New Enzymatic Method of Chiral Amino Acid Synthesis by Dynamic Kinetic Resolution of Amino Acid Amides: Use of Stereoselective Amino Acid Amidases in the Presence of α-Amino-ɛ-Caprolactam Racemase▿

PubMed Central

Yamaguchi, Shigenori; Komeda, Hidenobu; Asano, Yasuhisa

2007-01-01

d- and l-amino acids were produced from l- and d-amino acid amides by d-aminopeptidase from Ochrobactrum anthropi C1-38 and l-amino acid amidase from Pseudomonas azotoformans IAM 1603, respectively, in the presence of α-amino-ɛ-caprolactam racemase from Achromobacter obae as the catalyst by dynamic kinetic resolution of amino acid amides. PMID:17586677

Structural requirements of choline derivatives for 'conversion' of pneumococcal amidase. A new single-step procedure for purification of this autolysin.

PubMed

Sanz, J M; Lopez, R; Garcia, J L

1988-05-23

Tertiary amines appear to be the minimal structure needed to convert in vitro the inactive form (E-form) of pneumococcal amidase to the catalytic active form (C-form). Diethylethanolamine was one of the compounds that converted the E-form, a finding that has been used successfully to develop an affinity chromatography system in DEAE-cellulose for the rapid and efficient purification of lytic enzymes of pneumococcus and its bacteriophages.

Thin-Layer Polymer Wrapped Enzymes Encapsulated in Hierarchically Mesoporous Silica with High Activity and Enhanced Stability

NASA Astrophysics Data System (ADS)

Zhang, Fang; Wang, Meitao; Liang, Chao; Jiang, Huangyong; Shen, Jian; Li, Hexing

2014-03-01

A novel soft-hard cooperative approach was developed to synthesize bioactive mesoporous composite by pre-wrapping Penicillin G amidase with poly(acrylaimde) nanogel skin and subsequently incorporating such Penicillin G amidase nanocapsules into hierarchically mesoporous silica. The as-received bioactive mesoporous composite exhibited comparable activity and extraordinarily high stability in comparison with native Penicillin G amidase and could be used repetitively in the water-medium hydrolysis of penicillin G potassium salt. Furthermore, this strategy could be extended to the synthesis of multifunctional bioactive mesoporous composite by simultaneously introducing glucose oxidase nanocapsules and horseradish peroxidase nanocapsules into hierarchically mesoporous silica, which demonstrated a synergic effect in one-pot tandem oxidation reaction. Improvements in the catalytic performances were attributed to the combinational unique structure from soft polymer skin and hard inorganic mesoporous silica shell, which cooperatively helped enzyme molecules to retain their appropriate geometry and simultaneously decreased the enzyme-support negative interaction and mass transfer limitation under heterogeneous conditions.

Amidase encapsulated O-carboxymethyl chitosan nanoparticles for vaccine delivery.

PubMed

Smitha, K T; Sreelakshmi, M; Nisha, N; Jayakumar, R; Biswas, Raja

2014-02-01

This work reports the development of amidase encapsulated O-carboxymethyl chitosan nanoparticles (Ami-O-CMC NPs) of 300±50 nm size by ionic cross-linking method. The prepared Ami-O-CMC NPs had an encapsulation efficiency of 55.39%. Haemolysis assay and cytotoxicity studies proved the hemocompatibility and cytocompatibility of the prepared NPs. The sustained release of Ami from the NPs is expected to prolong its immunogenicity and in turn lead to development of better protective immunity against Staphylococcus aureus infections. Copyright © 2013 Elsevier B.V. All rights reserved.

Cloning and characterization of a novel amidase from Paracoccus sp. M-1, showing aryl acylamidase and acyl transferase activities.

PubMed

Shen, Weiliang; Chen, Honghong; Jia, Kaizhi; Ni, Jun; Yan, Xin; Li, Shunpeng

2012-05-01

A novel amidase gene, designated pamh, was cloned from Paracoccus sp. M-1. Site-directed mutagenesis and bioinformatic analysis showed that the PamH protein belonged to the amidase signature enzyme family. PamH was expressed in Escherichia coli, purified, and characterized. The molecular mass of PamH was determined to be 52 kDa with an isoelectric point of 5.13. PamH displayed its highest enzymatic activity at 45°C and at pH 8.0 and was stable within a pH range of 5.0-10.0. The PamH enzyme exhibited amidase activity, aryl acylamidase activity, and acyl transferase activity, allowing it to function across a very broad substrate spectrum. PamH was highly active on aromatic and short-chain aliphatic amides (benzamide and propionamide), moderately active on amino acid amides, and possessed weak urease activity. Of the anilides examined, only propanil was a good substrate for PamH. For propanil, the k (cat) and K (m) were 2.8 s(-1) and 158 μM, respectively, and the catalytic efficiency value (k (cat)/K (m)) was 0.018 μM(-1) s(-1). In addition, PamH was able to catalyze the acyl transfer reaction to hydroxylamine for both amide and anilide substrates, including acetamide, propanil, and 4-nitroacetanilide; the highest reaction rate was shown with isobutyramide. These characteristics make PamH an excellent candidate for environmental remediation and an important enzyme for the biosynthesis of novel amides.

Product-induced gene expression, a product-responsive reporter assay used to screen metagenomic libraries for enzyme-encoding genes.

PubMed

Uchiyama, Taku; Miyazaki, Kentaro

2010-11-01

A reporter assay-based screening method for enzymes, which we named product-induced gene expression (PIGEX), was developed and used to screen a metagenomic library for amidases. A benzoate-responsive transcriptional activator, BenR, was placed upstream of the gene encoding green fluorescent protein and used as a sensor. Escherichia coli sensor cells carrying the benR-gfp gene cassette fluoresced in response to benzoate concentrations as low as 10 μM but were completely unresponsive to the substrate benzamide. An E. coli metagenomic library consisting of 96,000 clones was grown in 96-well format in LB medium containing benzamide. The library cells were then cocultivated with sensor cells. Eleven amidase genes were recovered from 143 fluorescent wells; eight of these genes were homologous to known bacterial amidase genes while three were novel genes. In addition to their activity toward benzamide, the enzymes were active toward various substrates, including d- and l-amino acid amides, and displayed enantioselectivity. Thus, we demonstrated that PIGEX is an effective approach for screening novel enzymes based on product detection.

The Enzymology of 2-Hydroxyglutarate, 2-Hydroxyglutaramate and 2-Hydroxysuccinamate and Their Relationship to Oncometabolites

PubMed Central

Hariharan, Vivek A.; Denton, Travis T.; Paraszcszak, Sarah; McEvoy, Kyle; Jeitner, Thomas M.; Krasnikov, Boris F.; Cooper, Arthur J. L.

2017-01-01

Many enzymes make “mistakes”. Consequently, repair enzymes have evolved to correct these mistakes. For example, lactate dehydrogenase (LDH) and mitochondrial malate dehydrogenase (mMDH) slowly catalyze the reduction of 2-oxoglutarate (2-OG) to the oncometabolite l-2-hydroxyglutarate (l-2-HG). l-2-HG dehydrogenase corrects this error by converting l-2-HG to 2-OG. LDH also catalyzes the reduction of the oxo group of 2-oxoglutaramate (2-OGM; transamination product of l-glutamine). We show here that human glutamine synthetase (GS) catalyzes the amidation of the terminal carboxyl of both the l- and d- isomers of 2-HG. The reaction of 2-OGM with LDH and the reaction of l-2-HG with GS generate l-2-hydroxyglutaramate (l-2-HGM). We also show that l-2-HGM is a substrate of human ω-amidase. The product (l-2-HG) can then be converted to 2-OG by l-2-HG dehydrogenase. Previous work showed that 2-oxosuccinamate (2-OSM; transamination product of l-asparagine) is an excellent substrate of LDH. Finally, we also show that human ω-amidase converts the product of this reaction (i.e., l-2-hydroxysuccinamate; l-2-HSM) to l-malate. Thus, ω-amidase may act together with hydroxyglutarate dehydrogenases to repair certain “mistakes” of GS and LDH. The present findings suggest that non-productive pathways for nitrogen metabolism occur in mammalian tissues in vivo. Perturbations of these pathways may contribute to symptoms associated with hydroxyglutaric acidurias and to tumor progression. Finally, methods for the synthesis of l-2-HGM and l-2-HSM are described that should be useful in determining the roles of ω-amidase/4- and 5-C compounds in photorespiration in plants. PMID:28358347

The Enzymology of 2-Hydroxyglutarate, 2-Hydroxyglutaramate and 2-Hydroxysuccinamate and Their Relationship to Oncometabolites.

PubMed

Hariharan, Vivek A; Denton, Travis T; Paraszcszak, Sarah; McEvoy, Kyle; Jeitner, Thomas M; Krasnikov, Boris F; Cooper, Arthur J L

2017-03-30

Many enzymes make "mistakes". Consequently, repair enzymes have evolved to correct these mistakes. For example, lactate dehydrogenase (LDH) and mitochondrial malate dehydrogenase (mMDH) slowly catalyze the reduction of 2-oxoglutarate (2-OG) to the oncometabolite l-2-hydroxyglutarate (l-2-HG). l-2-HG dehydrogenase corrects this error by converting l-2-HG to 2-OG. LDH also catalyzes the reduction of the oxo group of 2-oxoglutaramate (2-OGM; transamination product of l-glutamine). We show here that human glutamine synthetase (GS) catalyzes the amidation of the terminal carboxyl of both the l- and d- isomers of 2-HG. The reaction of 2-OGM with LDH and the reaction of l-2-HG with GS generate l-2-hydroxyglutaramate (l-2-HGM). We also show that l-2-HGM is a substrate of human ω-amidase. The product (l-2-HG) can then be converted to 2-OG by l-2-HG dehydrogenase. Previous work showed that 2-oxosuccinamate (2-OSM; transamination product of l-asparagine) is an excellent substrate of LDH. Finally, we also show that human ω-amidase converts the product of this reaction (i.e., l-2-hydroxysuccinamate; l-2-HSM) to l-malate. Thus, ω-amidase may act together with hydroxyglutarate dehydrogenases to repair certain "mistakes" of GS and LDH. The present findings suggest that non-productive pathways for nitrogen metabolism occur in mammalian tissues in vivo. Perturbations of these pathways may contribute to symptoms associated with hydroxyglutaric acidurias and to tumor progression. Finally, methods for the synthesis of l-2-HGM and l-2-HSM are described that should be useful in determining the roles of ω-amidase/4- and 5-C compounds in photorespiration in plants.

Cell wall amidase AmiC1 is required for cellular communication and heterocyst development in the cyanobacterium Anabaena PCC 7120 but not for filament integrity.

PubMed

Berendt, Susanne; Lehner, Josef; Zhang, Yao Vincent; Rasse, Tobias M; Forchhammer, Karl; Maldener, Iris

2012-10-01

Filamentous cyanobacteria of the order Nostocales display typical properties of multicellular organisms. In response to nitrogen starvation, some vegetative cells differentiate into heterocysts, where fixation of N(2) takes place. Heterocysts provide a micro-oxic compartment to protect nitrogenase from the oxygen produced by the vegetative cells. Differentiation involves fundamental remodeling of the gram-negative cell wall by deposition of a thick envelope and by formation of a neck-like structure at the contact site to the vegetative cells. Cell wall-hydrolyzing enzymes, like cell wall amidases, are involved in peptidoglycan maturation and turnover in unicellular bacteria. Recently, we showed that mutation of the amidase homologue amiC2 gene in Nostoc punctiforme ATCC 29133 distorts filament morphology and function. Here, we present the functional characterization of two amiC paralogues from Anabaena sp. strain PCC 7120. The amiC1 (alr0092) mutant was not able to differentiate heterocysts or to grow diazotrophically, whereas the amiC2 (alr0093) mutant did not show an altered phenotype under standard growth conditions. In agreement, fluorescence recovery after photobleaching (FRAP) studies showed a lack of cell-cell communication only in the AmiC1 mutant. Green fluorescent protein (GFP)-tagged AmiC1 was able to complement the mutant phenotype to wild-type properties. The protein localized in the septal regions of newly dividing cells and at the neck region of differentiating heterocysts. Upon nitrogen step-down, no mature heterocysts were developed in spite of ongoing heterocyst-specific gene expression. These results show the dependence of heterocyst development on amidase function and highlight a pivotal but so far underestimated cellular process, the remodeling of peptidoglycan, for the biology of filamentous cyanobacteria.

Crystal structure analysis of a bacterial aryl acylamidase belonging to the amidase signature enzyme family

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

Lee, Saeyoung; Park, Eun-Hye; Ko, Hyeok-Jin

2015-11-13

The atomic structure of a bacterial aryl acylamidase (EC 3.5.1.13; AAA) is reported and structural features are investigated to better understand the catalytic profile of this enzyme. Structures of AAA were determined in its native form and in complex with the analgesic acetanilide, p-acetaminophenol, at 1.70 Å and 1.73 Å resolutions, respectively. The overall structural fold of AAA was identified as an α/β fold class, exhibiting an open twisted β-sheet core surrounded by α-helices. The asymmetric unit contains one AAA molecule and the monomeric form is functionally active. The core structure enclosing the signature sequence region, including the canonical Ser-cisSer-Lys catalytic triad,more » is conserved in all members of the Amidase Signature enzyme family. The structure of AAA in a complex with its ligand reveals a unique organization in the substrate-binding pocket. The binding pocket consists of two loops (loop1 and loop2) in the amidase signature sequence and one helix (α10) in the non-amidase signature sequence. We identified two residues (Tyr{sup 136} and Thr{sup 330}) that interact with the ligand via water molecules, and a hydrogen-bonding network that explains the catalytic affinity over various aryl acyl compounds. The optimum activity of AAA at pH > 10 suggests that the reaction mechanism employs Lys{sup 84} as the catalytic base to polarize the Ser{sup 187} nucleophile in the catalytic triad. - Highlights: • We determined the first structure of a bacterial aryl acylamidase (EC 3.5.1.13). • Structure revealed spatially distinct architecture of the substrate-binding pocket. • Hydrogen-bonding with Tyr{sup 136} and Thr{sup 330} mediates ligand-binding and substrate.« less

Characterization of a Thermostable d-Stereospecific Alanine Amidase from Brevibacillus borstelensis BCS-1

PubMed Central

Baek, Dae Heoun; Kwon, Seok-Joon; Hong, Seung-Pyo; Kwak, Mi-Sun; Lee, Mi-Hwa; Song, Jae Jun; Lee, Seung-Goo; Yoon, Ki-Hong; Sung, Moon-Hee

2003-01-01

A gene encoding a new thermostable d-stereospecific alanine amidase from the thermophile Brevibacillus borstelensis BCS-1 was cloned and sequenced. The molecular mass of the purified enzyme was estimated to be 199 kDa after gel filtration chromatography and about 30 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that the enzyme could be composed of a hexamer with identical subunits. The purified enzyme exhibited strong amidase activity towards d-amino acid-containing aromatic, aliphatic, and branched amino acid amides yet exhibited no enzyme activity towards l-amino acid amides, d-amino acid-containing peptides, and NH2-terminally protected amino acid amides. The optimum temperature and pH for the enzyme activity were 85°C and 9.0, respectively. The enzyme remained stable within a broad pH range from 7.0 to 10.0. The enzyme was inhibited by dithiothreitol, 2-mercaptoethanol, and EDTA yet was strongly activated by Co2+ and Mn2+. The kcat/Km for d-alaninamide was measured as 544.4 ± 5.5 mM−1 min−1 at 50°C with 1 mM Co2+. PMID:12571020

New generation of amino coumarin methyl sulfonate-based fluorogenic substrates for amidase assays in droplet-based microfluidic applications.

PubMed

Woronoff, Gabrielle; El Harrak, Abdeslam; Mayot, Estelle; Schicke, Olivier; Miller, Oliver J; Soumillion, Patrice; Griffiths, Andrew D; Ryckelynck, Michael

2011-04-15

Droplet-based microfluidics is a powerful tool for biology and chemistry as it allows the production and the manipulation of picoliter-size droplets acting as individual reactors. In this format, high-sensitivity assays are typically based on fluorescence, so fluorophore exchange between droplets must be avoided. Fluorogenic substrates based on the coumarin leaving group are widely used to measure a variety of enzymatic activities, but their application in droplet-based microfluidic systems is severely impaired by the fast transport of the fluorescent product between compartments. Here we report the synthesis of new amidase fluorogenic substrates based on 7-aminocoumarin-4-methanesulfonic acid (ACMS), a highly water-soluble dye, and their suitability for droplet-based microfluidics applications. Both substrate and product had the required spectral characteristics and remained confined in droplets from hours to days. As a model experiment, a phenylacetylated ACMS was synthesized and used as a fluorogenic substrate of Escherichia coli penicillin G acylase. Kinetic parameters (k(cat) and K(M)) measured in bulk and in droplets on-chip were very similar, demonstrating the suitability of this synthesis strategy to produce a variety of ACMS-based substrates for assaying amidase activities both in microtiter plate and droplet-based microfluidic formats. © 2011 American Chemical Society

A new acylamidase from Rhodococcus erythropolis TA37 can hydrolyze N-substituted amides.

PubMed

Lavrov, K V; Zalunin, I A; Kotlova, E K; Yanenko, A S

2010-08-01

A new acylamidase was isolated from Rhodococcus erythropolis TA37 and characterized. N-Substituted acrylamides (isopropyl acrylamide, N,N-dimethyl-aminopropyl acrylamide, and methylene-bis-acrylamide), acid para-nitroanilides (4'-nitroacetanilide, Gly-pNA, Ala-pNA, Leu-pNA), and N-acetyl derivatives of glycine, alanine, and leucine are good substrates for this enzyme. Aliphatic amides (acetamide, acrylamide, isobutyramide, n-butyramide, and valeramide) are also used as substrates but with less efficiency. The enzyme subunit mass by SDS-PAGE is 55 kDa. Maximal activity is exhibited at pH 7-8 and 55°C. The enzyme is stable for 15 h at 22°C and for 0.5 h at 45°C. The Michaelis constant (K(m)) is 0.25 mM with Gly-pNA and 0.55 mM with Ala-pNA. The acylamidase activity is suppressed by inhibitors of serine proteases (phenylmethylsulfonyl fluoride and diisopropyl fluorophosphate) but is not suppressed by inhibitors of aliphatic amidases (acetaldehyde and nitrophenyl disulfides). The N-terminal amino acid sequence of the acylamidase is highly homologous to those of two putative amidases detected from sequenced R. erythropolis genomes. It is suggested that the acylamidase together with the detected homologs forms a new class within the amidase signature family.

Role of Two Cell Wall Amidases in Septal Junction and Nanopore Formation in the Multicellular Cyanobacterium Anabaena sp. PCC 7120

PubMed Central

Bornikoel, Jan; Carrión, Alejandro; Fan, Qing; Flores, Enrique; Forchhammer, Karl; Mariscal, Vicente; Mullineaux, Conrad W.; Perez, Rebeca; Silber, Nadine; Wolk, C. Peter; Maldener, Iris

2017-01-01

Filamentous cyanobacteria have developed a strategy to perform incompatible processes in one filament by differentiating specialized cell types, N2-fixing heterocysts and CO2-fixing, photosynthetic, vegetative cells. These bacteria can be considered true multicellular organisms with cells exchanging metabolites and signaling molecules via septal junctions, involving the SepJ and FraCD proteins. Previously, it was shown that the cell wall lytic N-acetylmuramyl-L-alanine amidase, AmiC2, is essential for cell–cell communication in Nostoc punctiforme. This enzyme perforates the septal peptidoglycan creating an array of nanopores, which may be the framework for septal junction complexes. In Anabaena sp. PCC 7120, two homologs of AmiC2, encoded by amiC1 and amiC2, were identified and investigated in two different studies. Here, we compare the function of both AmiC proteins by characterizing different Anabaena amiC mutants, which was not possible in N. punctiforme, because there the amiC1 gene could not be inactivated. This study shows the different impact of each protein on nanopore array formation, the process of cell–cell communication, septal protein localization, and heterocyst differentiation. Inactivation of either amidase resulted in significant reduction in nanopore count and in the rate of fluorescent tracer exchange between neighboring cells measured by FRAP analysis. In an amiC1 amiC2 double mutant, filament morphology was affected and heterocyst differentiation was abolished. Furthermore, the inactivation of amiC1 influenced SepJ localization and prevented the filament-fragmentation phenotype that is characteristic of sepJ or fraC fraD mutants. Our findings suggest that both amidases are to some extent redundant in their function, and describe a functional relationship of AmiC1 and septal proteins SepJ and FraCD. PMID:28929086

Role of Two Cell Wall Amidases in Septal Junction and Nanopore Formation in the Multicellular Cyanobacterium Anabaena sp. PCC 7120.

PubMed

Bornikoel, Jan; Carrión, Alejandro; Fan, Qing; Flores, Enrique; Forchhammer, Karl; Mariscal, Vicente; Mullineaux, Conrad W; Perez, Rebeca; Silber, Nadine; Wolk, C Peter; Maldener, Iris

2017-01-01

Filamentous cyanobacteria have developed a strategy to perform incompatible processes in one filament by differentiating specialized cell types, N 2 -fixing heterocysts and CO 2 -fixing, photosynthetic, vegetative cells. These bacteria can be considered true multicellular organisms with cells exchanging metabolites and signaling molecules via septal junctions, involving the SepJ and FraCD proteins. Previously, it was shown that the cell wall lytic N -acetylmuramyl-L-alanine amidase, AmiC2, is essential for cell-cell communication in Nostoc punctiforme . This enzyme perforates the septal peptidoglycan creating an array of nanopores, which may be the framework for septal junction complexes. In Anabaena sp. PCC 7120, two homologs of AmiC2, encoded by amiC1 and amiC2 , were identified and investigated in two different studies. Here, we compare the function of both AmiC proteins by characterizing different Anabaena amiC mutants, which was not possible in N. punctiforme , because there the amiC1 gene could not be inactivated. This study shows the different impact of each protein on nanopore array formation, the process of cell-cell communication, septal protein localization, and heterocyst differentiation. Inactivation of either amidase resulted in significant reduction in nanopore count and in the rate of fluorescent tracer exchange between neighboring cells measured by FRAP analysis. In an amiC1 amiC2 double mutant, filament morphology was affected and heterocyst differentiation was abolished. Furthermore, the inactivation of amiC1 influenced SepJ localization and prevented the filament-fragmentation phenotype that is characteristic of sepJ or fraC fraD mutants. Our findings suggest that both amidases are to some extent redundant in their function, and describe a functional relationship of AmiC1 and septal proteins SepJ and FraCD.

A peptidoglycan recognition protein from Sciaenops ocellatus is a zinc amidase and a bactericide with a substrate range limited to Gram-positive bacteria.

PubMed

Li, Mo-Fei; Zhang, Min; Wang, Chun-Lin; Sun, Li

2012-02-01

Peptidoglycan recognition proteins (PGRPs) are a family of innate immune molecules that recognize bacterial peptidoglycan. PGRPs are highly conserved in invertebrates and vertebrates including fish. However, the biological function of teleost PGRP remains largely uninvestigated. In this study, we identified a PGRP homologue, SoPGLYRP-2, from red drum (Sciaenops ocellatus) and analyzed its activity and potential function. The deduced amino acid sequence of SoPGLYRP-2 is composed of 482 residues and shares 46-94% overall identities with known fish PGRPs. SoPGLYRP-2 contains at the C-terminus a single zinc amidase domain with conserved residues that form the catalytic site. Quantitative RT-PCR analysis detected SoPGLYRP-2 expression in multiple tissues, with the highest expression occurring in liver and the lowest expression occurring in brain. Experimental bacterial infection upregulated SoPGLYRP-2 expression in kidney, spleen, and liver in time-dependent manners. To examine the biological activity of SoPGLYRP-2, purified recombinant proteins representing the intact SoPGLYRP-2 (rSoPGLYRP-2) and the amidase domain (rSoPGLYRP-AD) were prepared from Escherichia coli. Subsequent analysis showed that rSoPGLYRP-2 and rSoPGLYRP-AD (i) exhibited comparable Zn(2+)-dependent peptidoglycan-lytic activity and were able to recognize and bind to live bacterial cells, (ii) possessed bactericidal effect against Gram-positive bacteria and slight bacteriostatic effect against Gram-negative bacteria, (iii) were able to block bacterial infection into host cells. These results indicate that SoPGLYRP-2 is a zinc-dependent amidase and a bactericide that targets preferentially at Gram-positive bacteria, and that SoPGLYRP-2 is likely to play a role in host innate immune defense during bacterial infection. Copyright © 2011 Elsevier Ltd. All rights reserved.

Structure-Function Analysis of Staphylococcus aureus Amidase Reveals the Determinants of Peptidoglycan Recognition and Cleavage*

PubMed Central

Büttner, Felix Michael; Zoll, Sebastian; Nega, Mulugeta; Götz, Friedrich; Stehle, Thilo

2014-01-01

The bifunctional major autolysin AtlA of Staphylococcus aureus cleaves the bacterium's peptidoglycan network (PGN) at two distinct sites during cell division. Deletion of the enzyme results in large cell clusters with disordered division patterns, indicating that AtlA could be a promising target for the development of new antibiotics. One of the two functions of AtlA is performed by the N-acetylmuramyl-l-alanine amidase AmiA, which cleaves the bond between the carbohydrate and the peptide moieties of PGN. To establish the structural requirements of PGN recognition and the enzymatic mechanism of cleavage, we solved the crystal structure of the catalytic domain of AmiA (AmiA-cat) in complex with a peptidoglycan-derived ligand at 1.55 Å resolution. The peptide stem is clearly visible in the structure, forming extensive contacts with protein residues by docking into an elongated groove. Less well defined electron density and the analysis of surface features indicate likely positions of the carbohydrate backbone and the pentaglycine bridge. Substrate specificity analysis supports the importance of the pentaglycine bridge for fitting into the binding cleft of AmiA-cat. PGN of S. aureus with l-lysine tethered with d-alanine via a pentaglycine bridge is completely hydrolyzed, whereas PGN of Bacillus subtilis with meso-diaminopimelic acid directly tethered with d-alanine is not hydrolyzed. An active site mutant, H370A, of AmiA-cat was completely inactive, providing further support for the proposed catalytic mechanism of AmiA. The structure reported here is not only the first of any bacterial amidase in which both the PGN component and the water molecule that carries out the nucleophilic attack on the carbonyl carbon of the scissile bond are present; it is also the first peptidoglycan amidase complex structure of an important human pathogen. PMID:24599952

A breakthrough in enzyme technology to fight penicillin resistance-industrial application of penicillin amidase.

PubMed

Buchholz, Klaus

2016-05-01

Enzymatic penicillin hydrolysis by penicillin amidase (also penicillin acylase, PA) represents a Landmark: the first industrially and economically highly important process using an immobilized biocatalyst. Resistance of infective bacteria to antibiotics had become a major topic of research and industrial activities. Solutions to this problem, the antibiotics resistance of infective microorganisms, required the search for new antibiotics, but also the development of derivatives, notably penicillin derivatives, that overcame resistance. An obvious route was to hydrolyse penicillin to 6-aminopenicillanic acid (6-APA), as a first step, for the introduction via chemical synthesis of various different side chains. Hydrolysis via chemical reaction sequences was tedious requiring large amounts of toxic chemicals, and they were cost intensive. Enzymatic hydrolysis using penicillin amidase represented a much more elegant route. The basis for such a solution was the development of techniques for enzyme immobilization, a highly difficult task with respect to industrial application. Two pioneer groups started to develop solutions to this problem in the late 1960s and 1970s: that of Günter Schmidt-Kastner at Bayer AG (Germany) and that of Malcolm Lilly of Imperial College London. Here, one example of this development, that at Bayer, will be presented in more detail since it illustrates well the achievement of a solution to the problems of industrial application of enzymatic processes, notably development of an immobilization method for penicillin amidase suitable for scale up to application in industrial reactors under economic conditions. A range of bottlenecks and technical problems of large-scale application had to be overcome. Data giving an inside view of this pioneer achievement in the early phase of the new field of biocatalysis are presented. The development finally resulted in a highly innovative and commercially important enzymatic process to produce 6-APA that created a new antibiotics industry and that opened the way for the establishment of over 100 industrial processes with immobilized biocatalysts worldwide today.

Structure-function analysis of Staphylococcus aureus amidase reveals the determinants of peptidoglycan recognition and cleavage.

PubMed

Büttner, Felix Michael; Zoll, Sebastian; Nega, Mulugeta; Götz, Friedrich; Stehle, Thilo

2014-04-18

The bifunctional major autolysin AtlA of Staphylococcus aureus cleaves the bacterium's peptidoglycan network (PGN) at two distinct sites during cell division. Deletion of the enzyme results in large cell clusters with disordered division patterns, indicating that AtlA could be a promising target for the development of new antibiotics. One of the two functions of AtlA is performed by the N-acetylmuramyl-l-alanine amidase AmiA, which cleaves the bond between the carbohydrate and the peptide moieties of PGN. To establish the structural requirements of PGN recognition and the enzymatic mechanism of cleavage, we solved the crystal structure of the catalytic domain of AmiA (AmiA-cat) in complex with a peptidoglycan-derived ligand at 1.55 Å resolution. The peptide stem is clearly visible in the structure, forming extensive contacts with protein residues by docking into an elongated groove. Less well defined electron density and the analysis of surface features indicate likely positions of the carbohydrate backbone and the pentaglycine bridge. Substrate specificity analysis supports the importance of the pentaglycine bridge for fitting into the binding cleft of AmiA-cat. PGN of S. aureus with l-lysine tethered with d-alanine via a pentaglycine bridge is completely hydrolyzed, whereas PGN of Bacillus subtilis with meso-diaminopimelic acid directly tethered with d-alanine is not hydrolyzed. An active site mutant, H370A, of AmiA-cat was completely inactive, providing further support for the proposed catalytic mechanism of AmiA. The structure reported here is not only the first of any bacterial amidase in which both the PGN component and the water molecule that carries out the nucleophilic attack on the carbonyl carbon of the scissile bond are present; it is also the first peptidoglycan amidase complex structure of an important human pathogen.

Shared strategies for β-lactam catabolism in the soil microbiome.

PubMed

Crofts, Terence S; Wang, Bin; Spivak, Aaron; Gianoulis, Tara A; Forsberg, Kevin J; Gibson, Molly K; Johnsky, Lauren A; Broomall, Stacey M; Rosenzweig, C Nicole; Skowronski, Evan W; Gibbons, Henry S; Sommer, Morten O A; Dantas, Gautam

2018-06-01

The soil microbiome can produce, resist, or degrade antibiotics and even catabolize them. While resistance genes are widely distributed in the soil, there is a dearth of knowledge concerning antibiotic catabolism. Here we describe a pathway for penicillin catabolism in four isolates. Genomic and transcriptomic sequencing revealed β-lactamase, amidase, and phenylacetic acid catabolon upregulation. Knocking out part of the phenylacetic acid catabolon or an apparent penicillin utilization operon (put) resulted in loss of penicillin catabolism in one isolate. A hydrolase from the put operon was found to degrade in vitro benzylpenicilloic acid, the β-lactamase penicillin product. To test the generality of this strategy, an Escherichia coli strain was engineered to co-express a β-lactamase and a penicillin amidase or the put operon, enabling it to grow using penicillin or benzylpenicilloic acid, respectively. Elucidation of additional pathways may allow bioremediation of antibiotic-contaminated soils and discovery of antibiotic-remodeling enzymes with industrial utility.

Second-Generation Non-Covalent NAAA Inhibitors are Protective in a Model of Multiple Sclerosis.

PubMed

Migliore, Marco; Pontis, Silvia; Fuentes de Arriba, Angel Luis; Realini, Natalia; Torrente, Esther; Armirotti, Andrea; Romeo, Elisa; Di Martino, Simona; Russo, Debora; Pizzirani, Daniela; Summa, Maria; Lanfranco, Massimiliano; Ottonello, Giuliana; Busquet, Perrine; Jung, Kwang-Mook; Garcia-Guzman, Miguel; Heim, Roger; Scarpelli, Rita; Piomelli, Daniele

2016-09-05

Palmitoylethanolamide (PEA) and oleoylethanolamide (OEA) are endogenous lipid mediators that suppress inflammation. Their actions are terminated by the intracellular cysteine amidase, N-acylethanolamine acid amidase (NAAA). Even though NAAA may offer a new target for anti-inflammatory therapy, the lipid-like structures and reactive warheads of current NAAA inhibitors limit the use of these agents as oral drugs. A series of novel benzothiazole-piperazine derivatives that inhibit NAAA in a potent and selective manner by a non-covalent mechanism are described. A prototype member of this class (8) displays high oral bioavailability, access to the central nervous system (CNS), and strong activity in a mouse model of multiple sclerosis (MS). This compound exemplifies a second generation of non-covalent NAAA inhibitors that may be useful in the treatment of MS and other chronic CNS disorders. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

[Cloning of new acylamidase gene from Rhodococcus erythropolis and its expression in Escherichia coli].

PubMed

Lavrov, K V; Ianenko, A S

2013-10-01

The gene for new Rhodococcus erythropolis TA37 acylamidase, which possesses unique substrate specificity, has been cloned and expressed in E. coli. Substrates for this enzyme are not only simple amides, such as acetamide and propionamide, but also N-substituted amides, such as 4'-nitroacetanilide. The 1431-bp gene was expressed in E. coli BL21 (DE3) cells on pET16b plasmid under the control of a promoter of the φ 10 gene from the T7 phage. The molecular mass of recombinant acylamidase in E. coli was 55 kDa, which corresponded to that of native acylamidase from Rhodococcus erythropolis TA37. Recombinant acylamidase was able to hydrolize N-substituted amides. A search of a nucleotide database and multiple alignment revealed that acylamidase belonged to the Amidase protein family PF01425, but its nucleotide and amino acid sequences differed significantly from those of the described amidases.

Hydrolysis of acetylthiocoline, o-nitroacetanilide and o-nitrotrifluoroacetanilide by fetal bovine serum acetylcholinesterase.

