochiai, Hiroshi; Takusagawa, Fusao; Pitot, Henry C; Mori, Hisashi; Sakurai, Hiroaki; Koizumi, Keiichi; Saiki, Ikuo; Oda, Hirofumi; Fujishita, Takashi; Miwa, Toshiro; Maruyama, Muneharu; Kobayashi, Masashi

2006-05-01

A cDNA clone similar to human serine dehydratase (SDH) is deposited in the GenBank/EMBL databases, but its structural and functional bases remain unknown. Despite the occurrence of mRNA, the expected protein level was found to be low in cultured cells. To learn about physicochemical properties of the protein, we expressed the cDNA in Escherichia coli, and compared the expressed protein with that of a hepatic SDH. The purified protein showed l-serine and l-threonine dehydratase activity, demonstrating to be an isoform of SDH. However, their Km and Vmax constants were different in a range of two-order. Removal of Pro128 from the hepatic SDH consisting of 328 residues, which is missing in the corresponding position of the isoform consisting of 329 residues, significantly changed the Michaelis constants and Kd value for pyridoxal 5'-phosphate, whereas addition of a proline residue to the isoform was without effect. These findings suggest the difference in the structures of the active sites of the two enzymes. Another striking feature was that the expressed level of the isoform in E. coli was 7-fold lower than that of the hepatic SDH. Substitution of Val for Leu287 in the isoform dramatically increased the protein level. The high yield of the mutated isoform was also confirmed by the in vitro transcription and translation experiment. The poor expression of the isoform could be explained by the more stable secondary structure of the mRNA than that of the hepatic SDH mRNA. The present findings may provide a clue as to why the protein level in cultured cells is low.

Vitamin B6 generated by obligate symbionts is critical for maintaining proline homeostasis and fecundity in tsetse flies.

PubMed

Michalkova, Veronika; Benoit, Joshua B; Weiss, Brian L; Attardo, Geoffrey M; Aksoy, Serap

2014-09-01

The viviparous tsetse fly utilizes proline as a hemolymph-borne energy source. In tsetse, biosynthesis of proline from alanine involves the enzyme alanine-glyoxylate aminotransferase (AGAT), which requires pyridoxal phosphate (vitamin B6) as a cofactor. This vitamin can be synthesized by tsetse's obligate symbiont, Wigglesworthia glossinidia. In this study, we examined the role of Wigglesworthia-produced vitamin B6 for maintenance of proline homeostasis, specifically during the energetically expensive lactation period of the tsetse's reproductive cycle. We found that expression of agat, as well as genes involved in vitamin B6 metabolism in both host and symbiont, increases in lactating flies. Removal of symbionts via antibiotic treatment of flies (aposymbiotic) led to hypoprolinemia, reduced levels of vitamin B6 in lactating females, and decreased fecundity. Proline homeostasis and fecundity recovered partially when aposymbiotic tsetse were fed a diet supplemented with either yeast or Wigglesworthia extracts. RNA interference-mediated knockdown of agat in wild-type flies reduced hemolymph proline levels to that of aposymbiotic females. Aposymbiotic flies treated with agat short interfering RNA (siRNA) remained hypoprolinemic even upon dietary supplementation with microbial extracts or B vitamins. Flies infected with parasitic African trypanosomes display lower hemolymph proline levels, suggesting that the reduced fecundity observed in parasitized flies could result from parasite interference with proline homeostasis. This interference could be manifested by competition between tsetse and trypanosomes for vitamins, proline, or other factors involved in their synthesis. Collectively, these results indicate that the presence of Wigglesworthia in tsetse is critical for the maintenance of proline homeostasis through vitamin B6 production. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Structure of human Fe-S assembly subcomplex reveals unexpected cysteine desulfurase architecture and acyl-ACP-ISD11 interactions.

PubMed

Cory, Seth A; Van Vranken, Jonathan G; Brignole, Edward J; Patra, Shachin; Winge, Dennis R; Drennan, Catherine L; Rutter, Jared; Barondeau, David P

2017-07-03

In eukaryotes, sulfur is mobilized for incorporation into multiple biosynthetic pathways by a cysteine desulfurase complex that consists of a catalytic subunit (NFS1), LYR protein (ISD11), and acyl carrier protein (ACP). This NFS1-ISD11-ACP (SDA) complex forms the core of the iron-sulfur (Fe-S) assembly complex and associates with assembly proteins ISCU2, frataxin (FXN), and ferredoxin to synthesize Fe-S clusters. Here we present crystallographic and electron microscopic structures of the SDA complex coupled to enzyme kinetic and cell-based studies to provide structure-function properties of a mitochondrial cysteine desulfurase. Unlike prokaryotic cysteine desulfurases, the SDA structure adopts an unexpected architecture in which a pair of ISD11 subunits form the dimeric core of the SDA complex, which clarifies the critical role of ISD11 in eukaryotic assemblies. The different quaternary structure results in an incompletely formed substrate channel and solvent-exposed pyridoxal 5'-phosphate cofactor and provides a rationale for the allosteric activator function of FXN in eukaryotic systems. The structure also reveals the 4'-phosphopantetheine-conjugated acyl-group of ACP occupies the hydrophobic core of ISD11, explaining the basis of ACP stabilization. The unexpected architecture for the SDA complex provides a framework for understanding interactions with acceptor proteins for sulfur-containing biosynthetic pathways, elucidating mechanistic details of eukaryotic Fe-S cluster biosynthesis, and clarifying how defects in Fe-S cluster assembly lead to diseases such as Friedreich's ataxia. Moreover, our results support a lock-and-key model in which LYR proteins associate with acyl-ACP as a mechanism for fatty acid biosynthesis to coordinate the expression, Fe-S cofactor maturation, and activity of the respiratory complexes.

Estradiol promotes pentose phosphate pathway addiction and cell survival via reactivation of Akt in mTORC1 hyperactive cells.

PubMed

Sun, Y; Gu, X; Zhang, E; Park, M-A; Pereira, A M; Wang, S; Morrison, T; Li, C; Blenis, J; Gerbaudo, V H; Henske, E P; Yu, J J

2014-05-15

Lymphangioleiomyomatosis (LAM) is a female-predominant interstitial lung disease that can lead to respiratory failure. LAM cells typically have inactivating TSC2 mutations, leading to mTORC1 activation. The gender specificity of LAM suggests that estradiol contributes to disease development, yet the underlying pathogenic mechanisms are not completely understood. Using metabolomic profiling, we identified an estradiol-enhanced pentose phosphate pathway signature in Tsc2-deficient cells. Estradiol increased levels of cellular NADPH, decreased levels of reactive oxygen species, and enhanced cell survival under oxidative stress. Mechanistically, estradiol reactivated Akt in TSC2-deficient cells in vitro and in vivo, induced membrane translocation of glucose transporters (GLUT1 or GLUT4), and increased glucose uptake in an Akt-dependent manner. (18)F-FDG-PET imaging demonstrated enhanced glucose uptake in xenograft tumors of Tsc2-deficient cells from estradiol-treated mice. Expression array study identified estradiol-enhanced transcript levels of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the pentose phosphate pathway. Consistent with this, G6PD was abundant in xenograft tumors and lung metastatic lesions of Tsc2-deficient cells from estradiol-treated mice. Molecular depletion of G6PD attenuated estradiol-enhanced survival in vitro, and treatment with 6-aminonicotinamide, a competitive inhibitor of G6PD, reduced lung colonization of Tsc2-deficient cells. Collectively, these data indicate that estradiol promotes glucose metabolism in mTORC1 hyperactive cells through the pentose phosphate pathway via Akt reactivation and G6PD upregulation, thereby enhancing cell survival under oxidative stress. Interestingly, a strong correlation between estrogen exposure and G6PD was also found in breast cancer cells. Targeting the pentose phosphate pathway may have therapeutic benefit for LAM and possibly other hormonally dependent neoplasms.

Purification of lipase produced by a new source of Bacillus in submerged fermentation using an aqueous two-phase system.

PubMed

Barbosa, José Murillo P; Souza, Ranyere L; Fricks, Alini T; Zanin, Gisella Maria; Soares, Cleide Mara F; Lima, Alvaro S

2011-12-15

This work discusses the application of an aqueous two-phase system for the purification of lipases produced by Bacillus sp. ITP-001 using polyethylene glycol (PEG) and potassium phosphate. In the first step, the protein content was precipitated with ammonium sulphate (80% saturation). The enzyme remained in the aqueous solution and was dialyzed against ultra-pure water for 18 h and used to prepare an aqueous two-phase system (PEG/potassium phosphate). The use of different molecular weights of PEG to purify the lipase was investigated; the best purification factor (PF) was obtained using PEG 20,000g/mol, however PEG 8000 was used in the next tests due to lower viscosity. The influence of PEG and potassium phosphate concentrations on the enzyme purification was then studied: the highest FP was obtained with 20% of PEG and 18% of potassium phosphate. NaCl was added to increase the hydrophobicity between the phases, and also increased the purification factor. The pH value and temperature affected the enzyme partitioning, with the best purifying conditions achieved at pH 6.0 and 4°C. The molecular mass of the purified enzyme was determined to be approximately 54 kDa by SDS-PAGE. According to the results the best combination for purifying the enzyme is PEG 8000g/mol and potassium phosphate (20/18%) with 6% of NaCl at pH 6.0 and 4°C (201.53 fold). The partitioning process of lipase is governed by the entropy contribution. Copyright © 2011 Elsevier B.V. All rights reserved.

Effects of polyamines and calcium and sodium ions on smooth muscle cytoskeleton-associated phosphatidylinositol (4)-phosphate 5-kinase.

