Purification of yeast alcohol dehydrogenase by using immobilized metal affinity cryogels.

PubMed

Akduman, Begüm; Uygun, Murat; Uygun, Deniz Aktaş; Akgöl, Sinan; Denizli, Adil

2013-12-01

In this study, poly(2-hydroxyethyl methacrylate-glycidylmethacrylate) [poly(HEMA-GMA)] cryogels were prepared by radical cryocopolymerization of HEMA with GMA as a functional comonomer and N,N'-methylene-bisacrylamide (MBAAm) as a crosslinker. Iminodiacetic acid (IDA) functional groups were attached via ring opening of the epoxy group on the poly(HEMA-GMA) cryogels and then Zn(II) ions were chelated with these structures. Characterization of cryogels was performed by FTIR, SEM, EDX and swelling studies. These cryogels have interconnected pores of 30-50 μm size. The equilibrium swelling degree of Zn(II) chelated poly(HEMA-GMA)-IDA cryogels was approximately 600%. Zn(II) chelated poly(HEMA-GMA)-IDA cryogels were used in the adsorption of alcohol dehydrogenase from aqueous solutions and adsorption was performed in continuous system. The effects of pH, alcohol dehydrogenase concentration, temperature, and flow rate on adsorption were investigated. The maximum amount of alcohol dehydrogenase adsorption was determined to be 9.94 mg/g cryogel at 1.0mg/mL alcohol dehydrogenase concentration and in acetate buffer at pH5.0 with a flow rate of 0.5 mL/min. Desorption of adsorbed alcohol dehydrogenase was carried out by using 1.0M NaCI at pH8.0 phosphate buffer and desorption yield was found to be 93.5%. Additionally, these cryogels were used for purification of alcohol dehydrogenase from yeast with a single-step. The purity of desorbed alcohol dehydrogenase was shown by silver-stained SDS-PAGE. This purification process can successfully be used for the purification of alcohol dehydrogenase from unclarified yeast homogenates and this work is the first report about the usage of the cryogels for purification of alcohol dehydrogenase. © 2013 Elsevier B.V. All rights reserved.

Multiple alcohol dehydrogenases but no functional acetaldehyde dehydrogenase causing excessive acetaldehyde production from ethanol by oral streptococci.

PubMed

Pavlova, Sylvia I; Jin, Ling; Gasparovich, Stephen R; Tao, Lin

2013-07-01

Ethanol consumption and poor oral hygiene are risk factors for oral and oesophageal cancers. Although oral streptococci have been found to produce excessive acetaldehyde from ethanol, little is known about the mechanism by which this carcinogen is produced. By screening 52 strains of diverse oral streptococcal species, we identified Streptococcus gordonii V2016 that produced the most acetaldehyde from ethanol. We then constructed gene deletion mutants in this strain and analysed them for alcohol and acetaldehyde dehydrogenases by zymograms. The results showed that S. gordonii V2016 expressed three primary alcohol dehydrogenases, AdhA, AdhB and AdhE, which all oxidize ethanol to acetaldehyde, but their preferred substrates were 1-propanol, 1-butanol and ethanol, respectively. Two additional dehydrogenases, S-AdhA and TdhA, were identified with specificities to the secondary alcohol 2-propanol and threonine, respectively, but not to ethanol. S. gordonii V2016 did not show a detectable acetaldehyde dehydrogenase even though its adhE gene encodes a putative bifunctional acetaldehyde/alcohol dehydrogenase. Mutants with adhE deletion showed greater tolerance to ethanol in comparison with the wild-type and mutant with adhA or adhB deletion, indicating that AdhE is the major alcohol dehydrogenase in S. gordonii. Analysis of 19 additional strains of S. gordonii, S. mitis, S. oralis, S. salivarius and S. sanguinis showed expressions of up to three alcohol dehydrogenases, but none showed detectable acetaldehyde dehydrogenase, except one strain that showed a novel ALDH. Therefore, expression of multiple alcohol dehydrogenases but no functional acetaldehyde dehydrogenase may contribute to excessive production of acetaldehyde from ethanol by certain oral streptococci.

Multiple alcohol dehydrogenases but no functional acetaldehyde dehydrogenase causing excessive acetaldehyde production from ethanol by oral streptococci

PubMed Central

Pavlova, Sylvia I.; Jin, Ling; Gasparovich, Stephen R.

2013-01-01

Ethanol consumption and poor oral hygiene are risk factors for oral and oesophageal cancers. Although oral streptococci have been found to produce excessive acetaldehyde from ethanol, little is known about the mechanism by which this carcinogen is produced. By screening 52 strains of diverse oral streptococcal species, we identified Streptococcus gordonii V2016 that produced the most acetaldehyde from ethanol. We then constructed gene deletion mutants in this strain and analysed them for alcohol and acetaldehyde dehydrogenases by zymograms. The results showed that S. gordonii V2016 expressed three primary alcohol dehydrogenases, AdhA, AdhB and AdhE, which all oxidize ethanol to acetaldehyde, but their preferred substrates were 1-propanol, 1-butanol and ethanol, respectively. Two additional dehydrogenases, S-AdhA and TdhA, were identified with specificities to the secondary alcohol 2-propanol and threonine, respectively, but not to ethanol. S. gordonii V2016 did not show a detectable acetaldehyde dehydrogenase even though its adhE gene encodes a putative bifunctional acetaldehyde/alcohol dehydrogenase. Mutants with adhE deletion showed greater tolerance to ethanol in comparison with the wild-type and mutant with adhA or adhB deletion, indicating that AdhE is the major alcohol dehydrogenase in S. gordonii. Analysis of 19 additional strains of S. gordonii, S. mitis, S. oralis, S. salivarius and S. sanguinis showed expressions of up to three alcohol dehydrogenases, but none showed detectable acetaldehyde dehydrogenase, except one strain that showed a novel ALDH. Therefore, expression of multiple alcohol dehydrogenases but no functional acetaldehyde dehydrogenase may contribute to excessive production of acetaldehyde from ethanol by certain oral streptococci. PMID:23637459

In situ rat brain and liver spontaneous chemiluminescence after acute ethanol intake.

PubMed

Boveris, A; Llesuy, S; Azzalis, L A; Giavarotti, L; Simon, K A; Junqueira, V B; Porta, E A; Videla, L A; Lissi, E A

1997-09-19

The influence of acute ethanol administration on the oxidative stress status of rat brain and liver was assessed by in situ spontaneous organ chemiluminescence (CL). Brain and liver CL was significantly increased after acute ethanol administration to fed rats, a response that is time-dependent and evidenced at doses higher than 1 g/kg. Ethanol-induced CL development is faster in liver compared with brain probably due to the greater ethanol metabolic capacity of the liver, whereas the net enhancement in brain light emission at 3 h after ethanol treatment is higher than that of the liver, which could reflect the greater susceptibility of brain to oxidative stress. The effect of ethanol on brain and liver CL seems to be mediated by acetaldehyde, due to its abolishment by the alcohol dehydrogenase inhibitor 4-methylpyrazole and exacerbation by the aldehyde dehydrogenase inhibitor disulfiram. In brain, these findings were observed in the absence of changes in the activity of superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, and glucose-6-phosphate dehydrogenase. However, the content of brain glutathione was significantly decreased by 31%, by ethanol, thus establishing an enhanced oxidative stress in this tissue.

Methoxyflurane enhances allyl alcohol hepatotoxicity in rats. Possible involvement of increased acrolein formation.

PubMed

Kershaw, W C; Barsotti, D A; Leonard, T B; Dent, J G; Lage, G L

1989-01-01

The effect of methoxyflurane anesthesia on allyl alcohol-induced hepatotoxicity and the metabolism of allyl alcohol was studied in male rats. Hepatotoxicity was assessed by the measurement of serum alanine aminotransferase activity and histopathological examination. Allyl alcohol-induced hepatotoxicity was enhanced when allyl alcohol (32 mg/kg) was administered 4 hr before or up to 8 days after a single 10-min exposure to methoxyflurane vapors. The possibility that methoxyflurane increases alcohol dehydrogenase-dependent oxidation of allyl alcohol to acrolein, the proposed toxic metabolite, was evaluated by measuring the rate of acrolein formation in the presence of allyl alcohol and liver cytosol. The effect of methoxyflurane on alcohol dehydrogenase activity in liver cytosol was also assessed by measuring the rate of NAD+ utilization in the presence of ethyl alcohol or allyl alcohol. Alcohol dehydrogenase activity and rate of acrolein formation were elevated in methoxyflurane-pretreated rats. The results suggest that a modest increase in alcohol dehydrogenase activity and rate of acrolein formation markedly enhances allyl alcohol-induced hepatotoxicity.

Acamprosate attenuates the handling induced convulsions during alcohol withdrawal in Swiss Webster mice

PubMed Central

Farook, Justin M.; Krazem, Ali; Littleton, John M.; Barron, Susan

2008-01-01

In the present study, we examined the effects of acamprosate for its ability to reduce handling induced convulsions (HICs) during alcohol withdrawal. Diazepam was used as a positive control. Swiss Webster male mice received three daily IP injections of alcohol (2.5 g/kg) or alcohol (2.5 g/kg) + methylpyrazole (4-MP) (9 mg/kg). (4-MP, being an alcohol dehydrogenase inhibitor slows down the breakdown of alcohol. 4-MP in combination with alcohol exhibits a dramatic increase in blood alcohol level compared to alcohol alone). Ten hours following the last alcohol injection, the mice were picked up by the tail and examined for their seizure susceptibility (HICs). Diazepam, a benzodiazepine known to reduce seizures during alcohol withdrawal, significantly reduced these HICs at doses of 0.25, 0.5 and 1mg/kg (p’s < 0.001). Acamprosate, an anti-relapse compound used clinically in newly abstinent alcoholics, also reduced these HICs at doses of 100, 200 and 300mg/kg (p’s < 0.05). This study supports the use of acamprosate during periods of alcohol withdrawal as well as during abstinence. PMID:18577392

Oxidation of methanol, ethylene glycol, and isopropanol with human alcohol dehydrogenases and the inhibition by ethanol and 4-methylpyrazole.

PubMed

Lee, Shou-Lun; Shih, Hsuan-Ting; Chi, Yu-Chou; Li, Yeung-Pin; Yin, Shih-Jiun

2011-05-30

Human alcohol dehydrogenases (ADHs) include multiple isozymes with broad substrate specificity and ethnic distinct allozymes. ADH catalyzes the rate-limiting step in metabolism of various primary and secondary aliphatic alcohols. The oxidation of common toxic alcohols, that is, methanol, ethylene glycol, and isopropanol by the human ADHs remains poorly understood. Kinetic studies were performed in 0.1M sodium phosphate buffer, at pH 7.5 and 25°C, containing 0.5 mM NAD(+) and varied concentrations of substrate. K(M) values for ethanol with recombinant human class I ADH1A, ADH1B1, ADH1B2, ADH1B3, ADH1C1, and ADH1C2, and class II ADH2 and class IV ADH4 were determined to be in the range of 0.12-57 mM, for methanol to be 2.0-3500 mM, for ethylene glycol to be 4.3-2600mM, and for isopropanol to be 0.73-3400 mM. ADH1B3 appeared to be inactive toward ethylene glycol, and ADH2 and ADH4, inactive with methanol. The variations for V(max) for the toxic alcohols were much less than that of the K(M) across the ADH family. 4-Methylpyrazole (4MP) was a competitive inhibitor with respect to ethanol for ADH1A, ADH1B1, ADH1B2, ADH1C1 and ADH1C2, and a noncompetitive inhibitor for ADH1B3, ADH2 and ADH4, with the slope inhibition constants (K(is)) for the whole family being 0.062-960 μM and the intercept inhibition constants (K(ii)), 33-3000 μM. Computer simulation studies using inhibition equations in the presence of alternate substrate ethanol and of dead-end inhibitor 4MP with the determined corresponding kinetic parameters for ADH family, indicate that the oxidation of the toxic alcohols up to 50mM are largely inhibited by 20 mM ethanol or by 50 μM 4MP with some exceptions. The above findings provide an enzymological basis for clinical treatment of methanol and ethylene glycol poisoning by 4MP or ethanol with pharmacogenetic perspectives. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

Digitalis metabolism and human liver alcohol dehydrogenase.

PubMed Central

Frey, W A; Vallee, B L

1980-01-01

Human liver alcohol dehydrogenase (alcohol: NAD" oxidoreductase, EC 1.1.1.1) catalyzes the oxidation of the 3 beta-OH group of digitoxigenin, digoxigenin, and gitoxigenin to their 3-keto derivatives, which have been characterized by high performance liquid chromatography and mass spectrometry. These studies have identified human liver alcohol dehydrogenase as the unknown NAD(H)-dependent liver enzyme specific for the free hydroxyl group at C3 of the cardiac genins; this hydroxyl is the critical site of the genins' enzymatic oxidation and concomitant pharmacological inactivation in humans. Several kinetic approaches have demonstrated that ethanol and the pharmacologically active components of the digitalis glycosides are oxidized with closely similar kcat/Km values at the same site on human liver alcohol dehydrogenase, for which they compete. Human liver alcohol dehydrogenase thereby becomes an important biochemical link in the metabolism, pharmacology, and toxicology of ethanol and these glycosides, structurally unrelated agents that are both used widely. Both the competition of ethanol with these cardiac sterols and the narrow margin of safety in the therapeutic use of digitalis derivatives would seem to place at increased risk those individuals who receive digitalis and simultaneously consume large amounts of ethanol or whose alcohol dehydrogenase function is impaired. PMID:6987673

Inhibition of 1,4-butanediol metabolism in human liver in vitro.

PubMed

Lenz, Daniel; Jübner, Martin; Bender, Katja; Wintermeyer, Annette; Beike, Justus; Rothschild, Markus A; Käferstein, Herbert

2011-06-01

The conversion of 1,4-butanediol (1,4-BD) to gamma-hydroxybutyric acid (GHB), a drug of abuse, is most probably catalyzed by alcohol dehydrogenase, and potentially by aldehyde dehydrogenase. The purpose of this study was to investigate the degradation of 1,4-BD in cytosolic supernatant of human liver in vitro, and to verify involvement of the suggested enzymes by means of gas chromatography-mass spectrometry. The coingestion of 1,4-BD and ethanol (EtOH) might cause complex pharmacokinetic interactions in humans. Therefore, the effect of EtOH on 1,4-BD metabolism by human liver was examined in vitro. Additionally, the influence of acetaldehyde (AL), which might inhibit the second step of 1,4-BD degradation, was investigated. In case of a 1,4-BD intoxication, the alcohol dehydrogenase inhibitor fomepizole (4-methylpyrazole, FOM) has been discussed as an antidote preventing the formation of the central nervous system depressing GHB. Besides FOM, we tested pyrazole, disulfiram, and cimetidine as possible inhibitors of the formation of GHB from 1,4-BD catalyzed by human liver enzymes in vitro. The conversion of 1,4-BD to GHB was inhibited competitively by EtOH with an apparent K(i) of 0.56 mM. Therefore, the coingestion of 1,4-BD and EtOH might increase the concentrations and the effects of 1,4-BD itself. By contrast AL accelerated the formation of GHB. All antidotes showed the ability to inhibit the formation of GHB. In comparison FOM showed the highest inhibitory effectiveness. Furthermore, the results confirm strong involvement of ADH in 1,4-BD metabolism by human liver.

Chronic alcoholism in rats induces a compensatory response, preserving brain thiamine diphosphate, but the brain 2-oxo acid dehydrogenases are inactivated despite unchanged coenzyme levels.

PubMed

Parkhomenko, Yulia M; Kudryavtsev, Pavel A; Pylypchuk, Svetlana Yu; Chekhivska, Lilia I; Stepanenko, Svetlana P; Sergiichuk, Andrej A; Bunik, Victoria I

2011-06-01

Thiamine-dependent changes in alcoholic brain were studied using a rat model. Brain thiamine and its mono- and diphosphates were not reduced after 20 weeks of alcohol exposure. However, alcoholism increased both synaptosomal thiamine uptake and thiamine diphosphate synthesis in brain, pointing to mechanisms preserving thiamine diphosphate in the alcoholic brain. In spite of the unchanged level of the coenzyme thiamine diphosphate, activities of the mitochondrial 2-oxoglutarate and pyruvate dehydrogenase complexes decreased in alcoholic brain. The inactivation of pyruvate dehydrogenase complex was caused by its increased phosphorylation. The inactivation of 2-oxoglutarate dehydrogenase complex (OGDHC) correlated with a decrease in free thiols resulting from an elevation of reactive oxygen species. Abstinence from alcohol following exposure to alcohol reactivated OGDHC along with restoration of the free thiol content. However, restoration of enzyme activity occurred before normalization of reactive oxygen species levels. Hence, the redox status of cellular thiols mediates the action of oxidative stress on OGDHC in alcoholic brain. As a result, upon chronic alcohol consumption, physiological mechanisms to counteract the thiamine deficiency and silence pyruvate dehydrogenase are activated in rat brain, whereas OGDHC is inactivated due to impaired antioxidant ability. © 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.

Laboratory evolution of Pyrococcus furiosus alcohol dehydrogenase to improve the production of (2S,5S)-hexanediol at moderate temperatures

PubMed Central

Leferink, Nicole G. H.; Hendriks, Annemarie; Brouns, Stan J. J.; Hennemann, Hans-Georg; Dauβmann, Thomas; van der Oost, John

2008-01-01

There is considerable interest in the use of enantioselective alcohol dehydrogenases for the production of enantio- and diastereomerically pure diols, which are important building blocks for pharmaceuticals, agrochemicals and fine chemicals. Due to the need for a stable alcohol dehydrogenase with activity at low-temperature process conditions (30°C) for the production of (2S,5S)-hexanediol, we have improved an alcohol dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus (AdhA). A stable S-selective alcohol dehydrogenase with increased activity at 30°C on the substrate 2,5-hexanedione was generated by laboratory evolution on the thermostable alcohol dehydrogenase AdhA. One round of error-prone PCR and screening of ∼1,500 mutants was performed. The maximum specific activity of the best performing mutant with 2,5-hexanedione at 30°C was tenfold higher compared to the activity of the wild-type enzyme. A 3D-model of AdhA revealed that this mutant has one mutation in the well-conserved NADP(H)-binding site (R11L), and a second mutation (A180V) near the catalytic and highly conserved threonine at position 183. PMID:18452026

Contribution of liver alcohol dehydrogenase to metabolism of alcohols in rats.

PubMed

Plapp, Bryce V; Leidal, Kevin G; Murch, Bruce P; Green, David W

2015-06-05

The kinetics of oxidation of various alcohols by purified rat liver alcohol dehydrogenase (ADH) were compared with the kinetics of elimination of the alcohols in rats in order to investigate the roles of ADH and other factors that contribute to the rates of metabolism of alcohols. Primary alcohols (ethanol, 1-propanol, 1-butanol, 2-methyl-1-propanol, 3-methyl-1-butanol) and diols (1,3-propanediol, 1,3-butanediol, 1,4-butanediol, 1,5-pentanediol) were eliminated in rats with zero-order kinetics at doses of 5-20 mmol/kg. Ethanol was eliminated most rapidly, at 7.9 mmol/kgh. Secondary alcohols (2-propanol-d7, 2-propanol, 2-butanol, 3-pentanol, cyclopentanol, cyclohexanol) were eliminated with first order kinetics at doses of 5-10 mmol/kg, and the corresponding ketones were formed and slowly eliminated with zero or first order kinetics. The rates of elimination of various alcohols were inhibited on average 73% (55% for 2-propanol to 90% for ethanol) by 1 mmol/kg of 4-methylpyrazole, a good inhibitor of ADH, indicating a major role for ADH in the metabolism of the alcohols. The Michaelis kinetic constants from in vitro studies (pH 7.3, 37 °C) with isolated rat liver enzyme were used to calculate the expected relative rates of metabolism in rats. The rates of elimination generally increased with increased activity of ADH, but a maximum rate of 6±1 mmol/kg h was observed for the best substrates, suggesting that ADH activity is not solely rate-limiting. Because secondary alcohols only require one NAD(+) for the conversion to ketones whereas primary alcohols require two equivalents of NAD(+) for oxidation to the carboxylic acids, it appears that the rate of oxidation of NADH to NAD(+) is not a major limiting factor for metabolism of these alcohols, but the rate-limiting factors are yet to be identified. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Contribution of Liver Alcohol Dehydrogenase to Metabolism of Alcohols in Rats

PubMed Central

Plapp, Bryce V.; Leidal, Kevin G.; Murch, Bruce P.; Green, David W.

2015-01-01

The kinetics of oxidation of various alcohols by purified rat liver alcohol dehydrogenase (ADH) were compared with the kinetics of elimination of the alcohols in rats in order to investigate the roles of ADH and other factors that contribute to the rates of metabolism of alcohols. Primary alcohols (ethanol, 1-propanol, 1-butanol, 2-methyl-1-propanol, 3-methyl-1-butanol) and diols (1,3-propanediol, 1,3-butanediol, 1,4-butanediol, 1,5-pentanediol) were eliminated in rats with zero-order kinetics at doses of 5–20 mmole/kg. Ethanol was eliminated most rapidly, at 7.9 mmole/kg•h. Secondary alcohols (2-propanol-d7, 2-propanol, 2-butanol, 3-pentanol, cyclopentanol, cyclohexanol) were eliminated with first order kinetics at doses of 5–10 mmole/kg, and the corresponding ketones were formed and slowly eliminated with zero or first order kinetics. The rates of elimination of various alcohols were inhibited on average 73% (55% for 2-propanol to 90% for ethanol) by 1 mmole/kg of 4-methylpyrazole, a good inhibitor of ADH, indicating a major role for ADH in the metabolism of the alcohols. The Michaelis kinetic constants from in vitro studies (pH 7.3, 37 °C) with isolated rat liver enzyme were used to calculate the expected relative rates of metabolism in rats. The rates of elimination generally increased with increased activity of ADH, but a maximum rate of 6 ± 1 mmole/kg•h was observed for the best substrates, suggesting that ADH activity is not solely rate-limiting. Because secondary alcohols only require one NAD+ for the conversion to ketones whereas primary alcohols require two equivalents of NAD+ for oxidation to the carboxylic acids, it appears that the rate of oxidation of NADH to NAD+ is not a major limiting factor for metabolism of these alcohols, but the rate-limiting factors are yet to be identified. PMID:25641189

Ethanol metabolism by alcohol dehydrogenase or cytochrome P450 2E1 differentially impairs hepatic protein trafficking and growth hormone signaling.

PubMed

Doody, Erin E; Groebner, Jennifer L; Walker, Jetta R; Frizol, Brittnee M; Tuma, Dean J; Fernandez, David J; Tuma, Pamela L

2017-12-01

The liver metabolizes alcohol using alcohol dehydrogenase (ADH) and cytochrome P 450 2E1 (CYP2E1). Both enzymes metabolize ethanol into acetaldehyde, but CYP2E1 activity also results in the production of reactive oxygen species (ROS) that promote oxidative stress. We have previously shown that microtubules are hyperacetylated in ethanol-treated polarized, hepatic WIF-B cells and livers from ethanol-fed rats. We have also shown that enhanced protein acetylation correlates with impaired clathrin-mediated endocytosis, constitutive secretion, and nuclear translocation and that the defects are likely mediated by acetaldehyde. However, the roles of CYP2E1-generated metabolites and ROS in microtubule acetylation and these alcohol-induced impairments have not been examined. To determine if CYP2E1-mediated alcohol metabolism is required for enhanced acetylation and the trafficking defects, we coincubated cells with ethanol and diallyl sulfide (DAS; a CYP2E1 inhibitor) or N -acetyl cysteine (NAC; an antioxidant). Both agents failed to prevent microtubule hyperacetylation in ethanol-treated cells and also failed to prevent impaired secretion or clathrin-mediated endocytosis. Somewhat surprisingly, both DAS and NAC prevented impaired STAT5B nuclear translocation. Further examination of microtubule-independent steps of the pathway revealed that Jak2/STAT5B activation by growth hormone was prevented by DAS and NAC. These results were confirmed in ethanol-exposed HepG2 cells expressing only ADH or CYP2E1. Using quantitative RT-PCR, we further determined that ethanol exposure led to blunted growth hormone-mediated gene expression. In conclusion, we determined that alcohol-induced microtubule acetylation and associated defects in microtubule-dependent trafficking are mediated by ADH metabolism whereas impaired microtubule-independent Jak2/STAT5B activation is mediated by CYP2E1 activity. NEW & NOTEWORTHY Impaired growth hormone-mediated signaling is observed in ethanol-exposed hepatocytes and is explained by differential effects of alcohol dehydrogenase (ADH)- and cytochrome P 450 2E1 (CYP2E1)-mediated ethanol metabolism on the Jak2/STAT5B pathway. Copyright © 2017 the American Physiological Society.

Roles of the C-terminal domains of human dihydrodiol dehydrogenase isoforms in the binding of substrates and modulators: probing with chimaeric enzymes.

PubMed Central

Matsuura, K; Hara, A; Deyashiki, Y; Iwasa, H; Kume, T; Ishikura, S; Shiraishi, H; Katagiri, Y

1998-01-01

Human liver dihydrodiol dehydrogenase (DD; EC 1.3.1.20) exists in isoforms (DD1, DD2 and DD4) composed of 323 amino acids. DD1 and DD2 share 98% amino acid sequence identity, but show lower identities (approx. 83%) with DD4, in which a marked difference is seen in the C-terminal ten amino acids. DD4 exhibits unique catalytic properties, such as the ability to oxidize both (R)- and (S)-alicyclic alcohols equally, high dehydrogenase activity for bile acids, potent inhibition by steroidal anti-inflammatory drugs and activation by sulphobromophthalein and clofibric acid derivatives. In this study, we have prepared chimaeric enzymes, in which we exchanged the C-terminal 39 residues between the two enzymes. Compared with DD1, CDD1-4 (DD1 with the C-terminal sequence of DD4) had increased kcat/Km values for 3alpha-hydroxy-5beta-androstanes and bile acids of 3-9-fold and decreased values for the other substrates by 5-100-fold. It also became highly sensitive to DD4 inhibitors such as phenolphthalein and hexoestrol. Another chimaeric enzyme, CDD4-1 (DD4 with the C-terminal sequence of DD1), showed the same (S)-stereospecificity for the alicyclic alcohols as DD1, had decreased kcat/Km values for bile acids with 7beta- or 12alpha-hydroxy groups by more than 120-fold and was resistant to inhibition by betamethasone. In addition, the activation effects of sulphobromophthalein and bezafibrate decreased or disappeared for CDD4-1. The recombinant DD4 with the His314-->Pro (the corresponding residue of DD1) mutation showed intermediate changes in the properties between those of wild-type DD4 and CDD4-1. The results indicate that the binding of substrates, inhibitors and activators to the enzymes is controlled by residues in their C-terminal domains; multiple residues co-ordinately act as determinants for substrate specificity and inhibitor sensitivity. PMID:9820821

A Hepatocyte-Mimicking Antidote for Alcohol Intoxication.