PubMed

Montenegro, María F; Moral-Naranjo, María T; Muñoz-Delgado, Encarnación; Campoy, Francisco J; Vidal, Cecilio J

2009-04-01

Besides esterase activity, acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) hydrolyze o-nitroacetanilides through aryl acylamidase activity. We have reported that BuChE tetramers and monomers of human blood plasma differ in o-nitroacetanilide (ONA) hydrolysis. The homology in quaternary structure and folding of subunits in the prevalent BuChE species (G4(H)) of human plasma and AChE forms of fetal bovine serum prompted us to study the esterase and amidase activities of fetal bovine serum AChE. The k(cat)/K(m) values for acetylthiocholine (ATCh), ONA and its trifluoro derivative N-(2-nitrophenyl)-trifluoroacetamide (F-ONA) were 398 x 10(6) M(-1) min(-1), 0.8 x 10(6) M(-1) min(-1), and 17.5 x 10(6) M(-1) min(-1), respectively. The lack of inhibition of amidase activity at high F-ONA concentrations makes it unlikely that there is a role for the peripheral anionic site (PAS) in F-ONA degradation, but the inhibition of ATCh, ONA and F-ONA hydrolysis by the PAS ligand fasciculin-2 points to the transit of o-nitroacetalinides near the PAS on their way to the active site. Sedimentation analysis confirmed substrate hydrolysis by tetrameric 10.9S AChE. As compared with esterase activity, amidase activity was less sensitive to guanidine hydrochloride. This reagent led to the formation of 9.3S tetramers with partially unfolded subunits. Their capacity to hydrolyze ATCh and F-ONA revealed that, despite the conformational change, the active site architecture and functionality of AChE were partially retained.

METABOLIC ENGINEERING TO DEVELOP A PATHWAY FOR THE SELECTIVE CLEAVAGE OF CARBON-NITROGEN BONDS

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

John J. Kilbane III

The objective of the project is to develop biochemical pathways for the selective cleavage of C-N bonds in molecules found in petroleum. The initial phase of the project will focus on the isolation or development of an enzyme capable of cleaving the C-N bond in aromatic amides, specifically 2-aminobiphenyl. The objective of the second phase of the research will be to construct a biochemical pathway for the selective removal of nitrogen from carbazole by combining the carA genes from Sphingomonas sp. GTIN11 with the gene(s) encoding an appropriate amidase. The objective of the final phase of the project will bemore » to develop derivative CN bond cleaving enzymes that have broader substrate ranges and to demonstrate the use of such strains to selectively remove nitrogen from petroleum. The project is on schedule and no major difficulties have been encountered. During the first year of the project (October, 2002-September, 2003) enrichment culture experiments have resulted in the isolation of promising cultures that may be capable of cleaving C-N bonds in aromatic amides, several amidase genes have been cloned and are currently undergoing directed evolution to obtain derivatives that can cleave C-N bonds in aromatic amides, and the carA genes from Sphingomonas sp. GTIN11, and Pseudomonas resinovorans CA10 were cloned in vectors capable of replicating in Escherichia coli. Future research will address expression of these genes in Rhodococcus erythropolis. Enrichment culture experiments and directed evolution experiments continue to be a main focus of research activity and further work is required to obtain an appropriate amidase that will selectively cleave C-N bonds in aromatic substrates. Once an appropriate amidase gene is obtained it must be combined with genes encoding an enzyme capable of converting carbazole to 2'aminobiphenyl-2,3-diol: specifically carA genes. The carA genes from two sources have been cloned and are ready for construction of C-N bond cleavage pathway. The construction of a new metabolic pathway to selectively remove nitrogen from carbazole and other molecules typically found in petroleum should lead to the development of a process to improve oil refinery efficiency by reducing the poisoning, by nitrogen, of catalysts used in the hydrotreating and catalytic cracking of petroleum.« less

Key role of amino acid residues in the dimerization and catalytic activation of the autolysin LytA, an important virulence factor in Streptococcus pneumoniae.

PubMed

Romero, Patricia; López, Rubens; García, Ernesto

2007-06-15

LytA, the main autolysin of Streptococcus pneumoniae, was the first member of the bacterial N-acetylmuramoyl-l-alanine amidase (NAM-amidase) family of proteins to be well characterized. This autolysin degrades the peptidoglycan bonds of pneumococcal cell walls after anchoring to the choline residues of the cell wall teichoic acids via its choline-binding module (ChBM). The latter is composed of seven repeats (ChBRs) of approximately 20 amino acid residues. The translation product of the lytA gene is the low-activity E-form of LytA (a monomer), which can be "converted" (activated) in vitro by choline into the fully active C-form at low temperature. The C-form is a homodimer with a boomerang-like shape. To study the structural requirements for the monomer-to-dimer modification and to clarify whether "conversion" is synonymous with dimerization, the biochemical consequences of replacing four key amino acid residues of ChBR6 and ChBR7 (the repeats involved in dimer formation) were determined. The results obtained with a collection of 21 mutated NAM-amidases indicate that Ile-315 is a key amino acid residue in both LytA activity and folding. Amino acids with a marginal position in the solenoid structure of the ChBM were of minor influence in dimer stability; neither the size, polarity, nor aromatic nature of the replacement amino acids affected LytA activity. In contrast, truncated proteins were drastically impaired in their activity and conversion capacity. The results indicate that dimerization and conversion are different processes, but they do not answer the questions of whether conversion can only be achieved after a dimer formation step.

Synthesis, biological evaluation, and 3D QSAR study of 2-methyl-4-oxo-3-oxetanylcarbamic acid esters as N-acylethanolamine acid amidase (NAAA) inhibitors.

PubMed

Ponzano, Stefano; Berteotti, Anna; Petracca, Rita; Vitale, Romina; Mengatto, Luisa; Bandiera, Tiziano; Cavalli, Andrea; Piomelli, Daniele; Bertozzi, Fabio; Bottegoni, Giovanni

2014-12-11

N-(2-Oxo-3-oxetanyl)carbamic acid esters have recently been reported to be noncompetitive inhibitors of the N-acylethanolamine acid amidase (NAAA) potentially useful for the treatment of pain and inflammation. In the present study, we further explored the structure-activity relationships of the carbamic acid ester side chain of 2-methyl-4-oxo-3-oxetanylcarbamic acid ester derivatives. Additional favorable features in the design of potent NAAA inhibitors have been found together with the identification of a single digit nanomolar inhibitor. In addition, we devised a 3D QSAR using the atomic property field method. The model turned out to be able to account for the structural variability and was prospectively validated by designing, synthesizing, and testing novel inhibitors. The fairly good agreement between predictions and experimental potency values points to this 3D QSAR model as the first example of quantitative structure-activity relationships in the field of NAAA inhibitors.

The level of aryl acylamidase activity displayed by human butyrylcholinesterase depends on its molecular distribution.

PubMed

Montenegro, M F; Moral-Naranjo, M T; Páez de la Cadena, M; Campoy, F J; Muñoz-Delgado, E; Vidal, C J

2008-09-25

Butyrylcholinesterase (BuChE) and acetylcholinesterase (AChE) display both esterase and aryl acylamidase (AAA) activities. Their AAA activity can be measured using o-nitroacetanilide (ONA). In human samples depleted of acetylcholinesterase, we noticed that the ratio of amidase to esterase activities varied depending on the source, despite both activities being due to BuChE. Searching for an explanation, we compared the activities of BuChE molecular forms in samples of human colon, kidney and serum, and observed that BuChE monomers (G(1)) hydrolyzed o-nitroacetanilide much faster than tetramers (G(4)). This fact suggested that association might cause differences in the AAA site between single and polymerized subunits. This and other post-translational modifications in BuChE subunits probably determine their level of AAA activity. The higher amidase activity of monomers could justify the presence of single BuChE subunits in cells as a way to preserve the AAA activity of BuChE, which could be lost by oligomerization.

LysK CHAP endopeptidase domain is required for lysis of live staphylococcal cells.

USDA-ARS?s Scientific Manuscript database

LysK is a staphylococcal bacteriophage endolysin composed of three domains, an N-terminal cysteine, histidine-dependent amidohydrolases/peptidases (CHAP) endopeptidase domain (cleaves between D-alanine of the stem peptide and glycine of the cross-bridge peptide) a mid-protein amidase 2 domain (N-ace...

Bacteriophages of the family siphoviridae contain amidase enzymes that lyse Clostridium perfringens

USDA-ARS?s Scientific Manuscript database

*Agtech-Danisco, current address In chickens Clostridium perfringens (Cp) is the etiologic agent of necrotic enteritis and causes gas gangrene along with being the third leading cause of bacterial food-borne gastroenteritis in humans. While the disease in poultry can be controlled by antibiotics, th...

BACTERIOPHAGES OF THE FAMILY SIPHOVIRIDAE CONTAIN AMIDASE ENZYMES THAT LYSE CLOSTRIDIUM PERFRINGENS

USDA-ARS?s Scientific Manuscript database

In chickens Clostridium perfringens (Cp) is the etiologic agent of necrotic enteritis and causes gas gangrene along with being the third leading cause of bacterial food-borne gastroenteritis in humans. While the disease in poultry can be controlled by antibiotics, there is increasing pressure to ban...

Recombinant expression of a putative prophage amidase cloned from the genome of Listeria monocytogenes that lyses the bacterium and its biofilm

USDA-ARS?s Scientific Manuscript database

Listeria monocytogenes is a Gram-positive, non-sporeforming, catalase-positive rod that is a major bacterial food-borne disease agent, causing listeriosis. Listeria can be associated with uncooked meats including poultry, uncooked vegetables, soft cheeses and unpasteurized milk. The bacterium can be...

Effect of mass transfer in a recirculation batch reactor system for immobilized penicillin amidase.

PubMed

Park, J M; Choi, C Y; Seong, B L; Han, M H

1982-10-01

The effect of external mass transfer resistance on the overall reaction rate of the immobilized whole cell penicillin amidase of E. coli in a recirculation batch reactor was investigated. The internal diffusional resistance was found negligible as indicated by the value of effectiveness factor, 0.95. The local environmental change in a column due to the pH drop was successfully overcome by employing buffer solution. The reaction rate was measured by pH-stat method and was found to follow the simple Michaelis-Menten law at the initial stage of the reaction. The values of the net reaction rate experimentally determined were used to calculate the substrate concentration at the external surface of the catalyst pellet and then to calculate the mass transfer coefficient, k(L), at various flow rates and substrate concentrations. The correlation proposed by Chilton and Colburn represented adequately the experimental data. The linear change of log j(D) at low log N(Re) with negative slope was ascribed to the fact that the external mass transfer approached the state of pure diffusion in the limit of zero superficial velocity.

N-Acetylanthranilate Amidase from Arthrobacter nitroguajacolicus Rü61a, an α/β-Hydrolase-Fold Protein Active towards Aryl-Acylamides and -Esters, and Properties of Its Cysteine-Deficient Variant▿ †

PubMed Central

Kolkenbrock, Stephan; Parschat, Katja; Beermann, Bernd; Hinz, Hans-Jürgen; Fetzner, Susanne

2006-01-01

N-acetylanthranilate amidase (Amq), a 32.8-kDa monomeric amide hydrolase, is involved in quinaldine degradation by Arthrobacter nitroguajacolicus Rü61a. Sequence analysis and secondary structure predictions indicated that Amq is related to carboxylesterases and belongs to the α/β-hydrolase-fold superfamily of enzymes; inactivation of (His6-tagged) Amq by phenylmethanesulfonyl fluoride and diethyl pyrocarbonate and replacement of conserved residues suggested a catalytic triad consisting of S155, E235, and H266. Amq is most active towards aryl-acetylamides and aryl-acetylesters. Remarkably, its preference for ring-substituted analogues was different for amides and esters. Among the esters tested, phenylacetate was hydrolyzed with highest catalytic efficiency (kcat/Km = 208 mM−1 s−1), while among the aryl-acetylamides, o-carboxy- or o-nitro-substituted analogues were preferred over p-substituted or unsubstituted compounds. Hydrolysis by His6Amq of primary amides, lactams, N-acetylated amino acids, azocoll, tributyrin, and the acylanilide and urethane pesticides propachlor, propham, carbaryl, and isocarb was not observed; propanil was hydrolyzed with 1% N-acetylanthranilate amidase activity. The catalytic properties of the cysteine-deficient variant His6AmqC22A/C63A markedly differed from those of His6Amq. The replacements effected some changes in Kms of the enzyme and increased kcats for most aryl-acetylesters and some aryl-acetylamides by factors of about three to eight while decreasing kcat for the formyl analogue N-formylanthranilate by several orders of magnitude. Circular dichroism studies indicated that the cysteine-to-alanine replacements resulted in significant change of the overall fold, especially an increase in α-helicity of the cysteine-deficient protein. The conformational changes may also affect the active site and may account for the observed changes in kinetic properties. PMID:17041061

Characterization of proacrosin/acrosin system after liquid storage and cryopreservation of turkey semen (Meleagris gallopavo).

PubMed

Słowińska, M; Liszewska, E; Dietrich, G J; Ciereszko, A

2012-09-15

This study was designed to identify the effect of liquid storage at 4 °C for 48 h and cryopreservation on the proacrosin/acrosin system of turkey spermatozoa. Anti-acrosin I antibodies were produced and used to demonstrate Western blot analysis profile of the proacrosin/acrosin system of sperm and seminal plasma and possible changes in the proacrosin/acrosin system of turkey sperm stored for 2.5, 24, and 48 h or cryopreserved. At the same time acrosin-like activity was examined by the measurement of amidase activity of sperm extracts, sperm suspension, and seminal plasma of turkey semen. A computer-assisted sperm analysis system was used to monitor the sperm motility characteristics of turkey sperm stored for 48 h or cryopreserved. Different profiles of the sperm proacrosin/acrosin system were observed regarding the presence or absence of inhibitors (p-nitrophenyl-p'-guanidine benzoate [NPGB] and Kazal family inhibitor) during the extraction process. When NPGB was present three main bands were observed with the molecular weight ranging from 66 to 35 kDa. Bands corresponding to acrosin I and II were not observed. In sperm extract without NPGB, three or four bands were observed with the molecular weight ranging from 41 to 30 kDa. The bands corresponding to acrosin I and II were observed. During liquid storage a decrease in sperm motility and an increase in sperm-extracted amidase activity were observed. After 24 and 48 h of storage, extracted amidase activity was higher than at 2.5 h by 24% and 31%, respectively. However, no changes in the Western blot analysis profiles of sperm extract and seminal plasma were visible during liquid storage. After cryopreservation a decrease in sperm motility and all sperm motility parameters were observed. In contrast to liquid storage, cryopreservation did not increase extracted amidase activity. However, changes in Western blot analysis profiles were visible in sperm extract and seminal plasma after cryopreservation. After freezing-thawing, additional bands appeared in sperm extract and seminal plasma. These bands were of different molecular weight regarding the presence or absence of NPGB. These data suggest that the mechanism of damage to the proacrosin/acrosin system is different for liquid storage and cryopreservation. Liquid storage seems to increase in the susceptibility of the proacrosin/acrosin system to be activated during extraction. Kazal inhibitors of turkey seminal plasma are involved in the control of proacrosin activation. The disturbances of the proacrosin/acrosin system of turkey spermatozoa can be related to a disturbance in the induction of the acrosome reaction. Our results may be important for a better understanding of the proacrosin/acrosin system of turkey spermatozoa and disturbance to this system during liquid storage and cryopreservation. Copyright © 2012 Elsevier Inc. All rights reserved.

Expression of a Clostridium perfringens genome-encoded putative N-acetylmuramoyl-L-alanine amidase as a potential antimicrobial to control the bacterium

USDA-ARS?s Scientific Manuscript database

Clostridium perfringens is a Gram-positive, spore-forming anaerobic bacterium that plays a substantial role in non-foodborne human, animal and avian diseases as well as human foodborne disease. Previously discovered C. perfringens bacteriophage lytic enzyme amino acid sequences were utilized to iden...

A chimeric LysK-lysostaphin fusion enzyme lysing Staphylococcus aureus cells: a study of both kinetics of inactivation and specifics of interaction with anionic polymers

USDA-ARS?s Scientific Manuscript database

A staphylolytic fusion protein (K-L) was created, harboring three unique lytic activities comprised of the LysK CHAP endopeptidase, and amidase domains, and the lysostaphin glycyl-glycine endopeptidase domain. To assess the potential of possible therapeutic applications, the kinetic behavior of K-L...

Recombinant Expression of a Genome-encoded N-acetylmuramoyl-L-alanine Amidase that Synergistically Lyses Listeria monocytogenes Biofilms with a Protease

USDA-ARS?s Scientific Manuscript database

Listeria monocytogenes plays a significant role in human food-borne disease caused by eating food contaminated with the bacterium and although incidence is low it is a leading cause of life-threatening, bacterial food-borne disease in humans. L. monocytogenes serotypes 1/2a and 4b can form mixed-cu...

Activity Augmentation of Amphioxus Peptidoglycan Recognition Protein BbtPGRP3 via Fusion with a Chitin Binding Domain

PubMed Central

Wang, Wen-Jie; Cheng, Wang; Luo, Ming; Yan, Qingyu; Yu, Hong-Mei; Li, Qiong; Cao, Dong-Dong; Huang, Shengfeng; Xu, Anlong; Mariuzza, Roy A.; Chen, Yuxing; Zhou, Cong-Zhao

2015-01-01

Peptidoglycan recognition proteins (PGRPs), which have been identified in most animals, are pattern recognition molecules that involve antimicrobial defense. Resulting from extraordinary expansion of innate immune genes, the amphioxus encodes many PGRPs of diverse functions. For instance, three isoforms of PGRP encoded by Branchiostoma belcheri tsingtauense, termed BbtPGRP1~3, are fused with a chitin binding domain (CBD) at the N-terminus. Here we report the 2.7 Å crystal structure of BbtPGRP3, revealing an overall structure of an N-terminal hevein-like CBD followed by a catalytic PGRP domain. Activity assays combined with site-directed mutagenesis indicated that the individual PGRP domain exhibits amidase activity towards both DAP-type and Lys-type peptidoglycans (PGNs), the former of which is favored. The N-terminal CBD not only has the chitin-binding activity, but also enables BbtPGRP3 to gain a five-fold increase of amidase activity towards the Lys-type PGNs, leading to a significantly broadened substrate spectrum. Together, we propose that modular evolution via domain shuffling combined with gene horizontal transfer makes BbtPGRP1~3 novel PGRPs of augmented catalytic activity and broad recognition spectrum. PMID:26479246

Characterization of Enterococcus faecium bacteriophage IME-EFm5 and its endolysin LysEFm5.

PubMed

Gong, Pengjuan; Cheng, Mengjun; Li, Xinwei; Jiang, Haiyan; Yu, Chuang; Kahaer, Nadire; Li, Juecheng; Zhang, Lei; Xia, Feifei; Hu, Liyuan; Sun, Changjiang; Feng, Xin; Lei, Liancheng; Han, Wenyu; Gu, Jingmin

2016-05-01

Due to the worldwide prevalence of antibiotic resistant strains, phages therapy has been revitalized recently. In this study, an Enterococcus faecium phage named IME-EFm5 was isolated from hospital sewage. Whole genomic sequence analysis demonstrated that IME-EFm5 belong to the Siphoviridae family, and has a double-stranded genome of 42,265bp (with a 35.51% G+C content) which contains 70 putative coding sequences. LysEFm5, the endolysin of IME-EFm5, contains an amidase domain in its N-terminal and has a wider bactericidal spectrum than its parental phage IME-EFm5, including 7 strains of vancomycin-resistant E. faecium. The mutagenesis analysis revealed that the zinc ion binding residues (H27, H132, and C140), E90, and T138 are required for the catalysis of LysEFm5. However, the antibacterial activity of LysEFm5 is zinc ion independent, which is inconsistent with most of other amidase members. The phage lysin LysEFm5 might be an alternative treatment strategy for infections caused by multidrug-resistant E. faecium. Copyright © 2016 Elsevier Inc. All rights reserved.

Enzyme immobilization techniques on poly(glycidyl methacrylate-co-ethylene dimethacrylate) carrier with penicillin amidase as model.

PubMed

Drobník, J; Saudek, V; Svec, F; Kálal, J; Vojtísek, V; Bárta, M

1979-08-01

Two types of bead-form macroporous carriers based on glycidyl methacrylate with ethylene dimethacrylate copolymers were used for the immobilization of penicillin amidase either directly or after chemical modification. Direct binding through oxirane groups, which is equally efficient at pH 4.2 and 7, is relatively slow and brings about an activity loss at low enzyme concentrations. The most efficient immobilization was achieved on glutaraldehyde-activated amino carrier, irrespective of whether the amino groups were formed by ammonia or 1,6-diaminohexane treatment of the original oxirane carrier. Hydrazine treatment gave lower immobilization yields. The same is true of the azide method independent of the length of the spacer. Most enzyme activity was preserved by coupling the carbodiimide-activated enzyme to the carrier with alkyl or arylamino groups at the end of a longer substituent. Immobilization on diazo-modified carrier gave average results. Rapid immobilization by a lysine-modified phosgene-treated carrier resulted in an activity loss. It is suggested that multipoint and very tight attachment of the enzyme molecule to the matrix decreased the activity. The immobilized activity is quite stable in solution and very stable upon lyophilization with sucrose.

Ligand-Binding Properties and Conformational Dynamics of Autolysin Repeat Domains in Staphylococcal Cell Wall Recognition

PubMed Central

Zoll, Sebastian; Schlag, Martin; Shkumatov, Alexander V.; Rautenberg, Maren; Svergun, Dmitri I.; Götz, Friedrich

2012-01-01

The bifunctional major autolysin Atl plays a key role in staphylococcal cell separation. Processing of Atl yields catalytically active amidase (AM) and glucosaminidase (GL) domains that are each fused to repeating units. The two repeats of AM (R1 and R2) target the enzyme to the septum, where it cleaves murein between dividing cells. We have determined the crystal structure of R2, which reveals that each repeat folds into two half-open β-barrel subunits. We further demonstrate that lipoteichoic acid serves as a receptor for the repeats and that this interaction depends on conserved surfaces in each subunit. Small-angle X-ray scattering of the mature amidase reveals the presence of flexible linkers separating the AM, R1, and R2 units. Different levels of flexibility for each linker provide mechanistic insights into the conformational dynamics of the full-length protein and the roles of its components in cell wall association and catalysis. Our analysis supports a model in which the repeats direct the catalytic AM domain to the septum, where it can optimally perform the final step of cell division. PMID:22609916

Molecular and functional characterization of peptidoglycan-recognition protein SC2 (PGRP-SC2) from Nile tilapia (Oreochromis niloticus) involved in the immune response to Streptococcus agalactiae.

PubMed

Gan, Zhen; Chen, Shannan; Hou, Jing; Huo, Huijun; Zhang, Xiaolin; Ruan, Baiye; Laghari, Zubair Ahmed; Li, Li; Lu, Yishan; Nie, Pin

2016-07-01

PGRP-SC2, the member of PGRP family, plays an important role in regulation of innate immune response. In this paper, a PGRP-SC2 gene of Nile tilapia, Oreochromis niloticus (designated as On-PGRP-SC2) was cloned and its expression pattern under the infection of Streptococcus agalactiae was investigated. Sequence analysis showed main structural features required for amidase activity were detected in the deduced amino acid sequence of On-PGRP-SC2. In healthy tilapia, the On-PGRP-SC2 transcripts could be detected in all the examined tissues, with the most abundant expression in the muscle. When infected with S. agalactiae, there was a clear time-dependent expression pattern of On-PGRP-SC2 in the spleen, head kidney and brain. The assays for the amidase activity suggested that recombinant On-PGRP-SC2 protein had a Zn(2+)-dependent PGN-degrading activity. Moreover, our works showed that recombinant On-PGRP-SC2 protein could significantly reduce bacterial load in target organs attacked by S. agalactiae. These findings indicated that On-PGRP-SC2 may play important roles in the immune response to S. agalactiae in Nile tilapia. Copyright © 2016 Elsevier Ltd. All rights reserved.

Molecular characterization of a novel bacterial aryl acylamidase belonging to the amidase signature enzyme family.

PubMed

Ko, Hyeok-Jin; Lee, Eun Woo; Bang, Won-Gi; Lee, Cheol-Koo; Kim, Kyoung Heon; Choi, In-Geol

2010-05-01

In seeking aryl acylamidase (EC 3.5.1.13) acting on an amide bond in p-acetaminophenol (Tylenol), we identified a novel gene encoding 496 residues of a protein. The gene revealed a conserved amidase signature region with a canonical catalytic triad. The gene was expressed in E. coli and characterized for its biochemical properties. The optimum pH and temperature for the activity on p-acetaminophenol were 10 and 37 degrees C, respectively. The half-life of enzyme activity at 37 degrees C was 192 h and 90% of its activity remained after 3 h incubation at 40 degrees C. Divalent metals was found to inhibit the activity of enzyme. The K (m) values for various aryl acylamides such as 4-nitroacetanilide, p-acetaminophenol, phenacetin, 4-chloroacetanilide and acetanilide were 0.10, 0.32, 0.83, 1.9 and 19 mM, respectively. The reverse reaction activity (amide synthesis) was also examined using various chain lengths (C(1) approximately C(4) and C(10)) of carboxylic donors and aniline as substrates. These kinetic parameters and substrate specificity in forward and reverse reaction indicated that the aryl acylamidase in this study has a preference for aryl substrate having polar functional groups and hydrophobic carboxylic donors.

Thermophilic archaeal enzymes and applications in biocatalysis.

PubMed

Littlechild, Jennifer A

2011-01-01

Thermophilic enzymes have advantages for their use in commercial applications and particularly for the production of chiral compounds to produce optically pure pharmaceuticals. They can be used as biocatalysts in the application of 'green chemistry'. The thermophilic archaea contain enzymes that have already been used in commercial applications such as the L-aminoacylase from Thermococcus litoralis for the resolution of amino acids and amino acid analogues. This enzyme differs from bacterial L-aminoacylases and has similarities to carboxypeptidases from other archaeal species. An amidase/γ-lactamase from Sulfolobus solfataricus has been used for the production of optically pure γ-lactam, the building block for antiviral carbocyclic nucleotides. This enzyme has similarities to the bacterial signature amidase family. An alcohol dehydrogenase from Aeropyrum pernix has been used for the production of optically pure alcohols and is related to the zinc-containing eukaryotic alcohol dehydrogenases. A transaminase and a dehalogenase from Sulfolobus species have also been studied. The archaeal transaminase is found in a pathway for serine synthesis which is found only in eukaryotes and not in bacteria. It can be used for the asymmetric synthesis of homochiral amines of high enantioselective purity. The L-2-haloacid dehalogenase has applications both in biocatalysis and in bioremediation. All of these enzymes have increased thermostability over their mesophilic counterparts.

Characterization of an Indole-3-Acetamide Hydrolase from Alcaligenes faecalis subsp. parafaecalis and Its Application in Efficient Preparation of Both Enantiomers of Chiral Building Block 2,3-Dihydro-1,4-Benzodioxin-2-Carboxylic Acid.

PubMed

Mishra, Pradeep; Kaur, Suneet; Sharma, Amar Nath; Jolly, Ravinder S

2016-01-01

Both the enantiomers of 2,3-dihydro-1,4-benzodioxin-2-carboxylic acid are valuable chiral synthons for enantiospecific synthesis of therapeutic agents such as (S)-doxazosin mesylate, WB 4101, MKC 242, 2,3-dihydro-2-hydroxymethyl-1,4-benzodioxin, and N-[2,4-oxo-1,3-thiazolidin-3-yl]-2,3-dihydro-1,4-benzodioxin-2-carboxamide. Pharmaceutical applications require these enantiomers in optically pure form. However, currently available methods suffer from one drawback or other, such as low efficiency, uncommon and not so easily accessible chiral resolving agent and less than optimal enantiomeric purity. Our interest in finding a biocatalyst for efficient production of enantiomerically pure 2,3-dihydro-1,4-benzodioxin-2-carboxylic acid lead us to discover an amidase activity from Alcaligenes faecalis subsp. parafaecalis, which was able to kinetically resolve 2,3-dihydro-1,4-benzodioxin-2-carboxyamide with E value of >200. Thus, at about 50% conversion, (R)-2,3-dihydro-1,4-benzodioxin-2-carboxylic acid was produced in >99% e.e. The remaining amide had (S)-configuration and 99% e.e. The amide and acid were easily separated by aqueous (alkaline)-organic two phase extraction method. The same amidase was able to catalyse, albeit at much lower rate the hydrolysis of (S)-amide to (S)-acid without loss of e.e. The amidase activity was identified as indole-3-acetamide hydrolase (IaaH). IaaH is known to catalyse conversion of indole-3-acetamide (IAM) to indole-3-acetic acid (IAA), which is phytohormone of auxin class and is widespread among plants and bacteria that inhabit plant rhizosphere. IaaH exhibited high activity for 2,3-dihydro-1,4-benzodioxin-2-carboxamide, which was about 65% compared to its natural substrate, indole-3-acetamide. The natural substrate for IaaH indole-3-acetamide shared, at least in part a similar bicyclic structure with 2,3-dihydro-1,4-benzodioxin-2-carboxamide, which may account for high activity of enzyme towards this un-natural substrate. To the best of our knowledge this is the first application of IaaH in production of industrially important molecules.

Deduced catalytic mechanism of d-amino acid amidase from Ochrobactrum anthropi SV3

PubMed Central

Okazaki, Seiji; Suzuki, Atsuo; Komeda, Hidenobu; Asano, Yasuhisa; Yamane, Takashi

2008-01-01

d-Amino acid amidase (DAA) from Ochrobactrum anthropi SV3 catalyzes d-stereospecific hydrolysis of amino acid amides. DAA has attracted attention as a catalyst for the stereospecific production of d-amino acids, although the mechanism that drives the reaction has not been clear. Previously, the structure of DAA was classified into two types, a substrate-bound state with an ordered Ω loop, and a ground state with a disordered Ω loop. Because the binding of the substrate facilitates ordering, this transition was regarded to be induced fit motion. The angles and distances of hydrogen bonds at Tyr149 Oη, Ser60 Oγ and Lys63 Nζ revealed that Tyr149 Oη donates an H atom to a water molecule in the substrate-bound state, and that Tyr149 Oη donates an H atom to Ser60 Oγ or Lys63 Nζ in the ground state. Taking into consideration the locations of the H atoms of Tyr149 Oη, Ser60 Oγ and Lys63 Nζ, a catalytic mechanism of DAA activity is presented, wherein a shift of an H atom at Tyr149 Oη in the substrate-bound versus the ground state plays a significant role in the reaction. This mechanism explains well why acylation proceeds and deacylation does not proceed in the substrate-bound state. PMID:18421151

α-Ketoglutaramate: An overlooked metabolite of glutamine and a biomarker for hepatic encephalopathy and inborn errors of the urea cycle

PubMed Central

Cooper, Arthur J. L.; Kuhara, Tomiko

2013-01-01

Glutamine metabolism is generally regarded as proceeding via glutaminase-catalyzed hydrolysis to glutamate and ammonia, followed by conversion of glutamate to α-ketoglutarate catalyzed by glutamate dehydrogenase or by a glutamate-linked aminotransferase (transaminase). However, another pathway exists for the conversion of glutamine to α-ketoglutarate that is often overlooked, but is widely distributed in nature. This pathway, referred to as the glutaminase II pathway, consists of a glutamine transaminase coupled to ω-amidase. Transamination of glutamine results in formation of the corresponding α-keto acid, namely, α-ketoglutaramate (KGM). KGM is hydrolyzed by ω-amidase to α-ketoglutarate and ammonia. The net glutaminase II reaction is: L-Glutamine + α-keto acid + H2O → α-ketoglutarate + L-amino acid + ammonia. In this mini-review the biochemical importance of the glutaminase II pathway is summarized, with emphasis on the key component KGM. Forty years ago it was noted that the concentration of KGM is increased in the cerebrospinal fluid (CSF) of patients with hepatic encephalopathy (HE) and that the level of KGM in the CSF correlates well with the degree of encephalopathy. In more recent work, we have shown that KGM is markedly elevated in the urine of patients with inborn errors of the urea cycle. It is suggested that KGM may be a useful biomarker for many hyperammonemic diseases including hepatic encephalopathy, inborn errors of the urea cycle, citrin deficiency and lysinuric protein intolerance. PMID:24234505

Lipoamidase activity in normal and mutagenized pancreatic cholesterol esterase (bile salt-stimulated lipase).

PubMed Central

Hui, D Y; Hayakawa, K; Oizumi, J

1993-01-01

Purified human milk lipoamidase was digested with endoproteinase Lys-C and the digested peptides were subjected to gasphase microsequence analysis. The sequencing of three isolated peptides of human milk lipoamidase revealed the identity of this protein with human milk bile salt-stimulated lipase (pancreatic cholesterol esterase). The identity of the cholesterol esterase with lipoamidase was confirmed by expressing a recombinant form of rat pancreatic cholesterol esterase and testing for lipoamidase activity of the recombinant protein. The results showed that the recombinant cholesterol esterase displayed both lipolytic and lipoamidase activities and was capable of hydrolysing triacetin and lipoyl-4-aminobenzoate (LPAB). The mechanisms of the esterase and amidase activities of the enzyme were further tested by determining enzyme activity in a mutagenized cholesterol esterase with a His435-->Gln435 substitution. This mutation has been shown previously to abolish enzyme activity against esterase substrates [DiPersio, Fontaine and Hui (1991) J. Biol. Chem. 266, 4033-4036]. We showed that the mutagenized protein was effective in hydrolysing the amidase substrate LPAB and displayed similar enzyme kinetics to those of the native enzyme. These data indicate that the mechanism for the cholesterol esterase hydrolysis of lipoamides is different from that of the hydrolysis of substrates with an ester linkage. The presence of an enzyme in the gastrointestinal tract capable of both ester and amide hydrolysis suggests an important role for this protein in the digestion and absorption processes. PMID:8471055

Transferred interbacterial antagonism genes augment eukaryotic innate immune function.