PubMed

Chen, H; Baron, C B; Griffiths, T; Greeley, P; Coburn, R F

1998-10-01

In many different cell types, including smooth muscle cells (Baron et al., 1989, Am. J. Physiol., 256: C375-383; Baron et al., J. Pharmacol. Exp. Ther. 266: 8-15), phosphatidylinositol (4)-phosphate 5-kinase plays a critical role in the regulation of membrane concentrations of phosphatidylinositol (4,5)-bisphosphate and formation of inositol (1,4,5)-trisphosphate. In unstimulated porcine trachealis smooth muscle, 70% of total cellular phosphatidylinositol (4)-phosphate 5-kinase activity was associated with cytoskeletal proteins and only trace activity was detectable in isolated sarcolemma. Using two different preparations, we studied cytoskeleton-associated phosphatidyl inositol (4)-phosphate 5-kinase under conditions that attempted to mimic the ionic and thermal cytoplasmic environment of living cells. The cytoskeleton-associated enzyme, studied using phosphatidylinositol (4)-phosphate substrate concentrations that produced phosphatidylinositol 4,5-bisphosphate at about 10% of the maximal rate, was sensitive to free [Mg2+], had an absolute requirement for phosphatidylserine, phosphatidic acid, or phosphatidylinositol, and included type I isoforms. At 0.5 mM free [Mg2+], physiological spermine concentrations, 0.2-0.4 mM, increased phosphatidylinositol (4)-phosphate 5-kinase activity two to four times compared to controls run without spermine. The EC50 for spermine-evoked increases in activity was 0.17 +/- 0.02 mM. Spermine-evoked enzyme activity was a function of both free [Mg2+] and substrate concentration. Cytoskeleton-associated phosphatidylinositol (4)-phosphate 5-kinase was inhibited by free [Ca2+] over a physiological range for cytoplasm--10(-8) to 10(-5) M, an effect independent of the presence of calmodulin. Na+ over the range 20 to 50 mM also inhibited this enzyme activated by 5 mM Mg2+ but had no effect on spermine-activated enzyme. Na+, Ca2+, and spermine appear to be physiological modulators of smooth muscle cytoskeleton-bound phosphatidylinositol (4)-phosphate 5-kinase.

Proteoliposomes harboring alkaline phosphatase and nucleotide pyrophosphatase as matrix vesicle biomimetics.

PubMed

Simão, Ana Maria S; Yadav, Manisha C; Narisawa, Sonoko; Bolean, Mayte; Pizauro, Joao Martins; Hoylaerts, Marc F; Ciancaglini, Pietro; Millán, José Luis

2010-03-05

We have established a proteoliposome system as an osteoblast-derived matrix vesicle (MV) biomimetic to facilitate the study of the interplay of tissue-nonspecific alkaline phosphatase (TNAP) and NPP1 (nucleotide pyrophosphatase/phosphodiesterase-1) during catalysis of biomineralization substrates. First, we studied the incorporation of TNAP into liposomes of various lipid compositions (i.e. in pure dipalmitoyl phosphatidylcholine (DPPC), DPPC/dipalmitoyl phosphatidylserine (9:1 and 8:2), and DPPC/dioctadecyl-dimethylammonium bromide (9:1 and 8:2) mixtures. TNAP reconstitution proved virtually complete in DPPC liposomes. Next, proteoliposomes containing either recombinant TNAP, recombinant NPP1, or both together were reconstituted in DPPC, and the hydrolysis of ATP, ADP, AMP, pyridoxal-5'-phosphate (PLP), p-nitrophenyl phosphate, p-nitrophenylthymidine 5'-monophosphate, and PP(i) by these proteoliposomes was studied at physiological pH. p-Nitrophenylthymidine 5'-monophosphate and PLP were exclusively hydrolyzed by NPP1-containing and TNAP-containing proteoliposomes, respectively. In contrast, ATP, ADP, AMP, PLP, p-nitrophenyl phosphate, and PP(i) were hydrolyzed by TNAP-, NPP1-, and TNAP plus NPP1-containing proteoliposomes. NPP1 plus TNAP additively hydrolyzed ATP, but TNAP appeared more active in AMP formation than NPP1. Hydrolysis of PP(i) by TNAP-, and TNAP plus NPP1-containing proteoliposomes occurred with catalytic efficiencies and mild cooperativity, effects comparable with those manifested by murine osteoblast-derived MVs. The reconstitution of TNAP and NPP1 into proteoliposome membranes generates a phospholipid microenvironment that allows the kinetic study of phosphosubstrate catabolism in a manner that recapitulates the native MV microenvironment.

Proteoliposomes Harboring Alkaline Phosphatase and Nucleotide Pyrophosphatase as Matrix Vesicle Biomimetics*

PubMed Central

Simão, Ana Maria S.; Yadav, Manisha C.; Narisawa, Sonoko; Bolean, Mayte; Pizauro, Joao Martins; Hoylaerts, Marc F.; Ciancaglini, Pietro; Millán, José Luis

2010-01-01

We have established a proteoliposome system as an osteoblast-derived matrix vesicle (MV) biomimetic to facilitate the study of the interplay of tissue-nonspecific alkaline phosphatase (TNAP) and NPP1 (nucleotide pyrophosphatase/phosphodiesterase-1) during catalysis of biomineralization substrates. First, we studied the incorporation of TNAP into liposomes of various lipid compositions (i.e. in pure dipalmitoyl phosphatidylcholine (DPPC), DPPC/dipalmitoyl phosphatidylserine (9:1 and 8:2), and DPPC/dioctadecyl-dimethylammonium bromide (9:1 and 8:2) mixtures. TNAP reconstitution proved virtually complete in DPPC liposomes. Next, proteoliposomes containing either recombinant TNAP, recombinant NPP1, or both together were reconstituted in DPPC, and the hydrolysis of ATP, ADP, AMP, pyridoxal-5′-phosphate (PLP), p-nitrophenyl phosphate, p-nitrophenylthymidine 5′-monophosphate, and PPi by these proteoliposomes was studied at physiological pH. p-Nitrophenylthymidine 5′-monophosphate and PLP were exclusively hydrolyzed by NPP1-containing and TNAP-containing proteoliposomes, respectively. In contrast, ATP, ADP, AMP, PLP, p-nitrophenyl phosphate, and PPi were hydrolyzed by TNAP-, NPP1-, and TNAP plus NPP1-containing proteoliposomes. NPP1 plus TNAP additively hydrolyzed ATP, but TNAP appeared more active in AMP formation than NPP1. Hydrolysis of PPi by TNAP-, and TNAP plus NPP1-containing proteoliposomes occurred with catalytic efficiencies and mild cooperativity, effects comparable with those manifested by murine osteoblast-derived MVs. The reconstitution of TNAP and NPP1 into proteoliposome membranes generates a phospholipid microenvironment that allows the kinetic study of phosphosubstrate catabolism in a manner that recapitulates the native MV microenvironment. PMID:20048161

Characterization and molecular modeling of Inositol 1,3,4 tris phosphate 5/6 kinase-2 from Glycine max (L) Merr.: comprehending its evolutionary conservancy at functional level.

PubMed

Marathe, Ashish; Krishnan, Veda; Mahajan, Mahesh M; Thimmegowda, Vinutha; Dahuja, Anil; Jolly, Monica; Praveen, Shelly; Sachdev, Archana

2018-01-01

Soybean genome encodes a family of four inositol 1,3,4 trisphosphate 5/6 kinases which belong to the ATP-GRASP group of proteins. Inositol 1,3,4 trisphosphate kinase-2 ( GmItpk2 ), catalyzing the ATP-dependent phosphorylation of Inositol 1,3,4 trisphosphate (IP3) to Inositol 1,3,4,5 tetra phosphate or Inositol 1,3,4,6 tetra phosphate, is a key enzyme diverting the flux of inositol phosphate pool towards phytate biosynthesis. Although considerable research on characterizing genes involved in phytate biosynthesis is accomplished at genomic and transcript level, characterization of the proteins is yet to be explored. In the present study, we report the isolation and expression of single copy Itpk 2 (948 bp) from Glycine max cv Pusa-16 predicted to encode 315 amino acid protein with an isoelectric point of 5.9. Sequence analysis revealed that Gm ITPK2 shared highest similarity (80%) with Phaseolus vulgaris. The predicted 3D model confirmed 12 α helices and 14 β barrel sheets with ATP-binding site close to β sheet present towards the C-terminus of the protein molecule. Spatio-temporal transcript profiling signified GmItpk2 to be seed specific, with higher transcript levels in the early stage of seed development. The present study using various molecular and bio-computational tools could, therefore, help in improving our understanding of this key enzyme and prove to be a potential target towards generating low phytate trait in nutritionally rich crop like soybean.

Inactivation of phosphorylase b by potassium ferrate, a new reactive analogue of the phosphate group.

PubMed

Lee, Y M; Benisek, W F

1976-03-25

Rabbit muscle phosphorylase b reacts with the phosphate-like reagent potassium ferrate, K2FeO4, a potent oxidizing agent. The reaction results in inactivation of the enzyme and abolition of the ability of the enzyme to bind 5'-AMP. Activating and nonactivating nucleotides which bind at the 5'-AMP binding site such as 5'-AMP, 2'-AMP, 3'-AMP, and 5'-IMP substantially protect the enzyme from inactivation by ferrate. One to two residues of tyrosine and approximately 1 residue of cysteine are modified by ferrate under the conditions employed. Tyrosine is protected by 5-AMP, whereas cysteine is not. The tyrosine modification is suggested as the inactivating chemical reaction. The location of the inactivating reaction is suggested to be in or near the 5'-AMP binding site. The structural and chemical properties of ferrate ion are discussed and compared to those of phosphate. Ferrate ion may be a reagent useful for phosphate group binding site-directed modification of proteins.

MicroCommentary: A New Role for Coenzyme F420 in Aflatoxin Reduction by Soil Mycobacteria

DOE Office of Scientific and Technical Information (OSTI.GOV)

Graham, David E

Hepatotoxic aflatoxins have found a worthy adversary in two new families of bacterial oxidoreductases. These enzymes use the reduced coenzyme F420 to initiate the degradation of furanocoumarin compounds, including the major mycotoxin products of Aspergillus flavus. Along with pyridoxalamine 5 -phosphate oxidases and aryl nitroreductases, these proteins form a large and versatile superfamily of flavin and deazaflavin-dependent oxidoreductases. F420-dependent members of this family appear to share a common mechanism of hydride transfer from the reduced deazaflavin to the electron-deficient ring systems of their substrates.