PubMed

Xu, Duo; Han, Hui; He, Yuxin; Lee, Harrison; Wu, Di; Liu, Fang; Liu, Xiangsheng; Liu, Yang; Lu, Yunfeng; Ji, Cheng

2018-04-11

Alcohol intoxication causes serious diseases, whereas current treatments are mostly supportive and unable to remove alcohol efficiently. Upon alcohol consumption, alcohol is sequentially oxidized to acetaldehyde and acetate by the endogenous alcohol dehydrogenase and aldehyde dehydrogenase, respectively. Inspired by the metabolism of alcohol, a hepatocyte-mimicking antidote for alcohol intoxication through the codelivery of the nanocapsules of alcohol oxidase (AOx), catalase (CAT), and aldehyde dehydrogenase (ALDH) to the liver, where AOx and CAT catalyze the oxidation of alcohol to acetaldehyde, while ALDH catalyzes the oxidation of acetaldehyde to acetate. Administered to alcohol-intoxicated mice, the antidote rapidly accumulates in the liver and enables a significant reduction of the blood alcohol concentration. Moreover, blood acetaldehyde concentration is maintained at an extremely low level, significantly contributing to liver protection. Such an antidote, which can eliminate alcohol and acetaldehyde simultaneously, holds great promise for the treatment of alcohol intoxication and poisoning and can provide therapeutic benefits. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Comparative Proteomic Analysis of Tolerance and Adaptation of Ethanologenic Saccharomyces cerevisiae to Furfural, a Lignocellulosic Inhibitory Compound▿ †

PubMed Central

Lin, Feng-Ming; Qiao, Bin; Yuan, Ying-Jin

2009-01-01

The molecular mechanism involved in tolerance and adaptation of ethanologenic Saccharomyces cerevisiae to inhibitors (such as furfural, acetic acid, and phenol) represented in lignocellulosic hydrolysate is still unclear. Here, 18O-labeling-aided shotgun comparative proteome analysis was applied to study the global protein expression profiles of S. cerevisiae under conditions of treatment of furfural compared with furfural-free fermentation profiles. Proteins involved in glucose fermentation and/or the tricarboxylic acid cycle were upregulated in cells treated with furfural compared with the control cells, while proteins involved in glycerol biosynthesis were downregulated. Differential levels of expression of alcohol dehydrogenases were observed. On the other hand, the levels of NADH, NAD+, and NADH/NAD+ were reduced whereas the levels of ATP and ADP were increased. These observations indicate that central carbon metabolism, levels of alcohol dehydrogenases, and the redox balance may be related to tolerance of ethanologenic yeast for and adaptation to furfural. Furthermore, proteins involved in stress response, including the unfolded protein response, oxidative stress, osmotic and salt stress, DNA damage and nutrient starvation, were differentially expressed, a finding that was validated by quantitative real-time reverse transcription-PCR to further confirm that the general stress responses are essential for cellular defense against furfural. These insights into the response of yeast to the presence of furfural will benefit the design and development of inhibitor-tolerant ethanologenic yeast by metabolic engineering or synthetic biology. PMID:19363068

Purification of acetaldehyde dehydrogenase and alcohol dehydrogenases from Thermoanaerobacter ethanolicus 39E and characterization of the secondary-alcohol dehydrogenase (2 degrees Adh) as a bifunctional alcohol dehydrogenase--acetyl-CoA reductive thioesterase.

PubMed Central

Burdette, D; Zeikus, J G

1994-01-01

The purification and characterization of three enzymes involved in ethanol formation from acetyl-CoA in Thermoanaerobacter ethanolicus 39E (formerly Clostridium thermohydrosulfuricum 39E) is described. The secondary-alcohol dehydrogenase (2 degrees Adh) was determined to be a homotetramer of 40 kDa subunits (SDS/PAGE) with a molecular mass of 160 kDa. The 2 degrees Adh had a lower catalytic efficiency for the oxidation of 1 degree alcohols, including ethanol, than for the oxidation of secondary (2 degrees) alcohols or the reduction of ketones or aldehydes. This enzyme possesses a significant acetyl-CoA reductive thioesterase activity as determined by NADPH oxidation, thiol formation and ethanol production. The primary-alcohol dehydrogenase (1 degree Adh) was determined to be a homotetramer of 41.5 kDa (SDS/PAGE) subunits with a molecular mass of 170 kDa. The 1 degree Adh used both NAD(H) and NADP(H) and displayed higher catalytic efficiencies for NADP(+)-dependent ethanol oxidation and NADH-dependent acetaldehyde (identical to ethanal) reduction than for NADPH-dependent acetaldehyde reduction or NAD(+)-dependent ethanol oxidation. The NAD(H)-linked acetaldehyde dehydrogenase was a homotetramer (360 kDa) of identical subunits (100 kDa) that readily catalysed thioester cleavage and condensation. The 1 degree Adh was expressed at 5-20% of the level of the 2 degrees Adh throughout the growth cycle on glucose. The results suggest that the 2 degrees Adh primarily functions in ethanol production from acetyl-CoA and acetaldehyde, whereas the 1 degree Adh functions in ethanol consumption for nicotinamide-cofactor recycling. Images Figure 1 PMID:8068002

Purification and characterization of an oxygen-labile, NAD-dependent alcohol dehydrogenase from Desulfovibrio gigas.

PubMed Central

Hensgens, C M; Vonck, J; Van Beeumen, J; van Bruggen, E F; Hansen, T A

1993-01-01

A NAD-dependent, oxygen-labile alcohol dehydrogenase was purified from Desulfovibrio gigas. It was decameric, with subunits of M(r) 43,000. The best substrates were ethanol (Km, 0.15 mM) and 1-propanol (Km, 0.28 mM). N-terminal amino acid sequence analysis showed that the enzyme belongs to the same family of alcohol dehydrogenases as Zymomonas mobilis ADH2 and Bacillus methanolicus MDH. Images PMID:8491707

Improved synthesis of chiral alcohols with Escherichia coli cells co-expressing pyridine nucleotide transhydrogenase, NADP+-dependent alcohol dehydrogenase and NAD+-dependent formate dehydrogenase.

PubMed

Weckbecker, Andrea; Hummel, Werner

2004-11-01

Recombinant pyridine nucleotide transhydrogenase (PNT) from Escherichia coli has been used to regenerate NAD+ and NADPH. The pnta and pntb genes encoding for the alpha- and beta-subunits were cloned and co-expressed with NADP+-dependent alcohol dehydrogenase (ADH) from Lactobacillus kefir and NAD+-dependent formate dehydrogenase (FDH) from Candida boidinii. Using this whole-cell biocatalyst, efficient conversion of prochiral ketones to chiral alcohols was achieved: 66% acetophenone was reduced to (R)-phenylethanol over 12 h, whereas only 19% (R)-phenylethanol was formed under the same conditions with cells containing ADH and FDH genes but without PNT genes. Cells that were permeabilized with toluene showed ketone reduction only if both cofactors were present.

OLITA: an alternative in the treatment of therapy-resistant chronic alcoholics. First evaluation of a new approach.

PubMed

Ehrenreich, H; Mangholz, A; Schmitt, M; Lieder, P; Völkel, W; Rüther, E; Poser, W

1997-01-01

The Outpatient Long-term Intensive Therapy for Alcoholics (OLITA) is a four-step program of care for severely affected chronic alcoholics which, after inpatient detoxification, extends over a total of 2 years. High-frequency short-term individual therapeutic contacts, initially daily, are followed by a slow tapering of individual contact frequency and resolve in a group session once weekly towards the end of the second abstinent year. Further elements of OLITA are: (a) induction of alcohol intolerance by the application of aldehyde dehydrogenase inhibitors; (b) introduction of control factors, i.e. controlled intake of deterrent medication and regular urine analysis for alcohol; and (c) allocation of responsibility to the patient with respect to the overall success of the therapeutic concept including his own physical rehabilitation. Thus far, 30 male alcoholic patients from two recruitment periods have been treated for 6-26 months with a success rate of 60% abstinent patients. In conclusion, OLITA, based on the gradual tapering of high-frequency therapeutic contacts, thus far unique among outpatient programs for alcoholics, represents a promising advance in the treatment of therapy-resistant chronic alcoholics.

Redox self-sufficient whole cell biotransformation for amination of alcohols.

PubMed

Klatte, Stephanie; Wendisch, Volker F

2014-10-15

Whole cell biotransformation is an upcoming tool to replace common chemical routes for functionalization and modification of desired molecules. In the approach presented here the production of various non-natural (di)amines was realized using the designed whole cell biocatalyst Escherichia coli W3110/pTrc99A-ald-adh-ta with plasmid-borne overexpression of genes for an l-alanine dehydrogenase, an alcohol dehydrogenase and a transaminase. Cascading alcohol oxidation with l-alanine dependent transamination and l-alanine dehydrogenase allowed for redox self-sufficient conversion of alcohols to the corresponding amines. The supplementation of the corresponding (di)alcohol precursors as well as amino group donor l-alanine and ammonium chloride were sufficient for amination and redox cofactor recycling in a resting buffer system. The addition of the transaminase cofactor pyridoxal-phosphate and the alcohol dehydrogenase cofactor NAD(+) was not necessary to obtain complete conversion. Secondary and cyclic alcohols, for example, 2-hexanol and cyclohexanol were not aminated. However, efficient redox self-sufficient amination of aliphatic and aromatic (di)alcohols in vivo was achieved with 1-hexanol, 1,10-decanediol and benzylalcohol being aminated best. Copyright © 2014 Elsevier Ltd. All rights reserved.

Ethanol Metabolism by HeLa Cells Transduced with Human Alcohol Dehydrogenase Isoenzymes: Control of the Pathway by Acetaldehyde Concentration†

PubMed Central

Matsumoto, Michinaga; Cyganek, Izabela; Sanghani, Paresh C.; Cho, Won Kyoo; Liangpunsakul, Suthat; Crabb, David W.

2010-01-01

Background Human class I alcohol dehydrogenase 2 isoenzymes (encoded by the ADH1B locus) have large differences in kinetic properties; however, individuals inheriting the alleles for the different isoenzymes exhibit only small differences in alcohol elimination rates. This suggests that other cellular factors must regulate the activity of the isoenzymes. Methods The activity of the isoenzymes expressed from ADH1B*1, ADH1B*2, and ADH1B*3 cDNAs was examined in stably transduced HeLa cell lines, including lines which expressed human low Km aldehyde dehydrogenase (ALDH2). The ability of the cells to metabolize ethanol was compared with that of HeLa cells expressing rat class I ADH (HeLa-rat ADH cells), rat hepatoma (H4IIEC3) cells, and rat hepatocytes. Results The isoenzymes had similar protein half-lives in the HeLa cells. Rat hepatocytes, H4IIEC3 cells, and HeLa-rat ADH cells oxidized ethanol much faster than the cells expressing the ADH1B isoenzymes. This was not explained by high cellular NADH levels or endogenous inhibitors; but rather because the activity of the β1 and β2 ADHs were constrained by the accumulation of acetaldehyde, as shown by the increased rate of ethanol oxidation by cell lines expressing β2 ADH plus ALDH2. Conclusion The activity of the human β2 ADH isoenzyme is sensitive to inhibition by acetaldehyde, which likely limits its activity in vivo. This study emphasizes the importance of maintaining a low steady–state acetaldehyde concentration in hepatocytes during ethanol metabolism. PMID:21166830

Inhibition of NAD glycohydrolase and ADP-ribosyl transferases by carbocyclic analogues of oxidized nicotinamide adenine dinucleotide.

PubMed

Slama, J T; Simmons, A M

1989-09-19

Analogues of oxidized nicotinamide adenine dinucleotide (NAD+) in which a 2,3-dihydroxycyclopentane ring replaces the beta-D-ribonucleotide ring of the nicotinamide riboside moiety of NAD+ have recently been synthesized [Slama, J. T., & Simmons, A. M. (1988) Biochemistry 27, 183]. Carbocyclic NAD+ analogues have been shown to inhibit NAD glycohydrolases and ADP-ribosyl transferases such as cholera toxin A subunit. In this study, the diastereomeric mixture of dinucleotides was separated, and the inhibitory capacity of each of the purified diastereomers was defined. The NAD+ analogue in which the D-dihydroxycyclopentane is substituted for the D-ribose is designated carba-NAD and was demonstrated to be a poor inhibitor of the Bungarus fasciatus venom NAD glycohydrolase. The diastereomeric dinucleotide pseudo-carbocyclic-NAD (psi-carba-NAD), containing L-dihydroxycyclopentane in place of the D-ribose of NAD+, was shown, however, to be a potent competitive inhibitor of the venom NAD glycohydrolase with an inhibitor dissociation constant (Ki) of 35 microM. This was surprising since psi-carba-NAD contains the carbocyclic analogue of the unnatural L-ribotide and was therefore expected to be a biologically inactive diastereomer. psi-Carba-NAD also competitively inhibited the insoluble brain NAD glycohydrolase from cow (Ki = 6.7 microM) and sheep (Ki = 31 microM) enzyme against which carba-NAD is ineffective. Sensitivity to psi-carba-NAD was found to parallel sensitivity to inhibition by isonicotinic acid hydrazide, another NADase inhibitor. psi-Carba-NAD is neither a substrate for nor an inhibitor of alcohol dehydrogenase, whereas carba-NAD is an efficient dehydrogenase substrate.(ABSTRACT TRUNCATED AT 250 WORDS)

Direct Electrochemical Addressing of Immobilized Alcohol Dehydrogenase for the Heterogeneous Bioelectrocatalytic Reduction of Butyraldehyde to Butanol.

PubMed

Schlager, S; Neugebauer, H; Haberbauer, M; Hinterberger, G; Sariciftci, N S

2015-03-01

Modified electrodes using immobilized alcohol dehydrogenase enzymes for the efficient electroreduction of butyraldehyde to butanol are presented as an important step for the utilization of CO 2 -reduction products. Alcohol dehydrogenase was immobilized, embedded in an alginate-silicate hybrid gel, on a carbon felt (CF) electrode. The application of this enzyme to the reduction of an aldehyde to an alcohol with the aid of the coenzyme nicotinamide adenine dinucleotide (NADH), in analogy to the final step in the natural reduction cascade of CO 2 to alcohol, has been already reported. However, the use of such enzymatic reductions is limited because of the necessity of providing expensive NADH as a sacrificial electron and proton donor. Immobilization of such dehydrogenase enzymes on electrodes and direct pumping of electrons into the biocatalysts offers an easy and efficient way for the biochemical recycling of CO 2 to valuable chemicals or alternative synthetic fuels. We report the direct electrochemical addressing of immobilized alcohol dehydrogenase for the reduction of butyraldehyde to butanol without consumption of NADH. The selective reduction of butyraldehyde to butanol occurs at room temperature, ambient pressure and neutral pH. Production of butanol was detected by using liquid-injection gas chromatography and was estimated to occur with Faradaic efficiencies of around 40 %.

Highly selective anti-Prelog synthesis of optically active aryl alcohols by recombinant Escherichia coli expressing stereospecific alcohol dehydrogenase.

PubMed

Li, Ming; Nie, Yao; Mu, Xiao Qing; Zhang, Rongzhen; Xu, Yan

2016-07-03

Biocatalytic asymmetric synthesis has been widely used for preparation of optically active chiral alcohols as the important intermediates and precursors of active pharmaceutical ingredients. However, the available whole-cell system involving anti-Prelog specific alcohol dehydrogenase is yet limited. A recombinant Escherichia coli system expressing anti-Prelog stereospecific alcohol dehydrogenase from Candida parapsilosis was established as a whole-cell system for catalyzing asymmetric reduction of aryl ketones to anti-Prelog configured alcohols. Using 2-hydroxyacetophenone as the substrate, reaction factors including pH, cell status, and substrate concentration had obvious impacts on the outcome of whole-cell biocatalysis, and xylose was found to be an available auxiliary substrate for intracellular cofactor regeneration, by which (S)-1-phenyl-1,2-ethanediol was achieved with an optical purity of 97%e.e. and yield of 89% under the substrate concentration of 5 g/L. Additionally, the feasibility of the recombinant cells toward different aryl ketones was investigated, and most of the corresponding chiral alcohol products were obtained with an optical purity over 95%e.e. Therefore, the whole-cell system involving recombinant stereospecific alcohol dehydrogenase was constructed as an efficient biocatalyst for highly enantioselective anti-Prelog synthesis of optically active aryl alcohols and would be promising in the pharmaceutical industry.

Activation of liver alcohol dehydrogenase by glycosylation.

PubMed Central

Tsai, C S; White, J H

1983-01-01

D-Fructose and D-glucose activate alcohol dehydrogenase from horse liver to oxidize ethanol. One mol of D-[U-14C]fructose or D-[U-14C]glucose is covalently incorporated per mol of the maximally activated enzyme. Amino acid and N-terminal analyses of the 14C-labelled glycopeptide isolated from a proteolytic digest of the [14C]glycosylated enzyme implicate lysine-315 as the site of the glycosylation. 13C-n.m.r.-spectroscopic studies indicate that D-[13C]glucose is covalently linked in N-glucosidic and Amadori-rearranged structures in the [13C]glucosylated alcohol dehydrogenase. Experimental results are consistent with the formation of the N-glycosylic linkage between glycose and lysine-315 of liver alcohol dehydrogenase in the initial step that results in an enhanced catalytic efficiency to oxidize ethanol. PMID:6342612

Enzymatic transformation of hydrocarbons by methanotrophic organisms

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

Patel, R.N.; Hou, C.T.

Soluble methane monooxygenase from a facultative methane-utilizing organism, Methylobacterium sp. CRL-26 or R6, catalyzed the NAD(P)H-dependent epoxidation/hydroxylation of a variety of hydrocarbons, including terminal alkenes, internal alkenes, substituted alkenes, branch-chain alkenes, alkanes (C1-C8), substituted alkanes, branch-chain alkanes, carbon monoxide, ether, cyclic and aromatic compounds. The NAD -linked dehydrogenases such as formate dehydrogenase or secondary alcohol dehydrogenase in the presence of formate or secondary alcohol, respectively, regenerated NAD/NADH required for the methane monooxygenase in a coupled enzymes reactions. Oxidation of secondary alcohols to the corresponding methylketones in methanotrophs is catalyzed by an NAD -dependent, zinc-containing, secondary alcohol hydrogenase. Primary alcohols weremore » oxidized to the corresponding aldehydes by a phenazine methosulfate-dependent, pyrollo quinoline quinone (methoxatin or PQQ) containing, methanol dehydrogenase. Oxidation of aldehydes (C1 to C10) to the corresponding carboxylic acids is catalyzed by a heme-containing aldehyde dehydrogenase. Methanotrophs have been considered potentially useful for single cell protein (SCP), amino acids, and biopolymer production at the expense of growth on cheap and readily available C1 compounds. 80 references, 1 figure, 6 tables.« less

Alcohol and aldehyde dehydrogenase polymorphisms in Chinese and Indian populations.

PubMed

Tan, Ene-Choo; Lim, Leslie; Leong, Jern-Yi; Lim, Jing-Yan; Lee, Arthur; Yang, Jun; Tan, Chay-Hoon; Winslow, Munidasa

2010-01-01

The association between two functional polymorphisms in alcohol dehydrogenase (ADH2/ADH1B) and aldehyde dehydrogenase (ALDH2) genes and alcohol dependence was examined in 182 Chinese and Indian patients undergoing treatment for alcohol dependence and 184 screened control subjects from Singapore. All subjects were screened by the Alcohol Use Disorders Identification Test (AUDIT). Patients were also administered the Severity of Alcohol Dependence Questionnaire (SADQ). Polymorphisms were genotyped by allele-specific polymerase chain reaction and selected genotypes confirmed by DNA sequencing or restriction fragment length polymorphism. Our results showed that frequencies of ADH1B*2 and ALDH2*2 were higher in controls compared to alcohol-dependent subjects for both Chinese and Indians. Frequencies of these two alleles were also higher in the 104 Chinese controls compared to the 80 Indian controls. None of the eight Chinese who were homozygous for both protective alleles was alcohol dependent. The higher frequencies of the protective alleles could explain the lower rate of alcohol dependence in Chinese.

Human class II (pi) alcohol dehydrogenase has a redox-specific function in norepinephrine metabolism.

PubMed Central

Mårdh, G; Dingley, A L; Auld, D S; Vallee, B L

1986-01-01

Studies of the function of human alcohol dehydrogenase (ADH) have revealed substrates that are virtually unique for class II ADH (pi ADH). It catalyzes the formation of the intermediary glycols of norepinephrine metabolism, 3,4-dihydroxyphenylglycol and 4-hydroxy-3-methoxyphenylglycol, from the corresponding aldehydes 3,4-dihydroxymandelaldehyde and 4-hydroxy-3-methoxymandelaldehyde with Km values of 55 and 120 microM and kcat/Km ratios of 14,000 and 17,000 mM-1 X min-1; these are from 60- to 210-fold higher than those obtained with class I ADH isozymes. The catalytic preference of class II ADH also extends to benzaldehydes. The kcat/Km values for the reduction of benzaldehyde, 3,4-dihydroxybenzaldehyde and 4-hydroxy-3-methoxybenzaldehyde by pi ADH are from 9- to 29-fold higher than those for a class I isozyme, beta 1 gamma 2 ADH. Furthermore, the norepinephrine aldehydes are potent inhibitors of alcohol (ethanol) oxidation by pi ADH. The high catalytic activity of pi ADH-catalyzed reduction of the aldehydes in combination with a possible regulatory function of the aldehydes in the oxidative direction leads to essentially "unidirectional" catalysis by pi ADH. These features and the presence of pi ADH in human liver imply a physiological role for pi ADH in the degradation of circulating epinephrine and norepinephrine. PMID:3466164

Identification of a mitochondrial alcohol dehydrogenase in Schizosaccharomyces pombe: new insights into energy metabolism

PubMed Central

Crichton, Paul G.; Affourtit, Charles; Moore, Anthony L.

2006-01-01

In the present study we have shown that mitochondria isolated from Schizosaccharomyces pombe exhibit antimycin A-sensitive oxygen uptake activity that is exclusively dependent on ethanol and is inhibited by trifluoroethanol, a potent inhibitor of ADH (alcohol dehydrogenase). Ethanol-dependent respiratory activity has, to our knowledge, not been reported in S. pombe mitochondria to date, which is surprising as it has been concluded previously that only one ADH gene, encoding a cytosolic enzyme, occurs in this yeast. Spectrophotometric enzyme assays reveal that ADH activity in isolated mitochondria is increased ∼16-fold by Triton X-100, which demonstrates that the enzyme is located in the matrix. Using genetic knockouts, we show conclusively that the novel mitochondrial ADH is encoded by adh4 and, as such, is unrelated to ADH isoenzymes found in mitochondria of other yeasts. By performing a modular-kinetic analysis of mitochondrial electron transfer, we furthermore show how ethanol-dependent respiratory activity (which involves oxidation of matrix-located NADH) compares with that observed when succinate or externally added NADH are used as substrates. This analysis reveals distinct kinetic differences between substrates which fully explain the lack of respiratory control generally observed during ethanol oxidation in yeast mitochondria. PMID:16999687

Changes in cinnamyl alcohol dehydrogenase activities from sugarcane cultivars inoculated with Sporisorium scitamineum sporidia.

PubMed

Santiago, Rocío; Alarcón, Borja; de Armas, Roberto; Vicente, Carlos; Legaz, María Estrella

2012-06-01

This study describes a method for determining cinnamyl alcohol dehydrogenase activity in sugarcane stems using reverse phase (RP) high-performance liquid chromatography to elucidate their possible lignin origin. Activity is assayed using the reverse mode, the oxidation of hydroxycinnamyl alcohols into hydroxycinnamyl aldehydes. Appearance of the reaction products, coniferaldehyde and sinapaldehyde is determined by measuring absorbance at 340 and 345 nm, respectively. Disappearance of substrates, coniferyl alcohol and sinapyl alcohol is measured at 263 and 273 nm, respectively. Isocratic elution with acetonitrile:acetic acid through an RP Mediterranea sea C18 column is performed. As case examples, we have examined two different cultivars of sugarcane; My 5514 is resistant to smut, whereas B 42231 is susceptible to the pathogen. Inoculation of sugarcane stems elicits lignification and produces significant increases of coniferyl alcohol dehydrogenase (CAD) and sinapyl alcohol dehydrogenase (SAD). Production of lignin increases about 29% in the resistant cultivar and only 13% in the susceptible cultivar after inoculation compared to uninoculated plants. Our results show that the resistance of My 5514 to smut is likely derived, at least in part, to a marked increase of lignin concentration by the activation of CAD and SAD. Copyright © Physiologia Plantarum 2012.

Cloning and sequencing of the alcohol dehydrogenase II gene from Zymomonas mobilis

DOEpatents

Ingram, Lonnie O.; Conway, Tyrrell

1992-01-01

The alcohol dehydrogenase II gene from Zymomonas mobilis has been cloned and sequenced. This gene can be expressed at high levels in other organisms to produce acetaldehyde or to convert acetaldehyde to ethanol.

Functional characterization of transmembrane intracellular pH regulators and mechanism of alcohol-induced intracellular acidosis in human umbilical cord blood stem cell-like cells.

PubMed

Tsai, Yi-Ting; Liu, Jah-Yao; Lee, Chung-Yi; Tsai, Chien-Sung; Chen, Ming-Hurng; Ou, Chien-Chih; Chen, Wei-Hwa; Loh, Shih-Hurng

2011-12-01

Changing intracellular pH (pHi) exerts considerable influence on many cellular functions. Different pHi regulators, such as the Na-H exchanger (NHE), Na/(Equation is included in full-text article.)symporter, and Cl/OH exchanger (CHE), have been identified in mature mammalian cells. The aims of the present study were to investigate the physiological mechanisms of pHi recovery and to further explore the effects of alcohol on the pHi in human umbilical cord blood CD34 stem cell-like cells (HUCB-CD34STs). HUCB-CD34STs were loaded with the pH-sensitive dye, 2',7'-bis(2-carboxethyl)-5(6)-carboxyfluorescein, to examine pHi. In isolated HUCB-CD34STs, we found that (1) the resting pHi is 7.03 ± 0.02; (2) 2 Na-dependent acid extruders and a Cl-dependent acid loading carrier exist and are functional; (3) alcohol functions in a concentration-dependent manner to reduce pHi and increase NHE activity, but it does not affect CHE activity; and (4) fomepizole, a specific alcohol dehydrogenase inhibitor, does not change the intracellular acidosis and NHE activity-induced by alcohol, whereas 3-amino-1, 2,4-trizole, a specific catalase inhibitor, entirely abolishes these effects. In conclusion, we demonstrate that 2 acid extruders and 1 acid loader (most likely NHE, NBC, and CHE, respectively) functionally existed in HUCB-CD34STs. Additionally, the intracellular acidosis is mainly caused by catalase-mediated alcohol metabolites, which provoke the activity of NHE.