PubMed

Chou, Seemay; Daugherty, Matthew D; Peterson, S Brook; Biboy, Jacob; Yang, Youyun; Jutras, Brandon L; Fritz-Laylin, Lillian K; Ferrin, Michael A; Harding, Brittany N; Jacobs-Wagner, Christine; Yang, X Frank; Vollmer, Waldemar; Malik, Harmit S; Mougous, Joseph D

2015-02-05

Horizontal gene transfer allows organisms to rapidly acquire adaptive traits. Although documented instances of horizontal gene transfer from bacteria to eukaryotes remain rare, bacteria represent a rich source of new functions potentially available for co-option. One benefit that genes of bacterial origin could provide to eukaryotes is the capacity to produce antibacterials, which have evolved in prokaryotes as the result of eons of interbacterial competition. The type VI secretion amidase effector (Tae) proteins are potent bacteriocidal enzymes that degrade the cell wall when delivered into competing bacterial cells by the type VI secretion system. Here we show that tae genes have been transferred to eukaryotes on at least six occasions, and that the resulting domesticated amidase effector (dae) genes have been preserved for hundreds of millions of years through purifying selection. We show that the dae genes acquired eukaryotic secretion signals, are expressed within recipient organisms, and encode active antibacterial toxins that possess substrate specificity matching extant Tae proteins of the same lineage. Finally, we show that a dae gene in the deer tick Ixodes scapularis limits proliferation of Borrelia burgdorferi, the aetiologic agent of Lyme disease. Our work demonstrates that a family of horizontally acquired toxins honed to mediate interbacterial antagonism confers previously undescribed antibacterial capacity to eukaryotes. We speculate that the selective pressure imposed by competition between bacteria has produced a reservoir of genes encoding diverse antimicrobial functions that are tailored for co-option by eukaryotic innate immune systems.

Discovery and Biochemical Characterization of PlyP56, PlyN74, and PlyTB40—Bacillus Specific Endolysins

PubMed Central

Etobayeva, Irina; Linden, Sara B.; Alem, Farhang; Rizkalla, Lucas; Temple, Louise; Hakami, Ramin M.

2018-01-01

Three Bacillus bacteriophage-derived endolysins, designated PlyP56, PlyN74, and PlyTB40, were identified, cloned, purified, and characterized for their antimicrobial properties. Sequence alignment reveals these endolysins have an N-terminal enzymatically active domain (EAD) linked to a C-terminal cell wall binding domain (CBD). PlyP56 has a Peptidase_M15_4/VanY superfamily EAD with a conserved metal binding motif and displays biological dependence on divalent ions for activity. In contrast, PlyN74 and PlyTB40 have T7 lysozyme-type Amidase_2 and carboxypeptidase T-type Amidase_3 EADs, respectively, which are members of the MurNAc-LAA superfamily, but are not homologs and thus do not have a shared protein fold. All three endolysins contain similar SH3-family CBDs. Although minor host range differences were noted, all three endolysins show relatively broad antimicrobial activity against members of the Bacillus cereus sensu lato group with the highest lytic activity against B. cereus ATCC 4342. Characterization studies determined the optimal lytic activity for these enzymes was at physiological pH (pH 7.0–8.0), over a broad temperature range (4–55 °C), and at low concentrations of NaCl (<50 mM). Direct comparison of lytic activity shows the PlyP56 enzyme to be twice as effective at lysing the cell wall peptidoglycan as PlyN74 or PlyTB40, suggesting PlyP56 is a good candidate for further antimicrobial development as well as bioengineering studies. PMID:29883383

A Novel Hydrolase Identified by Genomic-Proteomic Analysis of Phenylurea Herbicide Mineralization by Variovorax sp. Strain SRS16▿†

PubMed Central

Bers, Karolien; Leroy, Baptiste; Breugelmans, Philip; Albers, Pieter; Lavigne, Rob; Sørensen, Sebastian R.; Aamand, Jens; De Mot, René; Wattiez, Ruddy; Springael, Dirk

2011-01-01

The soil bacterial isolate Variovorax sp. strain SRS16 mineralizes the phenylurea herbicide linuron. The proposed pathway initiates with hydrolysis of linuron to 3,4-dichloroaniline (DCA) and N,O-dimethylhydroxylamine, followed by conversion of DCA to Krebs cycle intermediates. Differential proteomic analysis showed a linuron-dependent upregulation of several enzymes that fit into this pathway, including an amidase (LibA), a multicomponent chloroaniline dioxygenase, and enzymes associated with a modified chlorocatechol ortho-cleavage pathway. Purified LibA is a monomeric linuron hydrolase of ∼55 kDa with a Km and a Vmax for linuron of 5.8 μM and 0.16 nmol min−1, respectively. This novel member of the amidase signature family is unrelated to phenylurea-hydrolyzing enzymes from Gram-positive bacteria and lacks activity toward other tested phenylurea herbicides. Orthologues of libA are present in all other tested linuron-degrading Variovorax strains with the exception of Variovorax strains WDL1 and PBS-H4, suggesting divergent evolution of the linuron catabolic pathway in different Variovorax strains. The organization of the linuron degradation genes identified in the draft SRS16 genome sequence indicates that gene patchwork assembly is at the origin of the pathway. Transcription analysis suggests that a catabolic intermediate, rather than linuron itself, acts as effector in activation of the pathway. Our study provides the first report on the genetic organization of a bacterial pathway for complete mineralization of a phenylurea herbicide and the first report on a linuron hydrolase in Gram-negative bacteria. PMID:22003008

2-Pentadecyl-2-Oxazoline, the Oxazoline of Pea, Modulates Carrageenan-Induced Acute Inflammation

PubMed Central

Petrosino, Stefania; Campolo, Michela; Impellizzeri, Daniela; Paterniti, Irene; Allarà, Marco; Gugliandolo, Enrico; D’Amico, Ramona; Siracusa, Rosalba; Cordaro, Marika; Esposito, Emanuela; Di Marzo, Vincenzo; Cuzzocrea, Salvatore

2017-01-01

N-acylethanolamines (NAEs) involve a family of lipid molecules existent in animal and plant, with N-palmitoylethanolamide (PEA) that arouses great attention owing to its anti-inflammatory, analgesic and neuroprotective activities. Because PEA is produced on demand and exerts pleiotropic effects, the modulation of specific amidases for NAEs (and in particular NAE-hydrolyzing acid amidase NAAA, which is more selective for PEA) could be a condition to preserve its levels. Here we investigate the effect of 2-Pentadecyl-2-oxazoline (PEA-OXA) the oxazoline of PEA, on human recombinant NAAA in vitro and in an established model of Carrageenan (CAR)-induced rat paw inflammation. PEA-OXA dose-dependently significantly inhibited recombinant NAAA and, orally administered to rats (10 mg/kg), limiting histological damage, thermal hyperalgesia and the increase of infiltrating inflammatory cells after CAR injection in the rat right hindpaw, compared to ultramicronized PEA given orally at the same dose (10 mg/kg). These effects were accompanied by elevation of paw PEA levels. Moreover, PEA-OXA markedly reduced neutrophil infiltration and pro-inflammatory cytokine release and prevented CAR-induced IκB-α degradation, nuclear translocation of NF-κB p65, the increase of inducible nitric oxide synthase, cyclooxygenase-2, intercellular adhesion molecule-1, and mast cell activation. Experiments in PPAR-α knockout mice showed that the anti-inflammatory effects of PEA-OXA were not dependent on the presence of PPAR-α receptors. In conclusion, NAAA modulators as PEA-OXA could help to maximize the tissue availability of PEA by increasing its levels and anti-inflammatory effects. PMID:28611664

Acrylamide biodegradation ability and plant growth-promoting properties of Variovorax boronicumulans CGMCC 4969.

PubMed

Liu, Zhong-Hua; Cao, Yu-Min; Zhou, Qian-Wen; Guo, Kun; Ge, Feng; Hou, Jun-Yi; Hu, Si-Yi; Yuan, Sheng; Dai, Yi-Jun

2013-11-01

Species of the genus Variovorax are often isolated from nitrile or amide-containing organic compound-contaminated soil. However, there have been few biological characterizations of Variovorax and their contaminant-degrading enzymes. Previously, we reported a new soil isolate, Variovorax boronicumulans CGMCC 4969, and its nitrile hydratase that transforms the neonicotinoid insecticide thiacloprid into an amide metabolite. In this study, we showed that CGMCC 4969 is able to degrade acrylamide, a neurotoxicant and carcinogen in animals, during cell growth in a mineral salt medium as well as in its resting state. Resting cells rapidly hydrolyzed 600 mg/L acrylamide to acrylic acid with a half-life of 2.5 min. In in vitro tests, CGMCC 4969 showed plant growth-promoting properties; it produced a siderophore, ammonia, hydrogen cyanide, and the phytohormone salicylic acid. Interestingly, in soil inoculated with this strain, 200 mg/L acrylamide was completely degraded in 4 days. Gene cloning and overexpression in the Escherichia coli strain Rosetta (DE3) pLysS resulted in the production of an aliphatic amidase of 345 amino acids that hydrolyzed acrylamide into acrylic acid. The amidase contained a conserved catalytic triad, Glu59, Lys 134, and Cys166, and an "MRHGDISSS" amino acid sequence at the N-terminal region. Variovorax boronicumulans CGMCC 4969, which is able to use acrylamide for cell growth and rapidly degrade acrylamide in soil, shows promising plant growth-promoting properties. As such, it has the potential to be developed into an effective Bioaugmentation strategy to promote growth of field crops in acrylamide-contaminated soil.

Functional characterization of a short peptidoglycan recognition protein, PGRP5 in grass carp Ctenopharyngodon idella.

PubMed

Li, Jun Hua; Chang, Ming Xian; Xue, Na Na; Nie, P

2013-08-01

Peptidoglycan recognition proteins (PGRPs), which are evolutionarily conserved from insects to mammals, recognize bacterial peptidoglycan (PGN) and function in antibacterial innate immunity. In this study, a short-form PGRP, designated as gcPGRP5 was identified from grass carp Ctenopharyngodon idella. The deduced amino acid sequence of gcPGRP5 is composed of 180 residues with a conserved PGRP domain at the C-terminus. The gcPGRP5 gene consists of four exons and three introns, spacing approximately 2.3 kb in genomic sequence. Phylogenetic analysis demonstrated that the gcPGRP5 is clustered with other PGRP-S identified in teleost fish. The gcPGRP5 is constitutively expressed in all organs/tissues examined, and its expression was significantly induced in CIK cells treated with lipoteichoic acid (LTA), polyinosinic polycytidylic acid (Poly I:C) and PGN. Fluorescence analysis showed that gcPGRP5 is distributed in cytoplasm of CIK cells, and cell lysates from CIK cells transfected with pTurbo-gcPGRP5-GFP and ptGFP1-gcPGRP5 plasmids display the binding activity and peptidoglycan-lytic amidase activity toward Lys-PGN from Staphylococcus aureus and Dap-PGN from Bacillus subtilis. Furthermore, heat-shock protein70 (Hsp70), and MyD88, an adaptor molecule in Toll-like receptor pathway, had an increased expression in CIK cells overexpressed with gcPGRP5. It is thus indicated that gcPGRP5 exhibits amidase activity, and also possesses roles in anti-stress, and in Toll-like receptor signaling pathway. Copyright © 2013 Elsevier Ltd. All rights reserved.

Nitrile Metabolizing Yeasts

NASA Astrophysics Data System (ADS)

Bhalla, Tek Chand; Sharma, Monica; Sharma, Nitya Nand

Nitriles and amides are widely distributed in the biotic and abiotic components of our ecosystem. Nitrile form an important group of organic compounds which find their applications in the synthesis of a large number of compounds used as/in pharmaceutical, cosmetics, plastics, dyes, etc>. Nitriles are mainly hydro-lyzed to corresponding amide/acid in organic chemistry. Industrial and agricultural activities have also lead to release of nitriles and amides into the environment and some of them pose threat to human health. Biocatalysis and biotransformations are increasingly replacing chemical routes of synthesis in organic chemistry as a part of ‘green chemistry’. Nitrile metabolizing organisms or enzymes thus has assumed greater significance in all these years to convert nitriles to amides/ acids. The nitrile metabolizing enzymes are widely present in bacteria, fungi and yeasts. Yeasts metabolize nitriles through nitrilase and/or nitrile hydratase and amidase enzymes. Only few yeasts have been reported to possess aldoxime dehydratase. More than sixty nitrile metabolizing yeast strains have been hither to isolated from cyanide treatment bioreactor, fermented foods and soil. Most of the yeasts contain nitrile hydratase-amidase system for metabolizing nitriles. Transformations of nitriles to amides/acids have been carried out with free and immobilized yeast cells. The nitrilases of Torulopsis candida>and Exophiala oligosperma>R1 are enantioselec-tive and regiospecific respectively. Geotrichum>sp. JR1 grows in the presence of 2M acetonitrile and may have potential for application in bioremediation of nitrile contaminated soil/water. The nitrilase of E. oligosperma>R1 being active at low pH (3-6) has shown promise for the hydroxy acids. Immobilized yeast cells hydrolyze some additional nitriles in comparison to free cells. It is expected that more focus in future will be on purification, characterization, cloning, expression and immobilization of nitrile metabolizing enzymes of yeasts.

Characterization of Foodborne Strains of Staphylococcus aureus by Shotgun Proteomics: Functional Networks, Virulence Factors and Species-Specific Peptide Biomarkers

PubMed Central

Carrera, Mónica; Böhme, Karola; Gallardo, José M.; Barros-Velázquez, Jorge; Cañas, Benito; Calo-Mata, Pilar

2017-01-01

In the present work, we applied a shotgun proteomics approach for the fast and easy characterization of 20 different foodborne strains of Staphylococcus aureus (S. aureus), one of the most recognized foodborne pathogenic bacteria. A total of 644 non-redundant proteins were identified and analyzed via an easy and rapid protein sample preparation procedure. The results allowed the differentiation of several proteome datasets from the different strains (common, accessory, and unique datasets), which were used to determine relevant functional pathways and differentiate the strains into different Euclidean hierarchical clusters. Moreover, a predicted protein-protein interaction network of the foodborne S. aureus strains was created. The whole confidence network contains 77 nodes and 769 interactions. Most of the identified proteins were surface-associated proteins that were related to pathways and networks of energy, lipid metabolism and virulence. Twenty-seven virulence factors were identified, and most of them corresponded to autolysins, N-acetylmuramoyl-L-alanine amidases, phenol-soluble modulins, extracellular fibrinogen-binding proteins and virulence factor EsxA. Potential species-specific peptide biomarkers were screened. Twenty-one species-specific peptide biomarkers, belonging to eight different proteins (nickel-ABC transporter, N-acetylmuramoyl-L-alanine amidase, autolysin, clumping factor A, gram-positive signal peptide YSIRK, cysteine protease/staphopain, transcriptional regulator MarR, and transcriptional regulator Sar-A), were proposed to identify S. aureus. These results constitute the first major dataset of peptides and proteins of foodborne S. aureus strains. This repository may be useful for further studies, for the development of new therapeutic treatments for S. aureus food intoxications and for microbial source-tracking in foodstuffs. PMID:29312172

Pleiotropic Effects of Cell Wall Amidase LytA on Streptococcus pneumoniae Sensitivity to the Host Immune Response

PubMed Central

Ramos-Sevillano, Elisa; Urzainqui, Ana; Campuzano, Susana; Moscoso, Miriam; González-Camacho, Fernando; Domenech, Mirian; Rodríguez de Córdoba, Santiago; Sánchez-Madrid, Francisco; Brown, Jeremy S.; García, Ernesto

2014-01-01

The complement system is a key component of the host immune response for the recognition and clearance of Streptococcus pneumoniae. In this study, we demonstrate that the amidase LytA, the main pneumococcal autolysin, inhibits complement-mediated immunity independently of effects on pneumolysin by a complex process of impaired complement activation, increased binding of complement regulators, and direct degradation of complement C3. The use of human sera depleted of either C1q or factor B confirmed that LytA prevented activation of both the classical and alternative pathways, whereas pneumolysin inhibited only the classical pathway. LytA prevented binding of C1q and the acute-phase protein C-reactive protein to S. pneumoniae, thereby reducing activation of the classical pathway on the bacterial surface. In addition, LytA increased recruitment of the complement downregulators C4BP and factor H to the pneumococcal cell wall and directly cleaved C3b and iC3b to generate degradation products. As a consequence, C3b deposition and phagocytosis increased in the absence of LytA and were markedly enhanced for the lytA ply double mutant, confirming that a combination of LytA and Ply is essential for the establishment of pneumococcal pneumonia and sepsis in a murine model of infection. These data demonstrate that LytA has pleiotropic effects on complement activation, a finding which, in combination with the effects of pneumolysin on complement to assist with pneumococcal complement evasion, confirms a major role of both proteins for the full virulence of the microorganism during septicemia. PMID:25404032

A supramolecular complex between proteinases and beta-cyclodextrin that preserves enzymatic activity: physicochemical characterization.

PubMed

Denadai, Angelo M L; Santoro, Marcelo M; Lopes, Miriam T P; Chenna, Angélica; de Sousa, Frederico B; Avelar, Gabriela M; Gomes, Marco R Túlio; Guzman, Fanny; Salas, Carlos E; Sinisterra, Rubén D

2006-01-01

Cyclodextrins are suitable drug delivery systems because of their ability to subtly modify the physical, chemical, and biological properties of guest molecules through labile interactions by formation of inclusion and/or association complexes. Plant cysteine proteinases from Caricaceae and Bromeliaceae are the subject of therapeutic interest, because of their anti-inflammatory, antitumoral, immunogenic, and wound-healing properties. In this study, we analyzed the association between beta-cyclodextrin (betaCD) and fraction P1G10 containing the bioactive proteinases from Carica candamarcensis, and described the physicochemical nature of the solid-state self-assembled complexes by Fourier transform infrared (FTIR) spectroscopy, thermogravimetry (TG), differential scanning calorimetry (DSC), X-ray powder diffraction (XRD), and nuclear magnetic resonance (NMR), as well as in solution by circular dichroism (CD), isothermal titration calorimetry (ITC), and amidase activity. The physicochemical analyses suggest the formation of a complex between P1G10 and betaCD. Higher secondary interactions, namely hydrophobic interactions, hydrogen bonding and van der Waals forces were observed at higher P1G10 : betaCD mass ratios. These results provide evidence of the occurrence of strong solid-state supramolecular non-covalent interactions between P1G10 and betaCD. Microcalorimetric analysis demonstrates that complexation results in a favorable enthalpic contribution, as has already been described during formation of similar betaCD inclusion compounds. The amidase activity of the complex shows that the enzyme activity is not readily available at 24 hours after dissolution of the complex in aqueous buffer; the proteinase becomes biologically active by the second day and remains stable until day 16, when a gradual decrease occurs, with basal activity attained by day 29. The reported results underscore the potential for betaCDs as candidates for complexing cysteine proteinases, resulting in supramolecular arrays with sustained proteolytic activity.

The structural basis of the difference in sensitivity for PNGase F in the de-N-glycosylation of the native bovine pancreatic ribonucleases B and BS.

PubMed

Blanchard, Véronique; Frank, Martin; Leeflang, Bas R; Boelens, Rolf; Kamerling, Johannis P

2008-03-18

In glycoanalysis protocols, N-glycans from glycoproteins are most frequently released with peptide- N (4)-( N-acetyl-beta-glucosaminyl)asparagine amidase F (PNGase F). As the enzyme is an amidase, it cleaves the NH-CO linkage between the Asn side chain and the Asn-bound GlcNAc residue. Usually, the enzyme has a low activity, or is not active at all, on native glycoproteins. A typical example is native bovine pancreatic ribonuclease B (RNase B) with oligomannose-type N-glycans at Asn-34. However, native RNase BS, generated by subtilisin digestion of native RNase B, which comprises amino acid residues 21-124 of RNase B, is sensitive to PNGase F digestion. The same holds for carboxymethylated RNase B (RNase B (cm)). In this study, NMR spectroscopy and molecular modeling have been used to explain the differences in PNGase F activity for native RNase B, native RNase BS, and RNase B (cm). NMR analysis combined with literature data clearly indicated that the N-glycan at Asn-34 is more mobile in RNase BS than in RNase B. MD simulations showed that the region around Asn-34 in RNase B is not very flexible, whereby the alpha-helix of the amino acid residues 1-20 has a stabilizing effect. In RNase BS, the alpha-helix formed by amino acid residues 23-32 is significantly more flexible. Using these data, the possibilities for complex formation of both RNase B and RNase BS with PNGase F were studied, and a model for the RNase BS-PNGase F complex is proposed.

Development and validation of an automated enzyme assay for paracetamol (acetaminophen).

PubMed

Morris, H C; Overton, P D; Ramsay, J R; Campbell, R S; Hammond, P M; Atkinson, T; Price, C P

1990-02-28

A rapid, enzymatic assay for serum or plasma paracetamol has been developed with the potential for adaptation to a wide range of clinical analysers. The method involves the action of an amidase enzyme to produce 4-aminophenol from paracetamol, which in turn reacts with 8-hydroxyquinoline in the presence of manganese ions to form a blue dye. Two stable reagents are used and excellent precision is achieved over the drug concentration range 0-2.5 mmol/l. The method, which is complete within 6 min, has been validated using a Monarch centrifugal analyser and shows no significant interference from endogenous serum compounds, drugs or paracetamol metabolites.

In vitro bactericidal and bacteriolytic activity of ceragenin CSA-13 against planktonic cultures and biofilms of Streptococcus pneumoniae and other pathogenic streptococci.

PubMed

Moscoso, Miriam; Esteban-Torres, María; Menéndez, Margarita; García, Ernesto

2014-01-01

Ceragenin CSA-13, a cationic steroid, is here reported to show a concentration-dependent bactericidal/bacteriolytic activity against pathogenic streptococci, including multidrug-resistant Streptococcus pneumoniae. The autolysis promoted by CSA-13 in pneumococcal cultures appears to be due to the triggering of the major S. pneumoniae autolysin LytA, an N-acetylmuramoyl-L-alanine amidase. CSA-13 also disintegrated pneumococcal biofilms in a very efficient manner, although at concentrations slightly higher than those required for bactericidal activity on planktonic bacteria. CSA-13 has little hemolytic activity which should allow testing its antibacterial efficacy in animal models.

Method for metabolizing carbazole in petroleum

DOEpatents

Kayser, Kevin J.; Kilbane, II, John J.

2005-09-13

A method for selective cleavage of C--N bonds genes that encode for at least one enzyme suitable for conversion of carbazole to 2-aminobiphenyl-2,3-diol are combined with a gene encoding an amidase suitable for selectively cleaving a C--N bond in 2-aminobiphenyl-2,3-diol, forming an operon that encodes for cleavage of both C--N bonds of said carbazole. The operon is inserted into a host culture which, in turn, is contacted with the carbazole, resulting in selective cleavage of both C--N bonds of the carbazole. Also disclosed is a new microorganism that expresses a carbazole degradation trait constitutively and a method for degrading carbazole employing this microorganism.

PGRP-LB homolog acts as a negative modulator of immunity in maintaining the gut-microbe symbiosis of red palm weevil, Rhynchophorus ferrugineus Olivier.

PubMed

Dawadi, Bishnu; Wang, Xinghong; Xiao, Rong; Muhammad, Abrar; Hou, Youming; Shi, Zhanghong

2018-09-01

Many notorious insect pests live in the symbiotic associations with gut microbiota. However, the mechanisms underlying how they host their gut microbiota are unknown. Most gut bacteria can release peptidoglycan (PGN) which is an important antigen to activate the immune response. Therefore, how to keep the appropriate gut immune intensity to host commensals while to efficiently remove enteropathogens is vital for insect health. This study is aimed at elucidating the roles of an amidase PGRP, Rf PGRP-LB, in maintaining the gut-microbe symbiosis of Red palm weevil (RPW), Rhynchophorus ferrugineus Olivier. RfPGRP-LB is a secreted protein containing a typical PGRP domain. The existence of five conservative amino acid residues, being required for amidase activity, showed that RfPGRP-LB is a catalytic protein. Expression analysis revealed abundance of RfPGRP-LB transcripts in gut was dramatically higher than those in other tissues. RfPGRP-LB could be significantly induced against the infection of Escherichia coli. In vitro assays revealed that rRfPGRP-LB impaired the growth of E. coli and agglutinated bacteria cells obviously, suggesting RfPGRP-LB is a pathogen recognition receptor and bactericidal molecule. RfPGRP-LB knockdown reduced the persistence of E. coli in gut and load of indigenous gut microbiota significantly. Furthermore, the community structure of indigenous gut microbiota was also intensively altered by RfPGRP-LB silence. Higher levels of the antimicrobial peptide, attacin, were detected in guts of RfPGRP-LB silenced larvae than controls. Collectively, RfPGRP-LB plays multiple roles in modulating the homeostasis of RPW gut microbiota not only by acting as a negative regulator of mucosal immunity through PGN degradation but also as a bactericidal effector to prevent overgrowth of commensals and persistence of noncommensals. Copyright © 2018 Elsevier Ltd. All rights reserved.

Purification and Characterization of Allophanate Hydrolase (AtzF) from Pseudomonas sp. Strain ADP

PubMed Central

Shapir, Nir; Sadowsky, Michael J.; Wackett, Lawrence P.

2005-01-01

AtzF, allophanate hydrolase, is a recently discovered member of the amidase signature family that catalyzes the terminal reaction during metabolism of s-triazine ring compounds by bacteria. In the present study, the atzF gene from Pseudomonas sp. strain ADP was cloned and expressed as a His-tagged protein, and the protein was purified and characterized. AtzF had a deduced subunit molecular mass of 66,223, based on the gene sequence, and an estimated holoenzyme molecular mass of 260,000. The active protein did not contain detectable metals or organic cofactors. Purified AtzF hydrolyzed allophanate with a kcat/Km of 1.1 × 104 s−1 M−1, and 2 mol of ammonia was released per mol allophanate. The substrate range of AtzF was very narrow. Urea, biuret, hydroxyurea, methylcarbamate, and other structurally analogous compounds were not substrates for AtzF. Only malonamate, which strongly inhibited allophanate hydrolysis, was an alternative substrate, with a greatly reduced kcat/Km of 21 s−1 M−1. Data suggested that the AtzF catalytic cycle proceeds through a covalent substrate-enzyme intermediate. AtzF reacts with malonamate and hydroxylamine to generate malonohydroxamate, potentially derived from hydroxylamine capture of an enzyme-tethered acyl group. Three putative catalytically important residues, one lysine and two serines, were altered by site-directed mutagenesis, each with complete loss of enzyme activity. The identity of a putative serine nucleophile was probed using phenyl phosphorodiamidate that was shown to be a time-dependent inhibitor of AtzF. Inhibition was due to phosphoroamidation of Ser189 as shown by liquid chromatography/matrix-assisted laser desorption ionization mass spectrometry. The modified residue corresponds in sequence alignments to the nucleophilic serine previously identified in other members of the amidase signature family. Thus, AtzF affects the cleavage of three carbon-to-nitrogen bonds via a mechanism similar to that of enzymes catalyzing single-amide-bond cleavage reactions. AtzF orthologs appear to be widespread among bacteria. PMID:15901697

Improved penicillin amidase production using a genetically engineered mutant of escherichia coli ATCC 11105

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

Robas, N.; Zouheiry, H.; Branlant, G.

Penicillin G amidase (PGA) is a key enzyme for the industrial production of penicillin G derivatives used in therapeutics. Escherichia coli ATCC 11105 is the more commonly used strain for PGA production. To improve enzyme yield, the authors constructed various recombinant E. coli HB 101 and ATCC 11105 strains. For each strain, PGA production was determined for various concentrations of glucose and phenylacetic acid (PAA) in the medium. The E. coli strain, G271, was identified as the best performer (800 U NIPAB/L). This strain was obtained as follows: an E. coli ATCC 11105 mutant (E. coli G133) was first selectedmore » based on a low negative effect of glucose on PGA production. This mutant was then transformed with a pBR322 derivative containing the PGA gene. Various experiments were made to try to understand the reason for the high productivity of E. coli G271. The host strain, E. coli G133, was found to be mutated in one (or more) gene(s) whose product(s) act(s) in trans on the PGA gene expression. Its growth is not inhibited by high glucose concentration in the medium. Interestingly, whereas glucose still exerts some negative effect on the PGA production by E. coli G133, PGA production by its transformant (E. coli G271) is stimulated by glucose. The reason for this stimulation is discussed. Transformation of E. coli G133 with a pBR322 derivative containing the HindIII fragment of the PGA gene, showed that the performance of E. coli G271 depends both upon the host strain properties and the plasmid structure. Study of the production by the less efficient E. coli HB101 derivatives brought some light on the mechanism of regulation of the PGA gene.« less

Subcellular Localization of Rice Leaf Aryl Acylamidase Activity 1

PubMed Central

Gaynor, John J.; Still, Cecil C.

1983-01-01

The intracellular localization of aryl acylamidase (aryl-acylamide amidohydrolase, EC 3.5.1.13) in rice (Oryza sativa L. var Starbonnet) leaves was investigated. The enzyme hydrolyzes and detoxifies the herbicide propanil (3,4-dichloropropionanilide) thereby accounting for immunity of the rice plant to herbicidal action. Fractionation of mesophyll protoplasts by differential centrifugation yielded the highest specific activity of amidase in the crude mitochondrial fraction. Further separation of density gradients of the silica sol Percoll also indicated that this enzyme was mitochondrial. By the use of biochemical markers, the purified mitochondrial fraction was shown to be substantially free of contamination from nuclei, chloroplasts, golgi, and plasma membranes. Subfractionation of the purified mitochondria suggests that this enzyme is located on the outer membrane. PMID:16662987

[Human drug metabolizing enzymes. II. Conjugation enzymes].

PubMed

Vereczkey, L; Jemnitz, K; Gregus, Z

1998-09-01

In this review we focus on human conjugation enzymes (UDP-glucuronyltransferases, methyl-trasferases, N-acetyl-transferases, O-acetyl-transferases, Amidases/carboxyesterases, sulfotransferases, Glutation-S-transferases and the enzymes involved in the conjugation with amino acids) that participate in the metabolism of xenobiotics. Although conjugation reactions in most of the cases result in detoxication, more and more publications prove that the reactions catalysed by these enzymes very often lead to activated molecules that may attack macromolecules (proteins, RNAs, DNAs), resulting in toxicity (liver, neuro-, embryotoxicity, allergy, carcinogenecity). We have summarised the data available on these enzymes concerning their catalytic profile and specificity, inhibition, induction properties, their possible role in the generation of toxic compounds, their importance in clinical practice and drug development.

Metabolism of growth hormone releasing peptides.

PubMed

Thomas, Andreas; Delahaut, Philippe; Krug, Oliver; Schänzer, Wilhelm; Thevis, Mario

2012-12-04

New, potentially performance enhancing compounds have frequently been introduced to licit and illicit markets and rapidly distributed via worldwide operating Internet platforms. Developing fast analytical strategies to follow these new trends is one the most challenging issues for modern doping control analysis. Even if reference compounds for the active drugs are readily obtained, their unknown metabolism complicates effective testing strategies. Recently, a new class of small C-terminally amidated peptides comprising four to seven amino acid residues received considerable attention of sports drug testing authorities due to their ability to stimulate growth hormone release from the pituitary. The most promising candidates are the growth hormone releasing peptide (GHRP)-1, -2, -4, -5, -6, hexarelin, alexamorelin, and ipamorelin. With the exemption of GHRP-2, the entity of these peptides represents nonapproved pharmaceuticals; however, via Internet providers, all compounds are readily available. To date, only limited information on the metabolism of these substances is available and merely one metabolite for GHRP-2 is established. Therefore, a comprehensive in vivo (po and iv administration in rats) and in vitro (with human serum and recombinant amidase) study was performed in order to generate information on urinary metabolites potentially useful for routine doping controls. The urine samples from the in vivo experiments were purified by mixed-mode cation-exchange solid-phase extraction and analyzed by ultrahigh-performance liquid chromatography (UHPLC) separation followed by high-resolution/high-accuracy mass spectrometry. Combining the high resolution power of a benchtop Orbitrap mass analyzer for the first metabolite screening and the speed of a quadrupole/time-of-flight (Q-TOF) instrument for identification, urinary metabolites were screened by means of a sensitive full scan analysis and subsequently confirmed by high-accuracy product ion scan experiments. Two deuterium-labeled internal standards (triply deuterated GHRP-4 and GHRP-2 metabolite) were used to optimize the extraction and analysis procedure. Overall, 28 metabolites (at least three for each GHRP) were identified from the in vivo samples and main metabolites were confirmed by the human in vitro model. All identified metabolites were formed due to exopeptidase- (amino- or carboxy-), amidase-, or endopeptidase activity.

Bacterial cell motility of Burkholderia gut symbiont is required to colonize the insect gut.

PubMed

Lee, Jun Beom; Byeon, Jin Hee; Jang, Ho Am; Kim, Jiyeun Kate; Yoo, Jin Wook; Kikuchi, Yoshitomo; Lee, Bok Luel

2015-09-14

We generated a Burkholderia mutant, which is deficient of an N-acetylmuramyl-l-alanine amidase, AmiC, involved in peptidoglycan degradation. When non-motile ΔamiC mutant Burkholderia cells harboring chain form were orally administered to Riptortus insects, ΔamiC mutant cells were unable to establish symbiotic association. But, ΔamiC mutant complemented with amiC gene restored in vivo symbiotic association. ΔamiC mutant cultured in minimal medium restored their motility with single-celled morphology. When ΔamiC mutant cells harboring single-celled morphology were administered to the host insect, this mutant established normal symbiotic association, suggesting that bacterial motility is essential for the successful symbiosis between host insect and Burkholderia symbiont. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

A Novel Method for Relative Quantitation of N-Glycans by Isotopic Labeling Using 18O-Water

PubMed Central

Tao, Shujuan; Orlando, Ron

2014-01-01

Quantitation is an essential aspect of comprehensive glycomics study. Here, a novel isotopic-labeling method is described for N-glycan quantitation using 18O-water. The incorporation of the 18O-labeling into the reducing end of N-glycans is simply and efficiently achieved during peptide-N4-(N-acetyl-β-glucosaminyl) asparagine amidase F release. This process provides a 2-Da mass difference compared with the N-glycans released in 16O-water. A mathematical calculation method was also developed to determine the 18O/16O ratios from isotopic peaks. Application of this method to several standard glycoprotein mixtures and human serum demonstrated that this method can facilitate the relative quantitation of N-glycans over a linear dynamic range of two orders, with high accuracy and reproducibility. PMID:25365792

Distribution, industrial applications, and enzymatic synthesis of D-amino acids.