Biosynthesis of Nucleoside Diphosphoramidates in Campylobacter jejuni.

PubMed

Taylor, Zane W; Brown, Haley A; Holden, Hazel M; Raushel, Frank M

2017-11-21

Campylobacter jejuni is a pathogenic Gram-negative bacterium and a leading cause of food-borne gastroenteritis. C. jejuni produces a capsular polysaccharide (CPS) that contains a unique O-methyl phosphoramidate modification (MeOPN). Recently, the first step in the biosynthetic pathway for the assembly of the MeOPN modification to the CPS was elucidated. It was shown that the enzyme Cj1418 catalyzes the phosphorylation of the amide nitrogen of l-glutamine to form l-glutamine phosphate. In this investigation, the metabolic fate of l-glutamine phosphate was determined. The enzyme Cj1416 catalyzes the displacement of pyrophosphate from MgCTP by l-glutamine phosphate to form CDP-l-glutamine. The enzyme Cj1417 subsequently catalyzes the hydrolysis of CDP-l-glutamine to generate cytidine diphosphoramidate and l-glutamate. The structures of the two novel intermediates, CDP-l-glutamine and cytidine diphosphoramidate, were confirmed by 31 P nuclear magnetic resonance spectroscopy and mass spectrometry. It is proposed that the enzyme Cj1416 be named CTP:phosphoglutamine cytidylyltransferase and that the enzyme Cj1417 be named γ-glutamyl-CDP-amidate hydrolase.

Rheb Protein Binds CAD (Carbamoyl-phosphate Synthetase 2, Aspartate Transcarbamoylase, and Dihydroorotase) Protein in a GTP- and Effector Domain-dependent Manner and Influences Its Cellular Localization and Carbamoyl-phosphate Synthetase (CPSase) Activity*

PubMed Central

Sato, Tatsuhiro; Akasu, Hitomi; Shimono, Wataru; Matsu, Chisa; Fujiwara, Yuki; Shibagaki, Yoshio; Heard, Jeffrey J.; Tamanoi, Fuyuhiko; Hattori, Seisuke

2015-01-01

Rheb small GTPases, which consist of Rheb1 and Rheb2 (also known as RhebL1) in mammalian cells, are unique members of the Ras superfamily and play central roles in regulating protein synthesis and cell growth by activating mTOR. To gain further insight into the function of Rheb, we carried out a search for Rheb-binding proteins and found that Rheb binds to CAD protein (carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase), a multifunctional enzyme required for the de novo synthesis of pyrimidine nucleotides. CAD binding is more pronounced with Rheb2 than with Rheb1. Rheb binds CAD in a GTP- and effector domain-dependent manner. The region of CAD where Rheb binds is located at the C-terminal region of the carbamoyl-phosphate synthetase domain and not in the dihydroorotase and aspartate transcarbamoylase domains. Rheb stimulated carbamoyl-phosphate synthetase activity of CAD in vitro. In addition, an elevated level of intracellular UTP pyrimidine nucleotide was observed in Tsc2-deficient cells, which was attenuated by knocking down of Rheb. Immunostaining analysis showed that expression of Rheb leads to increased accumulation of CAD on lysosomes. Both a farnesyltransferase inhibitor that blocks membrane association of Rheb and knockdown of Rheb mislocalized CAD. These results establish CAD as a downstream effector of Rheb and suggest a possible role of Rheb in regulating de novo pyrimidine nucleotide synthesis. PMID:25422319

Rheb protein binds CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase) protein in a GTP- and effector domain-dependent manner and influences its cellular localization and carbamoyl-phosphate synthetase (CPSase) activity.

PubMed

Sato, Tatsuhiro; Akasu, Hitomi; Shimono, Wataru; Matsu, Chisa; Fujiwara, Yuki; Shibagaki, Yoshio; Heard, Jeffrey J; Tamanoi, Fuyuhiko; Hattori, Seisuke

2015-01-09

Rheb small GTPases, which consist of Rheb1 and Rheb2 (also known as RhebL1) in mammalian cells, are unique members of the Ras superfamily and play central roles in regulating protein synthesis and cell growth by activating mTOR. To gain further insight into the function of Rheb, we carried out a search for Rheb-binding proteins and found that Rheb binds to CAD protein (carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase), a multifunctional enzyme required for the de novo synthesis of pyrimidine nucleotides. CAD binding is more pronounced with Rheb2 than with Rheb1. Rheb binds CAD in a GTP- and effector domain-dependent manner. The region of CAD where Rheb binds is located at the C-terminal region of the carbamoyl-phosphate synthetase domain and not in the dihydroorotase and aspartate transcarbamoylase domains. Rheb stimulated carbamoyl-phosphate synthetase activity of CAD in vitro. In addition, an elevated level of intracellular UTP pyrimidine nucleotide was observed in Tsc2-deficient cells, which was attenuated by knocking down of Rheb. Immunostaining analysis showed that expression of Rheb leads to increased accumulation of CAD on lysosomes. Both a farnesyltransferase inhibitor that blocks membrane association of Rheb and knockdown of Rheb mislocalized CAD. These results establish CAD as a downstream effector of Rheb and suggest a possible role of Rheb in regulating de novo pyrimidine nucleotide synthesis. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Fructose 2,6-bisphosphate and 6-phosphofructo-2-kinase during liver regeneration.

PubMed Central

Rosa, J L; Ventura, F; Carreras, J; Bartrons, R

1990-01-01

Glycogen and fructose 2,6-bisphosphate levels in rat liver decreased quickly after partial hepatectomy. After 7 days the glycogen level was normalized and fructose 2,6-bisphosphate concentration still remained low. The 'active' (non-phosphorylated) form of 6-phosphofructo-2-kinase varied in parallel with fructose 2,6-bisphosphate levels, whereas the 'total' activity of the enzyme decreased only after 24 h, similarly to glucokinase. The response of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from hepatectomized rats (96 h) to sn-glycerol 3-phosphate and to cyclic AMP-dependent protein kinase was different from that of the enzyme from control animals and similar to that of the foetal isoenzyme. PMID:2173548

Metabolism of inositol(1,4,5)trisphosphate by a soluble enzyme fraction from pea (Pisum sativum) roots

DOE Office of Scientific and Technical Information (OSTI.GOV)

Drobak, B.K.; Watkins, P.A.C.; Roberts, K.

1991-02-01

Metabolism of the putative messenger molecule D-myo-inositol(1,4,5)trisphosphate (Ins(1,4,5)P{sub 3}) in plant cells has been studied using a soluble fraction from pea (pisum sativum) roots as enzyme source and (5-{sup 32}P)Ins(1,4,5)P{sub 3} and (2-{sup 3}H)Ins(1,4,5)P{sub 3} as tracers. Ins(1,4,5)P{sub 3} was rapidly converted into both lower and higher inositol phosphates. The major dephosphorylation product was inositol (4,5) bisphosphate (Ins(4,5)P{sub 2}) whereas inositol(1,4)bisphosphate (Ins(1,4)P{sub 2}) was only present in very small quantities throughout a 15 minute incubation period. In addition to these compounds, small amounts of nine other metabolites were produced including inositol and inositol(1,4,5,X)P{sub 4}. Dephosphorylation of Ins(1,4,5)P{sub 3} to Ins(4,5)P{submore » 2} was dependent on Ins(1,4,5)P{sub 3} concentration and was partially inhibited by the phosphohydrolase inhibitors 2,3-diphosphoglycerate, glucose 6-phosphate, and p-nitrophenylphosphate. Conversion of Ins(1,4,5)P{sub 3} to Ins(4,5)P{sub 2} and Ins(1,4,5,X)P{sub 4} was inhibited by 55 micromolar Ca{sup 2+}. This study demonstrates that enzymes are present in plant tissues which are capable of rapidly converting Ins(1,4,5)P{sub 3} and that pathways of inositol phosphate metabolism exist which may prove to be unique to the plant kingdom.« less

Loop-loop interactions govern multiple steps in indole-3-glycerol phosphate synthase catalysis

PubMed Central

Zaccardi, Margot J; O'Rourke, Kathleen F; Yezdimer, Eric M; Loggia, Laura J; Woldt, Svenja; Boehr, David D

2014-01-01

Substrate binding, product release, and likely chemical catalysis in the tryptophan biosynthetic enzyme indole-3-glycerol phosphate synthase (IGPS) are dependent on the structural dynamics of the β1α1 active-site loop. Statistical coupling analysis and molecular dynamic simulations had previously indicated that covarying residues in the β1α1 and β2α2 loops, corresponding to Arg54 and Asn90, respectively, in the Sulfolobus sulfataricus enzyme (ssIGPS), are likely important for coordinating functional motions of these loops. To test this hypothesis, we characterized site mutants at these positions for changes in catalytic function, protein stability and structural dynamics for the thermophilic ssIGPS enzyme. Although there were only modest changes in the overall steady-state kinetic parameters, solvent viscosity and solvent deuterium kinetic isotope effects indicated that these amino acid substitutions change the identity of the rate-determining step across multiple temperatures. Surprisingly, the N90A substitution had a dramatic effect on the general acid/base catalysis of the dehydration step, as indicated by the loss of the descending limb in the pH rate profile, which we had previously assigned to Lys53 on the β1α1 loop. These changes in enzyme function are accompanied with a quenching of ps-ns and µs-ms timescale motions in the β1α1 loop as measured by nuclear magnetic resonance studies. Altogether, our studies provide structural, dynamic and functional rationales for the coevolution of residues on the β1α1 and β2α2 loops, and highlight the multiple roles that the β1α1 loop plays in IGPS catalysis. Thus, substitution of covarying residues in the active-site β1α1 and β2α2 loops of indole-3-glycerol phosphate synthase results in functional, structural, and dynamic changes, highlighting the multiple roles that the β1α1 loop plays in enzyme catalysis and the importance of regulating the structural dynamics of this loop through noncovalent interactions with nearby structural elements. PMID:24403092

Purification and investigation of some kinetic properties of glucose-6-phosphate dehydrogenase from parsley (Petroselinum hortense) leaves.