Proton mediated control of biochemical reactions with bioelectronic pH modulation

NASA Astrophysics Data System (ADS)

Deng, Yingxin; Miyake, Takeo; Keene, Scott; Josberger, Erik E.; Rolandi, Marco

2016-04-01

In Nature, protons (H+) can mediate metabolic process through enzymatic reactions. Examples include glucose oxidation with glucose dehydrogenase to regulate blood glucose level, alcohol dissolution into carboxylic acid through alcohol dehydrogenase, and voltage-regulated H+ channels activating bioluminescence in firefly and jellyfish. Artificial devices that control H+ currents and H+ concentration (pH) are able to actively influence biochemical processes. Here, we demonstrate a biotransducer that monitors and actively regulates pH-responsive enzymatic reactions by monitoring and controlling the flow of H+ between PdHx contacts and solution. The present transducer records bistable pH modulation from an “enzymatic flip-flop” circuit that comprises glucose dehydrogenase and alcohol dehydrogenase. The transducer also controls bioluminescence from firefly luciferase by affecting solution pH.

Insight into the stereospecificity of short-chain thermus thermophilus alcohol dehydrogenase showing pro-S hydride transfer and prelog enantioselectivity.

PubMed

Pennacchio, Angela; Giordano, Assunta; Esposito, Luciana; Langella, Emma; Rossi, Mosè; Raia, Carlo A

2010-04-01

The stereochemistry of the hydride transfer in reactions catalyzed by NAD(H)-dependent alcohol dehydrogenase from Thermus thermophilus HB27 was determined by means of (1)H-NMR spectroscopy. The enzyme transfers the pro-S hydrogen of [4R-(2)H]NADH and exhibits Prelog specificity. Enzyme-substrate docking calculations provided structural details about the enantioselectivity of this thermophilic enzyme. These results give additional insights into the diverse active site architectures of the largely versatile short-chain dehydrogenase superfamily enzymes. A feasible protocol for the synthesis of [4R-(2)H]NADH with high yield was also set up by enzymatic oxidation of 2-propanol-d(8) catalyzed by Bacillus stearothermophilus alcohol dehydrogenase.

Kinetic mechanism of Escherichia coli isocitrate dehydrogenase and its inhibition by glyoxylate and oxaloacetate.

PubMed Central

Nimmo, H G

1986-01-01

The inhibition of Escherichia coli isocitrate dehydrogenase by glyoxylate and oxaloacetate was examined. The shapes of the progress curves in the presence of the inhibitors depended on the order of addition of the assay components. When isocitrate dehydrogenase or NADP+ was added last, the rate slowly decreased until a new, inhibited, steady state was obtained. When isocitrate was added last, the initial rate was almost zero, but the rate increased slowly until the same steady-state value was obtained. Glyoxylate and oxaloacetate gave competitive inhibition against isocitrate and uncompetitive inhibition against NADP+. Product-inhibition studies showed that isocitrate dehydrogenase obeys a compulsory-order mechanism, with coenzyme binding first. Glyoxylate and oxaloacetate bind to and dissociate from isocitrate dehydrogenase slowly. These observations can account for the shapes of the progress curves observed in the presence of the inhibitors. Condensation of glyoxylate and oxaloacetate produced an extremely potent inhibitor of isocitrate dehydrogenase. Analysis of the reaction by h.p.l.c. showed that this correlated with the formation of oxalomalate. This compound decomposed spontaneously in assay mixtures, giving 4-hydroxy-2-oxoglutarate, which was a much less potent inhibitor of the enzyme. Oxalomalate inhibited isocitrate dehydrogenase competitively with respect to isocitrate and was a very poor substrate for the enzyme. The data suggest that the inhibition of isocitrate dehydrogenase by glyoxylate and oxaloacetate is not physiologically significant. PMID:3521584

Structural and Kinetic Basis for Substrate Selectivity in Populus tremuloides Sinapyl Alcohol Dehydrogenase

PubMed Central

Bomati, Erin K.; Noel, Joseph P.

2005-01-01

We describe the three-dimensional structure of sinapyl alcohol dehydrogenase (SAD) from Populus tremuloides (aspen), a member of the NADP(H)-dependent dehydrogenase family that catalyzes the last reductive step in the formation of monolignols. The active site topology revealed by the crystal structure substantiates kinetic results indicating that SAD maintains highest specificity for the substrate sinapaldehyde. We also report substantial substrate inhibition kinetics for the SAD-catalyzed reduction of hydroxycinnamaldehydes. Although SAD and classical cinnamyl alcohol dehydrogenases (CADs) catalyze the same reaction and share some sequence identity, the active site topology of SAD is strikingly different from that predicted for classical CADs. Kinetic analyses of wild-type SAD and several active site mutants demonstrate the complexity of defining determinants of substrate specificity in these enzymes. These results, along with a phylogenetic analysis, support the inclusion of SAD in a plant alcohol dehydrogenase subfamily that includes cinnamaldehyde and benzaldehyde dehydrogenases. We used the SAD three-dimensional structure to model several of these SAD-like enzymes, and although their active site topologies largely mirror that of SAD, we describe a correlation between substrate specificity and amino acid substitution patterns in their active sites. The SAD structure thus provides a framework for understanding substrate specificity in this family of enzymes and for engineering new enzyme specificities. PMID:15829607

Structural and kinetic basis for substrate selectivity in Populus tremuloides sinapyl alcohol dehydrogenase.

PubMed

Bomati, Erin K; Noel, Joseph P

2005-05-01

We describe the three-dimensional structure of sinapyl alcohol dehydrogenase (SAD) from Populus tremuloides (aspen), a member of the NADP(H)-dependent dehydrogenase family that catalyzes the last reductive step in the formation of monolignols. The active site topology revealed by the crystal structure substantiates kinetic results indicating that SAD maintains highest specificity for the substrate sinapaldehyde. We also report substantial substrate inhibition kinetics for the SAD-catalyzed reduction of hydroxycinnamaldehydes. Although SAD and classical cinnamyl alcohol dehydrogenases (CADs) catalyze the same reaction and share some sequence identity, the active site topology of SAD is strikingly different from that predicted for classical CADs. Kinetic analyses of wild-type SAD and several active site mutants demonstrate the complexity of defining determinants of substrate specificity in these enzymes. These results, along with a phylogenetic analysis, support the inclusion of SAD in a plant alcohol dehydrogenase subfamily that includes cinnamaldehyde and benzaldehyde dehydrogenases. We used the SAD three-dimensional structure to model several of these SAD-like enzymes, and although their active site topologies largely mirror that of SAD, we describe a correlation between substrate specificity and amino acid substitution patterns in their active sites. The SAD structure thus provides a framework for understanding substrate specificity in this family of enzymes and for engineering new enzyme specificities.

Novel amide-based inhibitors of inosine 5'-monophosphate dehydrogenase.

PubMed

Watterson, Scott H; Liu, Chunjian; Dhar, T G Murali; Gu, Henry H; Pitts, William J; Barrish, Joel C; Fleener, Catherine A; Rouleau, Katherine; Sherbina, N Z; Hollenbaugh, Diane L; Iwanowicz, Edwin J

2002-10-21

A series of novel amide-based small molecule inhibitors of inosine monophosphate dehydrogenase (IMPDH) was explored. The synthesis and the structure-activity relationships (SARs) derived from in vitro studies are described.

Rapid synthesis of triazine inhibitors of inosine monophosphate dehydrogenase.

PubMed

Pitts, William J; Guo, Junqing; Dhar, T G Murali; Shen, Zhongqi; Gu, Henry H; Watterson, Scott H; Bednarz, Mark S; Chen, Bang Chi; Barrish, Joel C; Bassolino, Donna; Cheney, Daniel; Fleener, Catherine A; Rouleau, Katherine A; Hollenbaugh, Diane L; Iwanowicz, Edwin J

2002-08-19

A series of novel triazine-based small molecule inhibitors (IV) of inosine monophosphate dehydrogenase was prepared. The synthesis and the structure-activity relationships (SAR) derived from in vitro studies are described.

Proton mediated control of biochemical reactions with bioelectronic pH modulation

DOE PAGES

Deng, Yingxin; Miyake, Takeo; Keene, Scott; ...

2016-04-07

In Nature, protons (H +) can mediate metabolic process through enzymatic reactions. Examples include glucose oxidation with glucose dehydrogenase to regulate blood glucose level, alcohol dissolution into carboxylic acid through alcohol dehydrogenase, and voltage-regulated H + channels activating bioluminescence in firefly and jellyfish. Artificial devices that control H + currents and H + concentration (pH) are able to actively influence biochemical processes. Here, we demonstrate a biotransducer that monitors and actively regulates pH-responsive enzymatic reactions by monitoring and controlling the flow of H + between PdH x contacts and solution. The present transducer records bistable pH modulation from an “enzymaticmore » flip-flop” circuit that comprises glucose dehydrogenase and alcohol dehydrogenase. Furthermore, the transducer also controls bioluminescence from firefly luciferase by affecting solution pH.« less

Proton mediated control of biochemical reactions with bioelectronic pH modulation

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

Deng, Yingxin; Miyake, Takeo; Keene, Scott

In Nature, protons (H +) can mediate metabolic process through enzymatic reactions. Examples include glucose oxidation with glucose dehydrogenase to regulate blood glucose level, alcohol dissolution into carboxylic acid through alcohol dehydrogenase, and voltage-regulated H + channels activating bioluminescence in firefly and jellyfish. Artificial devices that control H + currents and H + concentration (pH) are able to actively influence biochemical processes. Here, we demonstrate a biotransducer that monitors and actively regulates pH-responsive enzymatic reactions by monitoring and controlling the flow of H + between PdH x contacts and solution. The present transducer records bistable pH modulation from an “enzymaticmore » flip-flop” circuit that comprises glucose dehydrogenase and alcohol dehydrogenase. Furthermore, the transducer also controls bioluminescence from firefly luciferase by affecting solution pH.« less

A survey of cyclic replacements for the central diamide moiety of inhibitors of inosine monophosphate dehydrogenase.

PubMed

Dhar, T G Murali; Liu, Chunjian; Pitts, William J; Guo, Junquing; Watterson, Scott H; Gu, Henry; Fleener, Catherine A; Rouleau, Katherine; Sherbina, N Z; Barrish, Joel C; Hollenbaugh, Diane; Iwanowicz, Edwin J

2002-11-04

A series of heterocyclic replacements for the central diamide moiety of 1, a potent small molecule inhibitor of inosine monophosphate dehydrogenase (IMPDH) were explored The synthesis and the structure-activity relationships (SARs), derived from in vitro studies, for these new series of inhibitors is given.

Recent advances in biotechnological applications of alcohol dehydrogenases.

PubMed

Zheng, Yu-Guo; Yin, Huan-Huan; Yu, Dao-Fu; Chen, Xiang; Tang, Xiao-Ling; Zhang, Xiao-Jian; Xue, Ya-Ping; Wang, Ya-Jun; Liu, Zhi-Qiang

2017-02-01

Alcohol dehydrogenases (ADHs), which belong to the oxidoreductase superfamily, catalyze the interconversion between alcohols and aldehydes or ketones with high stereoselectivity under mild conditions. ADHs are widely employed as biocatalysts for the dynamic kinetic resolution of racemic substrates and for the preparation of enantiomerically pure chemicals. This review provides an overview of biotechnological applications for ADHs in the production of chiral pharmaceuticals and fine chemicals.

The oxidative fermentation of ethanol in Gluconacetobacter diazotrophicus is a two-step pathway catalyzed by a single enzyme: alcohol-aldehyde Dehydrogenase (ADHa).

PubMed

Gómez-Manzo, Saúl; Escamilla, José E; González-Valdez, Abigail; López-Velázquez, Gabriel; Vanoye-Carlo, América; Marcial-Quino, Jaime; de la Mora-de la Mora, Ignacio; Garcia-Torres, Itzhel; Enríquez-Flores, Sergio; Contreras-Zentella, Martha Lucinda; Arreguín-Espinosa, Roberto; Kroneck, Peter M H; Sosa-Torres, Martha Elena

2015-01-07

Gluconacetobacter diazotrophicus is a N2-fixing bacterium endophyte from sugar cane. The oxidation of ethanol to acetic acid of this organism takes place in the periplasmic space, and this reaction is catalyzed by two membrane-bound enzymes complexes: the alcohol dehydrogenase (ADH) and the aldehyde dehydrogenase (ALDH). We present strong evidence showing that the well-known membrane-bound Alcohol dehydrogenase (ADHa) of Ga. diazotrophicus is indeed a double function enzyme, which is able to use primary alcohols (C2-C6) and its respective aldehydes as alternate substrates. Moreover, the enzyme utilizes ethanol as a substrate in a reaction mechanism where this is subjected to a two-step oxidation process to produce acetic acid without releasing the acetaldehyde intermediary to the media. Moreover, we propose a mechanism that, under physiological conditions, might permit a massive conversion of ethanol to acetic acid, as usually occurs in the acetic acid bacteria, but without the transient accumulation of the highly toxic acetaldehyde.

Similarity of Escherichia coli propanediol oxidoreductase (fucO product) and an unusual alcohol dehydrogenase from Zymomonas mobilis and Saccharomyces cerevisiae

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

Conway, T.; Ingram, L.O.

1989-07-01

The gene that encodes 1,2-propanediol oxidoreductase (fucO) from Escherichia coli was sequenced. The reading frame specified a protein of 383 amino acids (including the N-terminal methionine), with an aggregate molecular weight of 40,642. The induction of fucO transcription, which occurred in the presence of fucose, was confirmed by Northern blot analysis. In E. coli, the primary fucO transcript was approximately 2.1 kilobases in length. The 5{prime} end of the transcript began more than 0.7 kilobase upstream of the fucO start codon within or beyond the fucA gene. Propanediol oxidoreductase exhibited 41.7% identity with the iron-containing alcohol dehydrogenase II from Zymomonasmore » mobilis and 39.5% identity with ADH4 from Saccharomyces cerevisiae. These three proteins did not share homology with either short-chain or long-chain zinc-containing alcohol dehydrogenase enzymes. We propose that these three unusual alcohol dehydrogenases define a new family of enzymes.« less

The bifunctional alcohol and aldehyde dehydrogenase gene, adhE, is necessary for ethanol production in Clostridium thermocellum and Thermoanaerobacterium saccharolyticum.

PubMed

Lo, Jonathan; Zheng, Tianyong; Hon, Shuen; Olson, Daniel G; Lynd, Lee R

2015-04-01

Thermoanaerobacterium saccharolyticum and Clostridium thermocellum are anaerobic thermophilic bacteria being investigated for their ability to produce biofuels from plant biomass. The bifunctional alcohol and aldehyde dehydrogenase gene, adhE, is present in these bacteria and has been known to be important for ethanol formation in other anaerobic alcohol producers. This study explores the inactivation of the adhE gene in C. thermocellum and T. saccharolyticum. Deletion of adhE reduced ethanol production by >95% in both T. saccharolyticum and C. thermocellum, confirming that adhE is necessary for ethanol formation in both organisms. In both adhE deletion strains, fermentation products shifted from ethanol to lactate production and resulted in lower cell density and longer time to reach maximal cell density. In T. saccharolyticum, the adhE deletion strain lost >85% of alcohol dehydrogenase (ADH) activity. Aldehyde dehydrogenase (ALDH) activity did not appear to be affected, although ALDH activity was low in cell extracts. Adding ubiquinone-0 to the ALDH assay increased activity in the T. saccharolyticum parent strain but did not increase activity in the adhE deletion strain, suggesting that ALDH activity was inhibited. In C. thermocellum, the adhE deletion strain lost >90% of ALDH and ADH activity in cell extracts. The C. thermocellum adhE deletion strain contained a point mutation in the lactate dehydrogenase gene, which appears to deregulate its activation by fructose 1,6-bisphosphate, leading to constitutive activation of lactate dehydrogenase. Thermoanaerobacterium saccharolyticum and Clostridium thermocellum are bacteria that have been investigated for their ability to produce biofuels from plant biomass. They have been engineered to produce higher yields of ethanol, yet questions remain about the enzymes responsible for ethanol formation in these bacteria. The genomes of these bacteria encode multiple predicted aldehyde and alcohol dehydrogenases which could be responsible for alcohol formation. This study explores the inactivation of adhE, a gene encoding a bifunctional alcohol and aldehyde dehydrogenase. Deletion of adhE reduced ethanol production by >95% in both T. saccharolyticum and C. thermocellum, confirming that adhE is necessary for ethanol formation in both organisms. In strains without adhE, we note changes in biochemical activity, product formation, and growth. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Chronic alcohol binging injures the liver and other organs by reducing NAD⁺ levels required for sirtuin's deacetylase activity.

PubMed

French, Samuel W

2016-04-01

NAD(+) levels are markedly reduced when blood alcohol levels are high during binge drinking. This causes liver injury to occur because the enzymes that require NAD(+) as a cofactor such as the sirtuin de-acetylases cannot de-acetylate acetylated proteins such as acetylated histones. This prevents the epigenetic changes that regulate metabolic processes and which prevent organ injury such as fatty liver in response to alcohol abuse. Hyper acetylation of numerous regulatory proteins develops. Systemic multi-organ injury occurs when NAD(+) is reduced. For instance the Circadian clock is altered if NAD(+) is not available. Cell cycle arrest occurs due to up regulation of cell cycle inhibitors leading to DNA damage, mutations, apoptosis and tumorigenesis. NAD(+) is linked to aging in the regulation of telomere stability. NAD(+) is required for mitochondrial renewal. Alcohol dehydrogenase is present in every visceral organ in the body so that there is a systemic reduction of NAD(+) levels in all of these organs during binge drinking. Copyright © 2016. Published by Elsevier Inc.

Effects of Moderate Alcohol Consumption on Gene Expression Related to Colonic Inflammation and Antioxidant Enzymes in Rats

PubMed Central

Klarich, DawnKylee S.; Penprase, Jerrold; Cintora, Patricia; Medrano, Octavio; Erwin, Danielle; Brasser, Susan M.; Hong, Mee Young

2017-01-01

Excessive alcohol consumption is a risk factor associated with colorectal cancer; however, some studies have reported that moderate alcohol consumption may not contribute additional risk for developing colorectal cancer while others suggest that moderate alcohol consumption provides a protective effect that reduces colorectal cancer risk. The purpose of this study was to determine the effects of moderate voluntary alcohol (20% ethanol) intake on alternate days for 3 months in outbred Wistar rats on risk factors associated with colorectal cancer development. Colonic gene expression of cyclooxygenase-2, RelA, 8-oxoguanine DNA glycosylase 1, superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, glutathione-S-transferase M1, and aldehyde dehydrogenase 2 were determined. Blood alcohol content, liver function enzyme activities, and 8-oxo-deoxyguanosine DNA adducts were also assessed. Alcohol-treated rats were found to have significantly lower 8-oxo-deoxyguanosine levels in blood, a marker of DNA damage. Alanine aminotransferase and lactate dehydrogenase were both significantly lower in the alcohol group. Moderate alcohol significantly decreased cyclooxygenase-2 gene expression, an inflammatory marker associated with colorectal cancer risk. The alcohol group had significantly increased glutathione-S-transferase M1 expression, an antioxidant enzyme that helps detoxify carcinogens, such as acetaldehyde, and significantly increased aldehyde dehydrogenase 2 expression, which allows for greater acetaldehyde clearance. Increased expression of glutathione-S-transferase M1 and aldehyde dehydrogenase 2 likely contributed to reduce mucosal damage that is caused by acetaldehyde accumulation. These results indicate that moderate alcohol may reduce the risk for colorectal cancer development, which was evidenced by reduced inflammation activity and lower DNA damage after alcohol exposure. PMID:28599714

Enantiocomplementary Yarrowia lipolytica Oxidoreductases: Alcohol Dehydrogenase 2 and Short Chain Dehydrogenase/Reductase

PubMed Central

Napora-Wijata, Kamila; Strohmeier, Gernot A.; Sonavane, Manoj N.; Avi, Manuela; Robins, Karen; Winkler, Margit

2013-01-01

Enzymes of the non-conventional yeast Yarrowia lipolytica seem to be tailor-made for the conversion of lipophilic substrates. Herein, we cloned and overexpressed the Zn-dependent alcohol dehydrogenase ADH2 from Yarrowia lipolytica in Escherichia coli. The purified enzyme was characterized in vitro. The substrate scope for YlADH2 mediated oxidation and reduction was investigated spectrophotometrically and the enzyme showed a broader substrate range than its homolog from Saccharomyces cerevisiae. A preference for secondary compared to primary alcohols in oxidation direction was observed for YlADH2. 2-Octanone was investigated in reduction mode in detail. Remarkably, YlADH2 displays perfect (S)-selectivity and together with a highly (R)-selective short chain dehydrogenase/ reductase from Yarrowia lipolytica it is possible to access both enantiomers of 2-octanol in >99% ee with Yarrowia lipolytica oxidoreductases. PMID:24970175

Enantiocomplementary Yarrowia lipolytica Oxidoreductases: Alcohol Dehydrogenase 2 and Short Chain Dehydrogenase/Reductase.

PubMed

Napora-Wijata, Kamila; Strohmeier, Gernot A; Sonavane, Manoj N; Avi, Manuela; Robins, Karen; Winkler, Margit

2013-08-12

Enzymes of the non-conventional yeast Yarrowia lipolytica seem to be tailor-made for the conversion of lipophilic substrates. Herein, we cloned and overexpressed the Zn-dependent alcohol dehydrogenase ADH2 from Yarrowia lipolytica in Escherichia coli. The purified enzyme was characterized in vitro. The substrate scope for YlADH2 mediated oxidation and reduction was investigated spectrophotometrically and the enzyme showed a broader substrate range than its homolog from Saccharomyces cerevisiae. A preference for secondary compared to primary alcohols in oxidation direction was observed for YlADH2. 2-Octanone was investigated in reduction mode in detail. Remarkably, YlADH2 displays perfect (S)-selectivity and together with a highly (R)-selective short chain dehydrogenase/ reductase from Yarrowia lipolytica it is possible to access both enantiomers of 2-octanol in >99% ee with Yarrowia lipolytica oxidoreductases.

[Polymorphism of alcohol dehydrogenase gene ADH1B in eastern Slavic and Iranian-speaking populations].

PubMed

2005-11-01

Frequencies of alleles and genotypes for alcohol dehydrogenase gene ADH1B (arg47his polymorphism), associated with alcohol tolerance/sensitivity, were determined. It was demonstrated that the frequency of allele ADH1B*47his, corresponding to atypical alcohol dehydrogenase variant in Russians, Ukrainians, Iranians, and mountain-dwellers of the Pamirs constituted 3, 7, 24, and 22%, respectively. The frequencies established were consistent with the allele frequency distribution pattern among the populations of Eurasia. Russians and Ukrainians were indistinguishable from other European populations relative to the frequency of allele ADH1B*47his, and consequently, relative to specific features of ethanol metabolic pathways. The data obtained provide refinement of the geographic pattern of ADH1B*47his frequency distribution in Eurasia.

Novel guanidine-based inhibitors of inosine monophosphate dehydrogenase.

PubMed

Iwanowicz, Edwin J; Watterson, Scott H; Liu, Chunjian; Gu, Henry H; Mitt, Toomas; Leftheris, Katerina; Barrish, Joel C; Fleener, Catherine A; Rouleau, Katherine; Sherbina, N Z; Hollenbaugh, Diane L

2002-10-21

A series of novel guanidine-based small molecule inhibitors of inosine monophosphate dehydrogenase (IMPDH) was explored. IMPDH catalyzes the rate determining step in guanine nucleotide biosynthesis and is a target for anticancer, immunosuppressive and antiviral therapy. The synthesis and the structure-activity relationships (SARs), derived from in vitro studies, for this new series of inhibitors is given.

[Distribution of genotypes of alcohol dehydrogenase 2 and aldehyde dehydrogenase 2 in Japanese twin children].

PubMed

Qu, W; Yamagata, Z; Wu, D; Zhang, B; Zhang, Y

1999-03-01

In order to prevent alcohol related deseases, this study investigated the distribution of the genes controlling alcohol metabolism in Japan's twin. Restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR) technique was used to measure the control gene of alcohol metabolized enzymes and the genotypes of alcohol dehydrogenase 2 (ADH2) and aldehyde dehydrogenase 2 (ALDH2), which were distributed in Japan's twins. At the same time, according to the difference in genotypes, the sensitive individuals were screened from the study subjects. The distribution of ADH2 and ALDH2 genes were consistent with the Hardy-weinberg equation. The three genotypes of ADH2 gene were ADH2(1)/ADH2(1) (1.1%), ADH2(1)/ADH2(2) (44.6%) and ADH2(2)/ADH2(2) (54.3%). And those of ALDH2 gene were ALDH2(1)/ALDH2(1) (41.3%), ALDH2(1)/ALDH2(2) (39.1%) and ALDH2(2)/ALDH2(2) (19.6%). The frequency of ADH2 and ALDH2 genes was 0.255, 0.745 and 0.609, 0.391 respectively. Not only the distribution of genotypes of ADH2 and ALDH2 is known, but also the sensitive individuals are found, which can help prevent alcohol related disease.

Ultrafiltration-LC-MS combined with semi-preparative HPLC for the simultaneous screening and isolation of lactate dehydrogenase inhibitors from Belamcanda chinensis.