PubMed

Gao, Xiuzhen; Ma, Qinyuan; Zhu, Hailiang

2015-04-01

D-Amino acids exist widely in microbes, plants, animals, and food and can be applied in pharmaceutical, food, and cosmetics. Because of their widespread applications in industry, D-amino acids have recently received more and more attention. Enzymes including D-hydantoinase, N-acyl-D-amino acid amidohydrolase, D-amino acid amidase, D-aminopeptidase, D-peptidase, L-amino acid oxidase, D-amino acid aminotransferase, and D-amino acid dehydrogenase can be used for D-amino acids synthesis by kinetic resolution or asymmetric amination. In this review, the distribution, industrial applications, and enzymatic synthesis methods are summarized. And, among all the current enzymatic methods, D-amino acid dehydrogenase method not only produces D-amino acid by a one-step reaction but also takes environment and atom economics into consideration; therefore, it is deserved to be paid more attention.

Identification of NH4+-regulated genes of Herbaspirillum seropedicae by random insertional mutagenesis.

PubMed

Schwab, Stefan; Ramos, Humberto J; Souza, Emanuel M; Pedrosa, Fábio O; Yates, Marshall G; Chubatsu, Leda S; Rigo, Liu U

2007-05-01

Random mutagenesis using transposons with promoterless reporter genes has been widely used to examine differential gene expression patterns in bacteria. Using this approach, we have identified 26 genes of the endophytic nitrogen-fixing bacterium Herbaspirillum seropedicae regulated in response to ammonium content in the growth medium. These include nine genes involved in the transport of nitrogen compounds, such as the high-affinity ammonium transporter AmtB, and uptake systems for alternative nitrogen sources; nine genes coding for proteins responsible for restoring intracellular ammonium levels through enzymatic reactions, such as nitrogenase, amidase, and arginase; and a third group includes metabolic switch genes, coding for sensor kinases or transcription regulation factors, whose role in metabolism was previously unknown. Also, four genes identified were of unknown function. This paper describes their involvement in response to ammonium limitation. The results provide a preliminary profile of the metabolic response of Herbaspirillum seropedicae to ammonium stress.

Self-assembling choline mimicks with enhanced binding affinities to C-LytA protein

PubMed Central

Shi, Yang; Zhou, Hao; Zhang, Xiaoli; Wang, Jingyu; Long, Jiafu; Yang, Zhimou; Ding, Dan

2014-01-01

Streptococcus pneumoniae (pneumococcus) causes multiple illnesses in humans. Exploration of effective inhibitors with multivalent attachment sites for choline-binding modules is of great importance to reduce the pneumococcal virulence. In this work, we successfully developed two self-assembling choline mimicks, Ada-GFFYKKK' and Nap-GFFYKKK', which have the abilities to self-assemble into nanoparticles and nanofibers, respectively, yielding multivalent architectures. Additionally, the best characterized choline-binding module, C-terminal moiety of the pneumococcal cell-wall amidase LytA (C-LytA) was also produced with high purity. The self-assembling Ada-GFFYKKK' and Nap-GFFYKKK' show strong interactions with C-LytA, which possess much higher association constant values to the choline-binding modules as compared to the individual peptide Fmoc-K'. This study thus provides a self-assembly approach to yield inhibitors that are very promising for reducing the pneumococcal virulence. PMID:25315737

Structural insights into enzymatic degradation of oxidized polyvinyl alcohol.

PubMed

Yang, Yu; Ko, Tzu-Ping; Liu, Long; Li, Jianghua; Huang, Chun-Hsiang; Chan, Hsiu-Chien; Ren, Feifei; Jia, Dongxu; Wang, Andrew H-J; Guo, Rey-Ting; Chen, Jian; Du, Guocheng

2014-09-05

The ever-increasing production and use of polyvinyl alcohol (PVA) threaten our environment. Yet PVA can be assimilated by microbes in two steps: oxidation and cleavage. Here we report novel α/β-hydrolase structures of oxidized PVA hydrolase (OPH) from two known PVA-degrading organisms, Sphingopyxis sp. 113P3 and Pseudomonas sp. VM15C, including complexes with substrate analogues, acetylacetone and caprylate. The active site is covered by a lid-like β-ribbon. Unlike other esterase and amidase, OPH is unique in cleaving the CC bond of β-diketone, although it has a catalytic triad similar to that of most α/β-hydrolases. Analysis of the crystal structures suggests a double-oxyanion-hole mechanism, previously only found in thiolase cleaving β-ketoacyl-CoA. Three mutations in the lid region showed enhanced activity, with potential in industrial applications. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The SPOR Domain, a Widely Conserved Peptidoglycan Binding Domain That Targets Proteins to the Site of Cell Division.

PubMed

Yahashiri, Atsushi; Jorgenson, Matthew A; Weiss, David S

2017-07-15

Sporulation-related repeat (SPOR) domains are small peptidoglycan (PG) binding domains found in thousands of bacterial proteins. The name "SPOR domain" stems from the fact that several early examples came from proteins involved in sporulation, but SPOR domain proteins are quite diverse and contribute to a variety of processes that involve remodeling of the PG sacculus, especially with respect to cell division. SPOR domains target proteins to the division site by binding to regions of PG devoid of stem peptides ("denuded" glycans), which in turn are enriched in septal PG by the intense, localized activity of cell wall amidases involved in daughter cell separation. This targeting mechanism sets SPOR domain proteins apart from most other septal ring proteins, which localize via protein-protein interactions. In addition to SPOR domains, bacteria contain several other PG-binding domains that can exploit features of the cell wall to target proteins to specific subcellular sites. Copyright © 2017 American Society for Microbiology.

Occurrence of enzymes involved in biosynthesis of indole-3-acetic acid from indole-3-acetonitrile in plant-associated bacteria, Agrobacterium and Rhizobium.

PubMed Central

Kobayashi, M; Suzuki, T; Fujita, T; Masuda, M; Shimizu, S

1995-01-01

The occurrence of a hitherto unknown pathway involving the action of two enzymes, a nitrile hydratase and an amidase for the biosynthesis of indole-3-acetic acid was discovered in phytopathogenic bacteria Agrobacterium tumefaciens and in leguminous bacteria Rhizobium. The nitrile hydratase acting on indole-3-acetonitrile was purified to homogeneity through only two steps from the cell-free extract of A. tumefaciens. The molecular mass of the purified enzyme estimated by HPLC was about 102 kDa, and the enzyme consisted of four subunits identical in molecular mass. The enzyme exhibited a broad absorption spectrum in the visible range with absorption maxima at 408 nm and 705 nm, and it contained cobalt and iron. The enzyme stoichiometrically catalyzed the hydration of indole-3-acetonitrile into indole-3-acetamide with a specific activity of 13.7 mol per min per mg and a Km of 7.9 microM. Images Fig. 1 PMID:11607511

Saliva Microbiota Carry Caries-Specific Functional Gene Signatures

PubMed Central

Chang, Xingzhi; Yuan, Xiao; Tu, Qichao; Yuan, Tong; Deng, Ye; Hemme, Christopher L.; Van Nostrand, Joy; Cui, Xinping; He, Zhili; Chen, Zhenggang; Guo, Dawei; Yu, Jiangbo; Zhang, Yue; Zhou, Jizhong; Xu, Jian

2014-01-01

Human saliva microbiota is phylogenetically divergent among host individuals yet their roles in health and disease are poorly appreciated. We employed a microbial functional gene microarray, HuMiChip 1.0, to reconstruct the global functional profiles of human saliva microbiota from ten healthy and ten caries-active adults. Saliva microbiota in the pilot population featured a vast diversity of functional genes. No significant distinction in gene number or diversity indices was observed between healthy and caries-active microbiota. However, co-presence network analysis of functional genes revealed that caries-active microbiota was more divergent in non-core genes than healthy microbiota, despite both groups exhibited a similar degree of conservation at their respective core genes. Furthermore, functional gene structure of saliva microbiota could potentially distinguish caries-active patients from healthy hosts. Microbial functions such as Diaminopimelate epimerase, Prephenate dehydrogenase, Pyruvate-formate lyase and N-acetylmuramoyl-L-alanine amidase were significantly linked to caries. Therefore, saliva microbiota carried disease-associated functional signatures, which could be potentially exploited for caries diagnosis. PMID:24533043

Saliva microbiota carry caries-specific functional gene signatures.

PubMed

Yang, Fang; Ning, Kang; Chang, Xingzhi; Yuan, Xiao; Tu, Qichao; Yuan, Tong; Deng, Ye; Hemme, Christopher L; Van Nostrand, Joy; Cui, Xinping; He, Zhili; Chen, Zhenggang; Guo, Dawei; Yu, Jiangbo; Zhang, Yue; Zhou, Jizhong; Xu, Jian

2014-01-01

Human saliva microbiota is phylogenetically divergent among host individuals yet their roles in health and disease are poorly appreciated. We employed a microbial functional gene microarray, HuMiChip 1.0, to reconstruct the global functional profiles of human saliva microbiota from ten healthy and ten caries-active adults. Saliva microbiota in the pilot population featured a vast diversity of functional genes. No significant distinction in gene number or diversity indices was observed between healthy and caries-active microbiota. However, co-presence network analysis of functional genes revealed that caries-active microbiota was more divergent in non-core genes than healthy microbiota, despite both groups exhibited a similar degree of conservation at their respective core genes. Furthermore, functional gene structure of saliva microbiota could potentially distinguish caries-active patients from healthy hosts. Microbial functions such as Diaminopimelate epimerase, Prephenate dehydrogenase, Pyruvate-formate lyase and N-acetylmuramoyl-L-alanine amidase were significantly linked to caries. Therefore, saliva microbiota carried disease-associated functional signatures, which could be potentially exploited for caries diagnosis.

LytN, a Murein Hydrolase in the Cross-wall Compartment of Staphylococcus aureus, Is Involved in Proper Bacterial Growth and Envelope Assembly*

PubMed Central

Frankel, Matthew B.; Hendrickx, Antoni P. A.; Missiakas, Dominique M.; Schneewind, Olaf

2011-01-01

Cell cycle progression for the spherical microbe Staphylococcus aureus requires the coordinated synthesis and remodeling of peptidoglycan. The majority of these rearrangements takes place at the mid-cell, in a compartment designated the cross-wall. Secreted polypeptides endowed with a YSIRK-G/S signal peptide are directly delivered to the cross-wall compartment. One such YSIRK-containing protein is the murein hydrolase LytN. lytN mutations precipitate structural damage to the cross-wall and interfere with staphylococcal growth. Overexpression of lytN also affects growth and triggers rupture of the cross-wall. The lytN phenotype can be reversed by the controlled expression of lytN but not by adding purified LytN to staphylococcal cultures. LytN harbors LysM and CHAP domains, the latter of which functions as both an N-acetylmuramoyl-l-alanine amidase and d-alanyl-glycine endopeptidase. Thus, LytN secretion into the cross-wall promotes peptidoglycan separation and completion of the staphylococcal cell cycle. PMID:21784864

Influence of chronic oral intake of cannabis extract on oxidative and hydrolytic metabolism of xenobiotics in rat.

PubMed

Khanna, P; Gupta, M B; Gupta, G P; Sanwal, G G; Ali, B

1991-01-01

Dietary intake of petroleum ether extract of cannabis leaves by rats in doses of 158, 250 and 500 mg/kg in the first, second and third week, respectively, caused selective induction of hepatic microsomal carboxylesterases/amidases without affecting the renal hydrolytic activity. Acetanilide N-deacetylase, p-nitrophenylacetate (NPA) esterase and acetylsalicylic acid (ASA) esterase I and II (active at pH 5.5 and 7.4) were stimulated 125, 64, 82 and 60%, respectively, whereas the activities of procaine esterase and acetylaminofluorene (AAF) N-deacetylase remained unaltered. The hydrolysis of acetylcholine was also unchanged. Upon withdrawal of treatment microsomal hydrolytic activity receded to basal levels within 7 days. Curiously though, the two-fold induction of thiacetazone N-deacetylase (118%), a cytosolic hydrolase, remained largely undiminished (62%). An appraisal of the hepatic cytochrome P450 mediated oxidative metabolism revealed approximately three-fold induction of aromatic hydrocarbon hydroxylase (AHH) metabolizing benzo(a)pyrene whereas the N-demethylation of aminopyrene was unaffected. These activities were restored to normal when resin administration was discontinued.

Mass spectrometry characterization for N-glycosylation of immunoglobulin Y from hen egg yolk.

PubMed

Sheng, Long; He, Zhenjiao; Liu, Yaping; Ma, Meihu; Cai, Zhaoxia

2018-03-01

Immunoglobulin Y (IgY) is a new therapeutic antibody that exists in hen egg yolk. It is a glycoprotein, not much is known about its N-glycan structures, site occupancy and site-specific N-glycosylation. In this study, purified protein from hen egg yolk was identified as IgY based on SDS-PAGE and MALDI-TOF/TOF MS. N-glycan was released from IgY using peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine-amidase treatment, and the molecular weight of IgY was calculated using the difference between the molecular weight of IgY and deglycosylated IgY. Two potential N-Glycosylation sites (ASN 308 and ASN 409 ) were detected on IgY by nanoLC-ESI MS. Sugar chains were separated using normal phase liquid chromatography after fluorescence labeling, and 17 N-glycan structures were confirmed using ESI-MS. The sugar chain pattern contained high-mannose oligosaccharide, hybrid oligosaccharide and complex oligosaccharide. These results could lead to other important information regarding IgY glycosylation. Copyright © 2017 Elsevier B.V. All rights reserved.

The impact of N-glycosylation on conformation and stability of immunoglobulin Y from egg yolk.

PubMed

Sheng, Long; He, Zhenjiao; Chen, Jiahui; Liu, Yaofa; Ma, Meihu; Cai, Zhaoxia

2017-03-01

Immunoglobulin Y (IgY) is a new therapeutic antibody, and its applications in industry are very broad. To provide insight into the effects of N-glycosylation on IgY, its conformation and stability were studied. In this research, IgY was extracted from egg yolk and then digested by peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine-amidase. SDS-PAGE and infrared absorption spectrum showed that carbohydrates were distinctly reduced after enzymolysis. The circular dichroism spectrum indicated that the IgY molecule became more flexible and disordered after removal of N-glycan. The fluorescence intensity revealed that Trp residues were buried in a more hydrophobic environment after disposal of N-glycan. Storage stability decreased with the removal of oligosaccharide chains based on size-exclusion chromatography analysis. Deglycosylated IgY exhibited less resistance to guanidine hydrochloride-induced unfolding. After deglycosylation, IgY was more sensitive to pepsin. Therefore, N-glycosylation played an important role in the maintenance of the structure and stability of IgY. Copyright © 2016 Elsevier B.V. All rights reserved.

Degradation of the metal-cyano complex tetracyanonickelate (II) by Fusarium oxysporum N-10.

PubMed

Yanase, H; Sakamoto, A; Okamoto, K; Kita, K; Sato, Y

2000-03-01

A fungus with the ability to utilize a metalcyano compound, tetracyanonickelate (II) ¿K2[Ni (CN)4]; TCN¿, as its sole source of nitrogen was isolated from soil and identified as Fusarium oxysporum N-10. Both intact mycelia and cell-free extract of the strain catalyzed hydrolysis of TCN to formate and ammonia and produced formamide as an intermediate, thereby indicating that a hydratase and an amidase sequentially participated in the degradation of TCN. The enzyme catalyzing the hydration of TCN was purified approximately ten-fold from the cell-free extract of strain N-10 with a yield of 29%. The molecular mass of the active enzyme was estimated to be 160 kDa. The enzyme appears to exist as a homotetramer, each subunit having a molecular mass of 40 kDa. The enzyme also catalyzed the hydration of KCN, with a cyanide-hydrating activity 2 x 10(4) times greater than for TCN. The kinetic parameters for TCN and KCN indicated that hydratase isolated from F. oxysporum was a cyanide hydratase able to utilize a broad range of cyano compounds and nitriles as substrates.

Isolation and structures of glycoprotein-derived free oligosaccharides from the unfertilized eggs of Scyliorhinus caniculus. Characterization of the sequences galactose(alpha 1-4)galactose(beta 1-3)-N-acetylglucosamine and N-acetylneuraminic acid(alpha 2-6)galactose(beta 1-3)-N-acetylglucosamine.

PubMed

Plancke, Y; Delplace, F; Wieruszeski, J M; Maes, E; Strecker, G

1996-01-15

As previously reported [Ishii, K., Iwasaki, M., Inoue, S., Kenny, P. T. M., Komura, H. & Inoue, Y. (1989) J. Biol. Chem. 264, 1623-1630; Inoue, S., Iwasaki, M., Ishii, K., Kitajima, K. & Inoue, Y. (1989) J. Biol. Chem. 264, 18520-185261, the unfertilized eggs of two different species of fresh-water fish, Plecoglossus altivelis and Tribodolon hakonensis, contain relatively large amounts of free sialooligosaccharides. These oligosaccharides were found to derive from glycophosphoproteins, owing to the activity of a peptide - N4-(N-acetyl-beta-D-glucosaminyl)asparagine amidase [Iwasaki, M., Seko, A., Kitajima, K., Inoue, Y. & Inoue, S. (1992) J. Biol. Chem. 267, 24287-24296; Seko, A., Kitajima, K., Inoue, Y. & Inoue, S. (1991) J. Biol. Chem. 266, 22110-22114]. Here we describe a new type of free oligosaccharides, isolated from unfertilized eggs of Scyliorhinus caniculus. From the structural analysis, based upon 1H-NMR spectroscopy, the following glycan units are proposed.[Formula: see text

Enzymes in Fermented Fish.

PubMed

Giyatmi; Irianto, H E

Fermented fish products are very popular particularly in Southeast Asian countries. These products have unique characteristics, especially in terms of aroma, flavor, and texture developing during fermentation process. Proteolytic enzymes have a main role in hydrolyzing protein into simpler compounds. Fermentation process of fish relies both on naturally occurring enzymes (in the muscle or the intestinal tract) as well as bacteria. Fermented fish products processed using the whole fish show a different characteristic compared to those prepared from headed and gutted fish. Endogenous enzymes like trypsin, chymotrypsin, elastase, and aminopeptidase are the most involved in the fermentation process. Muscle tissue enzymes like cathepsins, peptidases, transaminases, amidases, amino acid decarboxylases, glutamic dehydrogenases, and related enzymes may also play a role in fish fermentation. Due to the decreased bacterial number during fermentation, contribution of microbial enzymes to proteolysis may be expected prior to salting of fish. Commercial enzymes are supplemented during processing for specific purposes, such as quality improvement and process acceleration. In the case of fish sauce, efforts to accelerate fermentation process and to improve product quality have been studied by addition of enzymes such as papain, bromelain, trypsin, pepsin, and chymotrypsin. © 2017 Elsevier Inc. All rights reserved.

Purification and Partial Characterization of a Novel Bacteriocin Synthesized by Lactobacillus paracasei HD1-7 Isolated from Chinese Sauerkraut Juice.

PubMed

Ge, Jingping; Sun, Yanyang; Xin, Xing; Wang, Ying; Ping, Wenxiang

2016-01-14

Bacteriocins have antimicrobial activities against food-spoiling bacteria and food-borne pathogens. Paracin 1.7, a bacteriocin synthesized by Lactobacillus paracasei HD1-7 isolated from Chinese sauerkraut juice, was studied. Following partial purification with ammonium sulfate precipitation, CM Sepharose Fast Flow, and Sephadex G-10 chromatography, the molecular weight of Paracin 1.7 was about 10 kDa based on Tricine-SDS-PAGE results. A 2.87 fold purified bacteriocin was produced, reaching a final yield of 39.93% and the specific activity of 1.56 × 10(3) AU/mg. The N-terminal amino acid sequence of Paracin 1.7 was VSNTFFA, and the LC/LTQ results revealed that the N-terminal amino acid sequence was similar to that of ABC-type oligopeptide transport system protein and N-acetylmuramoyl-L-alanine amidase. Paracin 1.7 was sensitive to protease K, had antimicrobial activities at a broad pH range (3.0-8.0), and was heat resistant (121 °C for 20 min). Paracin 1.7 from Lactobacillus paracasei HD1-7 is a novel bacteriocin that has potential applications in food preservation.

Purification and Partial Characterization of a Novel Bacteriocin Synthesized by Lactobacillus paracasei HD1-7 Isolated from Chinese Sauerkraut Juice

PubMed Central

Ge, Jingping; Sun, Yanyang; Xin, Xing; Wang, Ying; Ping, Wenxiang

2016-01-01

Bacteriocins have antimicrobial activities against food-spoiling bacteria and food-borne pathogens. Paracin 1.7, a bacteriocin synthesized by Lactobacillus paracasei HD1-7 isolated from Chinese sauerkraut juice, was studied. Following partial purification with ammonium sulfate precipitation, CM Sepharose Fast Flow, and Sephadex G-10 chromatography, the molecular weight of Paracin 1.7 was about 10 kDa based on Tricine-SDS-PAGE results. A 2.87 fold purified bacteriocin was produced, reaching a final yield of 39.93% and the specific activity of 1.56 × 103 AU/mg. The N-terminal amino acid sequence of Paracin 1.7 was VSNTFFA, and the LC/LTQ results revealed that the N-terminal amino acid sequence was similar to that of ABC-type oligopeptide transport system protein and N-acetylmuramoyl-L-alanine amidase. Paracin 1.7 was sensitive to protease K, had antimicrobial activities at a broad pH range (3.0–8.0), and was heat resistant (121 °C for 20 min). Paracin 1.7 from Lactobacillus paracasei HD1-7 is a novel bacteriocin that has potential applications in food preservation. PMID:26763314

A sensitive LC-MS/MS method for the simultaneous determination of amoxicillin and ambroxol in human plasma with segmental monitoring.

PubMed

Dong, Xin; Ding, Li; Cao, Xiaomei; Jiang, Liyuan; Zhong, Shuisheng

2013-04-01

Amoxicillin (AMO) degrades in plasma at room temperature and readily undergoes hydrolysis by the plasma amidase. In this paper, a novel, rapid and sensitive LC-MS/MS method operated in segmental and multiple reaction monitoring has been developed for the simultaneous determination of amoxicillin and ambroxol in human plasma. The degradation of amoxicillin in plasma was well prevented by immediate addition of 20 μL glacial acetic acid to 200 μL aliquot of freshly collected plasma samples before storage at -80°C. The sensitivity of the method was improved with segmental monitoring of the analytes, and lower limits of quantitation of 0.5 ng/mL for ambroxol and 5 ng/mL for amoxicillin were obtained. The sensitivity of our method was five times better than those of the existing methods. Furthermore, the mass response saturation problem with amoxicillin was avoided by diluting the deproteinized plasma samples with water before injection into the LC-MS/MS system. The method was successfully employed in a pharmacokinetic study of the compound amoxicillin and ambroxol hydrochloride tablets. Copyright © 2012 John Wiley & Sons, Ltd.

Total enzymatic synthesis of cholecystokinin CCK-5.

PubMed

Xiang, H; Xiang, G Y; Lu, Z M; Guo, L; Eckstein, H

2004-08-01

This paper describes the enzymatic synthesis of the C-terminal fragment H-Gly-Trp-Met-Asp-Phe-NH2 of cholecystokinin. Immobilized enzymes were used for the formation of all peptide bonds except thermolysin. Beginning the synthesis with phenylacetyl (PhAc) glycine carboxamidomethyl ester (OCam) and H-Trp-OMe by using immobilized papain as biocatalyst in buffered ethyl acetate, the dipeptide methyl ester was then coupled directly with Met-OEt.HCl by alpha-chymotrypsin/Celite 545 in a solvent free system. For the 3+2 coupling PhAc-Gly-Trp-Met-OEt had to be converted into its OCam ester. The other fragment H-Asp(OMe)-Phe-NH2 resulted from the coupling of Cbo-Asp(OMe)-OH with H-Phe-NH2.HCl and thermolysin as catalyst, followed by catalytic hydrogenation. Finally PhAc-Gly-Trp-Met-Asp-Phe-NH2 was obtained in a smooth reaction from PhAc-Gly-Trp-Met-OCam and H-Asp(OMe)-Phe-NH2 with alpha-chymotrypsin/Celite 545 in acetonitrile, followed by basic hydrolysis of the beta-methyl ester. The PhAc-group is removed with penicillin G amidase and CCK-5 is obtained in an overall isolated yield of 19.6%.

Purification and characterization of the enzymes involved in nicotinamide adenine dinucleotide degradation by Penicillium brevicompactum NRC 829.

PubMed

Ali, Thanaa Hamed; El-Ghonemy, Dina Helmy

2016-06-01

The present study was conducted to investigate a new pathway for the degradation of nicotinamide adenine dinucleotide (NAD) by Penicillium brevicompactum NRC 829 extracts. Enzymes involved in the hydrolysis of NAD, i.e. alkaline phosphatase, aminohydrolase and glycohydrolase were determined. Alkaline phosphatase was found to catalyse the sequential hydrolysis of two phosphate moieties of NAD molecule to nicotinamide riboside plus adenosine. Adenosine was then deaminated by aminohydrolase to inosine and ammonia. While glycohydrolase catalyzed the hydrolysis of the nicotinamide-ribosidic bond of NAD+ to produce nicotinamide and ADP-ribose in equimolar amounts, enzyme purification through a 3-step purification procedure revealed the existence of two peaks of alkaline phosphatases, and one peak contained deaminase and glycohydrolase activities. NAD deaminase was purified to homogeneity as estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis with an apparent molecular mass of 91 kDa. Characterization and determination of some of NAD aminohydrolase kinetic properties were conducted due to its biological role in the regulation of cellular NAD level. The results also revealed that NAD did not exert its feedback control on nicotinamide amidase produced by P. brevicompactum.

Glutamine metabolism and cycling in Neurospora crassa.

PubMed Central

Mora, J

1990-01-01

Evidence for the existence of a glutamine cycle in Neurospora crassa is reviewed. Through this cycle glutamine is converted into glutamate by glutamate synthase and catabolized by the glutamine transaminase-omega-amidase pathway, the products of which (2-oxoglutarate and ammonium) are the substrates for glutamate dehydrogenase-NADPH, which synthesizes glutamate. In the final step ammonium is assimilated into glutamine by the action of a glutamine synthetase (GS), which is formed by two distinct polypeptides, one catalytically very active (GS beta), and the other (GS alpha) less active but endowed with the capacity to modulate the activity of GS alpha. Glutamate synthase uses the amide nitrogen of glutamine to synthesize glutamate; glutamate dehydrogenase uses ammonium, and both are required to maintain the level of glutamate. The energy expended in the synthesis of glutamine drives the cycle. The glutamine cycle is not futile, because it is necessary to drive an effective carbon flow to support growth; in addition, it facilitates the allocation of nitrogen or carbon according to cellular demands. The glutamine cycle which dissipates energy links catabolism and anabolism and, in doing so, buffers variations in the nutrient supply and drives energy generation and carbon flow for optimal cell function. PMID:2145504

Reversed-phase high-performance liquid chromatographic method for the determination of peptidoglycan monomers and structurally related peptides and adamantyltripeptides.

PubMed

Krstanović, Marina; Frkanec, Ruza; Vranesić, Branka; Ljevaković, Durdica; Sporec, Vesna; Tomasić, Jelka

2002-06-25

The reversed-phase HPLC method using UV detection was developed for the determination of (a) immunostimulating peptidoglycan monomers represented by the basic structure GlcNAc-MurNAc-L-Ala-D-isoGln-meso-DAP(omegaNH(2))-D-Ala-D-Ala (PGM) and two more lipophilic derivatives, Boc-Tyr-PGM and (Ada-1-yl)-CH(2)-CO-PGM, (b) two diastereomeric immunostimulating adamantyltripeptides L- and D-(adamant-2-yl)-Gly-L-Ala-D-isoGln and (c) peptides obtained by the enzyme hydrolyses of peptidoglycans and related peptides. The enzymes used, N-acetylmuramyl-L-alanine amidase and an L,D-aminopeptidase are present in mammalian sera and are involved in the metabolism of peptidoglycans and related peptides. Appropriate solvent systems were chosen with regard to structure and lipophilicity of each compound. As well, different gradient systems within the same solvent system had to be applied in order to achieve satisfactory separation and retention time. HPLC separation was developed with the aim to use this method for the study of the stability of the tested compounds, the purity during preparation and isolation and for following the enzyme hydrolyses.

Peptidoglycan Hydrolases of Escherichia coli

PubMed Central

van Heijenoort, Jean

2011-01-01

Summary: The review summarizes the abundant information on the 35 identified peptidoglycan (PG) hydrolases of Escherichia coli classified into 12 distinct families, including mainly glycosidases, peptidases, and amidases. An attempt is also made to critically assess their functions in PG maturation, turnover, elongation, septation, and recycling as well as in cell autolysis. There is at least one hydrolytic activity for each bond linking PG components, and most hydrolase genes were identified. Few hydrolases appear to be individually essential. The crystal structures and reaction mechanisms of certain hydrolases having defined functions were investigated. However, our knowledge of the biochemical properties of most hydrolases still remains fragmentary, and that of their cellular functions remains elusive. Owing to redundancy, PG hydrolases far outnumber the enzymes of PG biosynthesis. The presence of the two sets of enzymes acting on the PG bonds raises the question of their functional correlations. It is difficult to understand why E. coli keeps such a large set of PG hydrolases. The subtle differences in substrate specificities between the isoenzymes of each family certainly reflect a variety of as-yet-unidentified physiological functions. Their study will be a far more difficult challenge than that of the steps of the PG biosynthesis pathway. PMID:22126997

Combination of a recombinant bacterium with organonitrile-degrading and biofilm-forming capability and a positively charged carrier for organonitriles removal.

PubMed

Li, Chunyan; Sun, Yueling; Yue, Zhenlei; Huang, Mingyan; Wang, Jinming; Chen, Xi; An, Xuejiao; Zang, Hailian; Li, Dapeng; Hou, Ning

2018-04-10

The immobilization of organonitrile-degrading bacteria via the addition of biofilm-forming bacteria represents a promising technology for the treatment of organonitrile-containing wastewater, but biofilm-forming bacteria simply mixed with degrading bacteria may reduce the biodegradation efficiency. Nitrile hydratase and amidase genes, which play critical roles in organonitriles degradation, were cloned and transformed into the biofilm-forming bacterium Bacillus subtilis N4 to construct a recombinant bacterium B. subtilis N4/pHTnha-ami. Modified polyethylene carriers with positive charge was applied to promote bacterial adherence and biofilm formation. The immobilized B. subtilis N4/pHTnha-ami was resistant to organonitriles loading shocks and could remove organic cyanide ion with a initial concentration of 392.6 mg/L for 24 h in a moving bed biofilm reactor. The imputed quorum-sensing signal and the high-throughput sequencing analysis of the biofilm indicated that B. subtilis N4/pHTnha-ami was successfully immobilized and became dominant. The successful application of the immobilized recombinant bacterium offers a novel strategy for the biodegradation of recalcitrant compounds. Copyright © 2018 Elsevier B.V. All rights reserved.

Complete genome sequence of 285P, a novel T7-like polyvalent E. coli bacteriophage.

PubMed

Xu, Bin; Ma, Xiangyu; Xiong, Hongyan; Li, Yafei

2014-06-01

Bacteriophages are considered potential biological agents for the control of infectious diseases and environmental disinfection. Here, we describe a novel T7-like polyvalent Escherichia coli bacteriophage, designated "285P," which can lyse several strains of E. coli. The genome, which consists of 39,270 base pairs with a G+C content of 48.73 %, was sequenced and annotated. Forty-three potential open reading frames were identified using bioinformatics tools. Based on whole-genome sequence comparison, phage 285P was identified as a novel strain of subgroup T7. It showed strongest sequence similarity to Kluyvera phage Kvp1. The phylogenetic analyses of both non-structural proteins (endonuclease gp3, amidase gp3.5, DNA primase/helicase gp4, DNA polymerase gp5, and exonuclease gp6) and structural protein (tail fiber protein gp17) led to the identification of 285P as T7-like phage. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analyses verified the annotation of the structural proteins (major capsid protein gp10a, tail protein gp12, and tail fiber protein gp17).

Characterization, Genome Sequence, and Analysis of Escherichia Phage CICC 80001, a Bacteriophage Infecting an Efficient L-Aspartic Acid Producing Escherichia coli.

PubMed

Xu, Youqiang; Ma, Yuyue; Yao, Su; Jiang, Zengyan; Pei, Jiangsen; Cheng, Chi

2016-03-01

Escherichia phage CICC 80001 was isolated from the bacteriophage contaminated medium of an Escherichia coli strain HY-05C (CICC 11022S) which could produce L-aspartic acid. The phage had a head diameter of 45-50 nm and a tail of about 10 nm. The one-step growth curve showed a latent period of 10 min and a rise period of about 20 min. The average burst size was about 198 phage particles per infected cell. Tests were conducted on the plaques, multiplicity of infection, and host range. The genome of CICC 80001 was sequenced with a length of 38,810 bp, and annotated. The key proteins leading to host-cell lysis were phylogenetically analyzed. One protein belonged to class II holin, and the other two belonged to the endopeptidase family and N-acetylmuramoyl-L-alanine amidase family, respectively. The genome showed the sequence identity of 82.7% with that of Enterobacteria phage T7, and carried ten unique open reading frames. The bacteriophage resistant E. coli strain designated CICC 11021S was breeding and its L-aspartase activity was 84.4% of that of CICC 11022S.

Pharmacological characterization of hydrolysis-resistant analogs of oleoylethanolamide with potent anorexiant properties.

PubMed

Astarita, Giuseppe; Di Giacomo, Barbara; Gaetani, Silvana; Oveisi, Fariba; Compton, Timothy R; Rivara, Silvia; Tarzia, Giorgio; Mor, Marco; Piomelli, Daniele

2006-08-01

Oleoylethanolamide (OEA) is an endogenous lipid mediator that reduces food intake, promotes lipolysis, and decreases body weight gain in rodents by activating peroxisome proliferator-activated receptor-alpha (PPAR-alpha). The biological effects of OEA are terminated by two intracellular lipid hydrolase enzymes, fatty-acid amide hydrolase and N-acylethanolamine-hydrolyzing acid amidase. In the present study, we describe OEA analogs that resist enzymatic hydrolysis, activate PPAR-alpha with high potency in vitro, and persistently reduce feeding when administered in vivo either parenterally or orally. The most potent of these compounds, (Z)-(R)-9-octadecenamide,N-(2-hydroxyethyl,1-methyl) (KDS-5104), stimulates transcriptional activity of PPAR-alpha with a half-maximal effective concentration (EC50) of 100 +/- 21 nM (n = 11). Parenteral administration of KDS-5104 in rats produces persistent dose-dependent prolongation of feeding latency and postmeal interval (half-maximal effective dose, ED50 = 2.4 +/- 1.8 mg kg(-1) i.p.; n = 18), as well as increased and protracted tissue exposure compared with OEA. Oral administration of the compound also results in a significant tissue exposure and reduction of food intake in free-feeding rats. These results suggest that the endogenous high-affinity PPAR-alpha agonist OEA may provide a scaffold for the discovery of novel orally active PPAR-alpha ligands.