PubMed

Coban, T Abdül Kadir; Ciftçi, Mehmet; Küfrevioğlu, O Irfan

2002-05-01

In this study, glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP+ oxidoreductase, EC 1.1.1.49; G6PD) was purified from parsley (Petroselinum hortense) leaves, and analysis of the kinetic behavior and some properties of the enzyme were investigated. The purification consisted of three steps: preparation of homogenate, ammonium sulfate fractionation, and DEAE-Sephadex A50 ion exchange chromatography. The enzyme was obtained with a yield of 8.79% and had a specific activity of 2.146 U (mg protein)(-1). The overall purification was about 58-fold. Temperature of +4 degrees C was maintained during the purification process. Enzyme activity was spectrophotometrically measured according to the Beutler method, at 340 nm. In order to control the purification of enzyme, SDS-polyacrylamide gel electrophoresis was carried out in 4% and 10% acrylamide for stacking and running gel, respectively. SDS-polyacrylamide gel electrophoresis showed a single band for enzyme. The molecular weight was found to be 77.6 kDa by Sephadex G-150 gel filtration chromatography. A protein band corresponding to a molecular weight of 79.3 kDa was obtained on SDS-polyacrylamide gel electrophoresis. For the enzymes, the stable pH, optimum pH, and optimum temperature were found to be 6.0, 8.0, and 60 degrees C, respectively. Moreover, KM and Vmax values for NADP+ and G6-P at optimum pH and 25 degrees C were determined by means of Lineweaver-Burk graphs. Additionally, effects of streptomycin sulfate and tetracycline antibiotics were investigated for the enzyme activity of glucose-6-phosphate dehydrogenase in vitro.

[Familial hypophosphatemic rickets].

PubMed

Reusz, G

2001-12-02

Familiar hypophosphatemic rickets (FHR) is characterized by isolated defect of renal phosphate reabsorption, hypophosphataemia, rickets and poor growth. In untreated cases parathyroid hormone and calcitriol levels are normal. FHR is caused by mutations of the PHEX gene encoding a zinc-binding metalloprotease enzyme. PHEX is expressed in bones and the parathyroid gland but not in the kidney. The gene product is involved in the inactivation of a phosphate regulating hormone (phosphatonin). The presence of this hormone through unknown mechanisms decreases the sodium-dependent phosphate cotransporter in the kidney resulting in impaired phosphate transport. In addition the PHEX gene product exerts autocrine and paracrine effects on the bone. Despite recent advances in the understanding of the pathomechanism, treatment of FHR is still symptomatic. It consists of active vitamin D analogues and oral phosphate supplementation. Nephrocalcinosis is a well-known, usually non-progressive side effect of the conventional therapy. As shown by pilot studies, poorly growing children with FHR may benefit from the positive effect of human recombinant growth hormone (rhGH). However, rhGH treatment could aggravate the already existing tendency to disproportionate growth resulting in the overgrowth of the trunk. The disturbed phosphate homeostasis persists during the whole life span of the FHR patients. It is therefore essential to provide lifelong care, to prevent late skeletal and dental consequences or to treat them if already established. That care should be done by the teamwork of the pediatrician, internist, orthopedist, dentist and the psychologist.

Structure of 3,4-dihydroxy-2-butanone 4-phosphate synthase from Methanococcus jannaschii in complex with divalent metal ions and the substrate ribulose 5-phosphate: implications for the catalytic mechanism.

PubMed

Steinbacher, Stefan; Schiffmann, Susanne; Richter, Gerald; Huber, Robert; Bacher, Adelbert; Fischer, Markus

2003-10-24

Skeletal rearrangements of carbohydrates are crucial for many biosynthetic pathways. In riboflavin biosynthesis ribulose 5-phosphate is converted into 3,4-dihydroxy-2-butanone 4-phosphate while its C4 atom is released as formate in a sequence of metal-dependent reactions. Here, we present the crystal structure of Methanococcus jannaschii 3,4-dihydroxy-2-butanone 4-phosphate synthase in complex with the substrate ribulose 5-phosphate at a dimetal center presumably consisting of non-catalytic zinc and calcium ions at 1.7-A resolution. The carbonyl group (O2) and two out of three free hydroxyl groups (OH3 and OH4) of the substrate are metal-coordinated. We correlate previous mutational studies on this enzyme with the present structural results. Residues of the first coordination sphere involved in metal binding are indispensable for catalytic activity. Only Glu-185 of the second coordination sphere cannot be replaced without complete loss of activity. It contacts the C3 hydrogen atom directly and probably initiates enediol formation in concert with both metal ions to start the reaction sequence. Mechanistic similarities to Rubisco acting on the similar substrate ribulose 1,5-diphosphate in carbon dioxide fixation as well as other carbohydrate (reducto-) isomerases are discussed.

Participation of glyceraldehyde-3-phosphate dehydrogenase in the regulation of 2,3-diphosphoglycerate level in erythrocytes.

PubMed

Fokina, K V; Yazykova, M Y; Danshina, P V; Schmalhausen, E V; Muronetz, V I

2000-04-01

Data are presented concerning the possible participation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in regulation of the glycolytic pathway and the level of 2,3-diphosphoglycerate in erythrocytes. Experimental support has been obtained for the hypothesis according to which a mild oxidation of GAPDH must result in acceleration of glycolysis and in decrease in the level of 2, 3-diphosphoglycerate due to the acyl phosphatase activity of the mildly oxidized enzyme. Incubation of erythrocytes in the presence of 1 mM hydrogen peroxide decreases 2,3-diphosphoglycerate concentration and causes accumulation of 3-phosphoglycerate. It is assumed that the acceleration of glycolysis in the presence of oxidative agents described previously by a number of authors could be attributed to the acyl phosphatase activity of GAPDH. A pH-dependent complexing of GAPDH and 3-phosphoglycerate kinase or 2, 3-diphosphoglycerate mutase is found to determine the fate of 1,3-diphosphoglycerate that serves as a substrate for the synthesis of 2,3-diphosphoglycerate as well as for the 3-phosphoglycerate kinase reaction in glycolysis. A withdrawal of the two-enzyme complexes from the erythrocyte lysates using Sepharose-bound anti-GAPDH antibodies prevents the pH-dependent accumulation of the metabolites. The role of GAPDH in the regulation of glycolysis and the level of 2,3-diphosphoglycerate in erythrocytes is discussed.

Metabolism of d-Arabinose: Origin of a d-Ribulokinase Activity in Escherichia coli1

PubMed Central

LeBlanc, Donald J.; Mortlock, Robert P.

1971-01-01

The kinase responsible for the phosphorylation of d-ribulose was purified 45.5-fold from a strain of Escherichia coli K-12 capable of growth on d-arabinose with no separation of d-ribulo- or l-fuculokinase activities. Throughout the purification, the ratios of activities remained essentially constant. A nonadditive effect of combining both substrates in an assay mixture; identical Km values for adenosine triphosphate with either l-fuculose or d-ribulose as substrate; and, the irreversible loss of activity on both substrates, after removal of magnesium ions from the enzyme preparation, suggest that the dual activity is due to the same enzyme. A fourfold greater affinity of the enzyme for l-fuculose than for d-ribulose, as well as a higher relative activity on l-fuculose, suggest that the natural substrate for this enzyme is l-fuculose. The product of the purified enzyme, with d-ribulose as substrate, was prepared. The ratio of total phosphorous to ribulose phosphate was 1.01:1, indicating that the product was ribulose monophosphate. The behavior of the kinase product in the cysteine-carbazole and orcinol reactions, as well as the results of periodate oxidation assays, provided evidence that it was not d-ribulose-5-phosphate. Reaction of this compound with a cell-free extract of E. coli possessing l-fuculose-l-phosphate aldolase activity resulted in the production of dihydroxyacetone phosphate and glycolaldehyde. The kinase product failed to reduce 2,3,5-triphenyltetrazolium and possessed a half-life of approximately 1.5 min in the presence of 1 n HCl at 100 C. These properties suggested that the phosphate group was attached to carbon atom 1 of d-ribulose. PMID:4323967

Reconstructed ancestral enzymes reveal that negative selection drove the evolution of substrate specificity in ADP-dependent kinases.

PubMed

Castro-Fernandez, Víctor; Herrera-Morande, Alejandra; Zamora, Ricardo; Merino, Felipe; Gonzalez-Ordenes, Felipe; Padilla-Salinas, Felipe; Pereira, Humberto M; Brandão-Neto, Jose; Garratt, Richard C; Guixe, Victoria

2017-09-22

One central goal in molecular evolution is to pinpoint the mechanisms and evolutionary forces that cause an enzyme to change its substrate specificity; however, these processes remain largely unexplored. Using the glycolytic ADP-dependent kinases of archaea, including the orders Thermococcales , Methanosarcinales , and Methanococcales , as a model and employing an approach involving paleoenzymology, evolutionary statistics, and protein structural analysis, we could track changes in substrate specificity during ADP-dependent kinase evolution along with the structural determinants of these changes. To do so, we studied five key resurrected ancestral enzymes as well as their extant counterparts. We found that a major shift in function from a bifunctional ancestor that could phosphorylate either glucose or fructose 6-phosphate (fructose-6-P) as a substrate to a fructose 6-P-specific enzyme was started by a single amino acid substitution resulting in negative selection with a ground-state mode against glucose and a subsequent 1,600-fold change in specificity of the ancestral protein. This change rendered the residual phosphorylation of glucose a promiscuous and physiologically irrelevant activity, highlighting how promiscuity may be an evolutionary vestige of ancestral enzyme activities, which have been eliminated over time. We also could reconstruct the evolutionary history of substrate utilization by using an evolutionary model of discrete binary characters, indicating that substrate uses can be discretely lost or acquired during enzyme evolution. These findings exemplify how negative selection and subtle enzyme changes can lead to major evolutionary shifts in function, which can subsequently generate important adaptive advantages, for example, in improving glycolytic efficiency in Thermococcales . © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Trehalose biosynthesis in Thermus thermophilus RQ-1: biochemical properties of the trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase.