PubMed

Li, Senlin; Li, Sainan; Tang, Ying; Liu, Chunming; Chen, Lina; Zhang, Yuchi

2016-12-01

Stroke represents the fourth leading cause of death in the USA and the second leading cause of death worldwide. Lactate dehydrogenase inhibitors are widely used in the treatment of ischemic stroke and natural products are considered a promising source of novel lactate dehydrogenase inhibitors. In this study, we used PC12 cells to determine the protective effect of extracts from the herb Belamcanda chinensis following toxic challenge. Using ultrafiltration high-performance liquid chromatography coupled with photo-diode array detection and electrospray ionization mass spectrometry, we screened and identified isoflavonoids from Belamcanda chinensis extracts. Semi-preparative high-performance liquid chromatography was then applied to separate and isolate the active constituents. Using these methods, we identified six major compounds in Belamcanda chinensis as lactate dehydrogenase inhibitors: tectoridin, iristectorin A, iridin, tectorigenin, irigenin, and irisflorentin, which were then isolated to >92% purity. This is the first report that Belamcanda chinensis extracts contain potent lactate dehydrogenase inhibitors. Our results demonstrate that the systematic isolation of bioactive components from Belamcanda chinensis guided by ultrafiltration high-performance liquid chromatography coupled with photo-diode array detection and electrospray ionization mass spectrometry represents a feasible and efficient technique that could be extended for the identification and isolation of other enzyme inhibitors. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

DOWNREGULATION OF CINNAMYL-ALCOHOL DEHYDROGENASE IN SWITCHGRASS BY RNA SILENCING RESULTS IN ENHANCED GLUCOSE RELEASE AFTER CELLULASE TREATMENT

USDA-ARS?s Scientific Manuscript database

Cinnamyl alcohol dehydrogenase (CAD), catalyzes the last step in monolignol biosynthesis and genetic evidence indicates CAD deficiency in grasses both decreases overall lignin, alters lignin structure and increases enzymatic recovery of sugars. To ascertain the effect of CAD downregulation in switch...

The metabolism of ethanol-derived acetaldehyde by alcohol dehydrogenase (EC 1.1.1.1) and aldehyde dehydrogenase (EC 1.2.1.3) in Drosophila melanogaster larvae.

PubMed Central

Heinstra, P W; Geer, B W; Seykens, D; Langevin, M

1989-01-01

Both aldehyde dehydrogenase (ALDH, EC 1.2.1.3) and the aldehyde dehydrogenase activity of alcohol dehydrogenase (ADH, EC 1.1.1.1) were found to coexist in Drosophila melanogaster larvae. The enzymes, however, showed different inhibition patterns with respect to pyrazole, cyanamide and disulphiram. ALDH-1 and ALDH-2 isoenzymes were detected in larvae by electrophoretic methods. Nonetheless, in tracer studies in vivo, more than 75% of the acetaldehyde converted to acetate by the ADH ethanol-degrading pathway appeared to be also catalysed by the ADH enzyme. The larval fat body probably was the major site of this pathway. Images Fig. 1. Fig. 2. PMID:2499314

Effects of cyanamide and clofibrate on the enzymes of ethanol oxydation and on ethanol consumption in the rat.

PubMed

Lamboeuf, Y; De Saint Blanquat, G

1980-01-01

The action of cyanamide and of clofibrate was studied on the voluntary drinking behaviour of alcohol intoxicated animals and also on the enzymes of ethyl oxydation. Both substances reduce alcohol consumption by about 35% and cause metabolic modifications: inhibition of aldehyde dehydrogenase and of catalase by cyanamide; activation of alcohol- and aldehyde-dehydrogenases by clofibrate. These effects are constantly found in most of the tissues of the rat. The results are discussed bearing in mind the relationships between alcohol metabolism, acetaldehyde metabolism, the toxicity of alcohol and the drinking behaviour.

Genes contributing to the development of alcoholism: an overview.

PubMed

Edenberg, Howard J

2012-01-01

Genetic factors (i.e., variations in specific genes) account for a substantial portion of the risk for alcoholism. However, identifying those genes and the specific variations involved is challenging. Researchers have used both case-control and family studies to identify genes related to alcoholism risk. In addition, different strategies such as candidate gene analyses and genome-wide association studies have been used. The strongest effects have been found for specific variants of genes that encode two enzymes involved in alcohol metabolism-alcohol dehydrogenase and aldehyde dehydrogenase. Accumulating evidence indicates that variations in numerous other genes have smaller but measurable effects.

GOLD HULL AND INTERNODE2 encodes a primarily multifunctional cinnamyl-alcohol dehydrogenase in rice.

PubMed

Zhang, Kewei; Qian, Qian; Huang, Zejun; Wang, Yiqin; Li, Ming; Hong, Lilan; Zeng, Dali; Gu, Minghong; Chu, Chengcai; Cheng, Zhukuan

2006-03-01

Lignin content and composition are two important agronomic traits for the utilization of agricultural residues. Rice (Oryza sativa) gold hull and internode phenotype is a classical morphological marker trait that has long been applied to breeding and genetics study. In this study, we have cloned the GOLD HULL AND INTERNODE2 (GH2) gene in rice using a map-based cloning approach. The result shows that the gh2 mutant is a lignin-deficient mutant, and GH2 encodes a cinnamyl-alcohol dehydrogenase (CAD). Consistent with this finding, extracts from roots, internodes, hulls, and panicles of the gh2 plants exhibited drastically reduced CAD activity and undetectable sinapyl alcohol dehydrogenase activity. When expressed in Escherichia coli, purified recombinant GH2 was found to exhibit strong catalytic ability toward coniferaldehyde and sinapaldehyde, while the mutant protein gh2 completely lost the corresponding CAD and sinapyl alcohol dehydrogenase activities. Further phenotypic analysis of the gh2 mutant plants revealed that the p-hydroxyphenyl, guaiacyl, and sinapyl monomers were reduced in almost the same ratio compared to the wild type. Our results suggest GH2 acts as a primarily multifunctional CAD to synthesize coniferyl and sinapyl alcohol precursors in rice lignin biosynthesis.

Clostridium species strain BOH3 tolerates and transforms inhibitors from horticulture waste hydrolysates.

PubMed

Yan, Yu; He, Jianzhong

2017-08-01

Conversion of lignocellulosic hydrolysate to biofuels is impeded by the toxic effects of inhibitors that are generated during pretreatment and hydrolysis processes. Here we describe a wild-type Clostridium sp. strain BOH3 with high tolerance to the lignocellulose-derived inhibitors and its capability to transform these inhibitors. Strain BOH3 is capable of tolerating over 60 mM furfural, 60 mM hydroxymethylfurfural, and 6.6 mM vanillin, respectively, and is able to convert 53.74 ± 0.37 mM furfural into furfuryl alcohol within 90 h. The high furfural tolerance and its biotransformation by strain BOH3, which is correlated to the high transcription levels of two short-chain dehydrogenase/reductases, enable strain BOH3 to produce 5.15 ± 0.52 g/L butanol from dilute sulfuric acid pretreated horticultural waste hydrolysate (HWH) that bypassed the detoxification step. The capability of strain BOH3 to produce butanol from un-detoxified HWH lays the foundation of cost-effective biofuel production from lignocellulosic materials.

The Activity of Class I-IV Alcohol Dehydrogenase Isoenzymes and Aldehyde Dehydrogenase in Bladder Cancer Cells.

PubMed

Orywal, Karolina; Jelski, Wojciech; Werel, Tadeusz; Szmitkowski, Maciej

2018-01-02

The aim of this study was to determine the differences in the activity of Alcohol Dehydrogenase (ADH) isoenzymes and Aldehyde Dehydrogenase (ALDH) in normal and cancerous bladder cells. Class III, IV of ADH and total ADH activity were measured by the photometric method and class I, II ADH and ALDH activity by the fluorometric method. Significantly higher total activity of ADH was found in both, low-grade and high-grade bladder cancer, in comparison to healthy tissues. The increased activity of total ADH in bladder cancer cells may be the cause of metabolic disorders in cancer cells, which may intensify carcinogenesis.

The Alcohol Dehydrogenase Kinetics Laboratory: Enhanced Data Analysis and Student-Designed Mini-Projects

ERIC Educational Resources Information Center

Silverstein, Todd P.

2016-01-01

A highly instructive, wide-ranging laboratory project in which students study the effects of various parameters on the enzymatic activity of alcohol dehydrogenase has been adapted for the upper-division biochemistry and physical biochemistry laboratory. Our two main goals were to provide enhanced data analysis, featuring nonlinear regression, and…

In vivo relationship between monoamine oxidase type B and alcohol dehydrogenase: effects of ethanol and phenylethylamine

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

Aliyu, S.U.; Upahi, L.

The role of acute ethanol and phenylethylamine on the brain and platelet monoamine oxidase activities, hepatic cytosolic alcohol dehydrogenase, redox state and motor behavior were studied in male rats. Ethanol on its own decreased the redox couple ratio, as well as, alcohol dehydrogenase activity in the liver while at the same time it increased brain and platelet monoamine oxidase activity due to lower Km with no change in Vmax. The elevation in both brain and platelet MAO activity was associated with ethanol-induced hypomotility in the rats. Co-administration of phenylethylamine and ethanol to the animals, caused antagonism of the ethanol-induced effectsmore » described above. The effects of phenylethylamine alone, on the above mentioned biochemical and behavioral indices, are more complex. Phenylethylamine on its own, like ethanol, caused reduction of the cytosolic redox, ratio and elevation of monoamine oxidase activity in the brain and platelets. However, in contrast to ethanol, this monoamine produced hypermotility and activation of the hepatic cytosolic alcohol dehydrogenase activity in the animals.« less

The Oxidative Fermentation of Ethanol in Gluconacetobacter diazotrophicus Is a Two-Step Pathway Catalyzed by a Single Enzyme: Alcohol-Aldehyde Dehydrogenase (ADHa)

PubMed Central

Gómez-Manzo, Saúl; Escamilla, José E.; González-Valdez, Abigail; López-Velázquez, Gabriel; Vanoye-Carlo, América; Marcial-Quino, Jaime; de la Mora-de la Mora, Ignacio; Garcia-Torres, Itzhel; Enríquez-Flores, Sergio; Contreras-Zentella, Martha Lucinda; Arreguín-Espinosa, Roberto; Kroneck, Peter M. H.; Sosa-Torres, Martha Elena

2015-01-01

Gluconacetobacter diazotrophicus is a N2-fixing bacterium endophyte from sugar cane. The oxidation of ethanol to acetic acid of this organism takes place in the periplasmic space, and this reaction is catalyzed by two membrane-bound enzymes complexes: the alcohol dehydrogenase (ADH) and the aldehyde dehydrogenase (ALDH). We present strong evidence showing that the well-known membrane-bound Alcohol dehydrogenase (ADHa) of Ga. diazotrophicus is indeed a double function enzyme, which is able to use primary alcohols (C2–C6) and its respective aldehydes as alternate substrates. Moreover, the enzyme utilizes ethanol as a substrate in a reaction mechanism where this is subjected to a two-step oxidation process to produce acetic acid without releasing the acetaldehyde intermediary to the media. Moreover, we propose a mechanism that, under physiological conditions, might permit a massive conversion of ethanol to acetic acid, as usually occurs in the acetic acid bacteria, but without the transient accumulation of the highly toxic acetaldehyde. PMID:25574602

Interaction of cytoplasmic dehydrogenases: quantitation of pathways of ethanol metabolism.

PubMed

Vind, C; Grunnet, N

1983-01-01

The interaction between xylitol, alcohol and lactate dehydrogenase has been studied in hepatocytes from rats by applying specifically tritiated substrates. A simple model, describing the metabolic fate of tritium from [2-3H] xylitol and (1R) [1-3H]ethanol is presented. The model allows calculation of the specific radioactivity of free, cytosolic NADH, based on transfer of tritium to lactate, glucose and water. From the initial labelling rate of lactate and the specific radioactivity of cytosolic NADH, we have determined the reversible flow through the lactate dehydrogenase catalyzed reaction to 1-5 mumol/min . g wet wt. The results suggest that xylitol, alcohol and lactate dehydrogenase share the same pool of NAD(H) in the cytoplasma. This finding allows estimation of the ethanol oxidation rate by the non-alcohol dehydrogenase pathways from the relative yield of tritium in water and glucose. The calculations are based on a comparison of the fate of the 1-pro-R hydrogen of ethanol and the hydrogen bound to carbon 2 of xylitol or carbon 2 of lactate under identical conditions.

Degradation pathway of 2-chloroethanol in Pseudomonas stutzeri strain JJ under denitrifying conditions.

PubMed

Dijk, John A; Gerritse, Jan; Schraa, Gosse; Stams, Alfons J M

2004-12-01

The pathway of 2-chloroethanol degradation in the denitrifying Pseudomonas stutzeri strain JJ was investigated. In cell-free extracts, activities of a phenazine methosulfate (PMS)-dependent chloroethanol dehydrogenase, an NAD-dependent chloroacetaldehyde dehydrogenase, and a chloroacetate dehalogenase were detected. This suggested that the 2-chloroethanol degradation pathway in this denitrifying strain is the same as found in aerobic bacteria that degrade chloroethanol. Activity towards primary alcohols, secondary alcohols, diols, and other chlorinated alcohols could be measured in cell-free extracts with chloroethanol dehydrogenase (CE-DH) activity. PMS and phenazine ethosulfate (PES) were used as primary electron acceptors, but not NAD, NADP or ferricyanide. Cells of strain JJ cultured in a continuous culture under nitrate limitation exhibited chloroethanol dehydrogenase activity that was a 12 times higher than in cells grown in batch culture. However, under chloroethanol-limiting conditions, CE-DH activity was in the same range as in batch culture. Cells grown on ethanol did not exhibit CE-DH activity. Instead, NAD-dependent ethanol dehydrogenase (E-DH) activity and PMS-dependent E-DH activity were detected.

3-cyanoindole-based inhibitors of inosine monophosphate dehydrogenase: synthesis and initial structure-activity relationships.

PubMed

Dhar, T G Murali; Shen, Zhongqi; Gu, Henry H; Chen, Ping; Norris, Derek; Watterson, Scott H; Ballentine, Shelley K; Fleener, Catherine A; Rouleau, Katherine A; Barrish, Joel C; Townsend, Robert; Hollenbaugh, Diane L; Iwanowicz, Edwin J

2003-10-20

A series of novel small molecule inhibitors of inosine monophosphate dehydrogenase (IMPDH), based upon a 3-cyanoindole core, were explored. IMPDH catalyzes the rate determining step in guanine nucleotide biosynthesis and is a target for anticancer, immunosuppressive and antiviral therapy. The synthesis and the structure-activity relationships (SAR), derived from in vitro studies, for this new series of inhibitors is given.

Effects of methylmercury and alcohol exposure in Drosophila melanogaster: Potential risks in neurodevelopmental disorders.

PubMed

Chauhan, Ved; Chauhan, Abha

2016-06-01

Extensive evidence suggests the role of oxidative stress in autism and other neurodevelopmental disorders. In this study, we investigated whether methylmercury (MeHg) and/or alcohol exposure has deleterious effects in Drosophila melanogaster (fruit flies). A diet containing different concentrations of MeHg in Drosophila induced free radical generation and increased lipid peroxidation (markers of oxidative stress) in a dose-dependent manner. This effect of MeHg on oxidative stress was enhanced by further exposure to alcohol. It was observed that alcohol alone could also induce free radical generation in flies. After alcohol exposure, MeHg did not affect the immobilization of flies, but it increased the recovery time in a concentration-dependent manner. MeHg significantly inhibited the activity of alcohol dehydrogenase (ADH) in a dose-dependent manner. Linear regression analysis showed a significant negative correlation between ADH activity and recovery time upon alcohol exposure in the flies fed a diet with MeHg. This relationship between ADH activity and recovery time after alcohol exposure was confirmed by adding 4-methyl pyrazole (an inhibitor of ADH) to the diet for the flies. These results suggest that consumption of alcohol by pregnant mothers who are exposed to MeHg may lead to increased oxidative stress and to increased length of time for alcohol clearance, which may have a direct impact on the development of the fetus, thereby increasing the risk of neurodevelopmental disorders. Published by Elsevier Ltd.

A Long-Chain Secondary Alcohol Dehydrogenase from Rhodococcus erythropolis ATCC 4277

PubMed Central

Ludwig, B.; Akundi, A.; Kendall, K.

1995-01-01

A NAD-dependent secondary alcohol dehydrogenase has been purified from the alkane-degrading bacterium, Rhodococcus erythropolis ATCC 4277. The enzyme was found to be active against a broad range of substrates, particularly long-chain secondary aliphatic alcohols. Although optimal activity was observed with linear 2-alcohols containing between 6 and 11 carbon atoms, secondary alcohols as long as 2-tetradecanol were oxidized at 25% of the rate seen with mid-range alcohols. The purified enzyme was specific for the S-(+) stereoisomer of 2-octanol and had a specific activity for 2-octanol of over 200 (mu)mol/min/mg of protein at pH 9 and 37(deg)C, 25-fold higher than that of any previously reported S-(+) secondary alcohol dehydrogenase. Linear primary alcohols containing between 3 and 13 carbon atoms were utilized 20- to 40-fold less efficiently than the corresponding secondary alcohols. The apparent K(infm) value for NAD(sup+) with 2-octanol as the substrate was 260 (mu)M, whereas the apparent K(infm) values for the 2-alcohols ranged from over 5 mM for 2-pentanol to less than 2 (mu)M for 2-tetradecanol. The enzyme showed moderate thermostability (half-life of 4 h at 60(deg)C) and could potentially be useful for the synthesis of optically pure stereoisomers of secondary alcohols. PMID:16535152

Association of polymorphisms in nicotinic acetylcholine receptor alpha 4 subunit gene (CHRNA4), mu-opioid receptor gene (OPRM1), and ethanol-metabolizing enzyme genes with alcoholism in Korean patients.

PubMed

Kim, Soon Ae; Kim, Jong-Woo; Song, Ji-Young; Park, Sunny; Lee, Hee Jae; Chung, Joo-Ho

2004-01-01

Findings obtained from several studies indicate that ethanol enhances the activity of alpha4beta2 neuronal nicotinic acetylcholine receptor and support the possibility that a polymorphism of the nicotinic acetylcholine receptor alpha4 subunit gene (CHRNA4) modulates enhancement of nicotinic receptor function by ethanol. To identify the association between the CfoI polymorphism of the CHRNA4 and alcoholism, we examined distribution of genotypes and allele frequencies in Korean patients diagnosed with alcoholism (n = 127) and Korean control subjects without alcoholism (n = 185) with polymerase chain reaction-restriction fragment length polymorphism methods. We were able to detect the association between the CfoI polymorphism of the CHRNA4 and alcoholism in Korean patients (genotype P = .023; allele frequency P = .047). The genotypes and allele frequencies of known polymorphisms in other alcoholism candidate genes, such as alcohol metabolism-related genes [alcohol dehydrogenase 2 (ADH2), aldehyde dehydrogenase 2 (ALDH2), alcohol dehydrogenase 3 (ADH3), and cytochrome P450 2E1 (CYP2E1)] and mu-opioid receptor gene (OPRM1), were studied. The polymorphisms of ADH2, ALDH2, and CYP2E1 were significantly different in Korean patients with alcoholism and Korean control subjects without alcoholism, but ADH3 and OPRM1 did not differ between the two groups.

Genetics of alcoholism.

PubMed

Edenberg, Howard J; Foroud, Tatiana

2014-01-01

Multiple lines of evidence strongly indicate that genetic factors contribute to the risk for alcohol use disorders (AUD). There is substantial heterogeneity in AUD, which complicates studies seeking to identify specific genetic factors. To identify these genetic effects, several different alcohol-related phenotypes have been analyzed, including diagnosis and quantitative measures related to AUDs. Study designs have used candidate gene analyses, genetic linkage studies, genomewide association studies (GWAS), and analyses of rare variants. Two genes that encode enzymes of alcohol metabolism have the strongest effect on AUD: aldehyde dehydrogenase 2 and alcohol dehydrogenase 1B each has strongly protective variants that reduce risk, with odds ratios approximately 0.2-0.4. A number of other genes important in AUD have been identified and replicated, including GABRA2 and alcohol dehydrogenases 1B and 4. GWAS have identified additional candidates. Rare variants are likely also to play a role; studies of these are just beginning. A multifaceted approach to gene identification, targeting both rare and common variations and assembling much larger datasets for meta-analyses, is critical for identifying the key genes and pathways important in AUD. © 2014 Elsevier B.V. All rights reserved.

Geraniol dehydrogenase, the key enzyme in biosynthesis of the alarm pheromone, from the astigmatid mite Carpoglyphus lactis (Acari: Carpoglyphidae).

PubMed

Noge, Koji; Kato, Makiko; Mori, Naoki; Kataoka, Michihiko; Tanaka, Chihiro; Yamasue, Yuji; Nishida, Ritsuo; Kuwahara, Yasumasa

2008-06-01

Geraniol dehydrogenase (GeDH), which plays an important role in the biosynthesis of neral, an alarm pheromone, was purified from the astigmatid mite Carpoglyphus lactis. The enzyme was obtained in an apparently homogeneous and active form after 1879-fold purification through seven steps of chromatography. Car. lactis GeDH was determined to be a monomer in its active form with a relative molecular mass of 42 800, which is a unique subunit structure in comparison with already established alcohol dehydrogenases. Car. lactis GeDH oxidized geraniol into geranial in the presence of NAD+. NADP+ was ineffective as a cofactor, suggesting that Car. lactis GeDH is an NAD+-dependent alcohol dehydrogenase. The optimal pH and temperature for geraniol oxidation were determined to be pH 9.0 and 25 degrees C, respectively. The Km values for geraniol and NAD+ were 51.0 microm and 59.5 microm, respectively. Car. lactis GeDH was shown to selectively oxidize geraniol, whereas its geometrical isomer, nerol, was inert as a substrate. The high specificity for geraniol suggests that Car. lactis GeDH specializes in the alarm pheromone biosynthesis of Car. lactis. Car. lactis GeDH is composed of 378 amino acids. Structurally, Car. lactis GeDH showed homology with zinc-dependent alcohol dehydrogenases found in mammals and a mosquito (36.6-37.6% identical), and the enzyme was considered to be a member of the medium-chain dehydrogenase/reductase family, in view of the highly conserved sequences of zinc-binding and NAD+-binding sites. Phylogenetic analyses indicate that Car. lactis GeDH could be categorized as a new class, different from other established alcohol dehydrogenases.

GOLD HULL AND INTERNODE2 Encodes a Primarily Multifunctional Cinnamyl-Alcohol Dehydrogenase in Rice1

PubMed Central

Zhang, Kewei; Qian, Qian; Huang, Zejun; Wang, Yiqin; Li, Ming; Hong, Lilan; Zeng, Dali; Gu, Minghong; Chu, Chengcai; Cheng, Zhukuan

2006-01-01

Lignin content and composition are two important agronomic traits for the utilization of agricultural residues. Rice (Oryza sativa) gold hull and internode phenotype is a classical morphological marker trait that has long been applied to breeding and genetics study. In this study, we have cloned the GOLD HULL AND INTERNODE2 (GH2) gene in rice using a map-based cloning approach. The result shows that the gh2 mutant is a lignin-deficient mutant, and GH2 encodes a cinnamyl-alcohol dehydrogenase (CAD). Consistent with this finding, extracts from roots, internodes, hulls, and panicles of the gh2 plants exhibited drastically reduced CAD activity and undetectable sinapyl alcohol dehydrogenase activity. When expressed in Escherichia coli, purified recombinant GH2 was found to exhibit strong catalytic ability toward coniferaldehyde and sinapaldehyde, while the mutant protein gh2 completely lost the corresponding CAD and sinapyl alcohol dehydrogenase activities. Further phenotypic analysis of the gh2 mutant plants revealed that the p-hydroxyphenyl, guaiacyl, and sinapyl monomers were reduced in almost the same ratio compared to the wild type. Our results suggest GH2 acts as a primarily multifunctional CAD to synthesize coniferyl and sinapyl alcohol precursors in rice lignin biosynthesis. PMID:16443696

Determination of hydride transfer stereospecificity of NADH-dependent alcohol-aldehyde/ketone oxidoreductase from Sulfolobus solfataricus.

PubMed

Trincone, A; Lama, L; Rella, R; D'Auria, S; Raia, C A; Nicolaus, B

1990-10-18

This paper describes the determination of stereospecificity of hydride transfer reaction of an alcohol dehydrogenase isolated from the archaebacterium Sulfolobus solfataricus. The 1H-NMR and EI-MS data indicate that the enzyme transfers the pro-R hydrogen from coenzyme to substrate and is therefore an A-specific dehydrogenase.

Characterization of the increased binding of acetaldehyde to red blood cells in alcoholics.

PubMed

Hernández-Muñoz, R; Baraona, E; Blacksberg, I; Lieber, C S

1989-10-01

Using equilibrium dialysis, we found that acetaldehyde, at the levels commonly occurring after ethanol ingestion, did not bind detectably to plasma proteins, but there was significant binding to red blood cells, more in alcoholics than in nonalcoholics. The binding to red blood cells was inhibited by pyridoxal phosphate and N-ethylmaleimide, suggesting adduction to amino and thiol groups. Binding kinetics were consistent with at least two sites. The one with the highest affinity for acetaldehyde corresponded to hemoglobin. Its affinity and Bmax were not changed in alcoholics, but these binding sites accounted for only 44% of the sites available in the red blood cells of alcoholics and 80% of those in controls. Moreover, this binding was not inhibited by N-ethylmaleimide. There was no detectable binding to red cell ghosts. Nonprotein binding was then assessed by changes in NADH produced by the addition of protein-free fractions of the cells to an alcohol dehydrogenase system in equilibrium; this revealed a second binder of lower affinity, larger capacity and with sensitivity to both inhibitors. This binding (possibly due to thiazolidine formation with cysteine) was enhanced in alcoholics, whose red blood cell cysteine content was doubled. Levels of red blood cell cysteine and acetaldehyde remained high for 2 weeks after withdrawal. Because of the prolonged persistence after withdrawal, these changes may provide new markers of alcoholism.

Characteristics of butanol metabolism in alcohol dehydrogenase-deficient deermice.

PubMed Central

Alderman, J A; Kato, S; Lieber, C S

1989-01-01

Deermice lacking the low-Km alcohol dehydrogenase eliminated butan-1-ol, a substrate for microsomal oxidation but not for catalase, at 117 mumol/min per kg body wt. Microsomal fractions and hepatocytes metabolized butan-1-ol also (Vmax. = 6.7 nmol/min per nmol of cytochrome P-450, Km = 0.85 mM; Vmax. = 5.3 nmol/min per 10(6) cells, Km = 0.71 mM respectively). These results are consistent with alcohol oxidation by the microsomal system in these deermice. PMID:2930472

Dissociation of branched-chain alpha-keto acid dehydrogenase kinase (BDK) from branched-chain alpha-keto acid dehydrogenase complex (BCKDC) by BDK inhibitors.