Detoxification of toxins by bacillithiol in Staphylococcus aureus

PubMed Central

Newton, Gerald L.; Fahey, Robert C.

2012-01-01

Bacillithiol (BSH), an α-anomeric glycoside of l-cysteinyl-d-glucosaminyl-l-malate, is a major low-molecular-mass thiol found in bacteria such as Bacillus sp., Staphylococcus aureus and Deinococcus radiodurans. Like other low-molecular-mass thiols such as glutathione and mycothiol, BSH is likely to be involved in protection against environmental toxins including thiol-reactive antibiotics. We report here a BSH-dependent detoxification mechanism in S. aureus. When S. aureus Newman strain was treated with monobromobimane and monochlorobimane, the cellular BSH was converted to the fluorescent S-conjugate BS-bimane. A bacillithiol conjugate amidase activity acted upon the BS-bimane to produce Cys-bimane, which was then acetylated by an N-acetyltransferase to generate N-acetyl-Cys-bimane, a mercapturic acid. An S. aureus mutant lacking BSH did not produce mercapturic acid when treated with monobromobimane and monochlorobimane, confirming the involvement of bacillithiol. Furthermore, treatment of S. aureus Newman with rifamycin, the parent compound of the first-line anti-tuberculosis drug, rifampicin, indicated that this thiol-reactive antibiotic is also detoxified in a BSH-dependent manner, since mercapturic acids of rifamycin were observed in the culture medium. These data indicate that toxins and thiol-reactive antibiotics are detoxified to less potent mercapturic acids in a BSH-dependent manner and then exported out of the cell in S. aureus. PMID:22262099

Targeted Mutagenesis of the Mycobacterium smegmatis mca Gene, Encoding a Mycothiol-Dependent Detoxification Protein

PubMed Central

Rawat, Mamta; Uppal, Mandeep; Newton, Gerald; Steffek, Micah; Fahey, Robert C.; Av-Gay, Yossef

2004-01-01

Mycothiol (MSH), a functional analogue of glutathione (GSH) that is found exclusively in actinomycetes, reacts with electrophiles and toxins to form MSH-toxin conjugates. Mycothiol S-conjugate amidase (Mca) then catalyzes the hydrolysis of an amide bond in the S conjugates, producing a mercapturic acid of the toxin, which is excreted from the bacterium, and glucosaminyl inositol, which is recycled back to MSH. In this study, we have generated and characterized an allelic exchange mutant of the mca gene of Mycobacterium smegmatis. The mca mutant accumulates the S conjugates of the thiol-specific alkylating agent monobromobimane and the antibiotic rifamycin S. Introduction of M. tuberculosis mca epichromosomally or introduction of M. smegmatis mca integratively resulted in complementation of Mca activity and reduced levels of S conjugates. The mutation in mca renders the mutant strain more susceptible to electrophilic toxins, such as N-ethylmalemide, iodoacetamide, and chlorodinitrobenzene, and to several oxidants, such as menadione and plumbagin. Additionally we have shown that the mca mutant is also more susceptible to the antituberculous antibiotic streptomycin. Mutants disrupted in genes belonging to MSH biosynthesis are also more susceptible to streptomycin, providing further evidence that Mca detoxifies streptomycin in the mycobacterial cell in an MSH-dependent manner. PMID:15342574

Microbial nitrilases: versatile, spiral forming, industrial enzymes.

PubMed

Thuku, R N; Brady, D; Benedik, M J; Sewell, B T

2009-03-01

The nitrilases are enzymes that convert nitriles to the corresponding acid and ammonia. They are members of a superfamily, which includes amidases and occur in both prokaryotes and eukaryotes. The superfamily is characterized by having a homodimeric building block with a alpha beta beta alpha-alpha beta beta alpha sandwich fold and an active site containing four positionally conserved residues: cys, glu, glu and lys. Their high chemical specificity and frequent enantioselectivity makes them attractive biocatalysts for the production of fine chemicals and pharmaceutical intermediates. Nitrilases are also used in the treatment of toxic industrial effluent and cyanide remediation. The superfamily enzymes have been visualized as dimers, tetramers, hexamers, octamers, tetradecamers, octadecamers and variable length helices, but all nitrilase oligomers have the same basic dimer interface. Moreover, in the case of the octamers, tetradecamers, octadecamers and the helices, common principles of subunit association apply. While the range of industrially interesting reactions catalysed by this enzyme class continues to increase, research efforts are still hampered by the lack of a high resolution microbial nitrilase structure which can provide insights into their specificity, enantioselectivity and the mechanism of catalysis. This review provides an overview of the current progress in elucidation of structure and function in this enzyme class and emphasizes insights that may lead to further biotechnological applications.

Cloning and expression of a conjugated bile acid hydrolase gene from Lactobacillus plantarum by using a direct plate assay.

PubMed

Christiaens, H; Leer, R J; Pouwels, P H; Verstraete, W

1992-12-01

The conjugated bile acid hydrolase gene from the silage isolate Lactobacillus plantarum 80 was cloned and expressed in Escherichia coli MC1061. For the screening of this hydrolase gene within the gene bank, a direct plate assay developed by Dashkevicz and Feighner (M. P. Dashkevicz and S. D. Feighner, Appl. Environ. Microbiol. 53:331-336, 1989) was adapted to the growth requirements of E. coli. Because of hydrolysis and medium acidification, hydrolase-active colonies were surrounded with big halos of precipitated, free bile acids. This phenomenon was also obtained when the gene was cloned into a multicopy shuttle vector and subsequently reintroduced into the parental Lactobacillus strain. The cbh gene and surrounding regions were characterized by nucleotide sequence analysis. The deduced amino acid sequence was shown to have 52% similarity with a penicillin V amidase from Bacillus sphaericus. Preliminary characterization of the gene product showed that it is a cholylglycine hydrolase (EC 3.5.1.24) with only slight activity against taurine conjugates. The optimum pH was between 4.7 and 5.5. Optimum temperature ranged from 30 to 45 degrees C. Southern blot analysis indicated that the cloned gene has similarity with genomic DNA of bile acid hydrolase-active Lactobacillus spp. of intestinal origin.

Determination of multidrug resistance mechanisms in Clostridium perfringens type A isolates using RNA sequencing and 2D-electrophoresis.

PubMed

Ma, Yu-Hua; Ye, Gui-Sheng

2018-06-11

In this study, we screened differentially expressed genes in a multidrug-resistant isolate strain of Clostridium perfringens by RNA sequencing. We also separated and identified differentially expressed proteins (DEPs) in the isolate strain by two-dimensional electrophoresis (2-DE) and mass spectrometry (MS). The RNA sequencing results showed that, compared with the control strain, 1128 genes were differentially expressed in the isolate strain, and these included 227 up-regulated genes and 901 down-regulated genes. Bioinformatics analysis identified the following genes and gene categories that are potentially involved in multidrug resistance (MDR) in the isolate strain: drug transport, drug response, hydrolase activity, transmembrane transporter, transferase activity, amidase transmembrane transporter, efflux transmembrane transporter, bacterial chemotaxis, ABC transporter, and others. The results of the 2-DE showed that 70 proteins were differentially expressed in the isolate strain, 45 of which were up-regulated and 25 down-regulated. Twenty-seven DEPs were identified by MS and these included the following protein categories: ribosome, antimicrobial peptide resistance, and ABC transporter, all of which may be involved in MDR in the isolate strain of C. perfringens. The results provide reference data for further investigations on the drug resistant molecular mechanisms of C. perfringens.

Molecular cloning and characterization of a short peptidoglycan recognition protein from silkworm Bombyx mori.

PubMed

Yang, P-J; Zhan, M-Y; Ye, C; Yu, X-Q; Rao, X-J

2017-12-01

Peptidoglycan is the major bacterial component recognized by the insect immune system. Peptidoglycan recognition proteins (PGRPs) are a family of pattern-recognition receptors that recognize peptidoglycans and modulate innate immune responses. Some PGRPs retain N-acetylmuramoyl-L-alanine amidase (Enzyme Commission number: 3.5.1.28) activity to hydrolyse bacterial peptidoglycans. Others have lost the enzymatic activity and work only as immune receptors. They are all important modulators for innate immunity. Here, we report the cloning and functional analysis of PGRP-S4, a short-form PGRP from the domesticated silkworm, Bombyx mori. The PGRP-S4 gene encodes a protein of 199 amino acids with a signal peptide and a PGRP domain. PGRP-S4 was expressed in the fat body, haemocytes and midgut. Its expression level was significantly induced by bacterial challenges in the midgut. The recombinant PGRP-S4 bound bacteria and different peptidoglycans. In addition, it inhibited bacterial growth and hydrolysed an Escherichia coli peptidoglycan in the presence of Zn 2+ . Scanning electron microscopy showed that PGRP-S4 disrupted the bacterial cell surface. PGRP-S4 further increased prophenoloxidase activation caused by peptidoglycans. Taken together, our data suggest that B. mori PGRP-S4 has multiple functions in immunity. © 2017 The Royal Entomological Society.

Enzymatic preparation of optically pure (+)-2-azabicyclo[2.2.1]hept-5-en-3-one by (-)-γ-lactamase from Bradyrhizobium japonicum USDA 6.

PubMed

Zhu, Shaozhou; Ren, Lu; Yu, Songzhu; Gong, Cuiyu; Song, Dawei; Zheng, Guojun

2014-10-15

Whole cells of Bradyrhizobium japonicum USDA 6 showed both (+)-γ-lactamase activity and (-)-γ-lactamase activity. Insight into the genome of B. japonicum USDA 6 revealed two potential γ-lactamases: a type I (+)-γ-lactamase and a (-)-γ-lactamase, making it the first strain to contain two totally different enantioselective lactamases. Both recombinant enzymes could easily be used to prepare either optically pure (+)-γ-lactam ((+)-2-azabicyclo[2.2.1]hept-5-en-3-one) or optically pure (-)-γ-lactam ((-)-2-azabicyclo[2.2.1]hept-5-en-3-one), which are versatile synthetic building blocks for the synthesis of various carbocyclic nucleosides and carbocyclic sugar analogues. Bioinformatic analysis showed that the type I (+)-γ-lactamase belongs to the amidase signature family, with 504 amino acids; the (-)-γ-lactamase, which consists of 274 amino acids, belongs to the hydrolase family. Here, we report that B. japonicum USDA contains a (-)-γ-lactamase in addition to a (+)-γ-lactamase, and it is the (-)-γ-lactamase from this strain that is examined in detail in this Letter. Enzymatic synthesis of optically pure (+)-γ-lactam with nearly 50% isolated yield and >99% ee was achieved. Copyright © 2014 Elsevier Ltd. All rights reserved.

Identification of fibrinogen-binding proteins of Aspergillus fumigatus using proteomic approach.

PubMed

Upadhyay, Santosh Kumar; Gautam, Poonam; Pandit, Hrishikesh; Singh, Yogendra; Basir, Seemi Farhat; Madan, Taruna

2012-03-01

Aspergillus fumigatus, the main etiological agent for various forms of human aspergillosis, gets access to the respiratory system of human host by inhalation of airborne conidia. These conidia possibly adhere to extracellular matrix (ECM) proteins. Among the ECM proteins involved in adherence, fibrinogen is thought to be crucial. Here, we studied whether A. fumigatus three-week culture filtrate (3wcf) proteins promote binding of A. fumigatus to ECM proteins and promote fungal growth. We observed that incubation of ECM with 3wcf proteins led to dose- and time-dependent increase in adherence of conidia to the ECM. In order to identify the catalogue of fibrinogen-binding A. fumigatus proteins, we carried out fibrinogen affinity blotting using two-dimensional gel electrophoresed 3wcf proteins. A total of 15 fibrinogen-binding protein spots corresponding to 7 unique proteins were identified in 3wcf using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF-TOF). Among these, 4 proteins, namely, beta-glucosidase, alpha-mannosidase, pectate lyase A and oryzin precursor were predicted to have cell wall or extracellular localization, whereas amidase family protein and two hypothetical proteins did not display the signal sequence. This study reports seven novel fibrinogen-binding proteins of A. fumigatus, some of which could be further explored for targeting the adhesion phenomenon as antifungal strategy.

Secreted Immunomodulatory Proteins of Staphylococcus aureus Activate Platelets and Induce Platelet Aggregation.

PubMed

Binsker, Ulrike; Palankar, Raghavendra; Wesche, Jan; Kohler, Thomas P; Prucha, Josephine; Burchhardt, Gerhard; Rohde, Manfred; Schmidt, Frank; Bröker, Barbara M; Mamat, Uwe; Pané-Farré, Jan; Graf, Anica; Ebner, Patrick; Greinacher, Andreas; Hammerschmidt, Sven

2018-04-01

Staphylococcus aureus can cause bloodstream infections associated with infective endocarditis (IE) and disseminated intravascular coagulopathy (DIC). Both complications involve platelets. In view of an increasing number of antibiotic-resistant strains, new approaches to control systemic S. aureus infection are gaining importance. Using a repertoire of 52 recombinant S. aureus proteins in flow cytometry-based platelet activation and aggregation assays, we identified, in addition to the extracellular adherence protein Eap, three secreted staphylococcal proteins as novel platelet activating proteins. Eap and the chemotaxis inhibitory protein of S. aureus (CHIPS), the formyl peptide receptor-like 1 inhibitory protein (FLIPr) and the major autolysin Atl induced P-selectin expression in washed platelets and platelet-rich plasma. Similarly, AtlA, CHIPS and Eap induced platelet aggregation in whole blood. Fluorescence microscopy illustrated that P-selectin expression is associated with calcium mobilization and re-organization of the platelet actin cytoskeleton. Characterization of the functionally active domains of the major autolysin AtlA and Eap indicates that the amidase domain of Atl and the tandem repeats 3 and 4 of Eap are crucial for platelet activation. These results provide new insights in S. aureus protein interactions with platelets and identify secreted proteins as potential treatment targets in case of antibiotic-resistant S. aureus infection. Schattauer GmbH Stuttgart.

Staphylococcus haemolyticus prophage ΦSH2 endolysin relies on Cysteine, Histidine-dependent Amidohydrolases/Peptidases activity for lysis ‘from without’

PubMed Central

Schmelcher, Mathias; Korobova, Olga; Schischkova, Nina; Kiseleva, Natalia; Kopylov, Paul; Pryamchuk, Sergey; Donovan, David M.; Abaev, Igor

2014-01-01

Staphylococcus aureus is an important pathogen, with methicillin-resistant (MRSA) and multi-drug resistant strains becoming increasingly prevalent in both human and veterinary clinics. S. aureus causing bovine mastitis yields high annual losses to the dairy industry. Conventional treatment of mastitis by broad range antibiotics is often not successful and may contribute to development of antibiotic resistance. Bacteriophage endolysins present a promising new source of antimicrobials. The endolysin of prophage ΦSH2 of Staphylococcus haemolyticus strain JCSC1435 (ΦSH2 lysin) is a peptidoglycan hydrolase consisting of two catalytic domains (CHAP and amidase) and an SH3b cell wall binding domain. In this work, we demonstrated its lytic activity against live staphylococcal cells and investigated the contribution of each functional module to bacterial lysis by testing a series of deletion constructs in zymograms and turbidity reduction assays. The CHAP domain exhibited three-fold higher activity than the full length protein and optimum activity in physiological saline. This activity was further enhanced by the presence of bivalent calcium ions. The SH3b domain was shown to be required for full activity of the complete ΦSH2 lysin. The full length enzyme and the CHAP domain showed activity against multiple staphylococcal strains, including MRSA strains, mastitis isolates, and CoNS. PMID:23026556

Staphylococcus haemolyticus prophage ΦSH2 endolysin relies on cysteine, histidine-dependent amidohydrolases/peptidases activity for lysis 'from without'.

PubMed

Schmelcher, Mathias; Korobova, Olga; Schischkova, Nina; Kiseleva, Natalia; Kopylov, Paul; Pryamchuk, Sergey; Donovan, David M; Abaev, Igor

2012-12-31

Staphylococcus aureus is an important pathogen, with methicillin-resistant (MRSA) and multi-drug resistant strains becoming increasingly prevalent in both human and veterinary clinics. S. aureus causing bovine mastitis yields high annual losses to the dairy industry. Conventional treatment of mastitis by broad range antibiotics is often not successful and may contribute to development of antibiotic resistance. Bacteriophage endolysins present a promising new source of antimicrobials. The endolysin of prophage ΦSH2 of Staphylococcus haemolyticus strain JCSC1435 (ΦSH2 lysin) is a peptidoglycan hydrolase consisting of two catalytic domains (CHAP and amidase) and an SH3b cell wall binding domain. In this work, we demonstrated its lytic activity against live staphylococcal cells and investigated the contribution of each functional module to bacterial lysis by testing a series of deletion constructs in zymograms and turbidity reduction assays. The CHAP domain exhibited three-fold higher activity than the full length protein and optimum activity in physiological saline. This activity was further enhanced by the presence of bivalent calcium ions. The SH3b domain was shown to be required for full activity of the complete ΦSH2 lysin. The full length enzyme and the CHAP domain showed activity against multiple staphylococcal strains, including MRSA strains, mastitis isolates, and CoNS. Published by Elsevier B.V.

Molecular cloning and functional characterization of a short peptidoglycan recognition protein (HcPGRPS1) from the freshwater mussel, Hyriopsis cumingi.

PubMed

Yang, Ziyan; Li, Junhua; Li, Ying; Wu, Hongjuan; Wang, Xiaoyan

2013-12-01

Peptidoglycan recognition proteins (PGRPs), which are evolutionarily conserved from invertebrates to vertebrates, function as pattern-recognition and effector molecules in innate immunity. In the present study, a short-form PGRP, designated as HcPGRPS1 was identified from freshwater mussel Hyriopsis cumingi. The deduced amino acid sequence of HcPGRPS1 is composed of 235 residues which contains a conserved PGRP domain at the C-terminus. Sequence analysis showed that HcPGRPS1 shared high identities with other known PGRPs. The mRNA of HcPGRPS1 is constitutively expressed in a wide range of all tested tissues, with highest expression level in hepatopancreas, and its expression in tissues (gonad, nephridium, gill and foot) was up-regulated significantly after LPS or PGN stimulation (P<0.05). The recombinant protein of HcPGRPS1 exhibited binding activity and peptidoglycan-lytic amidase activity toward Lys-PGN from Staphylococcus aureus and DAP-PGN from Bacillus subtilis. Furthermore, recombinant HcPGRPS1 displayed strong antibacterial activity to both Gram-negative bacteria Escherichia coli, Aeromonas hydrophila, Aeromonas sobria and Gram-positive bacteria S. aureus in the presence of Zn(2+). These results suggested that HcPGRPS1 plays a multifunctional role in the defense and protection mechanisms of mussel innate immunity against infections. Copyright © 2013 Elsevier Ltd. All rights reserved.

Crystal Structures of Fatty Acid Amide Hydrolase Bound to the Carbamate Inhibitor URB597: Discovery of a Deacylating Water Molecule and Insight into Enzyme Inactivation

PubMed Central

Mileni, Mauro; Kamtekar, Satwik; Wood, David C.; Benson, Timothy E.; Cravatt, Benjamin F.; Stevens, Raymond C.

2010-01-01

The endocannabinoid system regulates a wide range of physiological processes including pain, inflammation, and cognitive/emotional states. URB597 is one of the best characterized covalent inhibitors of the endocannabinoid-degrading enzyme fatty acid amide hydrolase (FAAH). Here, we report the structure of the FAAH-URB597 complex at 2.3 Å resolution. The structure provides insights into mechanistic details of enzyme inactivation and experimental evidence of a previously uncharacterized active site water molecule that likely is involved in substrate deacylation. This water molecule is part of an extensive hydrogen-bonding network, and is coordinated indirectly to residues lining the cytosolic port of the enzyme. In order to corroborate our hypothesis concerning the role of this water molecule in FAAH’s catalytic mechanism, we determined the structure of FAAH conjugated to a urea-based inhibitor, PF-3845, to a higher resolution (2.4 Å) than previously reported. The higher resolution structure confirms the presence of the water molecule in a virtually identical location in the active site. Examination of the structures of serine hydrolases that are non-homologous to FAAH, such as elastase, trypsin, or chymotrypsin, shows a similarly positioned hydrolytic water molecule and suggest a functional convergence between the amidase signature enzymes and serine proteases. PMID:20493882

Use of enzyme inhibitors to evaluate the conversion pathways of ester and amide prodrugs: a case study example with the prodrug ceftobiprole medocaril.

PubMed

Eichenbaum, Gary; Skibbe, Jennifer; Parkinson, Andrew; Johnson, Mark D; Baumgardner, Dawn; Ogilvie, Brian; Usuki, Etsuko; Tonelli, Fred; Holsapple, Jeff; Schmitt-Hoffmann, Anne

2012-03-01

An approach was developed that uses enzyme inhibitors to support the assessment of the pathways that are responsible for the conversion of intravenously administered ester and amide prodrugs in different biological matrices. The methodology was applied to ceftobiprole medocaril (BAL5788), the prodrug of the cephalosporin antibiotic, ceftobiprole. The prodrug was incubated in plasma, postmitochondrial supernatant fractions from human liver (impaired and nonimpaired), kidney, and intestine as well as erythrocytes, in the presence and absence of different enzyme inhibitors (acetylcholinesterase, pseudocholinesterase, retinyl palmitoyl hydrolase, serine esterases, amidases, and cholinesterase). Hydrolysis was rapid, extensive, and not dependent on the presence of β-nicotinamide-adenine dinucleotide phosphate (reduced form) in all matrices tested, suggesting the involvement of carboxylesterases but not P450 enzymes. Hydrolysis in healthy human plasma was rapid and complete and only partially inhibited in the presence of paraoxonase inhibitors or in liver from hepatic impaired patients, suggesting involvement of nonparaoxonase pathways. The results demonstrate the utility of this approach in confirming the presence of multiple conversion pathways of intravenously administered prodrugs and in the case of BAL5788 demonstrated that this prodrug is unlikely to be affected by genetic polymorphisms, drug interactions, or other environmental factors that might inhibit or induce the enzymes involved in its conversion. Copyright © 2011 Wiley Periodicals, Inc.

A shifted repertoire of endocannabinoid genes in the zebrafish (Danio rerio).

PubMed

McPartland, J M; Glass, Michelle; Matias, Isabel; Norris, Ryan W; Kilpatrick, C William

2007-05-01

The zebrafish has served as a model organism for developmental biology. Sequencing its genome has expanded zebrafish research into physiology and drug-development testing. Several cannabinoid pharmaceuticals are in development, but expression of endocannabinoid receptors and enzymes remains unknown in this species. We conducted a bioinformatics analysis of the zebrafish genome using 17 human endocannabinoid genes as a reference set. Putative zebrafish orthologs were identified in filtered BLAST searches as reciprocal best hits. Orthology was confirmed by three in silico methods: phylogenetic testing, synteny analysis, and functional mapping. Zebrafish expressed orthologs of cannabinoid receptor 1, transient receptor potential channel vanilloid receptor 4, GPR55 receptor, fatty acid amide hydrolase 1, monoacylglycerol lipase, NAPE-selective phospholipase D, abhydrolase domain-containing protein 4, and diacylglycerol lipase alpha and beta; and paired paralogs of cannabinoid receptor 2, fatty acid amide hydrolase 2, peroxisome proliferator-activated receptor alpha, prostaglandin-endoperoxide synthase 2, and transient receptor potential cation channel subtype A1. Functional mapping suggested the orthologs of transient receptor potential vanilloid receptor 1 and peroxisome proliferator-activated receptor gamma lack specific amino acids critical for cannabinoid ligand binding. No orthologs of N-acylethanolamine acid amidase or protein tyrosine phosphatase, non-receptor type 22 were identified. In conclusion, the zebrafish genome expresses a shifted repertoire of endocannabinoid genes. In vitro analyses are warranted before using zebrafish for cannabinoid development testing.

A Thermophilic Phage Endolysin Fusion to a Clostridium perfringens-Specific Cell Wall Binding Domain Creates an Anti-Clostridium Antimicrobial with Improved Thermostability.

PubMed

Swift, Steven M; Seal, Bruce S; Garrish, Johnna K; Oakley, Brian B; Hiett, Kelli; Yeh, Hung-Yueh; Woolsey, Rebekah; Schegg, Kathleen M; Line, John Eric; Donovan, David M

2015-06-12

Clostridium perfringens is the third leading cause of human foodborne bacterial disease and is the presumptive etiologic agent of necrotic enteritis among chickens. Treatment of poultry with antibiotics is becoming less acceptable. Endolysin enzymes are potential replacements for antibiotics. Many enzymes are added to animal feed during production and are subjected to high-heat stress during feed processing. To produce a thermostabile endolysin for treating poultry, an E. coli codon-optimized gene was synthesized that fused the N-acetylmuramoyl-L-alanine amidase domain from the endolysin of the thermophilic bacteriophage ɸGVE2 to the cell-wall binding domain (CWB) from the endolysin of the C. perfringens-specific bacteriophage ɸCP26F. The resulting protein, PlyGVE2CpCWB, lysed C. perfringens in liquid and solid cultures. PlyGVE2CpCWB was most active at pH 8, had peak activity at 10 mM NaCl, 40% activity at 150 mM NaCl and was still 16% active at 600 mM NaCl. The protein was able to withstand temperatures up to 50° C and still lyse C. perfringens. Herein, we report the construction and characterization of a thermostable chimeric endolysin that could potentially be utilized as a feed additive to control the bacterium during poultry production.

Crystal structure of fatty acid amide hydrolase bound to the carbamate inhibitor URB597: discovery of a deacylating water molecule and insight into enzyme inactivation.

PubMed

Mileni, Mauro; Kamtekar, Satwik; Wood, David C; Benson, Timothy E; Cravatt, Benjamin F; Stevens, Raymond C

2010-07-23

The endocannabinoid system regulates a wide range of physiological processes including pain, inflammation, and cognitive/emotional states. URB597 is one of the best characterized covalent inhibitors of the endocannabinoid-degrading enzyme fatty acid amide hydrolase (FAAH). Here, we report the structure of the FAAH-URB597 complex at 2.3 A resolution. The structure provides insights into mechanistic details of enzyme inactivation and experimental evidence of a previously uncharacterized active site water molecule that likely is involved in substrate deacylation. This water molecule is part of an extensive hydrogen-bonding network and is coordinated indirectly to residues lining the cytosolic port of the enzyme. In order to corroborate our hypothesis concerning the role of this water molecule in FAAH's catalytic mechanism, we determined the structure of FAAH conjugated to a urea-based inhibitor, PF-3845, to a higher resolution (2.4 A) than previously reported. The higher-resolution structure confirms the presence of the water molecule in a virtually identical location in the active site. Examination of the structures of serine hydrolases that are non-homologous to FAAH, such as elastase, trypsin, or chymotrypsin, shows a similarly positioned hydrolytic water molecule and suggests a functional convergence between the amidase signature enzymes and serine proteases. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

Cloning, Characterization and Effect of TmPGRP-LE Gene Silencing on Survival of Tenebrio Molitor against Listeria monocytogenes Infection

PubMed Central

Tindwa, Hamisi; Patnaik, Bharat Bhusan; Kim, Dong Hyun; Mun, Seulgi; Jo, Yong Hun; Lee, Bok Luel; Lee, Yong Seok; Kim, Nam Jung; Han, Yeon Soo

2013-01-01

Peptidoglycan recognition proteins (PGRPs) are a family of innate immune molecules that recognize bacterial peptidoglycan. PGRP-LE, a member of the PGRP family, selectively binds to diaminopimelic acid (DAP)-type peptidoglycan to activate both the immune deficiency (Imd) and proPhenoloxidase (proPO) pathways in insects. A PGRP-LE-dependent induction of autophagy to control Listeria monocytogenes has also been reported. We identified and partially characterized a novel PGRP-LE homologue, from Tenebrio molitor and analyzed its functional role in the survival of the insect against infection by a DAP-type PGN containing intracellular pathogen, L. monocytogenes. The cDNA is comprised of an open reading frame (ORF) of 990 bp and encodes a polypeptide of 329 residues. TmPGRP-LE contains one PGRP domain, but lacks critical residues for amidase activity. Quantitative RT-PCR analysis showed a broad constitutive expression of the transcript at various stages of development spanning from larva to adult. RNAi mediated knockdown of the transcripts, followed by a challenge with L. monocytogenes, showed a significant reduction in survival rate of the larvae, suggesting a putative role of TmPGRP-LE in sensing and control of L. monocytogenes infection in T. molitor. These results implicate PGRP-LE as a defense protein necessary for survival of T. molitor against infection by L. monocytogenes. PMID:24240808

Cloning, characterization and effect of TmPGRP-LE gene silencing on survival of Tenebrio molitor against Listeria monocytogenes infection.

PubMed

Tindwa, Hamisi; Patnaik, Bharat Bhusan; Kim, Dong Hyun; Mun, Seulgi; Jo, Yong Hun; Lee, Bok Luel; Lee, Yong Seok; Kim, Nam Jung; Han, Yeon Soo

2013-11-14

Peptidoglycan recognition proteins (PGRPs) are a family of innate immune molecules that recognize bacterial peptidoglycan. PGRP-LE, a member of the PGRP family, selectively binds to diaminopimelic acid (DAP)-type peptidoglycan to activate both the immune deficiency (Imd) and proPhenoloxidase (proPO) pathways in insects. A PGRP-LE-dependent induction of autophagy to control Listeria monocytogenes has also been reported. We identified and partially characterized a novel PGRP-LE homologue, from Tenebrio molitor and analyzed its functional role in the survival of the insect against infection by a DAP-type PGN containing intracellular pathogen, L. monocytogenes. The cDNA is comprised of an open reading frame (ORF) of 990 bp and encodes a polypeptide of 329 residues. TmPGRP-LE contains one PGRP domain, but lacks critical residues for amidase activity. Quantitative RT-PCR analysis showed a broad constitutive expression of the transcript at various stages of development spanning from larva to adult. RNAi mediated knockdown of the transcripts, followed by a challenge with L. monocytogenes, showed a significant reduction in survival rate of the larvae, suggesting a putative role of TmPGRP-LE in sensing and control of L. monocytogenes infection in T. molitor. These results implicate PGRP-LE as a defense protein necessary for survival of T. molitor against infection by L. monocytogenes.

Contribution of the Staphylococcus aureus Atl AM and GL murein hydrolase activities in cell division, autolysis, and biofilm formation.

PubMed

Bose, Jeffrey L; Lehman, McKenzie K; Fey, Paul D; Bayles, Kenneth W

2012-01-01

The most prominent murein hydrolase of Staphylococcus aureus, AtlA, is a bifunctional enzyme that undergoes proteolytic cleavage to yield two catalytically active proteins, an amidase (AM) and a glucosaminidase (GL). Although the bifunctional nature of AtlA has long been recognized, most studies have focused on the combined functions of this protein in cell wall metabolism and biofilm development. In this study, we generated mutant derivatives of the clinical S. aureus isolate, UAMS-1, in which one or both of the AM and GL domains of AtlA have been deleted. Examination of these strains revealed that each mutant exhibited growth rates comparable to the parental strain, but showed clumping phenotypes and lysis profiles that were distinct from the parental strain and each other, suggesting distinct roles in cell wall metabolism. Given the known function of autolysis in the release of genomic DNA for use as a biofilm matrix molecule, we also tested the mutants in biofilm assays and found both AM and GL necessary for biofilm development. Furthermore, the use of enzymatically inactive point mutations revealed that both AM and GL must be catalytically active for S. aureus to form a biofilm. The results of this study provide insight into the relative contributions of AM and GL in S. aureus and demonstrate the contribution of Atl-mediated lysis in biofilm development.

Enzymatic analysis of α-ketoglutaramate—A biomarker for hyperammonemia

PubMed Central

Halámková, Lenka; Mailloux, Shay; Halámek, Jan; Cooper, Arthur J.L.; Katz, Evgeny

2012-01-01

Two enzymatic assays were developed for the analysis of α-ketoglutaramate (KGM)—an important biomarker of hepatic encephalopathy and other hyperammonemic diseases. In both procedures, KGM is first converted to α-ketoglutarate (KTG) via a reaction catalyzed by ω-amidase (AMD). In the first procedure, KTG generated in the AMD reaction initiates a biocatalytic cascade in which the concerted action of alanine transaminase and lactate dehydrogenase results in the oxidation of NADH. In the second procedure, KTG generated from KGM is reductively aminated, with the concomitant oxidation of NADH, in a reaction catalyzed by L-glutamic dehydrogenase. In both assays, the decrease in optical absorbance (λ=340 nm) corresponding to NADH oxidation is used to quantify concentrations of KGM. The two analytical procedures were applied to 50% (v/v) human serum diluted with aqueous solutions containing the assay components and spiked with concentrations of KGM estimated to be present in normal human plasma and in plasma from hyperammonemic patients. Since KTG is the product of AMD-catalyzed hydrolysis of KGM, in a separate study, this compound was used as a surrogate for KGM. Statistical analyses of samples mimicking the concentration of KGM assumed to be present in normal and pathological concentration ranges were performed. Both enzymatic assays for KGM were confirmed to discriminate between the predicted normal and pathophysiological concentrations of the analyte. The present study is the first step toward the development of a clinically useful probe for KGM analysis in biological fluids. PMID:23141304

Characterization of the Lytic Capability of a LysK-Like Endolysin, Lys-phiSA012, Derived from a Polyvalent Staphylococcus aureus Bacteriophage

PubMed Central

Nakamura, Tomohiro; Furusawa, Takaaki; Ohno, Hazuki; Takahashi, Hiromichi; Kitana, Junya; Usui, Masaru; Higuchi, Hidetoshi; Tamura, Yutaka

2018-01-01

Antibiotic-resistant bacteria (ARB) have spread widely and rapidly, with their increased occurrence corresponding with the increased use of antibiotics. Infections caused by Staphylococcus aureus have a considerable negative impact on human and livestock health. Bacteriophages and their peptidoglycan hydrolytic enzymes (endolysins) have received significant attention as novel approaches against ARB, including S. aureus. In the present study, we purified an endolysin, Lys-phiSA012, which harbors a cysteine/histidine-dependent amidohydrolase/peptidase (CHAP) domain, an amidase domain, and a SH3b cell wall binding domain, derived from a polyvalent S. aureus bacteriophage which we reported previously. We demonstrate that Lys-phiSA012 exhibits high lytic activity towards staphylococcal strains, including methicillin-resistant S. aureus (MRSA). Analysis of deletion mutants showed that only mutants possessing the CHAP and SH3b domains could lyse S. aureus, indicating that lytic activity of the CHAP domain depended on the SH3b domain. The presence of at least 1 mM Ca2+ and 100 µM Zn2+ enhanced the lytic activity of Lys-phiSA012 in a turbidity reduction assay. Furthermore, a minimum inhibitory concentration (MIC) assay showed that the addition of Lys-phiSA012 decreased the MIC of oxacillin. Our results suggest that endolysins are a promising approach for replacing current antimicrobial agents and may contribute to the proper use of antibiotics, leading to the reduction of ARB. PMID:29495305

Physiological aspects of fungi isolated from root nodules of faba bean (Vicia faba L.).