PubMed

Silva, Zélia; Alarico, Susana; da Costa, Milton S

2005-02-01

The genes for trehalose synthesis in Thermus thermophilus RQ-1, namely otsA [trehalose-phosphate synthase (TPS)], otsB [trehalose-phosphate phosphatase (TPP)], and treS [trehalose synthase (maltose converting) (TreS)] genes are structurally linked. The TPS/TPP pathway plays a role in osmoadaptation, since mutants unable to synthesize trehalose via this pathway were less osmotolerant, in trehalose-deprived medium, than the wild-type strain. The otsA and otsB genes have now been individually cloned and overexpressed in Escherichia coli and the corresponding recombinant enzymes purified. The apparent molecular masses of TPS and TPP were 52 and 26 kDa, respectively. The recombinant TPS utilized UDP-glucose, TDP-glucose, ADP-glucose, or GDP-glucose, in this order as glucosyl donors, and glucose-6-phosphate as the glucosyl acceptor to produce trehalose-6-phosphate (T6P). The recombinant TPP catalyzed the dephosphorylation of T6P to trehalose. This enzyme also dephosphorylated G6P, and this activity was enhanced by NDP-glucose. TPS had an optimal activity at about 98 degrees C and pH near 6.0; TPP had a maximal activity near 70 degrees C and at pH 7.0. The enzymes were extremely thermostable: at 100 degrees C, TPS had a half-life of 31 min, and TPP had a half-life of 40 min. The enzymes did not require the presence of divalent cations for activity; however, the presence of Co2+ and Mg2+ stimulates both TPS and TPP. This is the first report of the characterization of TPS and TPP from a thermophilic organism.

Network-level fossil of a phosphate-free biosphere

NASA Astrophysics Data System (ADS)

Goldford, J.; Hartman, H.; Smith, T. F.; Segre, D.

2017-12-01

The emergence of a metabolism capable of sustaining cellular life on early Earth is a major unresolved enigma. Such a transition from prebiotic chemistry to an organized biochemical network seemingly required the concurrent availability of multiple molecular components. One of these components, phosphate, carries several essential functions in present-day metabolism, most notably energy transduction through ATP. However, the ubiquity of phosphate in living systems today stands in sharp contrast with its poor geochemical availability, prompting previous efforts to search for plausible prebiotic sources. The alternative, intriguing possibility is that primitive life did not require phosphate. Here we explore this possibility by determining the feasibility and functional potential of a phosphate-independent metabolism amongst the set of all known biochemical reactions in the biosphere. Surprisingly, we identified a cryptic phosphate-independent core metabolism that can be generated from simple sets of compounds thought to be available on early Earth. This network can support the biosynthesis of a broad category of key biomolecules. The enzymes contained in this network display a striking enrichment for dependence on iron-sulfur and transition metal coenzymes, a fundamental cornerstone of early biochemistry. We furthermore show that phosphate-independent precursors of present-day cofactors could have helped overcome thermodynamic energy barriers, enabling the production of a rich set of biomolecules, including 15 out of the 20 amino acids, vitamins, pentoses and nucleobases. Altogether, our results suggest that present-day biochemical networks may contain vestiges of a very ancient past, and that a complex thioester-based metabolism could have predated the incorporation of phosphate and an RNA-based genetic system.

Enzymatic synthesis of rare sugars with L-rhamnulose-1-phosphate aldolase from Thermotoga maritima MSB8.

PubMed

Li, Zijie; Wu, Xiaoru; Cai, Li; Duan, Shenglin; Liu, Jia; Yuan, Peng; Nakanishi, Hideki; Gao, Xiao-Dong

2015-09-15

L-Rhamnulose-1-phosphate aldolase from a thermophilic source (Thermotoga maritima MSB8) (RhaDT.mari) was heterologously overexpressed in Escherichia coli and the stereoselectivity of this enzyme with or without Nus tag was investigated. We also applied this enzyme to the synthesis of rare sugars D-psicose, D-sorbose, L-tagatose and L-fructose using our one-pot four-enzyme system. To the best of our knowledge, this is the first use of RhaD from a thermophilic source for rare sugar synthesis and the temperature tolerance of this enzyme paves the path for large scale fermentation. Copyright © 2015 Elsevier Ltd. All rights reserved.

Structure and mechanisms of Escherichia coli aspartate transcarbamoylase.

PubMed

Lipscomb, William N; Kantrowitz, Evan R

2012-03-20

Enzymes catalyze a particular reaction in cells, but only a few control the rate of this reaction and the metabolic pathway that follows. One specific mechanism for such enzymatic control of a metabolic pathway involves molecular feedback, whereby a metabolite further down the pathway acts at a unique site on the control enzyme to alter its activity allosterically. This regulation may be positive or negative (or both), depending upon the particular system. Another method of enzymatic control involves the cooperative binding of the substrate, which allows a large change in enzyme activity to emanate from only a small change in substrate concentration. Allosteric regulation and homotropic cooperativity are often known to involve significant conformational changes in the structure of the protein. Escherichia coli aspartate transcarbamoylase (ATCase) is the textbook example of an enzyme that regulates a metabolic pathway, namely, pyrimidine nucleotide biosynthesis, by feedback control and by the cooperative binding of the substrate, L-aspartate. The catalytic and regulatory mechanisms of this enzyme have been extensively studied. A series of X-ray crystal structures of the enzyme in the presence and absence of substrates, products, and analogues have provided details, at the molecular level, of the conformational changes that the enzyme undergoes as it shifts between its low-activity, low-affinity form (T state) to its high-activity, high-affinity form (R state). These structural data provide insights into not only how this enzyme catalyzes the reaction between l-aspartate and carbamoyl phosphate to form N-carbamoyl-L-aspartate and inorganic phosphate, but also how the allosteric effectors modulate this activity. In this Account, we summarize studies on the structure of the enzyme and describe how these structural data provide insights into the catalytic and regulatory mechanisms of the enzyme. The ATCase-catalyzed reaction is regulated by nucleotide binding some 60 Å from the active site, inducing structural alterations that modulate catalytic activity. The delineation of the structure and function in this particular model system will help in understanding the molecular basis of cooperativity and allosteric regulation in other systems as well.

Determination of chondroitin-6-sulphate by a competitive enzyme immunoassay using a biotinylated antigen.

PubMed

Kähnert, H; Brinkmann, T; Gässler, N; Kleesiek, K

1994-04-01

A competitive enzyme immunoassay was developed to determine chondroitin-6-sulphate in body fluids and cell cultures. The assay uses a monoclonal anti-chondroitin-6-sulphate antibody, immobilised to microtitre plates, and it involves a competitive binding reaction between chondroitin-6-sulphate in the samples and the biotinylated antigen. This assay enables the quantification of chondroitin-6-sulphate in the low concentration range of 16-120 micrograms/l. The intra-assay and inter-assay coefficients of variation are below 6.5% and 9.0%, respectively. More than 90% of chondroitin-6-sulphate was recovered when added to 0.1 mol/l phosphate-buffered saline, an albumin solution (40 g/l in phosphate-buffered saline) and cell culture medium (containing 100 ml/l foetal calf serum). Chondroitin-6-sulphate was also determined in sera of healthy male (n = 90) and female (n = 90) blood donors. The normal range was 55-169 micrograms/l. In men the mean value was estimated at 102.2 +/- 37.1 micrograms/l and in women at 98.7 +/- 26.4 micrograms/l. No age or sex dependence was observed. The urine excretion of chondroitin-6-sulphate in men (n = 16) was 44.5 +/- 21.1 mg/kg creatinine (mean +/- standard deviation) and in females (n = 10) 53.5 +/- 21.3 mg/kg creatinine. The clearance rate in men was 0.41 +/- 0.22 ml x min-1 and in women 0.38 +/- 0.15 ml x min-1. No sex dependence was found. Furthermore, the enzyme immunoassay was modified to measure the specific incorporation of a radioactively labelled precursor ([14C]galactosamine) into chondroitin-6-sulphate.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of viscosity and osmotic stress on the reaction of human butyrylcholinesterase with cresyl saligenin phosphate, a toxicant related to aerotoxic syndrome: kinetic and molecular dynamics studies.

PubMed

Masson, Patrick; Lushchekina, Sofya; Schopfer, Lawrence M; Lockridge, Oksana

2013-09-15

CSP (cresyl saligenin phosphate) is an irreversible inhibitor of human BChE (butyrylcholinesterase) that has been involved in the aerotoxic syndrome. Inhibition under pseudo-first-order conditions is biphasic, reflecting a slow equilibrium between two enzyme states E and E'. The elementary constants for CSP inhibition of wild-type BChE and D70G mutant were determined by studying the dependence of inhibition kinetics on viscosity and osmotic pressure. Glycerol and sucrose were used as viscosogens. Phosphorylation by CSP is sensitive to viscosity and is thus strongly diffusion-controlled (kon≈10⁸ M⁻¹·min⁻¹). Bimolecular rate constants (ki) are about equal to kon values, making CSP one of the fastest inhibitors of BChE. Sucrose caused osmotic stress because it is excluded from the active-site gorge. This depleted the active-site gorge of water. Osmotic activation volumes, determined from the dependence of ki on osmotic pressure, showed that water in the gorge of the D70G mutant is more easily depleted than that in wild-type BChE. This demonstrates the importance of the peripheral site residue Asp⁷⁰ in controlling the active-site gorge hydration. MD simulations provided new evidence for differences in the motion of water within the gorge of wild-type and D70G enzymes. The effect of viscosogens/osmolytes provided information on the slow equilibrium E⇌E', indicating that alteration in hydration of a key catalytic residue shifts the equilibrium towards E'. MD simulations showed that glycerol molecules that substitute for water molecules in the enzyme active-site gorge induce a conformational change in the catalytic triad residue His⁴³⁸, leading to the less reactive form E'.