PubMed

Murakami, Taro; Matsuo, Masayuki; Shimizu, Ayako; Shimomura, Yoshiharu

2005-02-01

Branched-chain alpha-keto acid dehydrogenase kinase (BDK) phosphorylates and inactivates the branched-chain alpha-keto acid dehydrogenase complex (BCKDC), which is the rate-limiting enzyme in the branched-chain amino acid catabolism. BDK has been believed to be bound to the BCKDC. However, recent our studies demonstrated that protein-protein interaction between BDK and BCKDC is one of the factors to regulate BDK activity. Furthermore, only the bound form of BDK appears to have its activity. In the present study, we examined effects of BDK inhibitors on the amount of BDK bound to the BCKDC using rat liver extracts. The bound form of BDK in the extracts of liver from low protein diet-fed rats was measured by an immunoprecipitation pull down assay with or without BDK inhibitors. Among the BDK inhibitors. alpha-ketoisocaproate, alpha-chloroisocaproate, and a-ketoisovalerate released the BDK from the complex. Furthermore, the releasing effect of these inhibitors on the BDK appeared to depend on their inhibition constants. On the other hand, clofibric acid and thiamine pyrophosphate had no effect on the protein-protein interaction between two enzymes. These results suggest that the dissociation of the BDK from the BCKDC is one of the mechanisms responsible for the action of some inhibitors to BDK.

Ethanol and 4-methylpyrazole increase DNA adduct formation of furfuryl alcohol in FVB/N wild-type mice and in mice expressing human sulfotransferases 1A1/1A2.

PubMed

Sachse, Benjamin; Meinl, Walter; Glatt, Hansruedi; Monien, Bernhard H

2016-03-01

Furfuryl alcohol (FFA) is a carcinogenic food contaminant, which is formed by acid- and heat-catalyzed degradation of fructose and glucose. The activation by sulfotransferases (SULTs) yields a DNA reactive and mutagenic sulfate ester. The most prominent DNA adduct, N(2)-((furan-2-yl)methyl)-2'-deoxyguanosine (N(2)-MF-dG), was detected in FFA-treated mice and also in human tissue samples. The dominant pathway of FFA detoxification is the oxidation via alcohol dehydrogenases (ADHs) and aldehyde dehydrogenases (ALDHs). The activity of these enzymes may be greatly altered in the presence of inhibitors or competitive substrates. Here, we investigated the impact of ethanol and the ADH inhibitor 4-methylpyrazole (4MP) on the DNA adduct formation by FFA in wild-type and in humanized mice that were transgenic for human SULT1A1/1A2 and deficient in the mouse (m) Sult1a1 and Sult1d1 genes (h1A1/1A2/1a1(-)/1d1(-)). The administration of FFA alone led to hepatic adduct levels of 4.5 N(2)-MF-dG/10(8) nucleosides and 33.6 N(2)-MF-dG/10(8) nucleosides in male and female wild-type mice, respectively, and of 19.6 N(2)-MF-dG/10(8) nucleosides and 95.4 N(2)-MF-dG/10(8) nucleosides in male and female h1A1/1A2/1a1(-)/1d1(-) mice. The coadministration of 1.6g ethanol/kg body weight increased N(2)-MF-dG levels by 2.3-fold in male and by 1.7-fold in female wild-type mice and by 2.5-fold in male and by 1.5-fold in female h1A1/1A2/1a1(-)/1d1(-) mice. The coadministration of 100mg 4MP/kg body weight had a similar effect on the adduct levels. These findings indicate that modulators of the oxidative metabolism, e.g. the drug 4MP or consumption of alcoholic beverages, may increase the genotoxic effects of FFA also in humans. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Pharmacotherapy of alcoholism - an update on approved and off-label medications.

PubMed

Soyka, Michael; Müller, Christian A

2017-08-01

Only a few medications are available for the treatment of alcohol use disorders (AUDs). Areas covered: This paper discusses approved AUD medications, including the opioid antagonists naltrexone and nalmefene (the latter is licensed for reduction of alcohol consumption only), the putative glutamate receptor antagonist acamprosate and the aldehyde dehydrogenase inhibitor disulfiram. It also covers off-label medications of interest, including topiramate, gabapentin, ondansetron, varenicline, baclofen, sodium oxybate and antidepressants. Clinical implications, benefits and risks of treatment are discussed. Expert opinion: Acamprosate, naltrexone, nalmefene and disulfiram are the only approved 'alcohol-specific' drugs. Acamprosate and naltrexone have been evaluated in numerous clinical trials and represent evidence-based treatments in AUDs. Nalmefene use, however, is controversial. Supervised disulfiram is a second-line treatment approach. Compounds developed and licensed for different neuropsychiatric disorders are potential alternatives. Encouraging results have been reported for topiramate, gabapentin and also varenicline, which might be useful in patients with comorbid nicotine dependence. The GABA (γ-aminobutyric acid)-B receptor agonist baclofen has shown mixed results; it is currently licensed for the treatment of AUDs in France only. Gabapentin may be close to approval in the USA. Further studies of these novel treatment approaches in AUDs are needed.

Determination of the Subunit Molecular Mass and Composition of Alcohol Dehydrogenase by SDS-PAGE

ERIC Educational Resources Information Center

Nash, Barbara T.

2007-01-01

SDS-PAGE is a simple, rapid technique that has many uses in biochemistry and is readily adaptable to the undergraduate laboratory. It is, however, a technique prone to several types of procedural pitfalls. This article describes the use of SDS-PAGE to determine the subunit molecular mass and composition of yeast alcohol dehydrogenase employing…

A novel type of pathogen defense-related cinnamyl alcohol dehydrogenase.

PubMed

Logemann, E; Reinold, S; Somssich, I E; Hahlbrock, K

1997-08-01

We describe an aromatic alcohol dehydrogenase with properties indicating a novel type of function in the defense response of plants to pathogens. To obtain the enzyme free of contamination with possible isoforms, a parsley (Petroselinum crispum) cDNA comprising the entire coding region of the elicitor-responsive gene, ELI3, was expressed in Escherichia coli. In accord with large amino acid sequence similarities with established cinnamyl and benzyl alcohol dehydrogenases from other plants, the enzyme efficiently reduced various cinnamyl and benzyl aldehydes using NADPH as a co-substrate. Highest substrate affinities were observed for cinnamaldehyde, 4-coumaraldehyde and coniferaldehyde, whereas sinapaldehyde, one of the most efficient substrates of several previously analyzed cinnamyl alcohol dehydrogenases and a characteristic precursor molecule of angiosperm lignin, was not converted. A single form of ELI3 mRNA was strongly and rapidly induced in fungal elicitor-treated parsley cells. These results, together with earlier findings that the ELI3 gene is strongly activated both in elicitor-treated parsley cells and at fungal infection sites in parsley leaves, but not in lignifying tissue, suggest a specific role of this enzyme in pathogen defense-related phenylpropanoid metabolism.

Characterization and further stabilization of a new anti-prelog specific alcohol dehydrogenase from Thermus thermophilus HB27 for asymmetric reduction of carbonyl compounds.

PubMed

Rocha-Martín, Javier; Vega, Daniel; Bolivar, Juan M; Hidalgo, Aurelio; Berenguer, José; Guisán, José M; López-Gallego, Fernando

2012-01-01

The use of dehydrogenases in asymmetric chemistry has exponentially grown in the last decades facilitated by the genome mining. Here, a new short-chain alcohol dehydrogenase from Thermus thermophilus HB27 has been expressed, purified, characterized and stabilized by immobilization on solid supports. The enzyme catalyzes both oxidative and reductive reactions at neutral pH with a broad range of substrates. Its highest activity was found towards the reduction of 2,2',2″-trifluoroacetophenone (85 U/mg at 65 °C and pH 7). Moreover, the enzyme was stabilized more than 200-fold by multipoint covalent immobilization on agarose matrixes via glyoxyl chemistry. Such heterogeneous catalyst coupled to an immobilized cofactor recycling partner performed the quantitative asymmetric reduction of 2,2',2″-trifluoroacetophenone and rac-2-phenylpropanal to (S)-(+)-α-(trifluoromethyl)benzyl alcohol and (R)-2-phenyl-1-propanol with enantiomeric excesses of 96% and 71%, respectively. To our knowledge this is the first alcohol dehydrogenase from a thermophilic source with anti-Prelog selectivity for aryl ketones and that preferentially produces R-profens. Copyright © 2011 Elsevier Ltd. All rights reserved.

Tolerance to disulfiram induced by chronic alcohol intake in the rat.

PubMed

Tampier, Lutske; Quintanilla, María Elena; Israel, Yedy

2008-06-01

Disulfiram, an inhibitor of aldehyde dehydrogenase used in the treatment of alcoholism, is an effective medication when its intake is supervised by a third person. However, its therapeutic efficacy varies widely, in part due to the fact that disulfiram is a pro-drug that requires its transformation into an active form and because it shows a wide range of secondary effects which often prevent the use of doses that ensure full therapeutic effectiveness. In this preclinical study in rats we report the development of tolerance to disulfiram induced by the chronic ingestion of ethanol, an additional source of variation for the actions of disulfiram with possible therapeutic significance, We also addresses the likely mechanism of this effect. Wistar-derived rats bred for generations as high ethanol drinkers (UChB) were trained for either 3 days (Group A) or 30 days (Group B) to choose between ethanol (10% v/v) or water, which were freely available from 2 bottles on a 24-hour basis. Subsequently, animals in both groups were administered disulfiram or cyanamide (another inhibitor of aldehyde dehydrogenase) and ethanol intake in this free choice paradigm was determined. Animals were also administered a standard dose of 1 g ethanol/kg (i.p) and arterial blood acetaldehyde was measured. Disulfiram (12.5 and 25 mg/kg) and cyanamide (10 mg/kg) markedly inhibited ethanol intake (up to 60 to 70%) in animals that had ethanol access for only 3 days (Group A). However both drugs were inactive in inhibiting ethanol intake in animals that had consumed ethanol for 30 days (Group B). Following the injection of 1 g ethanol/kg, arterial blood acetaldehyde levels reached levels of 150 and 300 microM for disulfiram and cyanamide respectively, values which were virtually identical regardless of the length of prior ethanol intake of the animals. Chronic ethanol intake in high-drinker rats leads to marked tolerance to the aversive effects of disulfiram and cyanamide on ethanol intake despite the presence of consistently high levels of blood acetaldehyde. These findings may have implications for the use of disulfiram for the treatment of alcoholism in humans.

Furaldehyde substrate specificity and kinetics of Saccharomyces cerevisiae alcohol dehydrogenase 1 variants.

PubMed

Laadan, Boaz; Wallace-Salinas, Valeria; Carlsson, Åsa Janfalk; Almeida, João Rm; Rådström, Peter; Gorwa-Grauslund, Marie F

2014-08-09

A previously discovered mutant of Saccharomyces cerevisiae alcohol dehydrogenase 1 (Adh1p) was shown to enable a unique NADH-dependent reduction of 5-hydroxymethylfurfural (HMF), a well-known inhibitor of yeast fermentation. In the present study, site-directed mutagenesis of both native and mutated ADH1 genes was performed in order to identify the key amino acids involved in this substrate shift, resulting in Adh1p-variants with different substrate specificities. In vitro activities of the Adh1p-variants using two furaldehydes, HMF and furfural, revealed that HMF reduction ability could be acquired after a single amino acid substitution (Y295C). The highest activity, however, was reached with the double mutation S110P Y295C. Kinetic characterization with both aldehydes and the in vivo primary substrate acetaldehyde also enabled to correlate the alterations in substrate affinity with the different amino acid substitutions. We demonstrated the key role of Y295C mutation in HMF reduction by Adh1p. We generated and kinetically characterized a group of protein variants using two furaldehyde compounds of industrial relevance. Also, we showed that there is a threshold after which higher in vitro HMF reduction activities do not correlate any more with faster in vivo rates of HMF conversion, indicating other cell limitations in the conversion of HMF.

[Enzyme kinetic glucose determination by the glucose dehydrogenase method. Enzyme kinetic substrate determination using competitive inhibitors, II (author's transl)].

PubMed

Müller-Matthesius, R

1975-05-01

The sensitivity of enzyme kinetic substrate determinations can be improved with the aid of competitive inhibitors. As an example, the determination of glucose dehydrogenase in the presence of potassium thiocyanate is described. The method has the advantage of rapid operation with satisfactory precision.

Novel chiral tool, (R)-2-octanol dehydrogenase, from Pichia finlandica: purification, gene cloning, and application for optically active α-haloalcohols.

PubMed

Yamamoto, Hiroaki; Kudoh, Masatake

2013-09-01

A novel enantioselective alcohol dehydrogenase, (R)-2-octanol dehydrogenase (PfODH), was discovered among methylotrophic microorganisms. The enzyme was purified from Pichia finlandica and characterized. The molecular mass of the enzyme was estimated to be 83,000 and 30,000 by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively. The enzyme was an NAD(+)-dependent secondary alcohol dehydrogenase and showed a strict enantioselectivity, very broad substrate specificity, and high tolerance to SH reagents. A gene-encoding PfODH was cloned and sequenced. The gene consisted of 765 nucleotides, coding polypeptides of 254 amino acids. The gene was singly expressed and coexpressed together with a formate dehydrogenase as an NADH regenerator in an Escherichia coli. Ethyl (S)-4-chloro-3-hydroxybutanoate and (S)-2-chloro-1-phenylethanol were synthesized using a whole-cell biocatalyst in more than 99 % optical purity.

Oxidation of Two Hydroxylated Ochratoxin A Metabolites by Alcohol Dehydrogenase

PubMed Central

Syvertsen, Christian; Størmer, Fredrik C.

1983-01-01

(4R)-4-hydroxyochratoxin A, (4S)-4-hydroxyochratoxin A, and 10-hydroxyochratoxin A, all formed from ochratoxin A, were incubated with alcohol dehydrogenase in the presence of NAD. Only (4R)-4-hydroxyochratoxin A and 10-hydroxyochratoxin A acted as substrates for the enzyme. Km and turnover number for 10-hydroxyochratoxin A were 110 μM and 0.1 s−1, respectively. PMID:6347065

Alcohol Dehydrogenase of Bacillus strain for Measuring Alcohol Electrochemically

NASA Astrophysics Data System (ADS)

Iswantini, D.; Nurhidayat, N.; Ferit, H.

2017-03-01

Alcohol dehydrogenase (ADH) was applied to produce alcohol biosensor. The enzyme was collected from cultured Bacillus sp. in solid media. From 6 tested isolates, bacteria from fermented rice grain (TST.A) showed the highest oxidation current which was further applied as the bioreceptor. Various ethanol concentrations was measured based on the increase of maximum oxidation current value. However, a reduction value was happened when the ethanol concentration was higher than 5%. Comparing the result of spectrophotometry measurement, R2 value obtained from the biosensor measurement method was higher. The new proposed method resulted a wider detection range, from 0.1-5% of ethanol concentration. The result showed that biosensor method has big potency to be used as alcohol detector in foods or bevearages.

Use of Fomepizole in Pediatric Methanol Exposure: The First Case Report in Taiwan and a Literature Review.

PubMed

Chen, Tzu-Hua; Kuo, Chang-Hung; Huang, Chia-Tsuan; Wang, Wei-Li

2016-08-01

Methanol poisoning is rare in the pediatric population, but a delay in diagnosis and intervention may cause severe morbidity and mortality. The current therapy for methanol poisoning is ethanol or fomepizole, which acts as a competitive inhibitor of hepatic alcohol dehydrogenase to inhibit the production of toxic metabolites derived from the oxidation of methanol. However, clinical experience in pediatric methanol poisoning is limited, and the safety profiles of the antidotes have not been established in children, especially in Asian populations. This is the first case to describe the use of fomepizole in a child with methanol exposure in Taiwan. Copyright © 2013. Published by Elsevier B.V.

Differential accumulation of proteins in oil palms affected by fatal yellowing disease

PubMed Central

do Nascimento, Sidney Vasconcelos; Magalhães, Marcelo Murad; Cunha, Roberto Lisboa; Costa, Paulo Henrique de Oliveira; Alves, Ronnie Cley de Oliveira; de Oliveira, Guilherme Corrêa

2018-01-01

There is still no consensus on the true origin of fatal yellowing, one of the most important diseases affecting oil palm (Elaeis guineensis Jacq.) plantations. This study involved two-dimensional liquid chromatography coupled with tandem mass spectrometry (2D-UPLC-MSE) analyses to identify changes in protein profiles of oil palms affected by FY disease. Oil palm roots were sampled from two growing areas. Differential accumulation of proteins was assessed by comparing plants with and without symptoms and between plants at different stages of FY development. Most of the proteins identified with differential accumulation were those related to stress response and energy metabolism. The latter proteins include the enzymes alcohol dehydrogenase and aldehyde dehydrogenase, related to alcohol fermentation, which were identified in plants with and without symptoms. The presence of these enzymes suggests an anaerobic condition before or during FY. Transketolase, isoflavone reductase, cinnamyl alcohol dehydrogenase, caffeic acid 3-O-methyltransferase, S-adenosylmethionine synthase, aldehyde dehydrogenase and ferritin, among others, were identified as potential marker proteins and could be used to guide selection of FY-tolerant oil palm genotypes or to understand the source of this anomaly. When comparing different stages of FY, we observed high accumulation of alcohol dehydrogenase and other abiotic stress related-proteins at all disease stages. On the other hand, biological stress-related proteins were more accumulated at later stages of the disease. These results suggest that changes in abiotic factors can trigger FY development, creating conditions for the establishment of opportunistic pathogens. PMID:29621343

Inhibitors of inosine monophosphate dehydrogenase: SARs about the N-[3-Methoxy-4-(5-oxazolyl)phenyl moiety.

PubMed

Iwanowicz, Edwin J; Watterson, Scott H; Guo, Junqing; Pitts, William J; Murali Dhar, T G; Shen, Zhongqi; Chen, Ping; Gu, Henry H; Fleener, Catherine A; Rouleau, Katherine A; Cheney, Daniel L; Townsend, Robert M; Hollenbaugh, Diane L

2003-06-16

The first reported structure-activity relationships (SARs) about the N-[3-methoxy-4-(5-oxazolyl)phenyl moiety for a series of recently disclosed inosine monophosphate dehydrogenase (IMPDH) inhibitors are described. The syntheses and in vitro inhibitory values for IMPDH II, and T-cell proliferation (for select analogues) are given.

Cloning, expression, and sequence analysis of the Bacillus methanolicus C1 methanol dehydrogenase gene.

PubMed Central

de Vries, G E; Arfman, N; Terpstra, P; Dijkhuizen, L

1992-01-01

The gene (mdh) coding for methanol dehydrogenase (MDH) of thermotolerant, methylotroph Bacillus methanolicus C1 has been cloned and sequenced. The deduced amino acid sequence of the mdh gene exhibited similarity to those of five other alcohol dehydrogenase (type III) enzymes, which are distinct from the long-chain zinc-containing (type I) or short-chain zinc-lacking (type II) enzymes. Highly efficient expression of the mdh gene in Escherichia coli was probably driven from its own promoter sequence. After purification of MDH from E. coli, the kinetic and biochemical properties of the enzyme were investigated. The physiological effect of MDH synthesis in E. coli and the role of conserved sequence patterns in type III alcohol dehydrogenases have been analyzed and are discussed. Images PMID:1644761

Microorganisms and methods for producing pyruvate, ethanol, and other compounds

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

Reed, Jennifer L.; Zhang, Xiaolin

Microorganisms comprising modifications for producing pyruvate, ethanol, and other compounds. The microorganisms comprise modifications that reduce or ablate activity of one or more of pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase, phosphate acetyltransferase, acetate kinase, pyruvate oxidase, lactate dehydrogenase, cytochrome terminal oxidase, succinate dehydrogenase, 6-phosphogluconate dehydrogenase, glutamate dehydrogenase, pyruvate formate lyase, pyruvate formate lyase activating enzyme, and isocitrate lyase. The microorganisms optionally comprise modifications that enhance expression or activity of pyruvate decarboxylase and alcohol dehydrogenase. The microorganisms are optionally evolved in defined media to enhance specific production of one or more compounds. Methods of producing compounds with the microorganisms are provided.

Characterization of hamster NAD+-dependent 3(17)β-hydroxysteroid dehydrogenase belonging to the aldo-keto reductase 1C subfamily.

PubMed

Endo, Satoshi; Noda, Misato; Ikari, Akira; Tatematsu, Kenjiro; El-Kabbani, Ossama; Hara, Akira; Kitade, Yukio; Matsunaga, Toshiyuki

2015-11-01

The cDNAs for morphine 6-dehydrogenase (AKR1C34) and its homologous aldo-keto reductase (AKR1C35) were cloned from golden hamster liver, and their enzymatic properties and tissue distribution were compared. AKR1C34 and AKR1C35 similarly oxidized various xenobiotic alicyclic alcohols using NAD(+), but differed in their substrate specificity for hydroxysteroids and inhibitor sensitivity. While AKR1C34 showed 3α/17β/20α-hydroxysteroid dehydrogenase activities, AKR1C35 efficiently oxidized various 3β- and 17β-hydroxysteroids, including biologically active 3β-hydroxy-5α/β-dihydro-C19/C21-steroids, dehydroepiandrosterone and 17β-estradiol. AKR1C35 also differed from AKR1C34 in its high sensitivity to flavonoids, which inhibited competitively with respect to 17β-estradiol (Ki 0.11-0.69 μM). The mRNA for AKR1C35 was expressed liver-specific in male hamsters and ubiquitously in female hamsters, whereas the expression of the mRNA for AKR1C34 displayed opposite sexual dimorphism. Because AKR1C35 is the first 317Β-HYDROXYSTEROID DEHYDROGENASE IN THE AKR SUPERFAMILY: , we also investigated the molecular determinants for the 3β-hydroxysteroid dehydrogenase activity by replacement of Val54 and Cys310 in AKR1C35 with the corresponding residues in AKR1C34, Ala and Phe, respectively. The mutation of Val54Ala, but not Cys310Phe, significantly impaired this activity, suggesting that Val54 plays a critical role in recognition of the steroidal substrate. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Nucleic acid molecules conferring enhanced ethanol tolerance and microorganisms having enhanced tolerance to ethanol

DOEpatents

Brown, Steven; Guss, Adam; Yang, Shihui; Karpinets, Tatiana; Lynd, Lee; Shao, Xiongjun

2014-01-14

The present invention provides isolated nucleic acid molecules which encode a mutant acetaldehyde-CoA/alcohol dehydrogenase or mutant alcohol dehydrogenase and confer enhanced tolerance to ethanol. The invention also provides related expression vectors, genetically engineered microorganisms having enhanced tolerance to ethanol, as well as methods of making and using such genetically modified microorganisms for production of biofuels based on fermentation of biomass materials.

Estrogen and androgen-converting enzymes 17β-hydroxysteroid dehydrogenase and their involvement in cancer: with a special focus on 17β-hydroxysteroid dehydrogenase type 1, 2, and breast cancer

PubMed Central

Hilborn, Erik; Stål, Olle; Jansson, Agneta

2017-01-01

Sex steroid hormones such as estrogens and androgens are involved in the development and differentiation of the breast tissue. The activity and concentration of sex steroids is determined by the availability from the circulation, and on local conversion. This conversion is primarily mediated by aromatase, steroid sulfatase, and 17β-hydroxysteroid dehydrogenases. In postmenopausal women, this is the primary source of estrogens in the breast. Up to 70-80% of all breast cancers express the estrogen receptor-α, responsible for promoting the growth of the tissue. Further, 60-80% express the androgen receptor, which has been shown to have tissue protective effects in estrogen receptor positive breast cancer, and a more ambiguous response in estrogen receptor negative breast cancers. In this review, we summarize the function and clinical relevance in cancer for 17β-hydroxysteroid dehydrogenases 1, which facilitates the reduction of estrone to estradiol, dehydroepiandrosterone to androstendiol and dihydrotestosterone to 3α- and 3β-diol as well as 17β-hydroxysteroid dehydrogenases 2 which mediates the oxidation of estradiol to estrone, testosterone to androstenedione and androstendiol to dehydroepiandrosterone. The expression of 17β-hydroxysteroid dehydrogenases 1 and 2 alone and in combination has been shown to predict patient outcome, and inhibition of 17β-hydroxysteroid dehydrogenases 1 has been proposed to be a prime candidate for inhibition in patients who develop aromatase inhibitor resistance or in combination with aromatase inhibitors as a first line treatment. Here we review the status of inhibitors against 17β-hydroxysteroid dehydrogenases 1. In addition, we review the involvement of 17β-hydroxysteroid dehydrogenases 4, 5, 7, and 14 in breast cancer. PMID:28430630

Estrogen and androgen-converting enzymes 17β-hydroxysteroid dehydrogenase and their involvement in cancer: with a special focus on 17β-hydroxysteroid dehydrogenase type 1, 2, and breast cancer.

PubMed

Hilborn, Erik; Stål, Olle; Jansson, Agneta

2017-05-02

Sex steroid hormones such as estrogens and androgens are involved in the development and differentiation of the breast tissue. The activity and concentration of sex steroids is determined by the availability from the circulation, and on local conversion. This conversion is primarily mediated by aromatase, steroid sulfatase, and 17β-hydroxysteroid dehydrogenases. In postmenopausal women, this is the primary source of estrogens in the breast. Up to 70-80% of all breast cancers express the estrogen receptor-α, responsible for promoting the growth of the tissue. Further, 60-80% express the androgen receptor, which has been shown to have tissue protective effects in estrogen receptor positive breast cancer, and a more ambiguous response in estrogen receptor negative breast cancers. In this review, we summarize the function and clinical relevance in cancer for 17β-hydroxysteroid dehydrogenases 1, which facilitates the reduction of estrone to estradiol, dehydroepiandrosterone to androstendiol and dihydrotestosterone to 3α- and 3β-diol as well as 17β-hydroxysteroid dehydrogenases 2 which mediates the oxidation of estradiol to estrone, testosterone to androstenedione and androstendiol to dehydroepiandrosterone. The expression of 17β-hydroxysteroid dehydrogenases 1 and 2 alone and in combination has been shown to predict patient outcome, and inhibition of 17β-hydroxysteroid dehydrogenases 1 has been proposed to be a prime candidate for inhibition in patients who develop aromatase inhibitor resistance or in combination with aromatase inhibitors as a first line treatment. Here we review the status of inhibitors against 17β-hydroxysteroid dehydrogenases 1. In addition, we review the involvement of 17β-hydroxysteroid dehydrogenases 4, 5, 7, and 14 in breast cancer.

Purification and characterization of an anti-Prelog alcohol dehydrogenase from Oenococcus oeni that reduces 2-octanone to (R)-2-octanol.