PubMed

Omar, S A; Abd-Alla, M H

2000-03-01

The present study was made to isolate and assess some physiological characteristics of root nodule-colonizing fungi. During this study, 17 fungal species were isolated from root nodule samples taken from faba bean plants (Vicia faba L.) collected from different sites at Assiut area (Egypt). The growth of faba bean plants in pots was significantly promoted by soil inoculation with most fungi. Growth was checked in pots with inocula of Cladosporium cladosporioides, Fusarium moniliforme, F: oxysporium, F solani, Macrophominia phaseolina and Rhizoctonia solani which were added separately. All growth-promoting fungi were capable of producing cellulase, pectin lyase, polygalacturonase, protease, urease, amidase, acid phosphatase, alkaline phosphatase and arylsulfatase in growth medium supplemented with the corresponding substrates. Four fungal species, Aspergillus awamori, A. flavus, Penicillium chrysogenum and Trichoderma koningii showed the highest rates of enzyme formation. The effect of the addition of six trace elements to the growth media at 30 micromol/ml on enzyme production revealed some dependency on species, enzyme and metal ion. Cd2+, Hg2+ and Zn2+ generally inhibited enzyme activity. Cu(1+), Fe3+ and Al3+ showed a stimulatory effect. Fungicides (afugan and tilt) and herbicides (brominal and fusilade) at 50 ppm generally promoted enzyme activity, but insecticides (kelthane and fenvalerate) caused some inhibition to enzyme activities. Salinization of the growth media with NaCl strongly inhibited the enzymatic activity of all fungi at concentrations between 0.5 and 1.5%.

Aryl acylamidase activity exhibited by butyrylcholinesterase is higher in chick than in horse, but much lower than in fetal calf serum.

PubMed

Weitnauer, E; Robitzki, A; Layer, P G

1998-10-02

Several side activities have been attributed to butyrylcholinesterase (BChE), including aryl acylamidase (AAA) activity, which is an amidase-like activity with unknown physiological function splitting the artificial substrate o-nitroacetanilide. For avians, extensive developmental data have pointed to neurogenetic functions of BChE, however, a possible AAA activity of BChE has not been studied. In this study, we first compare the relative levels of AAA exhibited by BChE in whole sera from chick, fetal calves (FCS) and horse. Remarkably, FCS exhibits a 400-fold higher ratio of AAA/BChE than horse and 80-fold higher than chick serum. We then show that an immunoisolated preparation of BChE from chicken serum presents significant activity for AAA. Both in sera and with the purified enzyme, the AAA activity is fully inhibited by anticholinesterase drugs, showing that AAA activity is exclusively conveyed by the BChE molecule. Noticeably, AAA inhibition even occurs at lower drug concentrations than that of BChE activity itself. Moreover, AAA is sensitive to serotonin. These data indicate that (1) AAA is a general feature of serum BChE of vertebrates including avians, (2) AAA is effectively inhibited by cholinergic and serotonergic agents, and (3) AAA may have a developmental role, since it is much pronounced in a serum from fetal animals. Functionally, deamination of neuropeptides, a link between cholinergic and serotonergic neurotransmitter systems, and roles in lipoprotein metabolism could be relevant.

Bacterial SPOR domains are recruited to septal peptidoglycan by binding to glycan strands that lack stem peptides

PubMed Central

Yahashiri, Atsushi; Jorgenson, Matthew A.; Weiss, David S.

2015-01-01

Bacterial SPOR domains bind peptidoglycan (PG) and are thought to target proteins to the cell division site by binding to “denuded” glycan strands that lack stem peptides, but uncertainties remain, in part because septal-specific binding has yet to be studied in a purified system. Here we show that fusions of GFP to SPOR domains from the Escherichia coli cell-division proteins DamX, DedD, FtsN, and RlpA all localize to septal regions of purified PG sacculi obtained from E. coli and Bacillus subtilis. Treatment of sacculi with an amidase that removes stem peptides enhanced SPOR domain binding, whereas treatment with a lytic transglycosylase that removes denuded glycans reduced SPOR domain binding. These findings demonstrate unequivocally that SPOR domains localize by binding to septal PG, that the physiologically relevant binding site is indeed a denuded glycan, and that denuded glycans are enriched in septal PG rather than distributed uniformly around the sacculus. Accumulation of denuded glycans in the septal PG of both E. coli and B. subtilis, organisms separated by 1 billion years of evolution, suggests that sequential removal of stem peptides followed by degradation of the glycan backbone is an ancient feature of PG turnover during bacterial cell division. Linking SPOR domain localization to the abundance of a structure (denuded glycans) present only transiently during biogenesis of septal PG provides a mechanism for coordinating the function of SPOR domain proteins with the progress of cell division. PMID:26305949

Cloning and analysis of peptidoglycan recognition protein-LC and immune deficiency from the diamondback moth, Plutella xylostella.

PubMed

Zhan, Ming-Yue; Yang, Pei-Jin; Rao, Xiang-Jun

2018-02-01

Peptidoglycan (PGN) exists in both Gram-negative and Gram-positive bacteria as a component of the cell wall. PGN is an important target to be recognized by the innate immune system of animals. PGN recognition proteins (PGRP) are responsible for recognizing PGNs. In Drosophila melanogaster, PGRP-LC and IMD (immune deficiency) are critical for activating the Imd pathway. Here, we report the cloning and analysis of PGRP-LC and IMD (PxPGRP-LC and PxIMD) from diamondback moth, Plutella xylostella (L.), the insect pest of cruciferous vegetables. PxPGRP-LC gene consists of six exons encoding a polypeptide of 308 amino acid residues with a transmembrane region and a PGRP domain. PxIMD cDNA encodes a polypeptide of 251 amino acid residues with a death domain. Sequence comparisons indicate that they are characteristic of Drosophila PGRP-LC and IMD homologs. PxPGRP-LC and PxIMD were expressed in various tissues and developmental stages. Their mRNA levels were affected by bacterial challenges. The PGRP domain of PxPGRP-LC lacks key residues for the amidase activity, but it can recognize two types of PGNs. Overexpression of full-length and deletion mutants in Drosophila S2 cells induced expression of some antimicrobial peptide genes. These results indicate that PxPGRP-LC and PxIMD may be involved in the immune signaling of P. xylostella. This study provides a foundation for further studies of the immune system of P. xylostella. © 2017 Wiley Periodicals, Inc.

Influence of sulfur oxidation state and steric bulk upon trifluoromethyl ketone (TFK) binding kinetics to carboxylesterases and fatty acid amide hydrolase (FAAH)

PubMed Central

Wheelock, Craig E.; Nishi, Kosuke; Ying, Andy; Jones, Paul D.; Colvin, Michael E.; Olmstead, Marilyn M.; Hammock, Bruce D.

2009-01-01

Carboxylesterases metabolize numerous exogenous and endogenous ester-containing compounds including the chemotherapeutic agent CPT-11, anti-influenza viral agent oseltamivir and many agrochemicals. Trifluoromethyl ketone (TFK)-containing compounds with a sulfur atom beta to the ketone moiety are some of the most potent carboxylesterase and amidase inhibitors identified to date. This study examined the effects of alkyl chain length (i.e., steric effects) and sulfur oxidation state upon TFK inhibitor potency (IC50) and binding kinetics (ki). The selective carboxylesterase inhibitor benzil was used as a non-TFK containing control. These effects were examined using two commercial esterases (porcine and rabbit liver esterase) and two human recombinant esterases (hCE-1 and hCE-2) as well as human recombinant fatty acid amide hydrolase (FAAH). In addition, the inhibition mechanism was examined using a combination of 1H NMR, X-ray crystallography and ab initio calculations. Overall, the data show that while sulfur oxidation state profoundly affects both inhibitor potency and binding kinetics, the steric effects dominate and override the contributions of sulfur oxidation. In addition, the data suggest that inclusion of a sulfur atom beta to the ketone contributes an increase (~5-fold) in inhibitor potency due to effects upon ketone hydration and/or intramolecular hydrogen bond formation. These results provide further information on the nature of the TFK binding interaction and will be useful in increasing our understanding of this basic biochemical process. PMID:18023188

Synthesis, properties, and application in peptide chemistry of a magnetically separable and reusable biocatalyst

NASA Astrophysics Data System (ADS)

Liria, Cleber W.; Ungaro, Vitor A.; Fernandes, Raphaella M.; Costa, Natália J. S.; Marana, Sandro R.; Rossi, Liane M.; Machini, M. Teresa

2014-11-01

Enzyme-catalyzed chemical processes are selective, very productive, and generate little waste. Nevertheless, they may be optimized using enzymes bound to solid supports, which are particularly important for protease-mediated reactions since proteases undergo fast autolysis in solution. Magnetic nanoparticles are suitable supports for this purpose owing to their high specific surface area and to be easily separated from reaction media. Here we describe the immobilization of bovine α-chymotrypsin (αCT) on silica-coated superparamagnetic nanoparticles (Fe3O4@silica) and the characterization of the enzyme-nanoparticle hybrid (Fe3O4@silica-αCT) in terms of protein content, properties, recovery from reaction media, application, and reuse in enzyme-catalyzed peptide synthesis. The results revealed that (i) full acid hydrolysis of the immobilized protease followed by amino acid analysis of the hydrolyzate is a reliable method to determine immobilization yield; (ii) despite showing lower amidase activity and a lower K cat/ K m value for a specific substrate than free αCT, the immobilized enzyme is chemically and thermally more stable, magnetically recoverable from reaction media, and can be consecutively reused for ten cycles to catalyze the amide bond hydrolysis and ester hydrolysis of the protected dipeptide Z-Ala-Phe-OMe. Altogether, these properties indicate the potential of Fe3O4@silica-αCT to act as an efficient, suitably stable, and reusable catalyst in amino acid, peptide, and protein chemistry as well as in proteomic studies.

Elucidating the pH-Dependent Structural Transition of T7 Bacteriophage Endolysin.

PubMed

Sharma, Meenakshi; Kumar, Dinesh; Poluri, Krishna Mohan

2016-08-23

Bacteriophages are the most abundant and diverse biological entities on earth. Bacteriophage endolysins are unique peptidoglycan hydrolases and have huge potential as effective enzybiotics in various infectious models. T7 bacteriophage endolysin (T7L), also known as N-acetylmuramoyl-l-alanine amidase or T7 lysozyme, is a 17 kDa protein that lyses a range of Gram-negative bacteria by hydrolyzing the amide bond between N-acetylmuramoyl residues and the l-alanine of the peptidoglycan layer. Although the activity profiles of several of the T7 family members have been known for many years, the molecular basis for their pH-dependent differential activity is not clear. In this study, we explored the pH-induced structural, stability, and activity characteristics of T7L by applying a variety of biophysical techniques and protein nuclear magnetic resonance (NMR) spectroscopy. Our studies established a reversible structural transition of T7L below pH 6 and the formation of a partially denatured conformation at pH 3. This low-pH conformation is thermally stable and exposed its hydrophobic pockets. Further, NMR relaxation measurements and structural analysis unraveled that T7L is highly dynamic in its native state and a network of His residues are responsible for the observed pH-dependent conformational dynamics and transitions. As bacteriophage chimeric and engineered endolysins are being developed as novel therapeutics against multiple drug resistance pathogens, we believe that our results are of great help in designing these entities as broadband antimicrobial and/or antibacterial agents.

Sex-dependent alterations in motor and anxiety-like behavior of aged bacterial peptidoglycan sensing molecule 2 knockout mice.

PubMed

Arentsen, Tim; Khalid, Roksana; Qian, Yu; Diaz Heijtz, Rochellys

2018-01-01

Peptidoglycan recognition proteins (PGRPs) are key sensing-molecules of the innate immune system that specifically detect bacterial peptidoglycan (PGN) and its derivates. PGRPs have recently emerged as potential key regulators of normal brain development and behavior. To test the hypothesis that PGRPs play a role in motor control and anxiety-like behavior in later life, we used 15-month old male and female peptidoglycan recognition protein 2 (Pglyrp2) knockout (KO) mice. Pglyrp2 is an N-acetylmuramyl-l-alanine amidase that hydrolyzes PGN between the sugar backbone and the peptide chain (which is unique among the mammalian PGRPs). Using a battery of behavioral tests, we demonstrate that Pglyrp2 KO male mice display decreased levels of anxiety-like behavior compared with wild type (WT) males. In contrast, Pglyrp2 KO female mice show reduced rearing activity and increased anxiety-like behavior compared to WT females. In the accelerated rotarod test, however, Pglyrp2 KO female mice performed better compared to WT females (i.e., they had longer latency to fall off the rotarod). Further, Pglyrp2 KO male mice exhibited decreased expression levels of synaptophysin, gephyrin, and brain-derived neurotrophic factor in the frontal cortex, but not in the amygdala. Pglyrp2 KO female mice exhibited increased expression levels of spinophilin and alpha-synuclein in the frontal cortex, while exhibiting decreased expression levels of synaptophysin, gephyrin and spinophilin in the amygdala. Our findings suggest a novel role for Pglyrp2asa key regulator of motor and anxiety-like behavior in late life. Copyright © 2017. Published by Elsevier Inc.

Comparative genomics of 9 novel Paenibacillus larvae bacteriophages

PubMed Central

Stamereilers, Casey; LeBlanc, Lucy; Yost, Diane; Amy, Penny S.; Tsourkas, Philippos K.

2016-01-01

ABSTRACT American Foulbrood Disease, caused by the bacterium Paenibacillus larvae, is one of the most destructive diseases of the honeybee, Apis mellifera. Our group recently published the sequences of 9 new phages with the ability to infect and lyse P. larvae. Here, we characterize the genomes of these P. larvae phages, compare them to each other and to other sequenced P. larvae phages, and putatively identify protein function. The phage genomes are 38–45 kb in size and contain 68–86 genes, most of which appear to be unique to P. larvae phages. We classify P. larvae phages into 2 main clusters and one singleton based on nucleotide sequence identity. Three of the new phages show sequence similarity to other sequenced P. larvae phages, while the remaining 6 do not. We identified functions for roughly half of the P. larvae phage proteins, including structural, assembly, host lysis, DNA replication/metabolism, regulatory, and host-related functions. Structural and assembly proteins are highly conserved among our phages and are located at the start of the genome. DNA replication/metabolism, regulatory, and host-related proteins are located in the middle and end of the genome, and are not conserved, with many of these genes found in some of our phages but not others. All nine phages code for a conserved N-acetylmuramoyl-L-alanine amidase. Comparative analysis showed the phages use the “cohesive ends with 3′ overhang” DNA packaging strategy. This work is the first in-depth study of P. larvae phage genomics, and serves as a marker for future work in this area. PMID:27738559

Structural characterization of the N-glycans of gpMuc from Mucuna pruriens seeds.

PubMed

Di Patrizi, Lisa; Rosati, Floriana; Guerranti, Roberto; Pagani, Roberto; Gerwig, Gerrit J; Kamerling, Johannis P

2006-11-01

Mucuna pruriens seeds are used in some countries as a human prophylactic oral anti-snake remedy. Aqueous extracts of M. pruriens seeds possess in vivo activity against cobra and viper venoms, and protect mice against Echis carinatus venom. It was recently demonstrated that the seed immunogen generating the antibody that cross-reacts with the venom proteins is a multiform glycoprotein (gpMuc), and the immunogenic properties of gpMuc seemed to mainly reside in its glycan chains. In the present study, gpMuc was found to contain only N-glycans. Part of the N-glycans could be released with peptide-(N (4)-(N-acetyl-beta -glucosaminyl)asparagine amidase F (PNGase F-sensitive N-glycans); the PNGase F-resistant N-glycans were PNGase A-sensitive. The oligosaccharides released were analyzed by a combination of MALDI-TOF mass spectrometry, HPLC profiling of 2-aminobenzamide-labelled derivatives and (1)H NMR spectroscopy. The PNGase F-sensitive N-glycans comprised a mixture of oligomannose-type structures ranging from Man(5)GlcNAc(2) to Man(9)GlcNAc(2), and two xylosylated structures, Xyl(1)Man(3)GlcNAc(2) and Xyl(1)Man(4)GlcNAc(2). The PNGase A-sensitive N-glycans, containing (alpha 1-3)-linked fucose, were identified as Fuc(1)Xyl(1)Man(2)GlcNAc(2) and Fuc(1)Xyl(1)Man(3)GlcNAc(2). In view of the determined N-glycan ensemble, the immunoreactivity of gpMuc was ascribed to the presence of core (beta 1-2)-linked xylose- and core alpha (1-3)-linked fucose-modified N-glycan chains.

Antiacanthain A: New proteases isolated from Bromelia antiacantha Bertol. (Bromeliaceae).

PubMed

Vallés, Diego; Cantera, Ana M B

2018-07-01

Crude extract (CE) from pulp of Bromelia antiacantha Bertol. mature fruit, contains at least 3 cysteine proteases with proteolytic activity. By single step cation exchange chromatography (Hi-trap SP-HP) of partially purified CE, the protease with the lowest pI, Antiacanthain A (AntA), was isolated. It showed maximum activity at pH9, and 75% of remaining activity was maintained over a wide pH range (pH6-10). The AntA activity exhibits a constant increase up to 70°C. Maintains almost 100% of its activity at 45 at pH6 and 9. A 60% of AntA was active by titration with specific inhibitor, E64. Amidasic activity was studied with pyroglutamyl-phenyl-leucyl-paranitroaniline (PFLNA) substrate having higher AntA catalytic efficiency of (k cat /K m =470s -1 M -1 ) relative to stem bromelain (k cat /K m =305s -1 M -1 ). Esterase activity using p-nitrophenyl esters of N-α-CBZ-l-Lysine (z-L-LysONp) showed a 10-fold higher catalytic efficiency for AntA (k cat /K m =6376s -1 M -1 ) relative to stem bromelain (k cat /K m =688s -1 M -1 ). Incubation with 8M Urea did not affect AntA activity and remained unchanged for 18h, with 6M GndHCl resulted in a 41% decrease in activity after 30min incubation, maintained this activity 18h. AntA exhibits high sequence identity with proteases of the Bromeliaceae family. Copyright © 2018 Elsevier B.V. All rights reserved.

Expression, purification, and characterization of a bifunctional 99-kDa peptidoglycan hydrolase from Pediococcus acidilactici ATCC 8042.

PubMed

García-Cano, Israel; Campos-Gómez, Manuel; Contreras-Cruz, Mariana; Serrano-Maldonado, Carlos Eduardo; González-Canto, Augusto; Peña-Montes, Carolina; Rodríguez-Sanoja, Romina; Sánchez, Sergio; Farrés, Amelia

2015-10-01

Pediococcus acidilactici ATCC 8042 is a lactic acid bacteria that inhibits pathogenic microorganisms such as Staphylococcus aureus through the production of two proteins with lytic activity, one of 110 kDa and the other of 99 kDa. The 99-kDa one has high homology to a putative peptidoglycan hydrolase (PGH) enzyme reported in the genome of P. acidilactici 7_4, where two different lytic domains have been identified but not characterized. The aim of this work was the biochemical characterization of the recombinant enzyme of 99 kDa. The enzyme was cloned and expressed successfully and retains its activity against Micrococcus lysodeikticus. It has a higher N-acetylglucosaminidase activity, but the N-acetylmuramoyl-L-alanine amidase can also be detected spectrophotometrically. The protein was then purified using gel filtration chromatography. Antibacterial activity showed an optimal pH of 6.0 and was stable between 5.0 and 7.0. The optimal temperature for activity was 60 °C, and all activity was lost after 1 h of incubation at 70 °C. The number of strains susceptible to the recombinant 99-kDa enzyme was lower than that susceptible to the mixture of the 110- and 99-kDa PGHs of P. acidilactici, a result that suggests synergy between these two enzymes. This is the first PGH from LAB that has been shown to possess two lytic sites. The results of this study will aid in the design of new antibacterial agents from natural origin that can combat foodborne disease and improve hygienic practices in the industrial sector.

Structural and biochemical studies on Vibrio cholerae Hsp31 reveals a novel dimeric form and Glutathione-independent Glyoxalase activity

PubMed Central

Dey, Sanjay

2017-01-01

Vibrio cholerae experiences a highly hostile environment at human intestine which triggers the induction of various heat shock genes. The hchA gene product of V. cholerae O395, referred to a hypothetical intracellular protease/amidase VcHsp31, is one such stress-inducible homodimeric protein. Our current study demonstrates that VcHsp31 is endowed with molecular chaperone, amidopeptidase and robust methylglyoxalase activities. Through site directed mutagenesis coupled with biochemical assays on VcHsp31, we have confirmed the role of residues in the vicinity of the active site towards amidopeptidase and methylglyoxalase activities. VcHsp31 suppresses the aggregation of insulin in vitro in a dose dependent manner. Through crystal structures of VcHsp31 and its mutants, grown at various temperatures, we demonstrate that VcHsp31 acquires two (Type-I and Type-II) dimeric forms. Type-I dimer is similar to EcHsp31 where two VcHsp31 monomers associate in eclipsed manner through several intersubunit hydrogen bonds involving their P-domains. Type-II dimer is a novel dimeric organization, where some of the intersubunit hydrogen bonds are abrogated and each monomer swings out in the opposite directions centering at their P-domains, like twisting of wet cloth. Normal mode analysis (NMA) of Type-I dimer shows similar movement of the individual monomers. Upon swinging, a dimeric surface of ~400Å2, mostly hydrophobic in nature, is uncovered which might bind partially unfolded protein substrates. We propose that, in solution, VcHsp31 remains as an equilibrium mixture of both the dimers. With increase in temperature, transformation to Type-II form having more exposed hydrophobic surface, occurs progressively accounting for the temperature dependent increase of chaperone activity of VcHsp31. PMID:28235098

The N-end rule pathway and regulation by proteolysis

PubMed Central

Varshavsky, Alexander

2011-01-01

The N-end rule relates the regulation of the in vivo half-life of a protein to the identity of its N-terminal residue. Degradation signals (degrons) that are targeted by the N-end rule pathway include a set called N-degrons. The main determinant of an N-degron is a destabilizing N-terminal residue of a protein. In eukaryotes, the N-end rule pathway is a part of the ubiquitin system and consists of two branches, the Ac/N-end rule and the Arg/N-end rule pathways. The Ac/N-end rule pathway targets proteins containing Nα-terminally acetylated (Nt-acetylated) residues. The Arg/N-end rule pathway recognizes unacetylated N-terminal residues and involves N-terminal arginylation. Together, these branches target for degradation a majority of cellular proteins. For example, more than 80% of human proteins are cotranslationally Nt-acetylated. Thus, most proteins harbor a specific degradation signal, termed AcN-degron, from the moment of their birth. Specific N-end rule pathways are also present in prokaryotes and in mitochondria. Enzymes that produce N-degrons include methionine-aminopeptidases, caspases, calpains, Nt-acetylases, Nt-amidases, arginyl-transferases, and leucyl-transferases. Regulated degradation of specific proteins by the N-end rule pathway mediates a legion of physiological functions, including the sensing of heme, oxygen, and nitric oxide; selective elimination of misfolded proteins; the regulation of DNA repair, segregation, and condensation; the signaling by G proteins; the regulation of peptide import, fat metabolism, viral and bacterial infections, apoptosis, meiosis, spermatogenesis, neurogenesis, and cardiovascular development; and the functioning of adult organs, including the pancreas and the brain. Discovered 25 years ago, this pathway continues to be a fount of biological insights. PMID:21633985

Murein Lytic Enzyme TgaA of Bifidobacterium bifidum MIMBb75 Modulates Dendritic Cell Maturation through Its Cysteine- and Histidine-Dependent Amidohydrolase/Peptidase (CHAP) Amidase Domain

PubMed Central

Zanoni, Ivan; Balzaretti, Silvia; Miriani, Matteo; Taverniti, Valentina; De Noni, Ivano; Presti, Ilaria; Stuknyte, Milda; Scarafoni, Alessio; Arioli, Stefania; Iametti, Stefania; Bonomi, Francesco; Mora, Diego; Karp, Matti; Granucci, Francesca

2014-01-01

Bifidobacteria are Gram-positive inhabitants of the human gastrointestinal tract that have evolved close interaction with their host and especially with the host's immune system. The molecular mechanisms underlying such interactions, however, are largely unidentified. In this study, we investigated the immunomodulatory potential of Bifidobacterium bifidum MIMBb75, a bacterium of human intestinal origin commercially used as a probiotic. Particularly, we focused our attention on TgaA, a protein expressed on the outer surface of MIMBb75's cells and homologous to other known bacterial immunoactive proteins. TgaA is a peptidoglycan lytic enzyme containing two active domains: lytic murein transglycosylase (LT) and cysteine- and histidine-dependent amidohydrolase/peptidase (CHAP). We ran immunological experiments stimulating dendritic cells (DCs) with the B. bifidum MIMBb75 and TgaA, with the result that both the bacterium and the protein activated DCs and triggered interleukin-2 (IL-2) production. In addition, we observed that the heterologous expression of TgaA in Bifidobacterium longum transferred to the bacterium the ability to induce IL-2. Subsequently, immunological experiments performed using two purified recombinant proteins corresponding to the single domains LT and CHAP demonstrated that the CHAP domain is the immune-reactive region of TgaA. Finally, we also showed that TgaA-dependent activation of DCs requires the protein CD14, marginally involves TRIF, and is independent of Toll-like receptor 4 (TLR4) and MyD88. In conclusion, our study suggests that the bacterial CHAP domain is a novel microbe-associated molecular pattern actively participating in the cross talk mechanisms between bifidobacteria and the host's immune system. PMID:24814791

SpxB is a suicide gene of Streptococcus pneumoniae and confers a selective advantage in an in vivo competitive colonization model.

PubMed

Regev-Yochay, Gili; Trzcinski, Krzysztof; Thompson, Claudette M; Lipsitch, Marc; Malley, Richard

2007-09-01

The human bacterial pathogen Streptococcus pneumoniae dies spontaneously upon reaching stationary phase. The extent of S. pneumoniae death at stationary phase is unusual in bacteria and has been conventionally attributed to autolysis by the LytA amidase. In this study, we show that spontaneous pneumococcal death is due to hydrogen peroxide (H(2)O(2)), not LytA, and that the gene responsible for H(2)O(2) production (spxB) also confers a survival advantage in colonization. Survival of S. pneumoniae in stationary phase was significantly prolonged by eliminating H(2)O(2) in any of three ways: chemically by supplementing the media with catalase, metabolically by growing the bacteria under anaerobic conditions, or genetically by constructing DeltaspxB mutants that do not produce H(2)O(2). Likewise, addition of H(2)O(2) to exponentially growing S. pneumoniae resulted in a death rate similar to that of cells in stationary phase. While DeltalytA mutants did not lyse at stationary phase, they died at a rate similar to that of the wild-type strain. Furthermore, we show that the death process induced by H(2)O(2) has features of apoptosis, as evidenced by increased annexin V staining, decreased DNA content, and appearance as assessed by transmission electron microscopy. Finally, in an in vivo rat model of competitive colonization, the presence of spxB conferred a selective advantage over the DeltaspxB mutant, suggesting an explanation for the persistence of this gene. We conclude that a suicide gene of pneumococcus is spxB, which induces an apoptosis-like death in pneumococci and confers a selective advantage in nasopharyngeal cocolonization.

Comparative genomics of four closely related Clostridium perfringens bacteriophages reveals variable evolution among core genes with therapeutic potential

PubMed Central

2011-01-01

Background Because biotechnological uses of bacteriophage gene products as alternatives to conventional antibiotics will require a thorough understanding of their genomic context, we sequenced and analyzed the genomes of four closely related phages isolated from Clostridium perfringens, an important agricultural and human pathogen. Results Phage whole-genome tetra-nucleotide signatures and proteomic tree topologies correlated closely with host phylogeny. Comparisons of our phage genomes to 26 others revealed three shared COGs; of particular interest within this core genome was an endolysin (PF01520, an N-acetylmuramoyl-L-alanine amidase) and a holin (PF04531). Comparative analyses of the evolutionary history and genomic context of these common phage proteins revealed two important results: 1) strongly significant host-specific sequence variation within the endolysin, and 2) a protein domain architecture apparently unique to our phage genomes in which the endolysin is located upstream of its associated holin. Endolysin sequences from our phages were one of two very distinct genotypes distinguished by variability within the putative enzymatically-active domain. The shared or core genome was comprised of genes with multiple sequence types belonging to five pfam families, and genes belonging to 12 pfam families, including the holin genes, which were nearly identical. Conclusions Significant genomic diversity exists even among closely-related bacteriophages. Holins and endolysins represent conserved functions across divergent phage genomes and, as we demonstrate here, endolysins can have significant variability and host-specificity even among closely-related genomes. Endolysins in our phage genomes may be subject to different selective pressures than the rest of the genome. These findings may have important implications for potential biotechnological applications of phage gene products. PMID:21631945

SpxB Is a Suicide Gene of Streptococcus pneumoniae and Confers a Selective Advantage in an In Vivo Competitive Colonization Model▿

PubMed Central

Regev-Yochay, Gili; Trzcinski, Krzysztof; Thompson, Claudette M.; Lipsitch, Marc; Malley, Richard

2007-01-01

The human bacterial pathogen Streptococcus pneumoniae dies spontaneously upon reaching stationary phase. The extent of S. pneumoniae death at stationary phase is unusual in bacteria and has been conventionally attributed to autolysis by the LytA amidase. In this study, we show that spontaneous pneumococcal death is due to hydrogen peroxide (H2O2), not LytA, and that the gene responsible for H2O2 production (spxB) also confers a survival advantage in colonization. Survival of S. pneumoniae in stationary phase was significantly prolonged by eliminating H2O2 in any of three ways: chemically by supplementing the media with catalase, metabolically by growing the bacteria under anaerobic conditions, or genetically by constructing ΔspxB mutants that do not produce H2O2. Likewise, addition of H2O2 to exponentially growing S. pneumoniae resulted in a death rate similar to that of cells in stationary phase. While ΔlytA mutants did not lyse at stationary phase, they died at a rate similar to that of the wild-type strain. Furthermore, we show that the death process induced by H2O2 has features of apoptosis, as evidenced by increased annexin V staining, decreased DNA content, and appearance as assessed by transmission electron microscopy. Finally, in an in vivo rat model of competitive colonization, the presence of spxB conferred a selective advantage over the ΔspxB mutant, suggesting an explanation for the persistence of this gene. We conclude that a suicide gene of pneumococcus is spxB, which induces an apoptosis-like death in pneumococci and confers a selective advantage in nasopharyngeal cocolonization. PMID:17631628

In vitro characterization of PlySK1249, a novel phage lysin, and assessment of its antibacterial activity in a mouse model of Streptococcus agalactiae bacteremia.

PubMed

Oechslin, Frank; Daraspe, Jean; Giddey, Marlyse; Moreillon, Philippe; Resch, Grégory

2013-12-01

Beta-hemolytic Streptococcus agalactiae is the leading cause of bacteremia and invasive infections. These diseases are treated with β-lactams or macrolides, but the emergence of less susceptible and even fully resistant strains is a cause for concern. New bacteriophage lysins could be promising alternatives against such organisms. They hydrolyze the bacterial peptidoglycan at the end of the phage cycle, in order to release the phage progeny. By using a bioinformatic approach to screen several beta-hemolytic streptococci, a gene coding for a lysin was identified on a prophage carried by Streptococcus dysgalactiae subsp. equisimilis SK1249. The gene product, named PlySK1249, harbored an original three-domain structure with a central cell wall-binding domain surrounded by an N-terminal amidase and a C-terminal CHAP domain. Purified PlySK1249 was highly lytic and bactericidal for S. dysgalactiae (2-log10 CFU/ml decrease within 15 min). Moreover, it also efficiently killed S. agalactiae (1.5-log10 CFU/ml decrease within 15 min) but not several streptococcal commensal species. We further investigated the activity of PlySK1249 in a mouse model of S. agalactiae bacteremia. Eighty percent of the animals (n = 10) challenged intraperitoneally with 10(6) CFU of S. agalactiae died within 72 h, whereas repeated injections of PlySK1249 (45 mg/kg 3 times within 24 h) significantly protected the mice (P < 0.01). Thus, PlySK1249, which was isolated from S. dysgalactiae, demonstrated high cross-lytic activity against S. agalactiae both in vitro and in vivo. These encouraging results indicated that PlySK1249 might represent a good candidate to be developed as a new enzybiotic for the treatment of systemic S. agalactiae infections.

X-ray crystallography and its impact on understanding bacterial cell wall remodeling processes.

PubMed

Büttner, Felix Michael; Renner-Schneck, Michaela; Stehle, Thilo

2015-02-01

The molecular structure of matter defines its properties and function. This is especially true for biological macromolecules such as proteins, which participate in virtually all biochemical processes. A three dimensional structural model of a protein is thus essential for the detailed understanding of its physiological function and the characterization of essential properties such as ligand binding and reaction mechanism. X-ray crystallography is a well-established technique that has been used for many years, but it is still by far the most widely used method for structure determination. A particular strength of this technique is the elucidation of atomic details of molecular interactions, thus providing an invaluable tool for a multitude of scientific projects ranging from the structural classification of macromolecules over the validation of enzymatic mechanisms or the understanding of host-pathogen interactions to structure-guided drug design. In the first part of this review, we describe essential methodological and practical aspects of X-ray crystallography. We provide some pointers that should allow researchers without a background in structural biology to assess the overall quality and reliability of a crystal structure. To highlight its potential, we then survey the impact X-ray crystallography has had on advancing an understanding of a class of enzymes that modify the bacterial cell wall. A substantial number of different bacterial amidase structures have been solved, mostly by X-ray crystallography. Comparison of these structures highlights conserved as well as divergent features. In combination with functional analyses, structural information on these enzymes has therefore proven to be a valuable template not only for understanding their mechanism of catalysis, but also for targeted interference with substrate binding. Copyright © 2015 Elsevier GmbH. All rights reserved.