Characterization of Runella slithyformis HD-Pnk, a Bifunctional DNA/RNA End-Healing Enzyme Composed of an N-Terminal 2′,3′-Phosphoesterase HD Domain and a C-Terminal 5′-OH Polynucleotide Kinase Domain

PubMed Central

Munir, Annum

2016-01-01

ABSTRACT 5′- and 3′-end-healing reactions are key steps in nucleic acid break repair in which 5′-OH ends are phosphorylated by a polynucleotide kinase (Pnk) and 3′-PO4 or 2′,3′-cyclic-PO4 ends are hydrolyzed by a phosphoesterase to generate the 5′-PO4 and 3′-OH termini required for sealing by classic polynucleotide ligases. End-healing and sealing enzymes are present in diverse bacterial taxa, often organized as modular units within a single multifunctional polypeptide or as subunits of a repair complex. Here we identify and characterize Runella slithyformis HD-Pnk as a novel bifunctional end-healing enzyme composed of an N-terminal 2′,3′-phosphoesterase HD domain and a C-terminal 5′-OH polynucleotide kinase P-loop domain. HD-Pnk phosphorylates 5′-OH polynucleotides (9-mers or longer) in the presence of magnesium and any nucleoside triphosphate donor. HD-Pnk dephosphorylates RNA 2′,3′-cyclic phosphate, RNA 3′-phosphate, RNA 2′-phosphate, and DNA 3′-phosphate ends in the presence of a transition metal cofactor, which can be nickel, copper, or cobalt. HD-Pnk homologs are present in genera from 11 bacterial phyla and are often encoded in an operon with a putative ATP-dependent polynucleotide ligase. IMPORTANCE The present study provides insights regarding the diversity of nucleic acid repair strategies via the characterization of Runella slithyformis HD-Pnk as the exemplar of a novel clade of dual 5′- and 3′-end-healing enzymes that phosphorylate 5′-OH termini and dephosphorylate 2′,3′-cyclic-PO4, 3′-PO4, and 2′-PO4 ends. The distinctive feature of HD-Pnk is its domain composition, i.e., a fusion of an N-terminal HD phosphohydrolase module and a C-terminal P-loop polynucleotide kinase module. Homologs of Runella HD-Pnk with the same domain composition, same domain order, and similar polypeptide sizes are distributed widely among genera from 11 bacterial phyla. PMID:27895092

Dissimilar Deficiency of Glucose-6-Phosphate Dehydrogenase (G-6-PD) among the AFARS and the Somalis of Djibouti

DTIC Science & Technology

1991-01-01

DEFICIENCY OF GLUCOSE - 6 - PHOSPHATE DEHYDROGENASE (G- 6 ...the prevalence of deficient activity of the enzyme glucose - 6 - phosphate dehydrogenase (G- 6 -PD) among - Ces difficiences enzymatiques sant plus particu...Screening for glucose - 6 - 3 - CaosBy W.H. - Hematologic diseases. In : I lunter’s Tropical phosphate dehydrogenase (G- 6 -PD) deficiency by a simple

Insights into the evolution of enzyme substrate promiscuity after the discovery of (βα)₈ isomerase evolutionary intermediates from a diverse metagenome.

PubMed

Noda-García, Lianet; Juárez-Vázquez, Ana L; Ávila-Arcos, María C; Verduzco-Castro, Ernesto A; Montero-Morán, Gabriela; Gaytán, Paul; Carrillo-Tripp, Mauricio; Barona-Gómez, Francisco

2015-06-10

Current sequence-based approaches to identify enzyme functional shifts, such as enzyme promiscuity, have proven to be highly dependent on a priori functional knowledge, hampering our ability to reconstruct evolutionary history behind these mechanisms. Hidden Markov Model (HMM) profiles, broadly used to classify enzyme families, can be useful to distinguish between closely related enzyme families with different specificities. The (βα)8-isomerase HisA/PriA enzyme family, involved in L-histidine (HisA, mono-substrate) biosynthesis in most bacteria and plants, but also in L-tryptophan (HisA/TrpF or PriA, dual-substrate) biosynthesis in most Actinobacteria, has been used as model system to explore evolutionary hypotheses and therefore has a considerable amount of evolutionary, functional and structural knowledge available. We searched for functional evolutionary intermediates between the HisA and PriA enzyme families in order to understand the functional divergence between these families. We constructed a HMM profile that correctly classifies sequences of unknown function into the HisA and PriA enzyme sub-families. Using this HMM profile, we mined a large metagenome to identify plausible evolutionary intermediate sequences between HisA and PriA. These sequences were used to perform phylogenetic reconstructions and to identify functionally conserved amino acids. Biochemical characterization of one selected enzyme (CAM1) with a mutation within the functionally essential N-terminus phosphate-binding site, namely, an alanine instead of a glycine in HisA or a serine in PriA, showed that this evolutionary intermediate has dual-substrate specificity. Moreover, site-directed mutagenesis of this alanine residue, either backwards into a glycine or forward into a serine, revealed the robustness of this enzyme. None of these mutations, presumably upon functionally essential amino acids, significantly abolished its enzyme activities. A truncated version of this enzyme (CAM2) predicted to adopt a (βα)6-fold, and thus entirely lacking a C-terminus phosphate-binding site, was identified and shown to have HisA activity. As expected, reconstruction of the evolution of PriA from HisA with HMM profiles suggest that functional shifts involve mutations in evolutionarily intermediate enzymes of otherwise functionally essential residues or motifs. These results are in agreement with a link between promiscuous enzymes and intragenic epistasis. HMM provides a convenient approach for gaining insights into these evolutionary processes.

Environmentally relevant organophosphate triesters in herring gulls: In vitro biotransformation and kinetics and diester metabolite formation using a hepatic microsomal assay

DOE Office of Scientific and Technical Information (OSTI.GOV)

Greaves, Alana K.

The in vitro biotransformation and kinetics of six organophosphate triester (OPE) flame retardants were investigated in herring gulls (Larus argentatus) from the Great Lakes using a hepatic microsomal metabolism assay. Administration of each individual OPE (tri-n-butyl phosphate (TNBP), tris(2-butoxyethyl) phosphate (TBOEP), triphenyl phosphate (TPHP), triethyl phosphate (TEP), tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) and tris(2-chloroisopropyl) phosphate (TCIPP)) to the in vitro assay (concentration range 0.01 to 10 μM) resulted in rapid depletion with the exception of TEP. Following the Michaelis-Menten enzyme kinetics model, a preliminary 2-minute incubation period was used to estimate the V{sub max} (± SE) values (i.e., the maximal rate ofmore » reaction for a saturated enzyme system), which ranged from 5.0 ± 0.4 (TPHP) to 29 ± 18 pmol/min/mg protein (TBOEP), as well as the K{sub M} (± SE) values (i.e., the OPE concentration corresponding to one half of the V{sub max}), which ranged from 9.8 ± 1 (TPHP) to 189 ± 135 nM (TBOEP). Biotransformation assays over a 100-minute incubation period revealed that TNBP was metabolized most rapidly (with a depletion rate of 73 ± 4 pmol/min/mg protein), followed by TBOEP (53 ± 8 pmol/min/mg), TCIPP (27 ± 1 pmol/min/mg), TPHP (22 ± 2 pmol/min/mg) and TDCIPP (8 ± 1 pmol/min/mg). In vitro biotransformation of OP triesters was clearly structure-dependent where non-halogenated alkyl OP triesters were metabolized more rapidly than halogenated alkyl triesters. Halogenated OP triesters were transformed to their respective diesters more efficiently relative to non-halogenated OP triesters. To our knowledge, this is the first study to investigate OP triester metabolism and OP diester formation in an avian or wildlife model system, which is important to understand the fate and biological activity of OPEs in an exposed organism. - Highlights: • The metabolism and kinetics of 6 OPEs were examined in herring gull liver microsomes. • The metabolism of OPEs was rapid, with the exception of TEP (no metabolism observed). • Relative metabolism rates (pmol/min/mg protein): TNBP > TBOEP > TPHP > TDCIPP > TCIPP. • OPE metabolite (i.e., OP diesters) formation was shown for all 5 OP triesters. • Structure-dependent metabolism rates and OP diester formation were observed.« less

21 CFR 862.1315 - Galactose-1-phosphate uridyl transferase test system.

Code of Federal Regulations, 2010 CFR

2010-04-01

... of the enzyme galactose-1-phosphate uridyl transferase in erythrocytes (red blood cells... hereditary disease galactosemia (disorder of galactose metabolism) in infants. (b) Classification. Class II. ...

E2P phosphoforms of Na,K-ATPase. I. Comparison of phosphointermediates formed from ATP and Pi by their reactivity toward hydroxylamine and vanadate.

PubMed

Fedosova, N U; Cornelius, F; Klodos, I

1998-09-29

The properties of Na,K-ATPase phosphoenzymes formed either from ATP in the presence of Mg2+ and Na+ or from Pi in the absence of alkali cations were investigated by biochemical methods and spectrofluorometry employing the styryl dye RH421. We characterized the phosphoenzyme species by their reaction to N-methyl hydroxylamine, which attacks specifically the protein-phosphate bond. We studied reactions of the phospho- and dephospho-enzymes with vanadate, which is a transition-state analogue of phosphate in this enzyme. On the basis of substantial differences in the properties of the phosphoenzyme species formed either from ATP or Pi, especially in their reactivity to N-methyl hydroxylamine, it is suggested that the two phosphoenzyme species are two subconformations of the E2P phosphoform. Analysis of the RH421 fluorescence responses under a variety of experimental conditions and comparing different enzyme sources suggested that the increase of RH421 fluorescence induced by inorganic phosphate in the absence of alkali cations is associated with the formation of the covalent acyl-phosphate bond.

Regulatory insights into the production of UDP-N-acetylglucosamine by Lactobacillus casei

PubMed Central

Rodríguez-Díaz, Jesús; Rubio-del-Campo, Antonio; Yebra, María J.