PubMed

Meng, Fantao; Xu, Yan

2010-04-01

An anti-Prelog alcohol dehydrogenase from Oenococcus oeni that reduces 2-octanone to (R)-2-octanol was purified by 26-fold to homogeneity. The enzyme had a homodimeric structure consisting of 49 kDa subunits, required NADPH, but not NADH, as a cofactor and was a Zn-independent short-chain dehydrogenase. Aliphatic methyl ketones (chain length > or =6 carbon atoms) and aromatic methyl ketones were the preferred substrates for the enzyme, the best being 2-octanone. Maximum enzyme activity with 2-octanone was at 45 degrees C and at pH 8.0.

Recent advances in the study of 11β-Hydroxysteroid dehydrogenase type 2 (11β-HSD2)Inhibitors.

PubMed

Zhou, Chunchun; Ye, Fan; Wu, He; Ye, Hui; Chen, Quanxu

2017-06-01

11β-Hydroxysteroid dehydrogenase (11β-HSD), which interconverts hormonally active cortisol and inactive cortisone in multiple human tissues, has two distinct isoforms named 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) and 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2). 11β-HSD2 is an NAD + -dependent oxidase which lowers cortisol by converting it to cortisone while 11β-HSD1 mainly catalyzes the reduction which converts cortisone into cortisol. Selective inhibition of 11β-HSD2 is generally detrimental to health because the accumulation of cortisol can cause metabolic symptoms such as apparent mineralocorticoid excess (AME), fetal developmental defects and lower testosterone levels in males. There has been some advances on the study of 11β-HSD2 inhibitors and we think it necessary to make a summary of the characteristics and inhibiting properties of latest 11β-HSD2 inhibitors. As another review on 11β-HSD2 inhibitors has been issued on 2011 (see review (Ma et al., 2011)), this mini-review concerns advances during the last 5 years. Copyright © 2017 Elsevier B.V. All rights reserved.

Bioactivities of decoctions from Plectranthus species related to their traditional use on the treatment of digestive problems and alcohol intoxication.

PubMed

Brito, Elsa; Gomes, Emma; Falé, Pedro L; Borges, Carlos; Pacheco, Rita; Teixeira, Vitor; Machuqueiro, Miguel; Ascensão, Lia; Serralheiro, Maria Luisa M

2018-06-28

Decoctions of Plectranthus species are traditionally ingested after large meals for treatment of food digestion and alcohol abuse. This study aims at associating the digestion-related ethno-uses of Plectranthus species decoctions to molecular mechanism that might explain them: easing digestion (AChE inhibition) and treating hangover (ADH inhibition) MATERIAL AND METHODS: Decoctions from Plectranthus species were analysed for their alcohol dehydrogenase (ADH) inhibition and acetylcholinesterase (AChE) inhibition, related with alcohol metabolism and intestinal motility, respectively. Identification of the active components was carried out by LC-MS/MS and the docking studies were performed with AChE and the bioactive molecules detected. All decoctions inhibited ADH activity. This inhibition was correlated with their rosmarinic acid (RA) content, which showed an IC 50 value of 19 μg/mL, similar to the reference inhibitor CuCl 2 . The presence of RA also leads to most decoctions showing AChE inhibiting capacity. P. zuluensis decoction with an IC 50 of 80 μg/mL presented also medioresinol, an even better inhibitor of AChE, as indicated by molecular docking studies. Furthermore, all decoctions tested showed no toxicity towards two human cell lines, and a high capacity to quench free radicals (DPPH), which also play a helpful in the digestive process, related with their RA content. All activities presented by the RA-rich Plectranthus decoctions support their use in treating digestion disorders and P. barbatus could explain its use also for alleviating hangover symptoms. Medioresinol, which is present in P. zuluensis, exhibited a significant AChE inhibition and may provide, in the future, a new lead for bioactive compounds. Copyright © 2018 Elsevier B.V. All rights reserved.

Isolation and characterization of full-length putative alcohol dehydrogenase genes from polygonum minus

NASA Astrophysics Data System (ADS)

Hamid, Nur Athirah Abd; Ismail, Ismanizan

2013-11-01

Polygonum minus, locally named as Kesum is an aromatic herb which is high in secondary metabolite content. Alcohol dehydrogenase is an important enzyme that catalyzes the reversible oxidation of alcohol and aldehyde with the presence of NAD(P)(H) as co-factor. The main focus of this research is to identify the gene of ADH. The total RNA was extracted from leaves of P. minus which was treated with 150 μM Jasmonic acid. Full-length cDNA sequence of ADH was isolated via rapid amplification cDNA end (RACE). Subsequently, in silico analysis was conducted on the full-length cDNA sequence and PCR was done on genomic DNA to determine the exon and intron organization. Two sequences of ADH, designated as PmADH1 and PmADH2 were successfully isolated. Both sequences have ORF of 801 bp which encode 266 aa residues. Nucleotide sequence comparison of PmADH1 and PmADH2 indicated that both sequences are highly similar at the ORF region but divergent in the 3' untranslated regions (UTR). The amino acid is differ at the 107 residue; PmADH1 contains Gly (G) residue while PmADH2 contains Cys (C) residue. The intron-exon organization pattern of both sequences are also same, with 3 introns and 4 exons. Based on in silico analysis, both sequences contain "classical" short chain alcohol dehydrogenases/reductases ((c) SDRs) conserved domain. The results suggest that both sequences are the members of short chain alcohol dehydrogenase family.

Conversion of alcohols to enantiopure amines through dual enzyme hydrogen-borrowing cascades

PubMed Central

Mutti, Francesco G.; Knaus, Tanja; Scrutton, Nigel S.; Breuer, Michael; Turner, Nicholas J.

2016-01-01

α-Chiral amines are key intermediates for the synthesis of a plethora of chemical compounds on industrial scale. Here we present a biocatalytic hydrogen-borrowing amination of primary and secondary alcohols that allows for the efficient and environmentally benign production of enantiopure amines. The method relies on the combination of an alcohol dehydrogenase (ADHs from Aromatoleum sp., Lactobacillus sp. and Bacillus sp.) enzyme operating in tandem with an amine dehydrogenase (AmDHs engineered from Bacillus sp.) to aminate a structurally diverse range of aromatic and aliphatic alcohols (up to 96% conversion and 99% enantiomeric excess). Furthermore, primary alcohols are aminated with high conversion (up to 99%). This redox self-sufficient network possesses high atom efficiency, sourcing nitrogen from ammonium and generating water as the sole by-product. PMID:26404833

Heat-stable, FE-dependent alcohol dehydrogenase for aldehyde detoxification

DOEpatents

Elkins, James G.; Clarkson, Sonya

2018-04-24

The present invention relates to microorganisms and polypeptides for detoxifying aldehydes associated with industrial fermentations. In particular, a heat-stable, NADPH- and iron-dependent alcohol dehydrogenase was cloned from Thermoanaerobacter pseudethanolicus 39E and displayed activity against a number of aldehydes including inhibitory compounds that are produced during the dilute-acid pretreatment process of lignocellulosic biomass before fermentation to biofuels. Methods to use the microorganisms and polypeptides of the invention for improved conversion of bio mass to biofuel are provided as well as use of the enzyme in metabolic engineering strategies for producing longer-chain alcohols from sugars using thermophilic, fermentative microorganisms.

Cupriavidus necator JMP134 rapidly reduces furfural with a Zn-dependent alcohol dehydrogenase.

PubMed

Li, Qunrui; Metthew Lam, L K; Xun, Luying

2011-11-01

Ethanol is a renewable biofuel, and it can be produced from lignocellulosic biomass. The biomass is usually converted to hydrolysates that consist of sugar and sugar derivatives, such as furfural. Yeast ferments sugar to ethanol, but furfural higher than 3 mM is inhibitory. It can take several days for yeast cells to reduce furfural to non-inhibitory furfuryl alcohol before producing ethanol. Bioreduction of furfural to furfuryl alcohol before fermentation may relieve yeast from furfural toxicity. We observed that Cupriavidus necator JMP134, a strict aerobe, rapidly reduced 17 mM furfural to less than 3 mM within 14 min with cell turbidity of 1.0 at 600 nm at 50°C. The rapid reduction consumed ethanol. The "furfural reductase" (FurX) was purified, and it oxidized ethanol to acetaldehyde and reduced furfural to furfuryl alcohol with NAD(+) as the cofactor. The protein was identified with mass spectrometry fingerprinting to be a hypothetical protein belonging to Zn-dependent alcohol dehydrogenase family. The furX-inactivation mutant of C. necator JMP134 lost the ability to rapidly reduce furfural, and Escherichia coli producing recombinant FurX gained the ability. Thus, an alcohol dehydrogenase enabled bacteria to rapidly reduce furfural with ethanol as the reducing power.

A Novel Cofactor-binding Mode in Bacterial IMP Dehydrogenases Explains Inhibitor Selectivity*

PubMed Central

Makowska-Grzyska, Magdalena; Kim, Youngchang; Maltseva, Natalia; Osipiuk, Jerzy; Gu, Minyi; Zhang, Minjia; Mandapati, Kavitha; Gollapalli, Deviprasad R.; Gorla, Suresh Kumar; Hedstrom, Lizbeth; Joachimiak, Andrzej

2015-01-01

The steadily rising frequency of emerging diseases and antibiotic resistance creates an urgent need for new drugs and targets. Inosine 5′-monophosphate dehydrogenase (IMP dehydrogenase or IMPDH) is a promising target for the development of new antimicrobial agents. IMPDH catalyzes the oxidation of IMP to XMP with the concomitant reduction of NAD+, which is the pivotal step in the biosynthesis of guanine nucleotides. Potent inhibitors of bacterial IMPDHs have been identified that bind in a structurally distinct pocket that is absent in eukaryotic IMPDHs. The physiological role of this pocket was not understood. Here, we report the structures of complexes with different classes of inhibitors of Bacillus anthracis, Campylobacter jejuni, and Clostridium perfringens IMPDHs. These structures in combination with inhibition studies provide important insights into the interactions that modulate selectivity and potency. We also present two structures of the Vibrio cholerae IMPDH in complex with IMP/NAD+ and XMP/NAD+. In both structures, the cofactor assumes a dramatically different conformation than reported previously for eukaryotic IMPDHs and other dehydrogenases, with the major change observed for the position of the NAD+ adenosine moiety. More importantly, this new NAD+-binding site involves the same pocket that is utilized by the inhibitors. Thus, the bacterial IMPDH-specific NAD+-binding mode helps to rationalize the conformation adopted by several classes of prokaryotic IMPDH inhibitors. These findings offer a potential strategy for further ligand optimization. PMID:25572472

Miniature enzyme-based electrodes for detection of hydrogen peroxide release from alcohol-injured hepatocytes.

PubMed

Matharu, Zimple; Enomoto, James; Revzin, Alexander

2013-01-15

Alcohol insult to the liver sets off a complex sequence of inflammatory and fibrogenic responses. There is increasing evidence that hepatocytes play a key role in triggering these responses by producing inflammatory signals such as cytokines and reactive oxygen species (ROS). In the present study, we employed a cell culture/biosensor platform consisting of electrode arrays integrated with microfluidics to monitor extracellular H(2)O(2), one of the major ROS types, produced by primary rat hepatocytes during alcohol injury. The biosensor consisted of hydrogel microstructures with entrapped horseradish peroxidase (HRP) immobilized on an array of miniature gold electrodes. These arrays of sensing electrodes were integrated into microfluidic devices and modified with collagen (I) to promote hepatocyte adhesion. Once seeded into the microfluidic devices, hepatocytes were exposed to 100 mM ethanol and the signal at the working electrode was monitored by cyclic voltammetry (CV) over the course of 4 h. The CV experiments revealed that hepatocytes secreted up to 1.16 μM H(2)O(2) after 3 h of stimulation. Importantly, when hepatocytes were incubated with antioxidants or alcohol dehydrogenase inhibitor prior to alcohol exposure, the H(2)O(2) signal was decreased by ~5-fold. These experiments further confirmed that the biosensor was indeed monitoring oxidative stress generated by the hepatocytes and also pointed to one future use of this technology for screening hepatoprotective effects of antioxidants.

Bioenergetics of Stromal Cells as a Predictor of Aggressive Prostate Cancer

DTIC Science & Technology

2016-11-01

complex tissue preparations (human prostate and prostatic adenoma) and rat ventral prostate cells it was reported to exhibit high aerobic glycolysis [19...pyruvate dehydrogenase kinase), 2DG (inhibitor of hexokinase), or metformin (inhibitor of mitochondrial complex I) [41] as a therapeutic approach to... cyanide 4-(trifluoromethoxy) phenylhydrazone; GAPDH, Glyceraldehyde 3-phosphate dehydrogenase; GlyST, Glycolytic stress test; HPV, human papilloma virus

Polymorphisms in the promoter region of the human class II alcohol dehydrogenase (ADH4) gene affect both transcriptional activity and ethanol metabolism in Japanese subjects.

PubMed

Kimura, Yukiko; Nishimura, Fusae T; Abe, Shuntaro; Fukunaga, Tatsushige; Tanii, Hideji; Saijoh, Kiyofumi

2009-02-01

Class II alcohol dehydrogenase (pi-ADH), encoded by alcohol dehydrogenase (ADH4), is considered to contribute to ethanol (EtOH) oxidation in the liver at high concentration. Four single nucleotide polymorphisms (SNPs) were found in the promoter region of this gene. Analysis of genotype distribution in 102 unrelated Japanese subjects revealed that four loci were in strong linkage disequilibrium and could be classified into three haplotypes. The effects of these polymorphisms on transcriptional activity were investigated in HepG2 cells. Transcriptional activity was significantly higher in cells with the -136A allele than in those with the -136C allele. To investigate whether this difference in transcriptional activity caused a difference in EtOH elimination, previous data on blood EtOH changes after 0.4 g/kg body weight alcohol ingestion were analyzed. When analyzed based on aldehyde dehydrogenase-2 gene (ALDH2) (487)Glu/Lys genotype, the significantly lower level of EtOH at peak in subjects with -136C/A and -136A/A genotype compared with subjects with -136C/C genotype indicated that -136 bp was a suggestive locus for differences in EtOH oxidation. This effect was observed only in subjects with ALDH2 (487)Glu/Glu. These results suggested that the SNP at -136bp in the ADH4 promoter had an effect on transcriptional regulation, and that the higher activity of the -136A allele compared with the -136C allele caused a lower level of blood EtOH after alcohol ingestion; that is, individuals with the -136A allele may consume more EtOH and might have a higher risk for development of alcohol dependence than those without the -136A allele.

Extracellular ascorbate stabilization as a result of transplasma electron transfer in Saccharomyces cerevisiae.

PubMed

Santos-Ocaña, C; Navas, P; Crane, F L; Córdoba, F

1995-12-01

The presence of yeast cells in the incubation medium prevents the oxidation of ascrobate catalyzed by copper ions. Ethanol increases ascorbate retention. Pyrazole, an alcohol dehydrogenase inhibitor, prevents ascorbate stabilization by cells. Chelation of copper ions does not account for stabilization, since oxidation rates with broken or boiled cells or conditioned media are similar to control rates in the absence of cells. Protoplast integrity is needed to reach optimal values of stabilization. Chloroquine, a known inhibitor of plasma membrane redox systems, inhibits the ascorbate stabilization, the inhibition being partially reversed by coenzyme Q6. Chloroquine does not inhibit ferricyanide reduction. Growth of yeast in iron-deficient media to increase ferric ion reductase activity also increases the stabilization. In conclusion, extracellular ascorbate stabilization by yeast cells can reflect a coenzyme Q dependent transplasmalemma electron transfer which uses NADH as electron donor. Iron deficiency increases the ascorbate stabilization but the transmembrane ferricyanide reduction system can act independently of ascorbate stabilization.

Effects of Beverages on Alcohol Metabolism: Potential Health Benefits and Harmful Impacts

PubMed Central

Wang, Fang; Zhang, Yu-Jie; Zhou, Yue; Li, Ya; Zhou, Tong; Zheng, Jie; Zhang, Jiao-Jiao; Li, Sha; Xu, Dong-Ping; Li, Hua-Bin

2016-01-01

Nonalcoholic beverages are usually consumed accompanying alcoholic drinks, and their effects on alcohol metabolism are unclear in vivo. In this study, the effects of 20 nonalcoholic beverages on alcohol metabolism and liver injury caused by alcohol were evaluated in mice. Kunming mice were orally fed with alcohol (52%, v/v) and beverages. The concentrations of ethanol and acetaldehyde in blood as well as the activities of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) in liver were assessed to indicate alcohol metabolism. The levels of aspartate aminotransferase (AST) and alanine transaminase (ALT) in serum as well as the levels of malonaldehyde (MDA) and superoxide dismutase (SOD) in liver were measured to reflect the alcohol-induced liver injury. The results showed that the treatment of soda water, green tea and honey chrysanthemum tea could accelerate ethanol metabolism and prevent liver injuries caused by alcohol when companied with excessive alcohol drinking. They might be potential dietary supplements for the alleviation of harmful effects from excessive alcohol consumption. On the contrary, some beverages such as fresh orange juice and red bull are not advised to drink when companied with alcohol consumption due to their adverse effects on ethanol induced liver injury. PMID:27005619

Existence of a novel enzyme, pyrroloquinoline quinone-dependent polyvinyl alcohol dehydrogenase, in a bacterial symbiont, Pseudomonas sp. strain VM15C.

PubMed Central

Shimao, M; Ninomiya, K; Kuno, O; Kato, N; Sakazawa, C

1986-01-01

A novel enzyme, pyrroloquinoline quinone (PQQ)-dependent polyvinyl alcohol (PVA) dehydrogenase, was found in and partially purified from the membrane fraction of a PVA-degrading symbiont, Pseudomonas sp. strain VM15C. The enzyme required PQQ for PVA dehydrogenation with phenazine methosulfate, phenazine ethosulfate, and 2,6-dichlorophenolindophenol as electron acceptors and did not show PVA oxidase activity leading to H2O2 formation. The enzyme was active toward low-molecular-weight secondary alcohols rather than primary alcohols. A membrane-bound PVA oxidase was also present in cells of VM15C. Although the purified oxidase showed a substrate specificity similar to that of PQQ-dependent PVA dehydrogenase and about threefold-higher PVA-dehydrogenating activity with phenazine methosulfate or phenazine ethosulfate than PVA oxidase activity with H2O2 formation, it was shown that the enzyme does not contain PQQ as the coenzyme, and PQQ did not affect its activity. Incubation of the membrane fraction of cells with PVA caused a reduction in the cytochrome(s) of the fraction. Images PMID:3513704

Expression of a heat-stable NADPH-dependent alcohol dehydrogenase from Thermoanaerobacter pseudethanolicus 39E in Clostridium thermocellum 1313 results in increased hydroxymethylfurfural resistance

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

Kim, Sun -Ki; Groom, Joseph; Chung, Daehwan

Resistance to deconstruction is a major limitation to the use of lignocellulosic biomass as a substrate for the production of fuels and chemicals. Consolidated bioprocessing (CBP), the use of microbes for the simultaneous hydrolysis of lignocellulose into soluble sugars and fermentation of the resulting sugars to products of interest, is a potential solution to this obstacle. The pretreatment of plant biomass, however, releases compounds that are inhibitory to the growth of microbes used for CBP. Heterologous expression of the Thermoanaerobacter pseudethanolicus 39E bdhA gene, that encodes an alcohol dehydrogenase, in Clostridium thermocellum significantly increased resistance to furan derivatives at concentrationsmore » found in acid-pretreated biomass. The mechanism of detoxification of hydroxymethylfurfural was shown to be primarily reduction using NADPH as the cofactor. In addition, we report the construction of new expression vectors for homologous and heterologous expression in C. thermocellum. These vectors use regulatory signals from both C. bescii (the S-layer promoter) and C. thermocellum (the enolase promoter) shown to efficiently drive expression of the BdhA enzyme. Toxic compounds present in lignocellulose hydrolysates that inhibit cell growth and product formation are obstacles to the commercialization of fuels and chemicals from biomass. Lastly, expression of genes that reduce the effect of these inhibitors, such as furan derivatives, will serve to enable commercial processes using plant biomass for the production of fuels and chemicals.« less

Purification, Characterization, and Cloning of Cinnamyl Alcohol Dehydrogenase in Loblolly Pine (Pinus taeda L.) 1

PubMed Central

O'Malley, David M.; Porter, Stephanie; Sederoff, Ronald R.

1992-01-01

Cinnamyl alcohol dehydrogenase (CAD, EC 1.1.1. 195) has been purified to homogeneity from differentiating xylem tissue and developing seeds of loblolly pine (Pinus taeda L.). The enzyme is a dimer with a native molecular weight of 82,000 and a subunit molecular weight of 44,000, and is the only form of CAD involved in lignification in differentiating xylem. High levels of loblolly pine CAD enzyme were found in nonlignifying seed tissue. Characterization of the enzyme from both seeds and xylem demonstrated that the enzyme is the same in both tissues. The enzyme has a high affinity for coniferaldehyde (Km = 1.7 micromolar) compared with sinapaldehyde (Km in excess of 100 micromolar). Kinetic data strongly suggest that coniferin is a noncompetitive inhibitor of CAD enzyme activity. Protein sequences were obtained for the N-terminus (28 amino acids) and for two other peptides. Degenerate oligonucleotide primers based on the protein sequences were used to amplify by polymerase chain reaction a 1050 base pair DNA fragment from xylem cDNA. Nucleotide sequence from the cloned DNA fragment coded for the N-terminal protein sequence and an internal peptide of CAD. The N-terminal protein sequence has little similarity with the λCAD4 clone isolated from bean (MH Walter, J Grima-Pettenati, C Grand, AM Boudet, CJ Lamb [1988] Proc Natl Acad Sci USA 86:5546-5550), which has homology with malic enzyme. ImagesFigure 2Figure 3 PMID:16668801

Purification, Characterization, and Cloning of Cinnamyl Alcohol Dehydrogenase in Loblolly Pine (Pinus taeda L.).

PubMed

O'malley, D M; Porter, S; Sederoff, R R

1992-04-01

Cinnamyl alcohol dehydrogenase (CAD, EC 1.1.1. 195) has been purified to homogeneity from differentiating xylem tissue and developing seeds of loblolly pine (Pinus taeda L.). The enzyme is a dimer with a native molecular weight of 82,000 and a subunit molecular weight of 44,000, and is the only form of CAD involved in lignification in differentiating xylem. High levels of loblolly pine CAD enzyme were found in nonlignifying seed tissue. Characterization of the enzyme from both seeds and xylem demonstrated that the enzyme is the same in both tissues. The enzyme has a high affinity for coniferaldehyde (K(m) = 1.7 micromolar) compared with sinapaldehyde (K(m) in excess of 100 micromolar). Kinetic data strongly suggest that coniferin is a noncompetitive inhibitor of CAD enzyme activity. Protein sequences were obtained for the N-terminus (28 amino acids) and for two other peptides. Degenerate oligonucleotide primers based on the protein sequences were used to amplify by polymerase chain reaction a 1050 base pair DNA fragment from xylem cDNA. Nucleotide sequence from the cloned DNA fragment coded for the N-terminal protein sequence and an internal peptide of CAD. The N-terminal protein sequence has little similarity with the lambdaCAD4 clone isolated from bean (MH Walter, J Grima-Pettenati, C Grand, AM Boudet, CJ Lamb [1988] Proc Natl Acad Sci USA 86:5546-5550), which has homology with malic enzyme.

Expression of a heat-stable NADPH-dependent alcohol dehydrogenase from Thermoanaerobacter pseudethanolicus 39E in Clostridium thermocellum 1313 results in increased hydroxymethylfurfural resistance

DOE PAGES

Kim, Sun -Ki; Groom, Joseph; Chung, Daehwan; ...

2017-03-15

Resistance to deconstruction is a major limitation to the use of lignocellulosic biomass as a substrate for the production of fuels and chemicals. Consolidated bioprocessing (CBP), the use of microbes for the simultaneous hydrolysis of lignocellulose into soluble sugars and fermentation of the resulting sugars to products of interest, is a potential solution to this obstacle. The pretreatment of plant biomass, however, releases compounds that are inhibitory to the growth of microbes used for CBP. Heterologous expression of the Thermoanaerobacter pseudethanolicus 39E bdhA gene, that encodes an alcohol dehydrogenase, in Clostridium thermocellum significantly increased resistance to furan derivatives at concentrationsmore » found in acid-pretreated biomass. The mechanism of detoxification of hydroxymethylfurfural was shown to be primarily reduction using NADPH as the cofactor. In addition, we report the construction of new expression vectors for homologous and heterologous expression in C. thermocellum. These vectors use regulatory signals from both C. bescii (the S-layer promoter) and C. thermocellum (the enolase promoter) shown to efficiently drive expression of the BdhA enzyme. Toxic compounds present in lignocellulose hydrolysates that inhibit cell growth and product formation are obstacles to the commercialization of fuels and chemicals from biomass. Lastly, expression of genes that reduce the effect of these inhibitors, such as furan derivatives, will serve to enable commercial processes using plant biomass for the production of fuels and chemicals.« less

Conversion of alcohols to enantiopure amines through dual-enzyme hydrogen-borrowing cascades.

PubMed

Mutti, Francesco G; Knaus, Tanja; Scrutton, Nigel S; Breuer, Michael; Turner, Nicholas J

2015-09-25

α-Chiral amines are key intermediates for the synthesis of a plethora of chemical compounds at industrial scale. We present a biocatalytic hydrogen-borrowing amination of primary and secondary alcohols that allows for the efficient and environmentally benign production of enantiopure amines. The method relies on a combination of two enzymes: an alcohol dehydrogenase (from Aromatoleum sp., Lactobacillus sp., or Bacillus sp.) operating in tandem with an amine dehydrogenase (engineered from Bacillus sp.) to aminate a structurally diverse range of aromatic and aliphatic alcohols, yielding up to 96% conversion and 99% enantiomeric excess. Primary alcohols were aminated with high conversion (up to 99%). This redox self-sufficient cascade possesses high atom efficiency, sourcing nitrogen from ammonium and generating water as the sole by-product. Copyright © 2015, American Association for the Advancement of Science.

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

Pfleger, Brian F.; Youngquist, Tyler J.

Recombinant cells and methods for improved yield of fatty alcohols. The recombinant cells harbor a recombinant thioesterase gene, a recombinant acyl-CoA synthetase gene, and a recombinant acyl-CoA reductase gene. In addition, a gene product from one or more of an acyl-CoA dehydrogenase gene, an enoyl-CoA hydratase gene, a 3-hydroxyacyl-CoA dehydrogenase gene, and a 3-ketoacyl-CoA thiolase gene in the recombinant cells is functionally deleted. Culturing the recombinant cells produces fatty alcohols at high yields.