Responsiveness to acidity via metal ion regulators mediates virulence in the gastric pathogen Helicobacter pylori.

PubMed

Bury-Moné, Stéphanie; Thiberge, Jean-Michel; Contreras, Monica; Maitournam, Aboubakar; Labigne, Agnès; De Reuse, Hilde

2004-07-01

The virulence of pathogenic bacteria is dependent on their adaptation to and survival in the stressful conditions encountered in their hosts. Helicobacter pylori exclusively colonizes the acid stomach of primates, making it an ideal study model. Little is known about how H. pylori responds to the moderately acidic conditions encountered at its colonization site, the gastric mucus layer. Thus, we compared gene expression profiles of H. pylori 26695 grown at neutral and acidic pH, and validated the data for a selection of genes by real-time polymerase chain reaction, dot-blots or enzymatic assays. During growth in acidic conditions, 56 genes were upregulated and 45 genes downregulated. We found that acidity is a signal modulating the expression of several virulence factors. Regulation of genes related to metal ion homeostasis suggests protective mechanisms involving diminished transport and enhanced storage. Genes encoding subunits of the F0F1 ATPase and of a newly identified Na+/H+ antiporter (NhaC-HP0946) were downregulated, revealing that this bacterium uses original mechanisms to control proton entry. Five of the upregulated genes encoded proteins controlling intracellular ammonia synthesis, including urease, amidase and formamidase, underlining the major role of this buffering compound in the protection against acidity in H. pylori. Regulatory networks and transcriptome analysis as well as enzymatic assays implicated two metal-responsive transcriptional regulators (NikR and Fur) and an essential two-component response regulator (HP0166, OmpR-like) as effectors of the H. pylori acid response. Finally, a nikR-fur mutant is attenuated in the mouse model, emphasizing the link between response to acidity, metal metabolism and virulence in this gastric pathogen.

Evolutionary history, structural features and biochemical diversity of the NlpC/P60 superfamily of enzymes.

PubMed

Anantharaman, Vivek; Aravind, L

2003-01-01

Peptidoglycan is hydrolyzed by a diverse set of enzymes during bacterial growth, development and cell division. The N1pC/P60 proteins define a family of cell-wall peptidases that are widely represented in various bacterial lineages. Currently characterized members are known to hydrolyze D-gamma-glutamyl-meso-diaminopimelate or N-acetylmuramate-L-alanine linkages. Detailed analysis of the N1pC/P60 peptidases showed that these proteins define a large superfamily encompassing several diverse groups of proteins. In addition to the well characterized P60-like proteins, this superfamily includes the AcmB/LytN and YaeF/YiiX families of bacterial proteins, the amidase domain of bacterial and kinetoplastid glutathionylspermidine synthases (GSPSs), and several proteins from eukaryotes, phages, poxviruses, positive-strand RNA viruses, and certain archaea. The eukaryotic members include lecithin retinol acyltransferase (LRAT), nematode developmental regulator Egl-26, and candidate tumor suppressor H-rev107. These eukaryotic proteins, along with the bacterial YaeF/poxviral G6R family, show a circular permutation of the catalytic domain. We identified three conserved residues, namely a cysteine, a histidine and a polar residue, that are involved in the catalytic activities of this superfamily. Evolutionary analysis of this superfamily shows that it comprises four major families, with diverse domain architectures in each of them. Several related, but distinct, catalytic activities, such as murein degradation, acyl transfer and amide hydrolysis, have emerged in the N1pC/P60 superfamily. The three conserved catalytic residues of this superfamily are shown to be equivalent to the catalytic triad of the papain-like thiol peptidases. The predicted structural features indicate that the N1pC/P60 enzymes contain a fold similar to the papain-like peptidases, transglutaminases and arylamine acetyltransferases.

Purification and characterization of an amidohydrolase for N4-long-chain fatty acyl derivatives of 1-beta-D-arabinofuranosylcytosine from mouse liver microsomes.

PubMed

Hori, K; Tsuruo, T; Tsukagoshi, S; Sakurai, Y

1984-03-01

N4-Long-chain fatty acyl-1-beta-D-arabinofuranosylcytosine amidohydrolase, a metabolizing enzyme for N4-acyl derivatives of 1-beta-D-arabinofuranosylcytosine with long-chain fatty acids, was purified from mouse liver microsomes. The purification was accomplished by solubilization of liver microsomes with Triton X-100, diethylaminoethyl cellulose chromatography, gel filtrations, hydroxyapatite chromatography, and concanavalin A:Sepharose chromatography. On sodium dodecyl sulfate:polyacrylamide gel electrophoresis, the purified enzyme preparation produced a single protein band with a molecular weight of 54,000. The enzyme had an optimal pH of 9.0, and the Michaelis constant for N4-palmitoyl-1-beta-D-arabinofuranosylcytosine was 67 microM. The thiols such as dithiothreitol or 2-mercaptoethanol stabilized the enzyme and stimulated its activity. p-Chloromercuribenzoate, N-ethylmaleimide, diisopropylfluorophosphate, and phenylmethylsulfonyl fluoride strongly inhibited the reaction. Bovine serum albumin markedly stimulated the enzyme activity, whereas detergents such as Triton X-100, deoxycholate, and sodium dodecyl sulfate had little effect. The enzyme did not require monovalent or divalent cations. Among the series of N4-acyl derivatives of 1-beta-D-arabinofuranosylcytosine with different chain lengths of acyl residues, the purified enzyme preferentially hydrolyzed the derivatives with long-chain fatty acids (C12 to C18), and N4-palmitoyl-1-beta-D-arabinofuranosylcytosine was the most susceptible. The purified enzyme was inactive on various N-acylamino acids, amides, oligopeptides, proteins, N-acylsphingosines (ceramides), triglyceride, lecithin, and lysolecithin. These results suggest that N4-long-chain fatty acyl-1-beta-D-arabinofuranosylcytosine amidohydrolase may be a new type of linear amidase.

The First Paenibacillus larvae Bacteriophage Endolysin (PlyPl23) with High Potential to Control American Foulbrood.

PubMed

Oliveira, Ana; Leite, Marta; Kluskens, Leon D; Santos, Sílvio B; Melo, Luís D R; Azeredo, Joana

2015-01-01

Endolysins, which are peptidoglycan-degrading enzymes expressed during the terminal stage of the reproduction cycle of bacteriophages, have great potential to control Gram-positive pathogens. This work describes the characterization of a novel endolysin (PlyPl23) encoded on the genome of Paenibacillus larvae phage phiIBB_Pl23 with high potential to control American foulbrood. This bacterial disease, caused by P. larvae, is widespread in North America and Europe and causes important economic losses in apiculture. The restriction to antibiotic residues in honey imposed by the EU legislation hinders its therapeutic use to combat American foulbrood and enforces the development of alternative antimicrobial methods. The new endolysin described herein has an N-acetylmuramoyl-L-alanine amidase catalytic domain and exhibits a broad-spectrum activity against common P. larvae genotypes. Moreover, the enzyme displays high antimicrobial activity in a range of pH that matches environmental conditions (pH between 5.0 and 7.0), showing its feasible application in the field. At pH 7.0, a concentration of 0.2 μM of enzyme was enough to lyse 104 CFU.mL-1 of P. larvae in no more than 2 h. The presence of sucrose and of the substances present in the larvae gut content did not affect the enzyme activity. Interestingly, an increase of activity was observed when PlyPl23 was previously incubated in royal jelly. Furthermore, in vivo safety evaluation assays demonstrated that this enzyme is not toxic to the bee larvae. The present work describes for the first time an endolysin encoded in a P. larvae phage that presents high potential to integrate a commercial product to control the problematic American foulbrood.

Formation and hydrolysis of amide bonds by lipase A from Candida antarctica; exceptional features.

PubMed

Liljeblad, Arto; Kallio, Pauli; Vainio, Marita; Niemi, Jarmo; Kanerva, Liisa T

2010-02-21

Various commercial lyophilized and immobilized preparations of lipase A from Candida antarctica (CAL-A) were studied for their ability to catalyze the hydrolysis of amide bonds in N-acylated alpha-amino acids, 3-butanamidobutanoic acid (beta-amino acid) and its ethyl ester. The activity toward amide bonds is highly untypical of lipases, despite the close mechanistic analogy to amidases which normally catalyze the corresponding reactions. Most CAL-A preparations cleaved amide bonds of various substrates with high enantioselectivity, although high variations in substrate selectivity and catalytic rates were detected. The possible role of contaminant protein species on the hydrolytic activity toward these bonds was studied by fractionation and analysis of the commercial lyophilized preparation of CAL-A (Cat#ICR-112, Codexis). In addition to minor impurities, two equally abundant proteins were detected, migrating on SDS-PAGE a few kDa apart around the calculated size of CAL-A. Based on peptide fragment analysis and sequence comparison both bands shared substantial sequence coverage with CAL-A. However, peptides at the C-terminal end constituting a motile domain described as an active-site flap were not identified in the smaller fragment. Separated gel filtration fractions of the two forms of CAL-A both catalyzed the amide bond hydrolysis of ethyl 3-butanamidobutanoate as well as the N-acylation of methyl pipecolinate. Hydrolytic activity towards N-acetylmethionine was, however, solely confined to the fractions containing the truncated form of CAL-A. These fractions were also found to contain a trace enzyme impurity identified in sequence analysis as a serine carboxypeptidase. The possible role of catalytic impurities versus the function of CAL-A in amide bond hydrolysis is further discussed in the paper.

Contribution of Three Bile-Associated Loci, bsh, pva, and btlB, to Gastrointestinal Persistence and Bile Tolerance of Listeria monocytogenes

PubMed Central

Begley, Máire; Sleator, Roy D.; Gahan, Cormac G. M.; Hill, Colin

2005-01-01

Listeria monocytogenes must resist the deleterious actions of bile in order to infect and subsequently colonize the human gastrointestinal tract. The molecular mechanisms used by the bacterium to resist bile and the influence of bile on pathogenesis are as yet largely unexplored. This study describes the analysis of three genes—bsh, pva, and btlB—previously annotated as bile-associated loci in the sequenced L. monocytogenes EGDe genome (lmo2067, lmo0446, and lmo0754, respectively). Analysis of deletion mutants revealed a role for all three genes in resisting the acute toxicity of bile and bile salts, particularly glycoconjugated bile salts at low pH. Mutants were unaffected in the other stress responses examined (acid, salt, and detergents). Bile hydrolysis assays demonstrate that L. monocytogenes possesses only one bile salt hydrolase gene, namely, bsh. Transcriptional analyses and activity assays revealed that, although it is regulated by both PrfA and σB, the latter appears to play the greater role in modulating bsh expression. In addition to being incapable of bile hydrolysis, a sigB mutant was shown to be exquisitely sensitive to bile salts. Furthermore, increased expression of sigB was detected under anaerobic conditions and during murine infection. A gene previously annotated as a possible penicillin V amidase (pva) or bile salt hydrolase was shown to be required for resistance to penicillin V but not penicillin G but did not demonstrate a role in bile hydrolysis. Finally, animal (murine) studies revealed an important role for both bsh and btlB in the intestinal persistence of L. monocytogenes. PMID:15664931

Diversity of Innate Immune Recognition Mechanism for Bacterial Polymeric meso-Diaminopimelic Acid-type Peptidoglycan in Insects

PubMed Central

Yu, Yang; Park, Ji-Won; Kwon, Hyun-Mi; Hwang, Hyun-Ok; Jang, In-Hwan; Masuda, Akiko; Kurokawa, Kenji; Nakayama, Hiroshi; Lee, Won-Jae; Dohmae, Naoshi; Zhang, Jinghai; Lee, Bok Luel

2010-01-01

In Drosophila, the synthesis of antimicrobial peptides in response to microbial infections is under the control of the Toll and immune deficiency (Imd) signaling pathway. The Toll signaling pathway responds mainly to the lysine-type peptidoglycan of Gram-positive bacteria and fungal β-1,3-glucan, whereas the Imd pathway responds to the meso-diaminopimelic acid (DAP)-type peptidoglycan of Gram-negative bacteria and certain Gram-positive bacilli. Recently we determined the activation mechanism of a Toll signaling pathway biochemically using a large beetle, Tenebrio molitor. However, DAP-type peptidoglycan recognition mechanism and its signaling pathway are still unclear in the fly and beetle. Here, we show that polymeric DAP-type peptidoglycan, but not its monomeric form, formed a complex with Tenebrio peptidoglycan recognition protein-SA, and this complex activated the three-step proteolytic cascade to produce processed Spätzle, a Toll receptor ligand, and induced Drosophila defensin-like antimicrobial peptide in Tenebrio larvae similarly to polymeric lysine-type peptidoglycan. Monomeric DAP-type peptidoglycan induced Drosophila diptericin-like antimicrobial peptide in Tenebrio hemocytes. In addition, both polymeric and monomeric DAP-type peptidoglycans induced expression of Tenebrio peptidoglycan recognition protein-SC2, which is DAP-type peptidoglycan-selective N-acetylmuramyl-l-alanine amidase that functions as a DAP-type peptidoglycan scavenger, appearing to function as a negative regulator of the DAP-type peptidoglycan signaling by cleaving DAP-type peptidoglycan in Tenebrio larvae. Taken together, these results demonstrate that molecular recognition mechanism for polymeric DAP-type peptidoglycan is different between Tenebrio larvae and Drosophila adults, providing biochemical evidences of biological diversity of innate immune responses in insects. PMID:20702416

Identification of 15 candidate structured noncoding RNA motifs in fungi by comparative genomics.

PubMed

Li, Sanshu; Breaker, Ronald R

2017-10-13

With the development of rapid and inexpensive DNA sequencing, the genome sequences of more than 100 fungal species have been made available. This dataset provides an excellent resource for comparative genomics analyses, which can be used to discover genetic elements, including noncoding RNAs (ncRNAs). Bioinformatics tools similar to those used to uncover novel ncRNAs in bacteria, likewise, should be useful for searching fungal genomic sequences, and the relative ease of genetic experiments with some model fungal species could facilitate experimental validation studies. We have adapted a bioinformatics pipeline for discovering bacterial ncRNAs to systematically analyze many fungal genomes. This comparative genomics pipeline integrates information on conserved RNA sequence and structural features with alternative splicing information to reveal fungal RNA motifs that are candidate regulatory domains, or that might have other possible functions. A total of 15 prominent classes of structured ncRNA candidates were identified, including variant HDV self-cleaving ribozyme representatives, atypical snoRNA candidates, and possible structured antisense RNA motifs. Candidate regulatory motifs were also found associated with genes for ribosomal proteins, S-adenosylmethionine decarboxylase (SDC), amidase, and HexA protein involved in Woronin body formation. We experimentally confirm that the variant HDV ribozymes undergo rapid self-cleavage, and we demonstrate that the SDC RNA motif reduces the expression of SAM decarboxylase by translational repression. Furthermore, we provide evidence that several other motifs discovered in this study are likely to be functional ncRNA elements. Systematic screening of fungal genomes using a computational discovery pipeline has revealed the existence of a variety of novel structured ncRNAs. Genome contexts and similarities to known ncRNA motifs provide strong evidence for the biological and biochemical functions of some newly found ncRNA motifs. Although initial examinations of several motifs provide evidence for their likely functions, other motifs will require more in-depth analysis to reveal their functions.

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

Wang, Tianyu; University of Chinese Academy of Sciences, Beijing 100049; Ding, Jinjing

The structure of the Tse3–Tsi3 complex associated with the bacterial type VI secretion system of P. aeruginosa has been solved and refined at 1.9 Å resolution. The structural basis of the recognition of the muramidase effector and its inactivation by its cognate immunity protein is revealed. The type VI secretion system (T6SS) is a bacterial protein-export machine that is capable of delivering virulence effectors between Gram-negative bacteria. The T6SS of Pseudomonas aeruginosa transports two lytic enzymes, Tse1 and Tse3, to degrade cell-wall peptidoglycan in the periplasm of rival bacteria that are competing for niches via amidase and muramidase activities, respectively.more » Two cognate immunity proteins, Tsi1 and Tsi3, are produced by the bacterium to inactivate the two antibacterial effectors, thereby protecting its siblings from self-intoxication. Recently, Tse1–Tsi1 has been structurally characterized. Here, the structure of the Tse3–Tsi3 complex is reported at 1.9 Å resolution. The results reveal that Tse3 contains a C-terminal catalytic domain that adopts a soluble lytic transglycosylase (SLT) fold in which three calcium-binding sites were surprisingly observed close to the catalytic Glu residue. The electrostatic properties of the substrate-binding groove are also distinctive from those of known structures with a similar fold. All of these features imply that a unique catalytic mechanism is utilized by Tse3 in cleaving glycosidic bonds. Tsi3 comprises a single domain showing a β-sandwich architecture that is reminiscent of the immunoglobulin fold. Three loops of Tsi3 insert deeply into the groove of Tse3 and completely occlude its active site, which forms the structural basis of Tse3 inactivation. This work is the first crystallographic report describing the three-dimensional structure of the Tse3–Tsi3 effector–immunity pair.« less

Identification of a novel gene cluster in the upstream region of the S-layer gene sbpA involved in cell wall metabolism of Lysinibacillus sphaericus CCM 2177 and characterization of the recombinantly produced autolysin and pyruvyl transferase.

PubMed

Pleschberger, Magdalena; Hildner, Florian; Rünzler, Dominik; Gelbmann, Nicola; Mayer, Harald F; Sleytr, Uwe B; Egelseer, Eva M

2013-05-01

The S-layer protein SbpA of Lysinibacillus sphaericus CCM 2177 assembles into a square (p4) lattice structure and recognizes a pyruvylated secondary cell wall polymer (SCWP) as the proper anchoring structure to the rigid cell wall layer. Sequencing of 8,004 bp in the 5'-upstream region of the S-layer gene sbpA led to five ORFs-encoding proteins involved in cell wall metabolism. After cloning and heterologous expression of ORF1 and ORF5 in Escherichia coli, the recombinant autolysin rAbpA and the recombinant pyruvyl transferase rCsaB were isolated, purified, and correct folding was confirmed by circular dichroism. Although rAbpA encoded by ORF1 showed amidase activity, it could attack whole cells of Ly. sphaericus CCM 2177 only after complete extraction of the S-layer lattice. Despite the presence of three S-layer-homology motifs on the N-terminal part, rAbpA did not show detectable affinity to peptidoglycan-containing sacculi, nor to isolated SCWP. As the molecular mass of the autolysin lies above the molecular exclusion limit of the S-layer, AbpA is obviously trapped within the rigid cell wall layer by the isoporous protein lattice. Immunogold-labeling of ultrathin-sectioned whole cells of Ly. sphaericus CCM 2177 with a polyclonal rabbit antiserum raised against rCsaB encoded by ORF5, and cell fractionation experiments demonstrated that the pyruvyl transferase was located in the cytoplasm, but not associated with cell envelope components including the plasma membrane. In enzymatic assays, rCsaB clearly showed pyruvyl transferase activity. By using RT-PCR, specific transcripts for each ORF could be detected. Cotranscription could be confirmed for ORF2 and ORF3.

Determination of the mitigating effect of colon-specific bioreversible codrugs of mycophenolic acid and aminosugars in an experimental colitis model in Wistar rats

PubMed Central

Chopade, Shakuntala Santosh; Dhaneshwar, Suneela Sunil

2018-01-01

AIM To design colon-targeted codrugs of mycophenolic acid (MPA) and aminosugars as a safer option to mycophenolate mofetil (MMF) in the management of inflammatory bowel disease. METHODS Codrugs were synthesized by coupling MPA with aminosugars (D-glucosamine and D-galactosamine) using EDCI coupling. The structures were confirmed by infrared radiation, nuclear magnetic resonance, mass spectroscopy and elemental analysis. The release profile of codrugs was extensively studied in aqueous buffers, upper gastrointestinal homogenates, faecal matter and caecal homogenates (in vitro) and rat blood (in vitro). Anti-colitic activity was assessed in 2,4,6-trinitrobezenesulfonic acid-induced colitis in Wistar rats by the estimation of various demarcating parameters. Statistical evaluation was performed by applying one-way and two-way ANOVA when compared with the disease control. RESULTS The prodrugs resisted activation in HCl buffer (pH 1.2) and stomach homogenates of rats with negligible hydrolysis in phosphate buffer (pH 7.4) and intestinal homogenates. Incubation with colon homogenates (in vitro) produced 76% to 89% release of MPA emphasizing colon-specific activation of codrugs and the release of MPA and aminosugars at the site of action. In the in vitro studies, the prodrug of MPA with D-glucosamine (MGLS) was selected which resulted in 68% release of MPA in blood. in vitro studies on MGLS revealed its colon-specific activation after a lag time of 8 h which could be ascribed to the hydrolytic action of N-acyl amidases found in the colon. The synthesized codrugs markedly diminished disease activity score and revived the disrupted architecture of the colon that was comparable to MMF but superior to MPA. CONCLUSION The significant attenuating effect of prodrugs and individual aminosugars on colonic inflammation proved that the rationale of the codrug approach is valid. PMID:29563754

A Personal Retrospective: Elevating Anandamide (AEA) by Targeting Fatty Acid Amide Hydrolase (FAAH) and the Fatty Acid Binding Proteins (FABPs).

PubMed

Deutsch, Dale G

2016-01-01

This perspective was adapted from a Career Achievement Award talk given at the International Cannabinoid Research Society Symposium in Bukovina, Poland on June 27, 2016. As a biochemist working in the neurosciences, I was always fascinated with neurotransmitter inactivation. In 1993 we identified an enzyme activity that breaks down anandamide. We called the enzyme anandamide amidase, now called FAAH. We and other laboratories developed FAAH inhibitors that were useful reagents that also proved to have beneficial physiological effects and until recently, new generations of inhibitors were in clinical trials. Nearly all neurotransmitters are water soluble and as such, require a transmembrane protein transporter to pass through the lipid membrane for inactivation inside the cell. However, using model systems, we and others have shown that this is unnecessary for anandamide, an uncharged hydrophobic molecule that readily diffuses across the cellular membrane. Interestingly, its uptake is driven by the concentration gradient resulting from its breakdown mainly by FAAH localized in the endoplasmic reticulum. We identified the FABPs as intracellular carriers that "solubilize" anandamide, transporting anandamide to FAAH. Compounds that bind to FABPs block AEA breakdown, raising its level. The cannabinoids (THC and CBD) also were discovered to bind FABPs and this may be one of the mechanisms by which CBD works in childhood epilepsy, raising anandamide levels. Targeting FABPs may be advantageous since they have some tissue specificity and do not require reactive serine hydrolase inhibitors, as does FAAH, with potential for off-target reactions. At the International Cannabis Research Society Symposium in 1992, Raphe Mechoulam revealed that his laboratory isolated an endogenous lipid molecule that binds to the CB1 receptor (cannabinoid receptor type 1) and this became the milestone paper published in December of that year describing anandamide (AEA, Devane et al., 1992). As to be expected, this discovery raised the issues of AEA's synthesis and breakdown.

Gene regulation associated with sexual development and female fertility in different isolates of Trichoderma reesei.

PubMed

Dattenböck, Christoph; Tisch, Doris; Schuster, Andre; Monroy, Alberto Alonso; Hinterdobler, Wolfgang; Schmoll, Monika

2018-01-01

Trichoderma reesei is one of the most frequently used filamentous fungi in industry for production of homologous and heterologous proteins. The ability to use sexual crossing in this fungus was discovered several years ago and opens up new perspectives for industrial strain improvement and investigation of gene regulation. Here we investigated the female sterile strain QM6a in comparison to the fertile isolate CBS999.97 and backcrossed derivatives of QM6a, which have regained fertility (FF1 and FF2 strains) in both mating types under conditions of sexual development. We found considerable differences in gene regulation between strains with the CBS999.97 genetic background and the QM6a background. Regulation patterns of QM6a largely clustered with the backcrossed FF1 and FF2 strains. Differential regulation between QM6a and FF1/FF2 as well as clustering of QM6a patterns with those of CBS999.97 strains was also observed. Consistent mating type dependent regulation was limited to mating type genes and those involved in pheromone response, but included also nta1 encoding a putative N-terminal amidase previously not associated with development. Comparison of female sterile QM6a with female fertile strains showed differential expression in genes encoding several transcription factors, metabolic genes and genes involved in secondary metabolism. Evaluation of the functions of genes specifically regulated under conditions of sexual development and of genes with highest levels of transcripts under these conditions indicated a relevance of secondary metabolism for sexual development in T. reesei . Among others, the biosynthetic genes of the recently characterized SOR cluster are in this gene group. However, these genes are not essential for sexual development, but rather have a function in protection and defence against competitors during reproduction.

A Personal Retrospective: Elevating Anandamide (AEA) by Targeting Fatty Acid Amide Hydrolase (FAAH) and the Fatty Acid Binding Proteins (FABPs)

PubMed Central

Deutsch, Dale G.

2016-01-01

This perspective was adapted from a Career Achievement Award talk given at the International Cannabinoid Research Society Symposium in Bukovina, Poland on June 27, 2016. As a biochemist working in the neurosciences, I was always fascinated with neurotransmitter inactivation. In 1993 we identified an enzyme activity that breaks down anandamide. We called the enzyme anandamide amidase, now called FAAH. We and other laboratories developed FAAH inhibitors that were useful reagents that also proved to have beneficial physiological effects and until recently, new generations of inhibitors were in clinical trials. Nearly all neurotransmitters are water soluble and as such, require a transmembrane protein transporter to pass through the lipid membrane for inactivation inside the cell. However, using model systems, we and others have shown that this is unnecessary for anandamide, an uncharged hydrophobic molecule that readily diffuses across the cellular membrane. Interestingly, its uptake is driven by the concentration gradient resulting from its breakdown mainly by FAAH localized in the endoplasmic reticulum. We identified the FABPs as intracellular carriers that “solubilize” anandamide, transporting anandamide to FAAH. Compounds that bind to FABPs block AEA breakdown, raising its level. The cannabinoids (THC and CBD) also were discovered to bind FABPs and this may be one of the mechanisms by which CBD works in childhood epilepsy, raising anandamide levels. Targeting FABPs may be advantageous since they have some tissue specificity and do not require reactive serine hydrolase inhibitors, as does FAAH, with potential for off-target reactions. At the International Cannabis Research Society Symposium in 1992, Raphe Mechoulam revealed that his laboratory isolated an endogenous lipid molecule that binds to the CB1 receptor (cannabinoid receptor type 1) and this became the milestone paper published in December of that year describing anandamide (AEA, Devane et al., 1992). As to be expected, this discovery raised the issues of AEA's synthesis and breakdown. PMID:27790143

Phenotypic and genotypic characterization of peptidoglycan hydrolases of Lactobacillus sakei

PubMed Central

Najjari, Afef; Amairi, Houda; Chaillou, Stéphane; Mora, Diego; Boudabous, Abdellatif; Zagorec, Monique; Ouzari, Hadda

2015-01-01

Lactobacillus sakei, a lactic acid bacterium naturally found in fresh meat and sea products, is considered to be one of the most important bacterial species involved in meat fermentation and bio-preservation. Several enzymes of Lb. sakei species contributing to microbial safeguarding and organoleptic properties of fermented-meat were studied. However, the specific autolytic mechanisms and associated enzymes involved in Lb. sakei are not well understood. The autolytic phenotype of 22 Lb. sakei strains isolated from Tunisian meat and seafood products was evaluated under starvation conditions, at pH 6.5 and 8.5, and in the presence of different carbon sources. A higher autolytic rate was observed when cells were grown in the presence of glucose and incubated at pH 6.5. Almost all strains showed high resistance to mutanolysin, indicating a minor role of muramidases in Lb. sakei cell lysis. Using Micrococcus lysodeikticus cells as a substrate in activity gels zymogram, peptidoglycan hydrolase (PGH) patterns for all strains was characterized by two lytic bands of ∼80 (B1) and ∼70 kDa (B2), except for strain BMG.167 which harbored two activity signals at a lower MW. Lytic activity was retained in high salt and in acid/basic conditions and was active toward cells of Lb. sakei, Listeria monocytogenes, Listeria ivanovii and Listeria innocua. Analysis of five putative PGH genes found in the Lb. sakei 23 K model strain genome, indicated that one gene, lsa1437, could encode a PGH (N-acetylmuramoyl-L-alanine amidase) containing B1 and B2 as isoforms. According to this hypothesis, strain BMG.167 showed an allelic version of lsa1437 gene deleted of one of the five LysM domains, leading to a reduction in the MW of lytic bands and the high autolytic rate of this strain. Characterization of autolytic phenotype of Lb. sakei should expand the knowledge of their role in fermentation processes where they represent the dominant species. PMID:26843981

Complete Reconstitution of the Vancomycin-Intermediate Staphylococcus aureus Phenotype of Strain Mu50 in Vancomycin-Susceptible S. aureus

PubMed Central

Sekine, Miwa; Hishinuma, Tomomi; Aiba, Yoshifumi; Hiramatsu, Keiichi

2016-01-01

Complete reconstitution of the vancomycin-intermediate Staphylococcus aureus (VISA) phenotype of strain Mu50 was achieved by sequentially introducing mutations into six genes of vancomycin-susceptible S. aureus (VSSA) strain N315ΔIP. The six mutated genes were detected in VISA strain Mu50 but not in N315ΔIP. Introduction of the mutation Ser329Leu into vraS, encoding the sensor histidine kinase of the vraSR two-component regulatory (TCR) system, and another mutation, Glu146Lys, into msrR, belonging to the LytR-CpsA-Psr (LCP) family, increased the level of vancomycin resistance to that detected in heterogeneous vancomycin-intermediate S. aureus (hVISA) strain Mu3. Introduction of two more mutations, Asn197Ser into graR of the graSR TCR system and His481Tyr into rpoB, encoding the β subunit of RNA polymerase, converted the hVISA strain into a VISA strain with the same level of vancomycin resistance as Mu50. Surprisingly, however, the constructed quadruple mutant strain ΔIP4 did not have a thickened cell wall, a cardinal feature of the VISA phenotype. Subsequent study showed that cell wall thickening was an inducible phenotype in the mutant strain, whereas it was a constitutive one in Mu50. Finally, introduction of the Ala297Val mutation into fdh2, which encodes a putative formate dehydrogenase, or a 67-amino-acid sequence deletion into sle1 [sle1(Δ67aa)], encoding the hydrolase of N-acetylmuramyl-l-alanine amidase in the peptidoglycan, converted inducible cell wall thickening into constitutive cell wall thickening. sle1(Δ67aa) was found to cause a drastic decrease in autolysis activity. Thus, all six mutated genes required for acquisition of the VISA phenotype were directly or indirectly involved in the regulation of cell physiology. The VISA phenotype seemed to be achieved through multiple genetic events accompanying drastic changes in cell physiology. PMID:27067329

Effects of tumour necrosis factor α upon the metabolism of the endocannabinoid anandamide in prostate cancer cells.

PubMed

Karlsson, Jessica; Gouveia-Figueira, Sandra; Alhouayek, Mireille; Fowler, Christopher J

2017-01-01

Tumour necrosis factor α (TNFα) is involved in the pathogenesis of prostate cancer, a disease where disturbances in the endocannabinoid system are seen. In the present study we have investigated whether treatment of DU145 human prostate cancer cells affects anandamide (AEA) catabolic pathways. Additionally, we have investigated whether cyclooxygenase-2 (COX-2) can regulate the uptake of AEA into cells. Levels of AEA synthetic and catabolic enzymes were determined by qPCR. AEA uptake and hydrolysis in DU145 and RAW264.7 macrophage cells were assayed using AEA labeled in the arachidonic and ethanolamine portions of the molecule, respectively. Levels of AEA, related N-acylethanolamines (NAEs), prostaglandins (PG) and PG-ethanolamines (PG-EA) in DU145 cells and medium were quantitated by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis. TNFα treatment of DU145 cells increased mRNA levels of PTSG2 (gene of COX-2) and decreased the mRNA of the AEA synthetic enzyme N-acyl-phosphatidylethanolamine selective phospholipase D. mRNA levels of the AEA hydrolytic enzymes fatty acid amide hydrolase (FAAH) and N-acylethanolamine-hydrolyzing acid amidase were not changed. AEA uptake in both DU145 and RAW264.7 cells was inhibited by FAAH inhibition, but not by COX-2 inhibition, even in RAW264.7 cells where the expression of this enzyme had greatly been induced by lipopolysaccharide + interferon γ treatment. AEA and related NAEs were detected in DU145 cells, but PGs and PGE2-EA were only detected when the cells had been preincubated with 100 nM AEA. The data demonstrate that in DU145 cells, TNFα treatment changes the relative expression of the enzymes involved in the hydrolytic and oxygenation catabolic pathways for AEA. In RAW264.7 cells, COX-2, in contrast to FAAH, does not regulate the cellular accumulation of AEA. Further studies are necessary to determine the extent to which inflammatory mediators are involved in the abnormal endocannabinoid signalling system in prostate cancer.

A novel type of peptidoglycan-binding domain highly specific for amidated D-Asp cross-bridge, identified in Lactobacillus casei bacteriophage endolysins.