2012-01-01

UDP-N-acetylglucosamine (UDP-GlcNAc) is an important sugar nucleotide used as a precursor of cell wall components in bacteria, and as a substrate in the synthesis of oligosaccharides in eukaryotes. In bacteria UDP-GlcNAc is synthesized from the glycolytic intermediate D-fructose-6-phosphate (fructose-6P) by four successive reactions catalyzed by three enzymes: glucosamine-6-phosphate synthase (GlmS), phosphoglucosamine mutase (GlmM) and the bi-functional enzyme glucosamine-1-phosphate acetyltransferase/ N-acetylglucosamine-1-phosphate uridyltransferase (GlmU). We have previously reported a metabolic engineering strategy in Lactobacillus casei directed to increase the intracellular levels of UDP-GlcNAc by homologous overexpression of the genes glmS, glmM and glmU. One of the most remarkable features regarding the production of UDP-GlcNAc in L. casei was to find multiple regulation points on its biosynthetic pathway: (1) regulation by the NagB enzyme, (2) glmS RNA specific degradation through the possible participation of a glmS riboswitch mechanism, (3) regulation of the GlmU activity probably by end product inhibition and (4) transcription of glmU. PMID:22825354

Subcellular distribution of gluconeogenetic enzymes in germinating castor bean endosperm

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nishimura, M.; Beevers, H.

1979-07-01

The intracellular distribution of enzymes capable of catalyzing the reactions from oxaloacetate to sucrose in germinating castor bean endosperm has been studied by sucrose density gradient centrifugation. One set of glycolytic enzyme activities was detected in the plastids and another in the cytosol. The percentages of their activities in the plastids were less than 10% of total activities except for aldolase and fructose diphosphatase. The activities of several of the enzymes present in the plastids seem to be too low to account for the in vivo rate of gluconeogenesis whereas those in the cytosol are quite adequate. Furthermore, phosphoenolypyruvate carboxykinase,more » sucrose phosphate synthetase, and sucrose synthetase, which catalyze the first and final steps in the conversion of oxaloacetate to sucrose, were found only in the cytosol. It is deduced that in germinating castor bean endosperm the complete conversion of oxaloacetate to sucrose and CO/sub 2/ occurs in the cytosol. The plastids contain some enzymes of the pentose phosphate pathway, pyruvate dehydrogenase and fatty acid synthetase in addition to the set of glycolytic enzymes. This suggests that the role of the plastid in the endosperm of germinating castor bean is the production of fatty acids from sugar phosphates, as it is known to be in the endosperm during seed development.« less

D-ribulose-5-phosphate 3-epimerase: Cloning and heterologous expression of the spinach gene, and purification and characterization of the recombinant enzyme

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Y.R.; Hartman, F.C.; Lu, T.Y.S.

The authors have achieved, to their knowledge, the first high-level heterologous expression of the gene encoding D-ribulose-5-phosphate 3-epimerase from any source, thereby permitting isolation and characterization of the epimerase as found in photosynthetic organisms. The extremely labile recombinant spinach (Spinacia oleracea L.) enzyme was stabilized by DL-{alpha}-glycerophosphate or ethanol and destabilized by D-ribulose-5-phosphate or 2-mercaptoethanol. Despite this lability, the unprecedentedly high specific activity of the purified material indicates that the structural integrity of the enzyme is maintained throughout isolation. Ethylenediaminetetraacetate and divalent metal cations did not affect epimerase activity, thereby excluding a requirement for the latter in catalysis. As deducedmore » from the sequence of the cloned spinach gene and the electrophoretic mobility under denaturing conditions of the purified recombinant enzyme, its 25-kD subunit size was about the same as that of the corresponding epimerases of yeast and mammals. However, in contrast to these other species, the recombinant spinach enzyme was octameric rather than dimeric, as assessed by gel filtration and polyacrylamide gel electrophoresis under nondenaturing conditions. Western-blot analyses with antibodies to the purified recombinant enzyme confirmed that the epimerase extracted from spinach leaves is also octameric.« less

The effects of iron deficiency on rat liver enzymes.

PubMed Central

Bailey-Wood, R.; Blayney, L. M.; Muir, J. R.; Jacobs, A.

1975-01-01

The effect of iron deficiency on a number or iron containing enzymes in rat liver has been examined. In addition, 6-phosphogluconate dehydrogenase and glucose 6-phosphate dehydrogenase have been assayed. Of the mitochondrial electron transport reactions only succinate-cytochrome C reductase activity was decreased in iron deficient animals. Microsomal reductase enzymes associated with the NADPH-oxidase system were also markedly decreased although cytochrome P450 concentrations were unaffected. Both 6-phosphogluconate dehydrogenase and glucose 6-phosphate dehydrogenase were reduced in young iron deficient rats but the former had returned to control levels at the age of 14 weeks. PMID:172099

Trehalose 6-phosphate phosphatases of Pseudomonas aeruginosa.

PubMed

Cross, Megan; Biberacher, Sonja; Park, Suk-Youl; Rajan, Siji; Korhonen, Pasi; Gasser, Robin B; Kim, Jeong-Sun; Coster, Mark J; Hofmann, Andreas

2018-04-24

The opportunistic bacterium Pseudomonas aeruginosa has been recognized as an important pathogen of clinical relevance and is a leading cause of hospital-acquired infections. The presence of a glycolytic enzyme in Pseudomonas, which is known to be inhibited by trehalose 6-phosphate (T6P) in other organisms, suggests that these bacteria may be vulnerable to the detrimental effects of intracellular T6P accumulation. In the present study, we explored the structural and functional properties of trehalose 6-phosphate phosphatase (TPP) in P. aeruginosa in support of future target-based drug discovery. A survey of genomes revealed the existence of 2 TPP genes with either chromosomal or extrachromosomal location. Both TPPs were produced as recombinant proteins, and characterization of their enzymatic properties confirmed specific, magnesium-dependent catalytic hydrolysis of T6P. The 3-dimensional crystal structure of the chromosomal TPP revealed a protein dimer arising through β-sheet expansion of the individual monomers, which possess the overall fold of halo-acid dehydrogenases.-Cross, M., Biberacher, S., Park, S.-Y., Rajan, S., Korhonen, P., Gasser, R. B., Kim, J.-S., Coster, M. J., Hofmann, A. Trehalose 6-phosphate phosphatases of Pseudomonas aeruginosa.

Two novel DNA variants associated with glucose-6-phosphate dehydrogenase deficiency found in Argentine pediatric patients.

PubMed

Chaves, Alejandro; Eberle, Silvia Eandi; Defelipe, Lucas; Pepe, Carolina; Milanesio, Berenice; Aguirre, Fernando; Fernandez, Diego; Turjanski, Adrian; Feliú-Torres, Aurora

2016-07-01

The enzyme glucose-6-phosphate dehydrogenase (G6PD) catalyses the first step in the pentose phosphate pathway, producing nicotinamide adenine dinucleotide phosphate (NADPH). NADPH plays a crucial role in preventing oxidative damage to proteins and other molecules in cells, mostly red blood cells. G6PD deficiency has an x-linked pattern of inheritance in which hemizygous males are deficient, while females may or may not be deficient depending on the number of affected alleles. We report two novel DNA variants in the G6PD gene detected in two male probands with chronic nonspherocytic hemolytic anemia (CNSHA), who were referred for hematological evaluation. Probands and their relatives underwent clinical, biochemical, and molecular assessment. Two novel DNA variants, c.995C>T and c.1226C>A, were found in this study. At the protein level, they produce the substitution of Ser332Phe and Pro409Gln, respectively. These DNA variants were analyzed in the female relatives of probands for genetic counseling. The novel DNA variants were classified as class I based on the clinical, biochemical, and molecular evaluations performed. Copyright © 2016 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Pyrophosphate-Dependent Fructose-6-Phosphate 1-Phosphotransferase Induction and Attenuation of Hsp Gene Expression during Endosperm Modification in Quality Protein Maize1[C][W][OA

PubMed Central

Guo, Xiaomei; Ronhovde, Kyla; Yuan, Lingling; Yao, Bo; Soundararajan, Madhavan P.; Elthon, Thomas; Zhang, Chi; Holding, David R.

2012-01-01

Quality Protein Maize (QPM) is a hard-endosperm version of the high-lysine opaque2 (o2) maize (Zea mays) mutant, but the genes involved in modification of the soft o2 endosperm are largely unknown. Pyrophosphate-dependent fructose-6-phosphate 1-phosphotransferase (PFP) catalyzes the ATP-independent conversion of fructose-6-phosphate to fructose-1,6-bisphosphate in glycolysis. We found a large increase in transcript and protein levels of the α-regulatory subunit of PFP (PFPα) in QPM endosperm. In vitro enzyme assays showed a significant increase in forward PFP activity in developing endosperm extracts of QPM relative to the wild type and o2. An expressed retrogene version of PFPα of unknown function that was not up-regulated in QPM was also identified. The elevated expression levels of a number of ATP-requiring heat shock proteins (Hsps) in o2 endosperm are ameliorated in QPM. PFPα is also coinduced with Hsps in maize roots in response to heat, cold, and the unfolded protein response stresses. We propose that reduced ATP availability resulting from the generalized Hsp response in addition to the reduction of pyruvate, orthophosphate dikinase activity in o2 endosperm is compensated in part by increased PFP activity in QPM. PMID:22158678

Phosphatidylglycerolphosphate synthase expression in Schizosaccharomyces pombe is regulated by the phospholipid precursors inositol and choline.