Proteomic Profiling of Liver and Plasma in Chronic Ethanol Feeding Model of Hepatic Alcohol Dehydrogenase-Deficient Deer Mice.

PubMed

Bhopale, Kamlesh K; Amer, Samir M; Kaphalia, Lata; Soman, Kizhake V; Wiktorowicz, John E; Shakeel Ansari, Ghulam A; Kaphalia, Bhupendra S

2017-10-01

Chronic alcohol abuse, a major risk factor for such diseases as hepatitis and cirrhosis, impairs hepatic alcohol dehydrogenase (ADH; key ethanol [EtOH]-metabolizing enzyme). Therefore, differentially altered hepatic and plasma proteomes were identified in chronic EtOH feeding model of hepatic ADH-deficient (ADH - ) deer mice to understand the metabolic basis of alcoholic liver disease (ALD). ADH - deer mice were fed 3.5 g% EtOH via Lieber-DeCarli liquid diet daily for 3 months and histology of the liver assessed. Liver and plasma proteins were separated by 2-dimensional gel electrophoresis. The proteins differentially expressed were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Histology of the liver showed panlobular steatosis and infiltration of T lymphocytes. Using the criteria of ≥1.5 for fold change (p-value ≤0.05) with expectation value (E ≤10 -3 ) and protein score (≥64), 18 proteins in the livers and 5 in the plasma of EtOH-fed mice were differentially expressed and identified. Prolyl 4-hydroxylase, cytochrome b-5, endo A cytokeratin, ATP synthase, heat-shock 70 kD proteins, enoyl CoA hydratase, stress-70 protein, peroxiredoxin 1, and ornithine carbamoyl transferase were up-regulated in the livers. However, carbonic anhydrase 3, mitochondrial ATP synthase, aldolase 2, actin γ, laminin receptor, and carbamoyl phosphate synthase were down-regulated. Contrary to the increased expression of creatine kinase M-type, a decreased expression of serine protease inhibitor A3A precursor, sulfated glycoprotein-2 (clusterin), and apolipoprotein E isoforms were found in the plasma of EtOH group. Chronic EtOH feeding in ADH - deer mice causes steatosis and infiltration of T lymphocytes in the livers along with increased expression of proteins involved in endoplasmic reticulum (ER) stress, fibrosis, fatty acid β oxidation and biogenesis, and decreased expression of proteins involved in ATP synthesis, carbohydrate metabolism, in cell regulation and architecture. Reduced expression of various carrier proteins as found in the plasma of EtOH group has a biomarker potential. Copyright © 2017 by the Research Society on Alcoholism.

De Novo-Synthesized Retinoic Acid in Ovarian Antral Follicles Enhances FSH-Mediated Ovarian Follicular Cell Differentiation and Female Fertility

PubMed Central

Kawai, Tomoko; Yanaka, Noriyuki; Richards, JoAnne S.

2016-01-01

Retinoic acid (RA) is the active form of vitamin A and is synthesized from retinol by two key enzymes, alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH). As the physiological precursor of RA, retinol impacts female reproductive functions and fertility. The expression of Adh1 and Adh5 as well as Aldh1a1 and Aldh1a7 are significantly increased in the ovaries of mice treated with equine chorionic gonadotropin/FSH. The RA receptor is expressed and localized in granulosa cells and is activated by endogenous RA as indicated by LacZ expression in granulosa cells of RA-responsive transgene-LacZ transgenic mice (RA reporter mice). Coinjection of the ADH inhibitor, 4-methylpyrazole, with equine chorionic gonadotropin significantly decreases the number and developmental competence of oocytes ovulated in response to human chorionic gonadotropin/LH as compared with controls. Injections of RA completely reverse the effects of the inhibitor of ovulation and oocyte development. When mice were fed a retinol-free, vitamin A-deficient diet that significantly reduced the serum levels of retinol, the expression of the LH receptor (Lhcgr) was significantly lower in the ovaries of the vitamin A-deficient mice, and injections of human chorionic gonadotropin failed to induce genes controlling ovulation. These results indicate that ovarian de novo biosynthesis of RA is required for the follicular expression of Lhcgr in granulosa cells and their ability to respond to the ovulatory LH surge. PMID:27022678

Simultaneous genotyping of single-nucleotide polymorphisms in alcoholism-related genes using duplex and triplex allele-specific PCR with two-step thermal cycles.

PubMed

Shirasu, Naoto; Kuroki, Masahide

2014-01-01

We developed a time- and cost-effective multiplex allele-specific polymerase chain reaction (AS-PCR) method based on the two-step PCR thermal cycles for genotyping single-nucleotide polymorphisms in three alcoholism-related genes: alcohol dehydrogenase 1B, aldehyde dehydrogenase 2 and μ-opioid receptor. Applying MightyAmp(®) DNA polymerase with optimized AS-primers and PCR conditions enabled us to achieve effective and selective amplification of the target alleles from alkaline lysates of a human hair root, and simultaneously to determine the genotypes within less than 1.5 h using minimal lab equipment.

AAV Gene Therapy for Alcoholism: Inhibition of Mitochondrial Aldehyde Dehydrogenase Enzyme Expression in Hepatoma Cells.

PubMed

Sanchez, Anamaria C; Li, Chengwen; Andrews, Barbara; Asenjo, Juan A; Samulski, R Jude

2017-09-01

Most ethanol is broken down in the liver in two steps by alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH2) enzymes, which metabolize down ethanol into acetaldehyde and then acetate. Some individuals from the Asian population who carry a mutation in the aldehyde dehydrogenase gene (ALDH2*2) cannot metabolize acetaldehyde as efficiently, producing strong effects, including facial flushing, dizziness, hypotension, and palpitations. This results in an aversion to alcohol intake and protection against alcoholism. The large prevalence of this mutation in the human population strongly suggests that modulation of ALDH2 expression by genetic technologies could result in a similar phenotype. scAAV2 vectors encoding ALDH2 small hairpin RNA (shRNA) were utilized to validate this hypothesis by silencing ALDH2 gene expression in human cell lines. Human cell lines HEK-293 and HepG2 were transduced with scAAV2/shRNA, showing a reduction in ALDH2 RNA and protein expression with the two viral concentration assayed (1 × 10 4 and 1 × 10 5 vg/cell) at two different time points. In both cell lines, ALDH2 RNA levels were reduced by 90% and protein expression was inhibited by 90% and 52%, respectively, 5 days post infection. Transduced HepG2 VL17A cells (ADH+) exposed to ethanol resulted in a 50% increase in acetaldehyde levels. These results suggest that gene therapy could be a useful tool for the treatment of alcoholism by knocking down ALDH2 expression using shRNA technology delivered by AAV vectors.

Physiological Regulation of Isocitrate Dehydrogenase and the Role of 2-Oxoglutarate in Prochlorococcus sp. Strain PCC 9511

PubMed Central

Diez, Jesús; Gómez-Baena, Guadalupe; Rangel-Zúñiga, Oriol Alberto; García-Fernández, José Manuel

2014-01-01

The enzyme isocitrate dehydrogenase (ICDH; EC 1.1.1.42) catalyzes the oxidative decarboxylation of isocitrate, to produce 2-oxoglutarate. The incompleteness of the tricarboxylic acids cycle in marine cyanobacteria confers a special importance to isocitrate dehydrogenase in the C/N balance, since 2-oxoglutarate can only be metabolized through the glutamine synthetase/glutamate synthase pathway. The physiological regulation of isocitrate dehydrogenase was studied in cultures of Prochlorococcus sp. strain PCC 9511, by measuring enzyme activity and concentration using the NADPH production assay and Western blotting, respectively. The enzyme activity showed little changes under nitrogen or phosphorus starvation, or upon addition of the inhibitors DCMU, DBMIB and MSX. Azaserine, an inhibitor of glutamate synthase, induced clear increases in the isocitrate dehydrogenase activity and icd gene expression after 24 h, and also in the 2-oxoglutarate concentration. Iron starvation had the most significant effect, inducing a complete loss of isocitrate dehydrogenase activity, possibly mediated by a process of oxidative inactivation, while its concentration was unaffected. Our results suggest that isocitrate dehydrogenase responds to changes in the intracellular concentration of 2-oxoglutarate and to the redox status of the cells in Prochlorococcus. PMID:25061751

Metabolism of 2-phenylethylamine and phenylacetaldehyde by precision-cut guinea pig fresh liver slices.

PubMed

Panoutsopoulos, Georgios I; Kouretas, Demetrios; Gounaris, Elias G; Beedham, Christine

2004-01-01

2-Phenylethylamine is an endogenous constituent of human brain and is implicated in cerebral transmission. It is also found in certain foodstuffs and may cause toxic side-effects in susceptible individuals. Metabolism of 2-phenylethylamine to phenylacetaldehyde is catalyzed by monoamine oxidase and the oxidation of the reactive aldehyde to its acid derivative is catalyzed mainly by aldehyde dehydrogenase and perhaps aldehyde oxidase, with xanthine oxidase having minimal transformation. The present investigation examines the metabolism of 2-phenylethylamine to phenylacetaldehyde in liver slices and compares the relative contribution of aldehyde oxidase, xanthine oxidase and aldehyde dehydrogenase activity in the oxidation of phenylacetaldehyde with precision-cut fresh liver slices in the presence/absence of specific inhibitors of each enzyme. In liver slices, phenylacetaldehyde was rapidly converted to phenylacetic acid. Phenylacetic acid was the main metabolite of 2-phenylethylamine, via the intermediate phenylacetaldehyde. Phenylacetic acid formation was completely inhibited by disulfiram (specific inhibitor of aldehyde dehydrogenase), whereas isovanillin (specific inhibitor of aldehyde oxidase) inhibited acid formation to a lesser extent and allopurinol (specific inhibitor of xanthine oxidase) had little or no effect. Therefore, in liver slices, phenylacetaldehyde is rapidly oxidized by aldehyde dehydrogenase and aldehyde oxidase with little or no contribution from xanthine oxidase.

STRUCTURE TOXICITY IN RELATIONSHIPS FOR A,B-UNSATURATED ALCOHOLS IN FISH

EPA Science Inventory

Previous toxicity testing with fathead minnows (Pimephales promelas) indicated that some unsaturated acetylenic and allylic alcohols can be metabolically activated, via alcohol dehydrogenase, to highly toxic a,B-unsaturated aldehydes and ketones or allene derivatives. lthough sev...

Protective effect of heat-treated cucumber (Cucumis sativus L.) juice on alcohol detoxification in experimental rats.

PubMed

Bajpai, Vivek K; Kim, Na-Hyung; Kim, Ji-Eun; Kim, Kangmin; Kang, Sun Chul

2016-05-01

In this study, heat-treated cucumber juice was assessed for its protective effect on blood alcohol levels and hepatic alcohol metabolic enzyme system in experimental rats. Initially, during detoxification of alcohol, all groups were orally dosed to 22% alcohol (6ml/kg body weight) along with different concentrations of heat-treated cucumber juice (10, 100 and 500mg/kg) and commercial goods for hangover-removal on sale (2ml/kg). Cucumber juice was dosed before 30 min, and simultaneously after 30min of alcohol administration, and its hepatoprotective effect on blood alcohol levels and hepatic alcohol metabolic enzyme system in experimental rats was evaluated. As a result, after 7h, remarkable reduction was found in the blood alcohol levels for all concentrations of cucumber juice treatment. Treatment with cucumber juice resulted in increasing dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) enzymatic activities in rat liver at 9h after alcohol administration thereby stimulated blood alcohol metabolism as compared with control group. The effect of heat-treated cucumber juice on alcohol detoxification was observed only in the rats treated before 30min from alcohol administration. These findings indicate that heat-treated cucumber juice has significant protective effect on alcohol detoxification in experimental rats, suggesting its usefulness in the treatment of liver injury caused by alcohol consumption.

Polymorphisms of alcohol dehydrogenase-1B and aldehyde dehydrogenase-2 and the blood and salivary ethanol and acetaldehyde concentrations of Japanese alcoholic men.

PubMed

Yokoyama, Akira; Tsutsumi, Eri; Imazeki, Hiromi; Suwa, Yoshihide; Nakamura, Chizu; Yokoyama, Tetsuji

2010-07-01

The effects of genetic polymorphism of aldehyde dehydrogenase-2 (ALDH2) on alcohol metabolism are striking in nonalcoholics, and the effects of genetic polymorphism of alcohol dehydrogenase-1B (ADH1B) are modest at most, whereas genetic polymorphisms of both strongly affect the susceptibility to alcoholism and upper aerodigestive tract (UADT) cancer of drinkers. We evaluated associations between ADH1B/ADH1C/ALDH2 genotypes and the blood and salivary ethanol and acetaldehyde levels of 168 Japanese alcoholic men who came to our hospital for the first time in the morning and had been drinking until the day before. The ethanol levels in their blood and saliva were similar, but the acetaldehyde levels in their saliva were much higher than in their blood, probably because of acetaldehyde production by oral bacteria. Blood and salivary ethanol and acetaldehyde levels were both significantly higher in the subjects with the less active ADH1B*1/*1 genotype than in the ADH1B*2 carriers, but none of the levels differed according to ALDH2 genotype. Significant linkage disequilibrium was detected between the ADH1B and ADH1C genotypes, but ADH1C genotype did not affect the blood or salivary ethanol or acetaldehyde levels. High blood acetaldehyde levels were found even in the active ALDH2*1/*1 alcoholics, which were comparable with the levels of the inactive heterozygous ALDH2*1/*2 alcoholics with less active ADH1B*1/*1. The slope of the increase in blood acetaldehyde level as the blood ethanol level increased was significantly steeper in alcoholics with inactive heterozygous ALDH2*1/*2 plus ADH1B*2 allele than with any other genotype combinations, but the slopes of the increase in salivary acetaldehyde level as the salivary ethanol level increased did not differ between the groups of subjects with any combinations of ALDH2 and ADH1B genotypes. The ADH1B/ALDH2 genotype affected the blood and salivary ethanol and acetaldehyde levels of nonabstinent alcoholics in a different manner from nonalcoholics, and clear effects of ADH1B genotype and less clear effects of ALDH2 were observed in the alcoholics. Alterations in alcohol metabolism as a result of alcoholism may modify the gene effects, and these findings provide some clues in regard to associations between the genotypes and the risks of alcoholism and UADT cancer.

Alcohol Dehydrogenase and Ethanol in the Stems of Trees 1

PubMed Central

Kimmerer, Thomas W.; Stringer, Mary A.

1988-01-01

Anaerobic fermentation in plants is usually thought to be a transient phenomenon, brought about by environmental limitations to oxygen availability, or by structural constraints to oxygen transport. The vascular cambium of trees is separated from the air by the outer bark and secondary phloem, and we hypothesized that the cambium may experience sufficient hypoxia to induce anaerobic fermentation. We found high alcohol dehydrogenase activity in the cambium of several tree species. Mean activity of alcohol dehydrogenase in Populus deltoides was 165 micromoles NADH oxidized per minute per gram fresh weight in May. Pyruvate decarboxylase activity was also present in the cambium of P. deltoides, with mean activity of 26 micromoles NADH oxidized per minute per gram fresh weight in May. Lactate dehydrogenase activity was not present in any tree species we examined. Contrary to our expectation, alcohol dehydrogenase activity was inversely related to bark thickness in Acer saccharum and unrelated to bark thickness in two Populus species. Bark thickness may be less important in limiting oxygen availability to the cambium than is oxygen consumption by rapidly respiring phloem and cambium in actively growing trees. Ethanol was present in the vascular cambium of all species examined, with mean concentrations of 35 to 143 nanomoles per gram fresh weight, depending on species. Ethanol was also present in xylem sap and may have been released from the cambium into the transpiration stream. The presence in the cambium of the enzymes necessary for fermentation as well as the products of fermentation is evidence that respiration in the vascular cambium of trees may be oxygen-limited, but other biosynthetic origins of ethanol have not been ruled out. PMID:16666209

Discovery and Optimization of Allosteric Inhibitors of Mutant Isocitrate Dehydrogenase 1 (R132H IDH1) Displaying Activity in Human Acute Myeloid Leukemia Cells.

PubMed

Jones, Stuart; Ahmet, Jonathan; Ayton, Kelly; Ball, Matthew; Cockerill, Mark; Fairweather, Emma; Hamilton, Nicola; Harper, Paul; Hitchin, James; Jordan, Allan; Levy, Colin; Lopez, Ruth; McKenzie, Eddie; Packer, Martin; Plant, Darren; Simpson, Iain; Simpson, Peter; Sinclair, Ian; Somervaille, Tim C P; Small, Helen; Spencer, Gary J; Thomson, Graeme; Tonge, Michael; Waddell, Ian; Walsh, Jarrod; Waszkowycz, Bohdan; Wigglesworth, Mark; Wiseman, Daniel H; Ogilvie, Donald

2016-12-22

A collaborative high throughput screen of 1.35 million compounds against mutant (R132H) isocitrate dehydrogenase IDH1 led to the identification of a novel series of inhibitors. Elucidation of the bound ligand crystal structure showed that the inhibitors exhibited a novel binding mode in a previously identified allosteric site of IDH1 (R132H). This information guided the optimization of the series yielding submicromolar enzyme inhibitors with promising cellular activity. Encouragingly, one compound from this series was found to induce myeloid differentiation in primary human IDH1 R132H AML cells in vitro.

Proteins Differentially Expressed in the Pancreas of Hepatic Alcohol Dehydrogenase-Deficient Deer Mice Fed Ethanol For 3 Months.

PubMed

Bhopale, Kamlesh K; Amer, Samir M; Kaphalia, Lata; Soman, Kizhake V; Wiktorowicz, John E; Shakeel Ansari, Ghulam A; Kaphalia, Bhupendra S

2017-07-01

The aim of this study was to identify differentially expressed proteins in the pancreatic tissue of hepatic alcohol dehydrogenase-deficient deer mice fed ethanol to understand metabolic basis and mechanism of alcoholic chronic pancreatitis. Mice were fed liquid diet containing 3.5 g% ethanol daily for 3 months, and differentially expressed pancreatic proteins were identified by protein separation using 2-dimensional gel electrophoresis and identification by mass spectrometry. Nineteen differentially expressed proteins were identified by applying criteria established for protein identification in proteomics. An increased abundance was found for ribosome-binding protein 1, 60S ribosomal protein L31-like isoform 1, histone 4, calcium, and adenosine triphosphate (ATP) binding proteins and the proteins involved in antiapoptotic processes and endoplasmic reticulum function, stress, and/or homeostasis. Low abundance was found for endoA cytokeratin, 40S ribosomal protein SA, amylase 2b isoform precursor, serum albumin, and ATP synthase subunit β and the proteins involved in cell motility, structure, and conformation. Chronic ethanol feeding in alcohol dehydrogenase-deficient deer mice differentially expresses pancreatic functional and structural proteins, which can be used to develop biomarker(s) of alcoholic chronic pancreatitis, particularly amylase 2b precursor, and 60 kDa heat shock protein and those involved in ATP synthesis and blood osmotic pressure.

A novel cofactor-binding mode in bacterial IMP dehydrogenases explains inhibitor selectivity

DOE PAGES

Makowska-Grzyska, Magdalena; Kim, Youngchang; Maltseva, Natalia; ...

2015-01-09

The steadily rising frequency of emerging diseases and antibiotic resistance creates an urgent need for new drugs and targets. Inosine 5'-monophosphate dehydrogenase (IMP dehydrogenase or IMPDH) is a promising target for the development of new antimicrobial agents. IMPDH catalyzes the oxidation of IMP to XMP with the concomitant reduction of NAD +, which is the pivotal step in the biosynthesis of guanine nucleotides. Potent inhibitors of bacterial IMPDHs have been identified that bind in a structurally distinct pocket that is absent in eukaryotic IMPDHs. The physiological role of this pocket was not understood. Here, we report the structures of complexesmore » with different classes of inhibitors of Bacillus anthracis, Campylobacter jejuni, and Clostridium perfringens IMPDHs. These structures in combination with inhibition studies provide important insights into the interactions that modulate selectivity and potency. We also present two structures of the Vibrio cholerae IMPDH in complex with IMP/NAD + and XMP/NAD +. In both structures, the cofactor assumes a dramatically different conformation than reported previously for eukaryotic IMPDHs and other dehydrogenases, with the major change observed for the position of the NAD+ adenosine moiety. More importantly, this new NAD +-binding site involves the same pocket that is utilized by the inhibitors. Thus, the bacterial IMPDH-specific NAD +-binding mode helps to rationalize the conformation adopted by several classes of prokaryotic IMPDH inhibitors. As a result, these findings offer a potential strategy for further ligand optimization.« less

A novel cofactor-binding mode in bacterial IMP dehydrogenases explains inhibitor selectivity.

PubMed

Makowska-Grzyska, Magdalena; Kim, Youngchang; Maltseva, Natalia; Osipiuk, Jerzy; Gu, Minyi; Zhang, Minjia; Mandapati, Kavitha; Gollapalli, Deviprasad R; Gorla, Suresh Kumar; Hedstrom, Lizbeth; Joachimiak, Andrzej

2015-02-27

The steadily rising frequency of emerging diseases and antibiotic resistance creates an urgent need for new drugs and targets. Inosine 5'-monophosphate dehydrogenase (IMP dehydrogenase or IMPDH) is a promising target for the development of new antimicrobial agents. IMPDH catalyzes the oxidation of IMP to XMP with the concomitant reduction of NAD(+), which is the pivotal step in the biosynthesis of guanine nucleotides. Potent inhibitors of bacterial IMPDHs have been identified that bind in a structurally distinct pocket that is absent in eukaryotic IMPDHs. The physiological role of this pocket was not understood. Here, we report the structures of complexes with different classes of inhibitors of Bacillus anthracis, Campylobacter jejuni, and Clostridium perfringens IMPDHs. These structures in combination with inhibition studies provide important insights into the interactions that modulate selectivity and potency. We also present two structures of the Vibrio cholerae IMPDH in complex with IMP/NAD(+) and XMP/NAD(+). In both structures, the cofactor assumes a dramatically different conformation than reported previously for eukaryotic IMPDHs and other dehydrogenases, with the major change observed for the position of the NAD(+) adenosine moiety. More importantly, this new NAD(+)-binding site involves the same pocket that is utilized by the inhibitors. Thus, the bacterial IMPDH-specific NAD(+)-binding mode helps to rationalize the conformation adopted by several classes of prokaryotic IMPDH inhibitors. These findings offer a potential strategy for further ligand optimization. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

A comparative multidimensional LC-MS proteomic analysis reveals mechanisms for furan aldehyde detoxification in Thermoanaerobacter pseudethanolicus 39E

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

Clarkson, Sonya M.; Hamilton-Brehm, Scott D.; Giannone, Richard J.

Background: Chemical and physical pretreatment of lignocellulosic biomass improves substrate reactivity for increased microbial biofuel production, but also restricts growth via the release of furan aldehydes such as furfural and 5-hydroxymethylfurfural (5-HMF). The physiological effects of these inhibitors on thermophilic, fermentative bacteria is important to understand; especially as cellulolytic strains are being developed for consolidated bioprocessing (CBP) of lignocellulosic feedstocks. Identifying mechanisms for detoxification of aldehydes in naturally resistant strains such as Thermoanaerobacter spp. may also enable improvements in candidate CBP microorganisms. Results: T. pseudethanolicus 39E, an anaerobic, saccharolytic thermophile, was found to grow readily in the presence of 30more » mM furfural and 20 mM 5-HMF and reduce these aldehydes to their respective alcohols in situ. The proteomes of T. pseudethanolicus 39E grown in the presence or absence of 15 mM furfural were compared to identify upregulated enzymes potentially responsible for the observed reduction. A total of 225 proteins were differentially regulated in response to the 15 mM furfural treatment with 152 upregulated vs. 73 downregulated. Only 86 proteins exhibited a 2-fold change in abundance in either direction. Of these, 53 were upregulated in the presence of furfural and 33 were downregulated. Two oxidoreductases were upregulated at least 2-fold by furfural and were targeted for further investigation: Teth39_1597, encodes a predicted butanol dehydrogenase (BdhA) and Teth39_1598, a predicted aldo/keto reductase (AKR). Both genes were cloned from T. pseudethanolicus 39E, with the respective enzymes overexpressed in E. coli and specific activities determined against a variety of aldehydes. BdhA showed significant activity with all aldehydes tested, including furfural and 5-HMF, using NADPH as the cofactor. AKR also showed significant activity with NADPH, but only with four carbon butyr- and isobutyraldehydes. Conclusions: Thermoanaerobacter pseudethanolicus 39E displays intrinsic tolerance to the common pretreatment inhibitors furfural and 5-HMF. Multidimensional proteomics analysis was used as an effective tool to identify putative mechanisms for detoxification of furfural and 5-HMF. T. pseudethanolicus was found to upregulate an NADPH-dependent alcohol dehydrogenase 6.8-fold in response to furfural. In vitro enzyme assays confirmed the reduction of furfural and 5-HMF to their respective alcohols.« less

A comparative multidimensional LC-MS proteomic analysis reveals mechanisms for furan aldehyde detoxification in Thermoanaerobacter pseudethanolicus 39E

DOE PAGES

Clarkson, Sonya M.; Hamilton-Brehm, Scott D.; Giannone, Richard J.; ...

2014-12-03

Background: Chemical and physical pretreatment of lignocellulosic biomass improves substrate reactivity for increased microbial biofuel production, but also restricts growth via the release of furan aldehydes such as furfural and 5-hydroxymethylfurfural (5-HMF). The physiological effects of these inhibitors on thermophilic, fermentative bacteria is important to understand; especially as cellulolytic strains are being developed for consolidated bioprocessing (CBP) of lignocellulosic feedstocks. Identifying mechanisms for detoxification of aldehydes in naturally resistant strains such as Thermoanaerobacter spp. may also enable improvements in candidate CBP microorganisms. Results: T. pseudethanolicus 39E, an anaerobic, saccharolytic thermophile, was found to grow readily in the presence of 30more » mM furfural and 20 mM 5-HMF and reduce these aldehydes to their respective alcohols in situ. The proteomes of T. pseudethanolicus 39E grown in the presence or absence of 15 mM furfural were compared to identify upregulated enzymes potentially responsible for the observed reduction. A total of 225 proteins were differentially regulated in response to the 15 mM furfural treatment with 152 upregulated vs. 73 downregulated. Only 86 proteins exhibited a 2-fold change in abundance in either direction. Of these, 53 were upregulated in the presence of furfural and 33 were downregulated. Two oxidoreductases were upregulated at least 2-fold by furfural and were targeted for further investigation: Teth39_1597, encodes a predicted butanol dehydrogenase (BdhA) and Teth39_1598, a predicted aldo/keto reductase (AKR). Both genes were cloned from T. pseudethanolicus 39E, with the respective enzymes overexpressed in E. coli and specific activities determined against a variety of aldehydes. BdhA showed significant activity with all aldehydes tested, including furfural and 5-HMF, using NADPH as the cofactor. AKR also showed significant activity with NADPH, but only with four carbon butyr- and isobutyraldehydes. Conclusions: Thermoanaerobacter pseudethanolicus 39E displays intrinsic tolerance to the common pretreatment inhibitors furfural and 5-HMF. Multidimensional proteomics analysis was used as an effective tool to identify putative mechanisms for detoxification of furfural and 5-HMF. T. pseudethanolicus was found to upregulate an NADPH-dependent alcohol dehydrogenase 6.8-fold in response to furfural. In vitro enzyme assays confirmed the reduction of furfural and 5-HMF to their respective alcohols.« less

Preparation of convection interaction media isobutyl disc monolithic column and its application to purification of secondary alcohol dehydrogenase and alcohol oxidase.