PubMed

Regulski, Krzysztof; Courtin, Pascal; Kulakauskas, Saulius; Chapot-Chartier, Marie-Pierre

2013-07-12

Peptidoglycan hydrolases (PGHs) are responsible for bacterial cell lysis. Most PGHs have a modular structure comprising a catalytic domain and a cell wall-binding domain (CWBD). PGHs of bacteriophage origin, called endolysins, are involved in bacterial lysis at the end of the infection cycle. We have characterized two endolysins, Lc-Lys and Lc-Lys-2, identified in prophages present in the genome of Lactobacillus casei BL23. These two enzymes have different catalytic domains but similar putative C-terminal CWBDs. By analyzing purified peptidoglycan (PG) degradation products, we showed that Lc-Lys is an N-acetylmuramoyl-L-alanine amidase, whereas Lc-Lys-2 is a γ-D-glutamyl-L-lysyl endopeptidase. Remarkably, both lysins were able to lyse only Gram-positive bacterial strains that possess PG with D-Ala(4)→D-Asx-L-Lys(3) in their cross-bridge, such as Lactococcus casei, Lactococcus lactis, and Enterococcus faecium. By testing a panel of L. lactis cell wall mutants, we observed that Lc-Lys and Lc-Lys-2 were not able to lyse mutants with a modified PG cross-bridge, constituting D-Ala(4)→L-Ala-(L-Ala/L-Ser)-L-Lys(3); moreover, they do not lyse the L. lactis mutant containing only the nonamidated D-Asp cross-bridge, i.e. D-Ala(4)→D-Asp-L-Lys(3). In contrast, Lc-Lys could lyse the ampicillin-resistant E. faecium mutant with 3→3 L-Lys(3)-D-Asn-L-Lys(3) bridges replacing the wild-type 4→3 D-Ala(4)-D-Asn-L-Lys(3) bridges. We showed that the C-terminal CWBD of Lc-Lys binds PG containing mainly D-Asn but not PG with only the nonamidated D-Asp-containing cross-bridge, indicating that the CWBD confers to Lc-Lys its narrow specificity. In conclusion, the CWBD characterized in this study is a novel type of PG-binding domain targeting specifically the D-Asn interpeptide bridge of PG.

Bifidobacterium breve C50 secretes lipoprotein with CHAP domain recognized in aggregated form by TLR2.

PubMed

Scuotto, Angelo; Djorie, Serge; Colavizza, Michel; Romond, Pierre-Charles; Romond, Marie-Bénédicte

2014-12-01

Extracellular components secreted by Bifidobacterium breve C50 can induce maturation, high IL-10 production and prolonged survival of dendritic cells via a TLR2 pathway. In this study, the components were isolated from the supernatant by gel filtration chromatography. Antibodies raised against the major compounds with molecular weight above 600 kDa (Bb C50BC) also recognized compounds of lower molecular weight (200–600 kDa). TLR2 and TLR6 bound to the components already recognized by the antibodies. Trypsin digestion of Bb C50BC released three major peptides whose sequences displayed close similarities to a putative secreted protein with a CHAP amidase domain from B. breve. The 1300-bp genomic region corresponding to the hypothetical protein was amplified by PCR. The deduced polypeptide started with an N-terminal signal sequence of 45 amino acids, containing the lipobox motif (LAAC) with the cysteine in position 25, and 2 positively charged residues within the first 14 residues of the signal sequence. Lipid detection in Bb C50BC by GC/MS further supported the implication of a lipoprotein. Sugars were also detected in Bb C50BC. Close similarity with the glucan-binding protein B from Bifidobacterium animalis of two released peptides from Bb C50BC protein suggested that glucose moieties, possibly in glucan form, could be bound to the lipoprotein. Finally, heating at 100 °C for 5 min led to the breakdown of Bb C50BC in compounds of molecular weight below 67 kDa, which suggested that Bb C50BC was an aggregate. One might assume that a basic unit was formed by the lipoprotein bound putatively to glucan. Besides the other sugars and hexosamines recognized by galectin 1 were localized at the surface of the Bb C50BC aggregate. In conclusion, the extracellular components secreted by B. breve C50 were constituted of a lipoprotein putatively associated with glucose moieties and acting in an aggregating form as an agonist of TLR2/TLR6.

Endocannabinoids as endometrial inflammatory markers in lactating Holstein cows.

PubMed

Bonsale, R; Seyed Sharifi, R; Dirandeh, E; Hedayat, N; Mojtahedin, A; Ghorbanalinia, M; Abolghasemi, A

2018-06-01

The objective of this study was to consider endocannabinoid system as inflammatory markers in bovine endometrium to better understand the role of this system in regulating many of the functions that are related to inflammatory condition. At day 26 post-partum, fourteen cows were divided into two groups depending on the inflammatory condition: 1- subclinical endometritis (n = 7, with purulent or mucopurulent uterine discharge detectable in the vagina) and 2- healthy (n = 7, No (muco)) purulent discharge. Blood samples were collected at 26 and 30 days relative to calving to determine plasma tumour necrosis factor (TNF) and lipopolysaccharide-binding protein (LBP) concentrations; moreover, uterine biopsy was carried out on day 26 post-partum to measure mRNA abundance of TNF, interleukin-1B (IL1B), interleukin-6 (IL-6), C-X-C motif chemokine ligand 8 (CXCL8), endocannabinoid receptor (CNR2), N-acyl phosphatidylethanolamine phospholipase D (NAPEPLD), fatty acid amide hydrolase (FAAH), N-acylethanolamine acid amidase (NAAA) and monoglyceride lipase (MGLL) by real-time PCR. Results showed mean plasma concentrations of TNF and LBP were lower in healthy cows compared to subclinical endometritis cows (p < .05). Relative mRNA expression for NAAA and FAAH was decreased (p < .05), and relative mRNA expression for CNR2 and NAPEPLD increased in cows with subclinical endometritis compared to healthy cows. In conclusion, relative mRNA expression of TNF, IL1B and CXCL8 and plasma concentration of LBP increased during inflammatory condition along with decreased endocannabinoids hydrolyzing enzyme (NAAA and FAAH), increased enzymes that synthesize endocannabinoids (NAPEPLD) and relative gene expression of the endocannabinoid receptor; together, these contribute to increased endocannabinoids levels during inflammation. Overall, we provide evidence that endocannabinoid system is altered in endometrium tissue during inflammation through increased mRNA expression of CNR2 and synthesis enzyme and decreased mRNA expression of hydrolyzing enzymes interfere with pro-cytokine production and signalling, which may interfere with the onset and progression of inflammation. © 2018 Blackwell Verlag GmbH.

Structures of enzyme-intermediate complexes of yeast Nit2: insights into its catalytic mechanism and different substrate specificity compared with mammalian Nit2.

PubMed

Liu, Hejun; Gao, Yongxiang; Zhang, Mengying; Qiu, Xiaoting; Cooper, Arthur J L; Niu, Liwen; Teng, Maikun

2013-08-01

The Nit (nitrilase-like) protein subfamily constitutes branch 10 of the nitrilase superfamily. Nit proteins are widely distributed in nature. Mammals possess two members of the Nit subfamily, namely Nit1 and Nit2. Based on sequence similarity, yeast Nit2 (yNit2) is a homologue of mouse Nit1, a tumour-suppressor protein whose substrate specificity is not yet known. Previous studies have shown that mammalian Nit2 (also a putative tumour suppressor) is identical to ω-amidase, an enzyme that catalyzes the hydrolysis of α-ketoglutaramate (α-KGM) and α-ketosuccinamate (α-KSM) to α-ketoglutarate (α-KG) and oxaloacetate (OA), respectively. In the present study, crystal structures of wild-type (WT) yNit2 and of WT yNit2 in complex with α-KG and with OA were determined. In addition, the crystal structure of the C169S mutant of yNit2 (yNit2-C169S) in complex with an endogenous molecule of unknown structure was also solved. Analysis of the structures revealed that α-KG and OA are covalently bound to Cys169 by the formation of a thioester bond between the sulfhydryl group of the cysteine residue and the γ-carboxyl group of α-KG or the β-carboxyl group of OA, reflecting the presumed reaction intermediates. However, an enzymatic assay suggests that α-KGM is a relatively poor substrate of yNit2. Finally, a ligand was found in the active site of yNit2-C169S that may be a natural substrate of yNit2 or an endogenous regulator of enzyme activity. These crystallographic analyses provide information on the mode of substrate/ligand binding at the active site of yNit2 and insights into the catalytic mechanism. These findings suggest that yNit2 may have broad biological roles in yeast, especially in regard to nitrogen homeostasis, and provide a framework for the elucidation of the substrate specificity and biological role of mammalian Nit1.

Preclinical evaluation of novel urinary biomarkers of cadmium nephrotoxicity

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

Prozialeck, Walter C.; Edwards, Joshua R.; Vaidya, Vishal S.

2009-08-01

As a result of the widespread use of Cd in industry and its extensive dissemination in the environment, there has been considerable interest in the identification of early biomarkers of Cd-induced kidney injury. Kim-1 is a transmembrane glycoprotein that is not detectable in normal kidney, but is up-regulated and shed into the urine following ischemic or nephrotoxic injury. Recent studies utilizing a sub-chronic model of Cd exposure in the rat have shown that Kim-1 is an early urinary marker of Cd-induced kidney injury. Kim-1 was detected in the urine 4-5 weeks before the onset of proteinuria and 1-3 weeks beforemore » the appearance of urinary metallothionein and Clara cell protein 16, which are standard markers of Cd nephrotoxicity. In the present study, we have compared the time course for the appearance of Kim-1 in the urine with the time course for the appearance of alpha glutathione-S-transferase ({alpha}-GST), N-acetyl-{beta}-D-glucose amidase (NAG) and Cd, each of which have been used or proposed as urinary markers of Cd nephrotoxicity. Adult male Sprague-Dawley rats were given daily subcutaneous injections of 0.6 mg (5.36 {mu}moles)/kg Cd, 5 days per week for up to 12 weeks. One day each week, 24 h urine samples were collected and analyzed for protein, creatinine and the various markers. The results showed that significant levels of Kim-1 appeared in the urine as early as 6 weeks into the treatment protocol and then continued to rise for the remainder of the 12 week treatment period. By contrast, significant levels of {alpha}-GST and NAG did not appear in the urine until 8 and 12 weeks, respectively, while proteinuria was not evident until 10 weeks. The urinary excretion of Cd was below the level of detection until week 4 and then showed a slow, linear increase over the next 6 weeks before increasing markedly between weeks 10 and 12. These results provide additional evidence that Kim-1 is a sensitive biomarker of the early stages of Cd-induced proximal tubule injury.« less

Insights into substrate specificity of NlpC/P60 cell wall hydrolases containing bacterial SH3 domains

DOE PAGES

Xu, Qingping; Mengin-Lecreulx, Dominique; Liu, Xueqian W.; ...

2015-09-15

Bacterial SH3 (SH3b) domains are commonly fused with papain-like Nlp/P60 cell wall hydrolase domains. To understand how the modular architecture of SH3b and NlpC/P60 affects the activity of the catalytic domain, three putative NlpC/P60 cell wall hydrolases were biochemically and structurally characterized. In addition, these enzymes all have γ-d-Glu-A 2pm (A 2pm is diaminopimelic acid) cysteine amidase (ordl-endopeptidase) activities but with different substrate specificities. One enzyme is a cell wall lysin that cleaves peptidoglycan (PG), while the other two are cell wall recycling enzymes that only cleave stem peptides with an N-terminall-Ala. Their crystal structures revealed a highly conserved structuremore » consisting of two SH3b domains and a C-terminal NlpC/P60 catalytic domain, despite very low sequence identity. Interestingly, loops from the first SH3b domain dock into the ends of the active site groove of the catalytic domain, remodel the substrate binding site, and modulate substrate specificity. Two amino acid differences at the domain interface alter the substrate binding specificity in favor of stem peptides in recycling enzymes, whereas the SH3b domain may extend the peptidoglycan binding surface in the cell wall lysins. Remarkably, the cell wall lysin can be converted into a recycling enzyme with a single mutation.Peptidoglycan is a meshlike polymer that envelops the bacterial plasma membrane and bestows structural integrity. Cell wall lysins and recycling enzymes are part of a set of lytic enzymes that target covalent bonds connecting the amino acid and amino sugar building blocks of the PG network. These hydrolases are involved in processes such as cell growth and division, autolysis, invasion, and PG turnover and recycling. To avoid cleavage of unintended substrates, these enzymes have very selective substrate specificities. Our biochemical and structural analysis of three modular NlpC/P60 hydrolases, one lysin, and two recycling enzymes, show that they may have evolved from a common molecular architecture, where the substrate preference is modulated by local changes. These results also suggest that new pathways for recycling PG turnover products, such as tracheal cytotoxin, may have evolved in bacteria in the human gut microbiome that involve NlpC/P60 cell wall hydrolases.« less

Insights into substrate specificity of NlpC/P60 cell wall hydrolases containing bacterial SH3 domains

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

Xu, Qingping; Mengin-Lecreulx, Dominique; Liu, Xueqian W.

Bacterial SH3 (SH3b) domains are commonly fused with papain-like Nlp/P60 cell wall hydrolase domains. To understand how the modular architecture of SH3b and NlpC/P60 affects the activity of the catalytic domain, three putative NlpC/P60 cell wall hydrolases were biochemically and structurally characterized. In addition, these enzymes all have γ-d-Glu-A 2pm (A 2pm is diaminopimelic acid) cysteine amidase (ordl-endopeptidase) activities but with different substrate specificities. One enzyme is a cell wall lysin that cleaves peptidoglycan (PG), while the other two are cell wall recycling enzymes that only cleave stem peptides with an N-terminall-Ala. Their crystal structures revealed a highly conserved structuremore » consisting of two SH3b domains and a C-terminal NlpC/P60 catalytic domain, despite very low sequence identity. Interestingly, loops from the first SH3b domain dock into the ends of the active site groove of the catalytic domain, remodel the substrate binding site, and modulate substrate specificity. Two amino acid differences at the domain interface alter the substrate binding specificity in favor of stem peptides in recycling enzymes, whereas the SH3b domain may extend the peptidoglycan binding surface in the cell wall lysins. Remarkably, the cell wall lysin can be converted into a recycling enzyme with a single mutation.Peptidoglycan is a meshlike polymer that envelops the bacterial plasma membrane and bestows structural integrity. Cell wall lysins and recycling enzymes are part of a set of lytic enzymes that target covalent bonds connecting the amino acid and amino sugar building blocks of the PG network. These hydrolases are involved in processes such as cell growth and division, autolysis, invasion, and PG turnover and recycling. To avoid cleavage of unintended substrates, these enzymes have very selective substrate specificities. Our biochemical and structural analysis of three modular NlpC/P60 hydrolases, one lysin, and two recycling enzymes, show that they may have evolved from a common molecular architecture, where the substrate preference is modulated by local changes. These results also suggest that new pathways for recycling PG turnover products, such as tracheal cytotoxin, may have evolved in bacteria in the human gut microbiome that involve NlpC/P60 cell wall hydrolases.« less

Transcriptional Analysis and Subcellular Protein Localization Reveal Specific Features of the Essential WalKR System in Staphylococcus aureus

PubMed Central

Poupel, Olivier; Moyat, Mati; Groizeleau, Julie; Antunes, Luísa C. S.; Gribaldo, Simonetta; Msadek, Tarek; Dubrac, Sarah

2016-01-01

The WalKR two-component system, controlling cell wall metabolism, is highly conserved among Bacilli and essential for cell viability. In Staphylococcus aureus, walR and walK are followed by three genes of unknown function: walH, walI and walJ. Sequence analysis and transcript mapping revealed a unique genetic structure for this locus in S. aureus: the last gene of the locus, walJ, is transcribed independently, whereas transcription of the tetra-cistronic walRKHI operon occurred from two independent promoters located upstream from walR. Protein topology analysis and protein-protein interactions in E. coli as well as subcellular localization in S. aureus allowed us to show that WalH and WalI are membrane-bound proteins, which associate with WalK to form a complex at the cell division septum. While these interactions suggest that WalH and WalI play a role in activity of the WalKR regulatory pathway, deletion of walH and/or walI did not have a major effect on genes whose expression is strongly dependent on WalKR or on associated phenotypes. No effect of WalH or WalI was seen on tightly controlled WalKR regulon genes such as sle1 or saouhsc_00773, which encodes a CHAP-domain amidase. Of the genes encoding the two major S. aureus autolysins, AtlA and Sle1, only transcription of atlA was increased in the ΔwalH or ΔwalI mutants. Likewise, bacterial autolysis was not increased in the absence of WalH and/or WalI and biofilm formation was lowered rather than increased. Our results suggest that contrary to their major role as WalK inhibitors in B. subtilis, the WalH and WalI proteins have evolved a different function in S. aureus, where they are more accessory. A phylogenomic analysis shows a striking conservation of the 5 gene wal cluster along the evolutionary history of Bacilli, supporting the key importance of this signal transduction system, and indicating that the walH and walI genes were lost in the ancestor of Streptococcaceae, leading to their atypical 3 wal gene cluster, walRKJ. PMID:26999783

Identification of In Vivo-Expressed Immunogenic Proteins by Serological Proteome Analysis of the Bacillus anthracis Secretome▿ †

PubMed Central

Chitlaru, Theodor; Gat, Orit; Grosfeld, Haim; Inbar, Itzhak; Gozlan, Yael; Shafferman, Avigdor

2007-01-01

In a previous comparative proteomic study of Bacillus anthracis examining the influence of the virulence plasmids and of various growth conditions on the composition of the bacterial secretome, we identified 64 abundantly expressed proteins (T. Chitlaru, O. Gat, Y. Gozlan, N. Ariel, and A. Shafferman, J. Bacteriol. 188:3551-3571, 2006). Using a battery of sera from B. anthracis-infected animals, in the present study we demonstrated that 49 of these proteins are immunogenic. Thirty-eight B. anthracis immunogens are documented in this study for the first time. The relative immunogenicities of the 49 secreted proteins appear to span a >10,000-fold range. The proteins eliciting the highest humoral response in the course of infection include, in addition to the well-established immunogens protective antigen (PA), Sap, and EA1, GroEL (BA0267), AhpC (BA0345), MntA (BA3189), HtrA (BA3660), 2,3-cyclic nucleotide diesterase (BA4346), collagen adhesin (BAS5205), an alanine amidase (BA0898), and an endopeptidase (BA1952), as well as three proteins having unknown functions (BA0796, BA0799, and BA0307). Of these 14 highly potent secreted immunogens, 11 are known to be associated with virulence and pathogenicity in B. anthracis or in other bacterial pathogens. Combining the results reported here with the results of a similar study of the membranal proteome of B. anthracis (T. Chitlaru, N. Ariel, A. Zvi, M. Lion, B. Velan, A. Shafferman, and E. Elhanany, Proteomics 4:677-691, 2004) and the results obtained in a functional genomic search for immunogens (O. Gat, H. Grosfeld, N. Ariel, I. Inbar, G. Zaide, Y. Broder, A. Zvi, T. Chitlaru, Z. Altboum, D. Stein, S. Cohen, and A. Shafferman, Infect. Immun. 74:3987-4001, 2006), we generated a list of 84 in vivo-expressed immunogens for future evaluation for vaccine development, diagnostics, and/or therapeutic intervention. In a preliminary study, the efficacies of eight immunogens following DNA immunization of guinea pigs were compared to the efficacy of a PA DNA vaccine. All eight immunogens induced specific high antibody titers comparable to the titers elicited by PA; however, unlike PA, none of them provided protection against a lethal challenge (50 50% lethal doses) of virulent B. anthracis strain Vollum spores. PMID:17353282

Insights into Substrate Specificity of NlpC/P60 Cell Wall Hydrolases Containing Bacterial SH3 Domains

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

Xu, Qingping; Mengin-Lecreulx, Dominique; Liu, Xueqian W.

ABSTRACT Bacterial SH3 (SH3b) domains are commonly fused with papain-like Nlp/P60 cell wall hydrolase domains. To understand how the modular architecture of SH3b and NlpC/P60 affects the activity of the catalytic domain, three putative NlpC/P60 cell wall hydrolases were biochemically and structurally characterized. These enzymes all have γ-d-Glu-A 2pm (A 2pm is diaminopimelic acid) cysteine amidase (ordl-endopeptidase) activities but with different substrate specificities. One enzyme is a cell wall lysin that cleaves peptidoglycan (PG), while the other two are cell wall recycling enzymes that only cleave stem peptides with an N-terminall-Ala. Their crystal structures revealed a highly conserved structure consistingmore » of two SH3b domains and a C-terminal NlpC/P60 catalytic domain, despite very low sequence identity. Interestingly, loops from the first SH3b domain dock into the ends of the active site groove of the catalytic domain, remodel the substrate binding site, and modulate substrate specificity. Two amino acid differences at the domain interface alter the substrate binding specificity in favor of stem peptides in recycling enzymes, whereas the SH3b domain may extend the peptidoglycan binding surface in the cell wall lysins. Remarkably, the cell wall lysin can be converted into a recycling enzyme with a single mutation. IMPORTANCEPeptidoglycan is a meshlike polymer that envelops the bacterial plasma membrane and bestows structural integrity. Cell wall lysins and recycling enzymes are part of a set of lytic enzymes that target covalent bonds connecting the amino acid and amino sugar building blocks of the PG network. These hydrolases are involved in processes such as cell growth and division, autolysis, invasion, and PG turnover and recycling. To avoid cleavage of unintended substrates, these enzymes have very selective substrate specificities. Our biochemical and structural analysis of three modular NlpC/P60 hydrolases, one lysin, and two recycling enzymes, show that they may have evolved from a common molecular architecture, where the substrate preference is modulated by local changes. These results also suggest that new pathways for recycling PG turnover products, such as tracheal cytotoxin, may have evolved in bacteria in the human gut microbiome that involve NlpC/P60 cell wall hydrolases.« less

Transcriptional Analysis and Subcellular Protein Localization Reveal Specific Features of the Essential WalKR System in Staphylococcus aureus.

PubMed

Poupel, Olivier; Moyat, Mati; Groizeleau, Julie; Antunes, Luísa C S; Gribaldo, Simonetta; Msadek, Tarek; Dubrac, Sarah

2016-01-01

The WalKR two-component system, controlling cell wall metabolism, is highly conserved among Bacilli and essential for cell viability. In Staphylococcus aureus, walR and walK are followed by three genes of unknown function: walH, walI and walJ. Sequence analysis and transcript mapping revealed a unique genetic structure for this locus in S. aureus: the last gene of the locus, walJ, is transcribed independently, whereas transcription of the tetra-cistronic walRKHI operon occurred from two independent promoters located upstream from walR. Protein topology analysis and protein-protein interactions in E. coli as well as subcellular localization in S. aureus allowed us to show that WalH and WalI are membrane-bound proteins, which associate with WalK to form a complex at the cell division septum. While these interactions suggest that WalH and WalI play a role in activity of the WalKR regulatory pathway, deletion of walH and/or walI did not have a major effect on genes whose expression is strongly dependent on WalKR or on associated phenotypes. No effect of WalH or WalI was seen on tightly controlled WalKR regulon genes such as sle1 or saouhsc_00773, which encodes a CHAP-domain amidase. Of the genes encoding the two major S. aureus autolysins, AtlA and Sle1, only transcription of atlA was increased in the ΔwalH or ΔwalI mutants. Likewise, bacterial autolysis was not increased in the absence of WalH and/or WalI and biofilm formation was lowered rather than increased. Our results suggest that contrary to their major role as WalK inhibitors in B. subtilis, the WalH and WalI proteins have evolved a different function in S. aureus, where they are more accessory. A phylogenomic analysis shows a striking conservation of the 5 gene wal cluster along the evolutionary history of Bacilli, supporting the key importance of this signal transduction system, and indicating that the walH and walI genes were lost in the ancestor of Streptococcaceae, leading to their atypical 3 wal gene cluster, walRKJ.

Characterization and complete genome sequence analysis of a novel virulent Siphoviridae phage against Staphylococcus aureus isolated from bovine mastitis in Xinjiang, China.

PubMed

Zhang, Qian; Xing, Shaozhen; Sun, Qiang; Pei, Guangqian; Cheng, Shi; Liu, Yannan; An, Xiaoping; Zhang, Xianglilan; Qu, Yonggang; Tong, Yigang

2017-06-01

Bovine mastitis is one of the most costly diseases in dairy cows worldwide. It can be caused by over 150 different microorganisms, where Staphylococcus aureus is the most frequently isolated and a major pathogen responsible for heavy economic losses in dairy industry. Although antibiotic therapy is most widely used, alternative treatments are necessary due to the increasing antibiotic resistance. Using phage for pathogen control is a promising tool in the fight against antibiotic resistance. Mainly using high-throughput sequencing, bioinformatics and our proposed phage termini identification method, we have isolated and characterized a novel virulent phage, designated as vB_SauS_IMEP5, from manure collected from dairy farms in Shihezi, Xinjiang, China, for use as a biocontrol agent against Staphylococcus aureus infections. Its latent period was about 30 min and its burst size was approximately 272PFU/cell. Phage vB_SauS_IMEP5 survives in a wide pH range between 3 and 12. A treatment at 70 °C for 20 min can inactive the phage. Morphological analysis of vB_SauS_IMEP5 revealed that phage vB_SauS_IMEP5 morphologically resembles phages in the family Siphoviridae. Among our tested multiplicity of infections (MOIs), the optimal multiplicity of infection (MOI) of this phage was determined to be 0.001, suggesting that phage vB_SauS_IMEP5 has high bacteriolytic potential and good efficiency for reducing bacterial growth. The complete genome of IME-P5 is a 44,677-bp, linear, double-stranded DNA, with a G+C content of 34.26%, containing 69 putative ORFs. The termini of genome were determined with next-generation sequencing data using our previously proposed termini identification method, which suggests that this phage has non-redundant termini with 9nt 3' protruding cohesive ends. The genomic and proteomic characteristics of IMEP5 demonstrate that this phage does not belong to any of the previously recognized Siphoviridae Staphylococcus phage groups, suggesting the creation of a new lineage, thus adding to the knowledge on the diversity of Staphylococcus phages. An N-acetylmuramoyl-L-alanine amidase gene and several conserved genes were predicted, while no virulence or antibiotic resistance genes were identified. This study isolated and characterized a novel S. aureus phage vB_SauS_IMEP5, and our findings suggest that this phage may be potentially utilized as a therapeutic or prophylactic candidate against S.aureus infections.

Exoproteome analysis reveals higher abundance of proteins linked to alkaline stress in persistent Listeria monocytogenes strains.

PubMed

Rychli, Kathrin; Grunert, Tom; Ciolacu, Luminita; Zaiser, Andreas; Razzazi-Fazeli, Ebrahim; Schmitz-Esser, Stephan; Ehling-Schulz, Monika; Wagner, Martin

2016-02-02

The foodborne pathogen Listeria monocytogenes, responsible for listeriosis a rare but severe infection disease, can survive in the food processing environment for month or even years. So-called persistent L. monocytogenes strains greatly increase the risk of (re)contamination of food products, and are therefore a great challenge for food safety. However, our understanding of the mechanism underlying persistence is still fragmented. In this study we compared the exoproteome of three persistent strains with the reference strain EGDe under mild stress conditions using 2D differential gel electrophoresis. Principal component analysis including all differentially abundant protein spots showed that the exoproteome of strain EGDe (sequence type (ST) 35) is distinct from that of the persistent strain R479a (ST8) and the two closely related ST121 strains 4423 and 6179. Phylogenetic analyses based on multilocus ST genes showed similar grouping of the strains. Comparing the exoproteome of strain EGDe and the three persistent strains resulted in identification of 22 differentially expressed protein spots corresponding to 16 proteins. Six proteins were significantly increased in the persistent L. monocytogenes exoproteomes, among them proteins involved in alkaline stress response (e.g. the membrane anchored lipoprotein Lmo2637 and the NADPH dehydrogenase NamA). In parallel the persistent strains showed increased survival under alkaline stress, which is often provided during cleaning and disinfection in the food processing environments. In addition, gene expression of the proteins linked to stress response (Lmo2637, NamA, Fhs and QoxA) was higher in the persistent strain not only at 37 °C but also at 10 °C. Invasion efficiency of EGDe was higher in intestinal epithelial Caco2 and macrophage-like THP1 cells compared to the persistent strains. Concurrently we found higher expression of proteins involved in virulence in EGDe e.g. the actin-assembly-inducing protein ActA and the surface virulence associated protein SvpA. Furthermore proteins involved in cell wall modification, such as the lipoteichonic acid primase LtaP and the N-acetylmuramoyl-l-alanine amidase (Lmo2591) are more abundant in EGDe than in the persistent strains and could indirectly contribute to virulence. In conclusion this study provides information about a set of proteins that could potentially support survival of L. monocytogenes in abiotic niches in food processing environments. Based on these data, a more detailed analysis of the role of the identified proteins under stresses mimicking conditions in food producing environment is essential for further elucidate the mechanism of the phenomenon of persistence of L. monocytogenes. Copyright © 2015 Elsevier B.V. All rights reserved.

The proteolytic system of pineapple stems revisited: Purification and characterization of multiple catalytically active forms.

PubMed

Matagne, André; Bolle, Laetitia; El Mahyaoui, Rachida; Baeyens-Volant, Danielle; Azarkan, Mohamed

2017-06-01

Crude pineapple proteases extract (aka stem bromelain; EC 3.4.22.4) is an important proteolytic mixture that contains enzymes belonging to the cysteine proteases of the papain family. Numerous studies have been reported aiming at the fractionation and characterization of the many molecular species present in the extract, but more efforts are still required to obtain sufficient quantities of the various purified protease forms for detailed physicochemical, enzymatic and structural characterization. In this work, we describe an efficient strategy towards the purification of at least eight enzymatic forms. Thus, following rapid fractionation on a SP-Sepharose FF column, two sub-populations with proteolytic activity were obtained: the unbound (termed acidic) and bound (termed basic) bromelain fractions. Following reversible modification with monomethoxypolyethylene glycol (mPEG), both fractions were further separated on Q-Sepharose FF and SP-Sepharose FF, respectively. This procedure yielded highly purified molecular species, all titrating ca. 1 mol of thiol group per mole of enzyme, with distinct biochemical properties. N-terminal sequencing allowed identifying at least eight forms with proteolytic activity. The basic fraction contained previously identified species, i.e. basic bromelain forms 1 and 2, ananain forms 1 and 2, and comosain (MEROPS identifier: C01.027). Furthermore, a new proteolytic species, showing similarities with basic bomelain forms 1 and 2, was discovered and termed bromelain form 3. The two remaining species were found in the acidic bromelain fraction and were arbitrarily named acidic bromelain forms 1 and 2. Both, acidic bromelain forms 1, 2 and basic bromelain forms 1, 2 and 3 are glycosylated, while ananain forms 1 and 2, and comosain are not. The eight protease forms display different amidase activities against the various substrates tested, namely small synthetic chromogenic compounds (DL-BAPNA and Boc-Ala-Ala-Gly-pNA), fluorogenic compounds (like Boc-Gln-Ala-Arg-AMC, Z-Arg-Arg-AMC and Z-Phe-Arg-AMC), and proteins (azocasein and azoalbumin), suggesting a specific organization of their catalytic residues. All forms are completely inhibited by specific cysteine and cysteine/serine protease inhibitors, but not by specific serine and aspartic protease inhibitors, with the sole exception of pepstatin A that significantly affects acidic bromelain forms 1 and 2. For all eight protease forms, inhibition is also observed with 1,10-phenanthrolin, a metalloprotease inhibitor. Metal ions (i.e. Mn 2+ , Mg 2+ and Ca 2+ ) showed various effects depending on the protease under consideration, but all of them are totally inhibited in the presence of Zn 2+ . Mass spectrometry analyses revealed that all forms have a molecular mass of ca. 24 kDa, which is characteristic of enzymes belonging to the papain-like proteases family. Far-UV CD spectra analysis further supported this analysis. Interestingly, secondary structure calculation proves to be highly reproducible for all cysteine proteases of the papain family tested so far (this work; see also Azarkan et al., 2011; Baeyens-Volant et al., 2015) and thus can be used as a test for rapid identification of the classical papain fold. Copyright © 2017 Elsevier Ltd. All rights reserved.

METABOLIC ENGINEERING TO DEVELOP A PATHWAY FOR THE SELECTIVE CLEAVAGE OF CARBON-NITROGEN BONDS

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

John J. Kilbane II

The objective of the project is to develop biochemical pathways for the selective cleavage of C-N bonds in molecules found in petroleum. The initial phase of the project was focused on the isolation or development of an enzyme capable of cleaving the C-N bond in aromatic amides, specifically 2-aminobiphenyl. The objective of the second phase of the research will be to construct a biochemical pathway for the selective removal of nitrogen from carbazole by combining the carA genes from Sphingomonas sp. GTIN11 with the gene(s) encoding an appropriate deaminase. The objective of the final phase of the project will bemore » to develop derivative C-N bond cleaving enzymes that have broader substrate ranges and to demonstrate the use of such strains to selectively remove nitrogen from petroleum. During the first year of the project (October, 2002-September, 2003) enrichment culture experiments resulted in the isolation of microbial cultures that utilize aromatic amides as sole nitrogen sources, several amidase genes were cloned and were included in directed evolution experiments to obtain derivatives that can cleave C-N bonds in aromatic amides, and the carA genes from Sphingomonas sp. GTIN11, and Pseudomonas resinovorans CA10 were cloned in vectors capable of replicating in Escherichia coli. During the second year of the project (October, 2003-September, 2004) enrichment culture experiments succeeded in isolating a mixed bacterial culture that can utilize 2-aminobiphenyl as a sole nitrogen source, directed evolution experiments were focused on the aniline dioxygenase enzyme that is capable of deaminating aniline, and expression vectors were constructed to enable the expression of genes encoding C-N bond cleaving enzymes in Rhodococcus hosts. The construction of a new metabolic pathway to selectively remove nitrogen from carbazole and other molecules typically found in petroleum should lead to the development of a process to improve oil refinery efficiency by reducing the poisoning, by nitrogen, of catalysts used in the hydrotreating and catalytic cracking of petroleum. Aromatic compounds such as carbazole are representative of the difficult-to-treat organonitrogen compounds most commonly encountered in petroleum. There are two C-N bonds in carbazole and the construction of a metabolic pathway for the removal of nitrogen from carbazole will require enzymes capable cleaving both C-N bonds. A multi-component enzyme, carbazole dioxygenase, which can selectively cleave the first C-N bond has been identified and the genes that encode this enzyme have been cloned, sequenced, and are being expressed in Rhodococcus erythropolis, a bacterial culture that tolerates exposure to petroleum. An enzyme capable of selectively cleaving the second C-N bond in carbazole has not yet been identified, but enrichment culture experiments have recently succeeded in isolating a bacterial culture that is a likely candidate and may possess a suitable enzyme. Research in the near future will verify if a suitable enzyme for the cleavage of the second C-N bond in carbazole has indeed been found, then the genes encoding a suitable enzyme will be identified, cloned, and sequenced. Ultimately genes encoding enzymes for selective cleavage of both C-N bonds in carbazole will be assembled into a new metabolic pathway and the ability of the resulting bacterial culture to remove nitrogen from petroleum will be determined.« less