PubMed Central

Karkhoff-Schweizer, R R; Kelly, B L; Greenberg, M L

1991-01-01

The enzyme phosphatidylglycerolphosphate synthase (PGPS; CDP-diacylglycerol glycerol 3-phosphate 3-phosphatidyltransferase; EC 2.7.8.5) catalyzes the committed step in the cardiolipin biosynthetic pathway. To study the regulation of PGPS in Schizosaccharomyces pombe, we characterized the enzyme biochemically. Maximum activity occurred in the presence of 6 mM Triton X-100 at pH 7.5. The apparent Km values for CDP-diacylglycerol and glycerol 3-phosphate were 130 and 26 microM, respectively. Optimal activity was at 35 degrees C, and enzyme activity was labile above 40 degrees C. Thioreactive agents were inhibitory to PGPS activity. To determine whether S. pombe PGPS is regulated by phospholipid precursors, we examined the time-dependent expression of PGPS upon inositol and choline starvation. Starvation for inositol resulted in a threefold increase in PGPS expression in wild-type cells. In cho1 and cho2 mutants, which are blocked in phosphatidylcholine synthesis, starvation for choline resulted in transient derepression of PGPS expression. In choline auxotrophs starved for inositol, PGPS was derepressed 2.5- to 3-fold in the presence of choline and less or not at all in the absence of choline. This is the first description of PGPS regulation in S. pombe and the first demonstration of inositol-mediated regulation in the inositol-requiring yeast species. PMID:1655700

Accessing non-natural reactivity by irradiating nicotinamide-dependent enzymes with light

NASA Astrophysics Data System (ADS)

Emmanuel, Megan A.; Greenberg, Norman R.; Oblinsky, Daniel G.; Hyster, Todd K.

2016-12-01

Enzymes are ideal for use in asymmetric catalysis by the chemical industry, because their chemical compositions can be tailored to a specific substrate and selectivity pattern while providing efficiencies and selectivities that surpass those of classical synthetic methods. However, enzymes are limited to reactions that are found in nature and, as such, facilitate fewer types of transformation than do other forms of catalysis. Thus, a longstanding challenge in the field of biologically mediated catalysis has been to develop enzymes with new catalytic functions. Here we describe a method for achieving catalytic promiscuity that uses the photoexcited state of nicotinamide co-factors (molecules that assist enzyme-mediated catalysis). Under irradiation with visible light, the nicotinamide-dependent enzyme known as ketoreductase can be transformed from a carbonyl reductase into an initiator of radical species and a chiral source of hydrogen atoms. We demonstrate this new reactivity through a highly enantioselective radical dehalogenation of lactones—a challenging transformation for small-molecule catalysts. Mechanistic experiments support the theory that a radical species acts as an intermediate in this reaction, with NADH and NADPH (the reduced forms of nicotinamide adenine nucleotide and nicotinamide adenine dinucleotide phosphate, respectively) serving as both a photoreductant and the source of hydrogen atoms. To our knowledge, this method represents the first example of photo-induced enzyme promiscuity, and highlights the potential for accessing new reactivity from existing enzymes simply by using the excited states of common biological co-factors. This represents a departure from existing light-driven biocatalytic techniques, which are typically explored in the context of co-factor regeneration.

Pyrophosphate-Dependent ATP Formation from Acetyl Coenzyme A in Syntrophus aciditrophicus , a New Twist on ATP Formation

DOE Office of Scientific and Technical Information (OSTI.GOV)

James, Kimberly L.; Ríos-Hernández, Luis A.; Wofford, Neil Q.

Syntrophus aciditrophicusis a model syntrophic bacterium that degrades key intermediates in anaerobic decomposition, such as benzoate, cyclohexane-1-carboxylate, and certain fatty acids, to acetate when grown with hydrogen-/formate-consuming microorganisms. ATP formation coupled to acetate production is the main source for energy conservation byS. aciditrophicus. However, the absence of homologs for phosphate acetyltransferase and acetate kinase in the genome ofS. aciditrophicusleaves it unclear as to how ATP is formed, as most fermentative bacteria rely on these two enzymes to synthesize ATP from acetyl coenzyme A (CoA) and phosphate. Here, we combine transcriptomic, proteomic, metabolite, and enzymatic approaches to show thatS. aciditrophicususes AMP-forming, acetyl-CoA synthetase (Acs1)more » for ATP synthesis from acetyl-CoA.acs1mRNA and Acs1 were abundant in transcriptomes and proteomes, respectively, ofS. aciditrophicusgrown in pure culture and coculture. Cell extracts ofS. aciditrophicushad low or undetectable acetate kinase and phosphate acetyltransferase activities but had high acetyl-CoA synthetase activity under all growth conditions tested. Both Acs1 purified fromS. aciditrophicusand recombinantly produced Acs1 catalyzed ATP and acetate formation from acetyl-CoA, AMP, and pyrophosphate. High pyrophosphate levels and a high AMP-to-ATP ratio (5.9 ± 1.4) inS. aciditrophicuscells support the operation of Acs1 in the acetate-forming direction. Thus,S. aciditrophicushas a unique approach to conserve energy involving pyrophosphate, AMP, acetyl-CoA, and an AMP-forming, acetyl-CoA synthetase. We find bacteria use two enzymes, phosphate acetyltransferase and acetate kinase, to make ATP from acetyl-CoA, while acetate-forming archaea use a single enzyme, an ADP-forming, acetyl-CoA synthetase, to synthesize ATP and acetate from acetyl-CoA.Syntrophus aciditrophicusapparently relies on a different approach to conserve energy during acetyl-CoA metabolism, as its genome does not have homologs to the genes for phosphate acetyltransferase and acetate kinase. Here, we show thatS. aciditrophicususes an alternative approach, an AMP-forming, acetyl-CoA synthetase, to make ATP from acetyl-CoA. AMP-forming, acetyl-CoA synthetases were previously thought to function only in the activation of acetate to acetyl-CoA.« less

Pyrophosphate-Dependent ATP Formation from Acetyl Coenzyme A in Syntrophus aciditrophicus , a New Twist on ATP Formation

DOE PAGES

James, Kimberly L.; Ríos-Hernández, Luis A.; Wofford, Neil Q.; ...

2016-08-16

Syntrophus aciditrophicusis a model syntrophic bacterium that degrades key intermediates in anaerobic decomposition, such as benzoate, cyclohexane-1-carboxylate, and certain fatty acids, to acetate when grown with hydrogen-/formate-consuming microorganisms. ATP formation coupled to acetate production is the main source for energy conservation byS. aciditrophicus. However, the absence of homologs for phosphate acetyltransferase and acetate kinase in the genome ofS. aciditrophicusleaves it unclear as to how ATP is formed, as most fermentative bacteria rely on these two enzymes to synthesize ATP from acetyl coenzyme A (CoA) and phosphate. Here, we combine transcriptomic, proteomic, metabolite, and enzymatic approaches to show thatS. aciditrophicususes AMP-forming, acetyl-CoA synthetase (Acs1)more » for ATP synthesis from acetyl-CoA.acs1mRNA and Acs1 were abundant in transcriptomes and proteomes, respectively, ofS. aciditrophicusgrown in pure culture and coculture. Cell extracts ofS. aciditrophicushad low or undetectable acetate kinase and phosphate acetyltransferase activities but had high acetyl-CoA synthetase activity under all growth conditions tested. Both Acs1 purified fromS. aciditrophicusand recombinantly produced Acs1 catalyzed ATP and acetate formation from acetyl-CoA, AMP, and pyrophosphate. High pyrophosphate levels and a high AMP-to-ATP ratio (5.9 ± 1.4) inS. aciditrophicuscells support the operation of Acs1 in the acetate-forming direction. Thus,S. aciditrophicushas a unique approach to conserve energy involving pyrophosphate, AMP, acetyl-CoA, and an AMP-forming, acetyl-CoA synthetase. We find bacteria use two enzymes, phosphate acetyltransferase and acetate kinase, to make ATP from acetyl-CoA, while acetate-forming archaea use a single enzyme, an ADP-forming, acetyl-CoA synthetase, to synthesize ATP and acetate from acetyl-CoA.Syntrophus aciditrophicusapparently relies on a different approach to conserve energy during acetyl-CoA metabolism, as its genome does not have homologs to the genes for phosphate acetyltransferase and acetate kinase. Here, we show thatS. aciditrophicususes an alternative approach, an AMP-forming, acetyl-CoA synthetase, to make ATP from acetyl-CoA. AMP-forming, acetyl-CoA synthetases were previously thought to function only in the activation of acetate to acetyl-CoA.« less

Consortium inoculum of five thermo-tolerant phosphate solubilizing Actinomycetes for multipurpose biofertilizer preparation

PubMed Central

Nandimath, Arusha P.; Karad, Dilip D.; Gupta, Shantikumar G.; Kharat, Arun S.

2017-01-01

Background and Objectives: Alkaline pH of the soil facilitates the conversion of phosphate present in phosphate fertilizer applied in the field to insoluble phosphate which is not available to plants. Problem of soluble phosphate deficiency arises, primarily due to needless use of phosphate fertilizer. We sought to biofertilizer with the thermo-tolerant phosphate solubilizing actinomycetes consortium that could convert insoluble phosphate to soluble phosphate at wider temperature range. Materials and Methods: In the present investigation consortium of five thermo-tolerant phosphate solubilizing actinomycetes was applied for preparation of inoculum to produce multipurpose bio-fertilizer. Phosphates solubilizing thermo-tolerant 32 actinomycetes strains were processed for identification with the use of PIBWIN software and were screened for phosphate solubilizing activity. Results: Amongst these five actinomycetes were selected on the basis of their ability to produce cellulase, chitinase, pectinase, protease, lipase, amylase and phosphate solubilizing enzymes. Ability to produce these enzymes at 28°C and 50°C were examined. Biofertilizer was prepared by using agricultural waste as a raw material. While preparation of bio-fertilizer the pH decreased from 7.5 to 4.3 and temperature increased up to 74°C maximum at the end of 4th week and in subsequent week it started to decline gradually till it reached around 50°C, which was found to be stable up to eighth week. This thermo-tolerant actinomycetes consortium released soluble phosphate of up to 46.7 μg ml−1. Conclusion: As the mesophilic organisms die out at high temperature of composting hence thormo-tolerant actinomycetes would be the better substitute for preparation of phosphate solubilizing bio-fertilizer with added potential to degrade complex macromolecules in composting. PMID:29296275