PubMed

Isobe, Kimiyasu; Kawakami, Yoshimitsu

2007-03-09

A convection interaction media (trade name CIM, BIA Separation, Ljubljana, Slovenia) isobutyl monolithic disc was prepared by incubating a CIM epoxy monolithic disc with isobutylamine, and it was then applied to the purification of secondary alcohol dehydrogenase (S-ADH) and primary alcohol oxidase (P-AOD). Both enzymes were adsorbed on this column and eluted with high purity. Thus, S-ADH was purified to an electrophoretically homogeneous state by four column chromatographies using CIM DEAE-8 and CIM C4-8 tube monolithic columns, blue-Sepharose column and CIM isobutyl disc monolithic column. P-AOD was also purified to an electrophoretically homogeneous state by three column chromatographies of CIM DEAE-8 tube, CIM C4-8 tube and CIM isobutyl disc columns.

The Structural Basis of Cryptosporidium-Specific IMP Dehydrogenase Inhibitor Selectivity

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

MacPherson, Iain S.; Kirubakaran, Sivapriya; Gorla, Suresh Kumar

2010-03-29

Cryptosporidium parvum is a potential biowarfare agent, an important AIDS pathogen, and a major cause of diarrhea and malnutrition. No vaccines or effective drug treatment exist to combat Cryptosporidium infection. This parasite relies on inosine 5{prime}-monophosphate dehydrogenase (IMPDH) to obtain guanine nucleotides, and inhibition of this enzyme blocks parasite proliferation. Here, we report the first crystal structures of CpIMPDH. These structures reveal the structural basis of inhibitor selectivity and suggest a strategy for further optimization. Using this information, we have synthesized low-nanomolar inhibitors that display 10{sup 3} selectivity for the parasite enzyme over human IMPDH2.

Determining the roles of the three alcohol dehydrogenases (AdhA, AdhB and AdhE) in Thermoanaerobacter ethanolicus during ethanol formation.

PubMed

Zhou, Jilai; Shao, Xiongjun; Olson, Daniel G; Murphy, Sean Jean-Loup; Tian, Liang; Lynd, Lee R

2017-05-01

Thermoanaerobacter ethanolicus is a promising candidate for biofuel production due to the broad range of substrates it can utilize and its high ethanol yield compared to other thermophilic bacteria, such as Clostridium thermocellum. Three alcohol dehydrogenases, AdhA, AdhB and AdhE, play key roles in ethanol formation. To study their physiological roles during ethanol formation, we deleted them separately and in combination. Previously, it has been thought that both AdhB and AdhE were bifunctional alcohol dehydrogenases. Here we show that AdhE has primarily acetyl-CoA reduction activity (ALDH) and almost no acetaldehyde reduction (ADH) activity, whereas AdhB has no ALDH activity and but high ADH activity. We found that AdhA and AdhB have similar patterns of activity. Interestingly, although deletion of both adhA and adhB reduced ethanol production, a single deletion of either one actually increased ethanol yields by 60-70%.

Ethanol production by recombinant hosts

DOEpatents

Fowler, David E.; Horton, Philip G.; Ben-Bassat, Arie

1996-01-01

Novel plasmids comprising genes which code for the alcohol dehydrogenase and pyruvate decarboxylase are described. Also described are recombinant hosts which have been transformed with genes coding for alcohol dehydrogenase and pyruvate. By virtue of their transformation with these genes, the recombinant hosts are capable of producing significant amounts of ethanol as a fermentation product. Also disclosed are methods for increasing the growth of recombinant hosts and methods for reducing the accumulation of undesirable metabolic products in the growth medium of these hosts. Also disclosed are recombinant host capable of producing significant amounts of ethanol as a fermentation product of oligosaccharides and plasmids comprising genes encoding polysaccharases, in addition to the genes described above which code for the alcohol dehydrogenase and pyruvate decarboxylase. Further, methods are described for producing ethanol from oligomeric feedstock using the recombinant hosts described above. Also provided is a method for enhancing the production of functional proteins in a recombinant host comprising overexpressing an adhB gene in the host. Further provided are process designs for fermenting oligosaccharide-containing biomass to ethanol.

Ethanol production by recombinant hosts

DOEpatents

Ingram, Lonnie O.; Beall, David S.; Burchhardt, Gerhard F. H.; Guimaraes, Walter V.; Ohta, Kazuyoshi; Wood, Brent E.; Shanmugam, Keelnatham T.

1995-01-01

Novel plasmids comprising genes which code for the alcohol dehydrogenase and pyruvate decarboxylase are described. Also described are recombinant hosts which have been transformed with genes coding for alcohol dehydrogenase and pyruvate. By virtue of their transformation with these genes, the recombinant hosts are capable of producing significant amounts of ethanol as a fermentation product. Also disclosed are methods for increasing the growth of recombinant hosts and methods for reducing the accumulation of undesirable metabolic products in the growth medium of these hosts. Also disclosed are recombinant host capable of producing significant amounts of ethanol as a fermentation product of oligosaccharides and plasmids comprising genes encoding polysaccharases, in addition to the genes described above which code for the alcohol dehydrogenase and pyruvate decarboxylase. Further, methods are described for producing ethanol from oligomeric feedstock using the recombinant hosts described above. Also provided is a method for enhancing the production of functional proteins in a recombinant host comprising overexpressing an adhB gene in the host. Further provided are process designs for fermenting oligosaccharide-containing biomass to ethanol.

Study of the Glutaminase Inhibitor CB-839 in Solid Tumors

ClinicalTrials.gov

2016-08-18

Solid Tumors; Triple-Negative Breast Cancer; Non Small Cell Lung Cancer; Renal Cell Carcinoma; Mesothelioma; Fumarate Hydratase (FH)-Deficient Tumors; Succinate Dehydrogenase (SDH)-Deficient Gastrointestinal Stromal Tumors (GIST); Succinate Dehydrogenase (SDH)-Deficient Non-gastrointestinal Stromal Tumors; Tumors Harboring Isocitrate Dehydrogenase-1 (IDH1) and IDH2 Mutations; Tumors Harboring Amplifications in the cMyc Gene

Characterization of an allylic/benzyl alcohol dehydrogenase from Yokenella sp. strain WZY002, an organism potentially useful for the synthesis of α,β-unsaturated alcohols from allylic aldehydes and ketones.

PubMed

Ying, Xiangxian; Wang, Yifang; Xiong, Bin; Wu, Tingting; Xie, Liping; Yu, Meilan; Wang, Zhao

2014-04-01

A novel whole-cell biocatalyst with high allylic alcohol-oxidizing activities was screened and identified as Yokenella sp. WZY002, which chemoselectively reduced the C=O bond of allylic aldehydes/ketones to the corresponding α,β-unsaturated alcohols at 30°C and pH 8.0. The strain also had the capacity of stereoselectively reducing aromatic ketones to (S)-enantioselective alcohols. The enzyme responsible for the predominant allylic/benzyl alcohol dehydrogenase activity was purified to homogeneity and designated YsADH (alcohol dehydrogenase from Yokenella sp.), which had a calculated subunit molecular mass of 36,411 Da. The gene encoding YsADH was subsequently expressed in Escherichia coli, and the purified recombinant YsADH protein was characterized. The enzyme strictly required NADP(H) as a coenzyme and was putatively zinc dependent. The optimal pH and temperature for crotonaldehyde reduction were pH 6.5 and 65°C, whereas those for crotyl alcohol oxidation were pH 8.0 and 55°C. The enzyme showed moderate thermostability, with a half-life of 6.2 h at 55°C. It was robust in the presence of organic solvents and retained 87.5% of the initial activity after 24 h of incubation with 20% (vol/vol) dimethyl sulfoxide. The enzyme preferentially catalyzed allylic/benzyl aldehydes as the substrate in the reduction of aldehydes/ketones and yielded the highest activity of 427 U mg(-1) for benzaldehyde reduction, while the alcohol oxidation reaction demonstrated the maximum activity of 79.9 U mg(-1) using crotyl alcohol as the substrate. Moreover, kinetic parameters of the enzyme showed lower Km values and higher catalytic efficiency for crotonaldehyde/benzaldehyde and NADPH than for crotyl alcohol/benzyl alcohol and NADP(+), suggesting the nature of being an aldehyde reductase.

Characterization of an Allylic/Benzyl Alcohol Dehydrogenase from Yokenella sp. Strain WZY002, an Organism Potentially Useful for the Synthesis of α,β-Unsaturated Alcohols from Allylic Aldehydes and Ketones

PubMed Central

Ying, Xiangxian; Wang, Yifang; Xiong, Bin; Wu, Tingting; Xie, Liping; Yu, Meilan

2014-01-01

A novel whole-cell biocatalyst with high allylic alcohol-oxidizing activities was screened and identified as Yokenella sp. WZY002, which chemoselectively reduced the C=O bond of allylic aldehydes/ketones to the corresponding α,β-unsaturated alcohols at 30°C and pH 8.0. The strain also had the capacity of stereoselectively reducing aromatic ketones to (S)-enantioselective alcohols. The enzyme responsible for the predominant allylic/benzyl alcohol dehydrogenase activity was purified to homogeneity and designated YsADH (alcohol dehydrogenase from Yokenella sp.), which had a calculated subunit molecular mass of 36,411 Da. The gene encoding YsADH was subsequently expressed in Escherichia coli, and the purified recombinant YsADH protein was characterized. The enzyme strictly required NADP(H) as a coenzyme and was putatively zinc dependent. The optimal pH and temperature for crotonaldehyde reduction were pH 6.5 and 65°C, whereas those for crotyl alcohol oxidation were pH 8.0 and 55°C. The enzyme showed moderate thermostability, with a half-life of 6.2 h at 55°C. It was robust in the presence of organic solvents and retained 87.5% of the initial activity after 24 h of incubation with 20% (vol/vol) dimethyl sulfoxide. The enzyme preferentially catalyzed allylic/benzyl aldehydes as the substrate in the reduction of aldehydes/ketones and yielded the highest activity of 427 U mg−1 for benzaldehyde reduction, while the alcohol oxidation reaction demonstrated the maximum activity of 79.9 U mg−1 using crotyl alcohol as the substrate. Moreover, kinetic parameters of the enzyme showed lower Km values and higher catalytic efficiency for crotonaldehyde/benzaldehyde and NADPH than for crotyl alcohol/benzyl alcohol and NADP+, suggesting the nature of being an aldehyde reductase. PMID:24509923

Suppression of Cellulase and Polygalacturonase and Induction of Alcohol Dehydrogenase Isoenzymes in Avocado Fruit Mesocarp Subjected to Low Oxygen Stress

PubMed Central

Kanellis, Angelos K.; Solomos, Theophanes; Roubelakis-Angelakis, Kalliopi A.

1991-01-01

Expression of polygalacturonase and cellulase, two hydrolytic enzymes of avocado (Persea americana, cv Hass) fruit which are synthesized de novo during ripening, and alcohol dehydrogenase, a known anaerobic protein, were studied under different O2 regimes. Low O2 concentrations (2.5-5.5%) diminished the accumulation of polygalacturonase and cellulase proteins and the expression of their isoenzymes. This pattern of change in cellulase protein was also reflected in the steady-state amount of its mRNA. In contrast, 7.5 and 10% O2 did not alter the changes observed in fruits ripened in air. On the other hand, alcohol dehydrogenase was induced in 2.5, 3.5, and 5.5% O2 but not in 7.5 or 10% O2. The recovery from the hypoxic stress upon returning the fruits back to air for 24 hours, was also a function of O2 tensions under which the fruits were kept. Thus, the synthesis of polygalacturonase and cellulase was directly related to O2 levels, while the activity of the isoenzymes of alcohol dehydrogenase was inversely related to O2 levels. The results indicate that hypoxia exerts both negative and positive effects on the expression of certain genes and that these effects are initiated at the same levels of O2. ImagesFigure 2Figure 3Figure 4Figure 5Figure 6Figure 7 PMID:16668163

Expression pattern of two paralogs encoding cinnamyl alcohol dehydrogenases in Arabidopsis. Isolation and characterization of the corresponding mutants.

PubMed

Sibout, Richard; Eudes, Aymerick; Pollet, Brigitte; Goujon, Thomas; Mila, Isabelle; Granier, Fabienne; Séguin, Armand; Lapierre, Catherine; Jouanin, Lise

2003-06-01

Studying Arabidopsis mutants of the phenylpropanoid pathway has unraveled several biosynthetic steps of monolignol synthesis. Most of the genes leading to monolignol synthesis have been characterized recently in this herbaceous plant, except those encoding cinnamyl alcohol dehydrogenase (CAD). We have used the complete sequencing of the Arabidopsis genome to highlight a new view of the complete CAD gene family. Among nine AtCAD genes, we have identified the two distinct paralogs AtCAD-C and AtCAD-D, which share 75% identity and are likely to be involved in lignin biosynthesis in other plants. Northern, semiquantitative restriction fragment-length polymorphism-reverse transcriptase-polymerase chain reaction and western analysis revealed that AtCAD-C and AtCAD-D mRNA and protein ratios were organ dependent. Promoter activities of both genes are high in fibers and in xylem bundles. However, AtCAD-C displayed a larger range of sites of expression than AtCAD-D. Arabidopsis null mutants (Atcad-D and Atcad-C) corresponding to both genes were isolated. CAD activities were drastically reduced in both mutants, with a higher impact on sinapyl alcohol dehydrogenase activity (6% and 38% of residual sinapyl alcohol dehydrogenase activities for Atcad-D and Atcad-C, respectively). Only Atcad-D showed a slight reduction in Klason lignin content and displayed modifications of lignin structure with a significant reduced proportion of conventional S lignin units in both stems and roots, together with the incorporation of sinapaldehyde structures ether linked at Cbeta. These results argue for a substantial role of AtCAD-D in lignification, and more specifically in the biosynthesis of sinapyl alcohol, the precursor of S lignin units.

Expression Pattern of Two Paralogs Encoding Cinnamyl Alcohol Dehydrogenases in Arabidopsis. Isolation and Characterization of the Corresponding Mutants1

PubMed Central

Sibout, Richard; Eudes, Aymerick; Pollet, Brigitte; Goujon, Thomas; Mila, Isabelle; Granier, Fabienne; Séguin, Armand; Lapierre, Catherine; Jouanin, Lise

2003-01-01

Studying Arabidopsis mutants of the phenylpropanoid pathway has unraveled several biosynthetic steps of monolignol synthesis. Most of the genes leading to monolignol synthesis have been characterized recently in this herbaceous plant, except those encoding cinnamyl alcohol dehydrogenase (CAD). We have used the complete sequencing of the Arabidopsis genome to highlight a new view of the complete CAD gene family. Among nine AtCAD genes, we have identified the two distinct paralogs AtCAD-C and AtCAD-D, which share 75% identity and are likely to be involved in lignin biosynthesis in other plants. Northern, semiquantitative restriction fragment-length polymorphism-reverse transcriptase-polymerase chain reaction and western analysis revealed that AtCAD-C and AtCAD-D mRNA and protein ratios were organ dependent. Promoter activities of both genes are high in fibers and in xylem bundles. However, AtCAD-C displayed a larger range of sites of expression than AtCAD-D. Arabidopsis null mutants (Atcad-D and Atcad-C) corresponding to both genes were isolated. CAD activities were drastically reduced in both mutants, with a higher impact on sinapyl alcohol dehydrogenase activity (6% and 38% of residual sinapyl alcohol dehydrogenase activities for Atcad-D and Atcad-C, respectively). Only Atcad-D showed a slight reduction in Klason lignin content and displayed modifications of lignin structure with a significant reduced proportion of conventional S lignin units in both stems and roots, together with the incorporation of sinapaldehyde structures ether linked at Cβ. These results argue for a substantial role of AtCAD-D in lignification, and more specifically in the biosynthesis of sinapyl alcohol, the precursor of S lignin units. PMID:12805615

Discovery of BI 135585, an in vivo efficacious oxazinanone-based 11β hydroxysteroid dehydrogenase type 1 inhibitor.

PubMed

Zhuang, Linghang; Tice, Colin M; Xu, Zhenrong; Zhao, Wei; Cacatian, Salvacion; Ye, Yuan-Jie; Singh, Suresh B; Lindblom, Peter; McKeever, Brian M; Krosky, Paula M; Zhao, Yi; Lala, Deepak; Kruk, Barbara A; Meng, Shi; Howard, Lamont; Johnson, Judith A; Bukhtiyarov, Yuri; Panemangalore, Reshma; Guo, Joan; Guo, Rong; Himmelsbach, Frank; Hamilton, Bradford; Schuler-Metz, Annette; Schauerte, Heike; Gregg, Richard; McGeehan, Gerard M; Leftheris, Katerina; Claremon, David A

2017-07-15

A potent, in vivo efficacious 11β hydroxysteroid dehydrogenase type 1 (11β HSD1) inhibitor (11j) has been identified. Compound 11j inhibited 11β HSD1 activity in human adipocytes with an IC 50 of 4.3nM and in primary human adipose tissue with an IC 80 of 53nM. Oral administration of 11j to cynomolgus monkey inhibited 11β HSD1 activity in adipose tissue. Compound 11j exhibited >1000× selectivity over other hydroxysteroid dehydrogenases, displays desirable pharmacodynamic properties and entered human clinical trials in 2011. Copyright © 2017 Elsevier Ltd. All rights reserved.

Methylotrophic Bacillus methanolicus Encodes Two Chromosomal and One Plasmid Born NAD+ Dependent Methanol Dehydrogenase Paralogs with Different Catalytic and Biochemical Properties

PubMed Central

Müller, Jonas E. N.; Kupper, Christiane E.; Schneider, Olha; Vorholt, Julia A.; Ellingsen, Trond E.; Brautaset, Trygve

2013-01-01

Bacillus methanolicus can utilize methanol as the sole carbon source for growth and it encodes an NAD+-dependent methanol dehydrogenase (Mdh), catalyzing the oxidation of methanol to formaldehyde. Recently, the genomes of the B. methanolicus strains MGA3 (ATCC53907) and PB1 (NCIMB13113) were sequenced and found to harbor three different putative Mdh encoding genes, each belonging to the type III Fe-NAD+-dependent alcohol dehydrogenases. In each strain, two of these genes are encoded on the chromosome and one on a plasmid; only one chromosomal act gene encoding the previously described activator protein ACT was found. The six Mdhs and the ACT proteins were produced recombinantly in Escherichia coli, purified, and characterized. All Mdhs required NAD+ as cosubstrate, were catalytically stimulated by ACT, exhibited a broad and different substrate specificity range and displayed both dehydrogenase and reductase activities. All Mdhs catalyzed the oxidation of methanol; however the catalytic activity for methanol was considerably lower than for most other alcohols tested, suggesting that these enzymes represent a novel class of alcohol dehydrogenases. The kinetic constants for the Mdhs were comparable when acting as pure enzymes, but together with ACT the differences were more pronounced. Quantitative PCR experiments revealed major differences with respect to transcriptional regulation of the paralogous genes. Taken together our data indicate that the repertoire of methanol oxidizing enzymes in thermotolerant bacilli is larger than expected with complex mechanisms involved in their regulation. PMID:23527128

Methylotrophic Bacillus methanolicus encodes two chromosomal and one plasmid born NAD+ dependent methanol dehydrogenase paralogs with different catalytic and biochemical properties.

PubMed

Krog, Anne; Heggeset, Tonje M B; Müller, Jonas E N; Kupper, Christiane E; Schneider, Olha; Vorholt, Julia A; Ellingsen, Trond E; Brautaset, Trygve

2013-01-01

Bacillus methanolicus can utilize methanol as the sole carbon source for growth and it encodes an NAD(+)-dependent methanol dehydrogenase (Mdh), catalyzing the oxidation of methanol to formaldehyde. Recently, the genomes of the B. methanolicus strains MGA3 (ATCC53907) and PB1 (NCIMB13113) were sequenced and found to harbor three different putative Mdh encoding genes, each belonging to the type III Fe-NAD(+)-dependent alcohol dehydrogenases. In each strain, two of these genes are encoded on the chromosome and one on a plasmid; only one chromosomal act gene encoding the previously described activator protein ACT was found. The six Mdhs and the ACT proteins were produced recombinantly in Escherichia coli, purified, and characterized. All Mdhs required NAD(+) as cosubstrate, were catalytically stimulated by ACT, exhibited a broad and different substrate specificity range and displayed both dehydrogenase and reductase activities. All Mdhs catalyzed the oxidation of methanol; however the catalytic activity for methanol was considerably lower than for most other alcohols tested, suggesting that these enzymes represent a novel class of alcohol dehydrogenases. The kinetic constants for the Mdhs were comparable when acting as pure enzymes, but together with ACT the differences were more pronounced. Quantitative PCR experiments revealed major differences with respect to transcriptional regulation of the paralogous genes. Taken together our data indicate that the repertoire of methanol oxidizing enzymes in thermotolerant bacilli is larger than expected with complex mechanisms involved in their regulation.

Geraniol and Geranial Dehydrogenases Induced in Anaerobic Monoterpene Degradation by Castellaniella defragrans

PubMed Central

Lüddeke, Frauke; Wülfing, Annika; Timke, Markus; Germer, Frauke; Weber, Johanna; Dikfidan, Aytac; Rahnfeld, Tobias; Linder, Dietmar; Meyerdierks, Anke

2012-01-01

Castellaniella defragrans is a Betaproteobacterium capable of coupling the oxidation of monoterpenes with denitrification. Geraniol dehydrogenase (GeDH) activity was induced during growth with limonene in comparison to growth with acetate. The N-terminal sequence of the purified enzyme directed the cloning of the corresponding open reading frame (ORF), the first bacterial gene for a GeDH (geoA, for geraniol oxidation pathway). The C. defragrans geraniol dehydrogenase is a homodimeric enzyme that affiliates with the zinc-containing benzyl alcohol dehydrogenases in the superfamily of medium-chain-length dehydrogenases/reductases (MDR). The purified enzyme most efficiently catalyzes the oxidation of perillyl alcohol (kcat/Km = 2.02 × 106 M−1 s−1), followed by geraniol (kcat/Km = 1.57 × 106 M−1 s−1). Apparent Km values of <10 μM are consistent with an in vivo toxicity of geraniol above 5 μM. In the genetic vicinity of geoA is a putative aldehyde dehydrogenase that was named geoB and identified as a highly abundant protein during growth with phellandrene. Extracts of Escherichia coli expressing geoB demonstrated in vitro a geranial dehydrogenase (GaDH) activity. GaDH activity was independent of coenzyme A. The irreversible formation of geranic acid allows for a metabolic flux from β-myrcene via linalool, geraniol, and geranial to geranic acid. PMID:22286981

Geraniol and geranial dehydrogenases induced in anaerobic monoterpene degradation by Castellaniella defragrans.

PubMed

Lüddeke, Frauke; Wülfing, Annika; Timke, Markus; Germer, Frauke; Weber, Johanna; Dikfidan, Aytac; Rahnfeld, Tobias; Linder, Dietmar; Meyerdierks, Anke; Harder, Jens

2012-04-01

Castellaniella defragrans is a Betaproteobacterium capable of coupling the oxidation of monoterpenes with denitrification. Geraniol dehydrogenase (GeDH) activity was induced during growth with limonene in comparison to growth with acetate. The N-terminal sequence of the purified enzyme directed the cloning of the corresponding open reading frame (ORF), the first bacterial gene for a GeDH (geoA, for geraniol oxidation pathway). The C. defragrans geraniol dehydrogenase is a homodimeric enzyme that affiliates with the zinc-containing benzyl alcohol dehydrogenases in the superfamily of medium-chain-length dehydrogenases/reductases (MDR). The purified enzyme most efficiently catalyzes the oxidation of perillyl alcohol (k(cat)/K(m) = 2.02 × 10(6) M(-1) s(-1)), followed by geraniol (k(cat)/K(m) = 1.57 × 10(6) M(-1) s(-1)). Apparent K(m) values of <10 μM are consistent with an in vivo toxicity of geraniol above 5 μM. In the genetic vicinity of geoA is a putative aldehyde dehydrogenase that was named geoB and identified as a highly abundant protein during growth with phellandrene. Extracts of Escherichia coli expressing geoB demonstrated in vitro a geranial dehydrogenase (GaDH) activity. GaDH activity was independent of coenzyme A. The irreversible formation of geranic acid allows for a metabolic flux from β-myrcene via linalool, geraniol, and geranial to geranic acid.

Metabolism of 2-phenylethylamine to phenylacetic acid, via the intermediate phenylacetaldehyde, by freshly prepared and cryopreserved guinea pig liver slices.

PubMed

Panoutsopoulos, Georgios I

2004-01-01

2-Phenylethylamine is an endogenous amine, which acts as a neuromodulator of dopaminergic responses. Exogenous 2-phenylethylamine is found in certain foodstuffs and may cause toxic side-effects in susceptible individuals. The present investigation examined the metabolism of 2-phenylethylamine to phenylacetic acid, via phenylacetaldehyde, in freshly prepared and cryopreserved liver slices. Additionally, it compared the relative contribution of aldehyde oxidase, xanthine oxidase and aldehyde dehydrogenase by using specific inhibitors for each oxidizing enzyme. In freshly prepared and cryopreserved liver slices, phenylacetic acid was the main metabolite of 2-phenylethalamine. In freshly prepared liver slices, phenylacetic acid was completely inhibited by disulfiram (inhibitor of aldehyde dehydrogenase), whereas isovanillin (inhibitor of aldehyde oxidase) inhibited acid formation to a lesser extent and allopurinol (inhibitor of xanthine oxidase) had no effect. In cryopreserved liver slices, isovanillin inhibited phenylacetic acid by 85%, whereas disulfiram inhibited acid formation to a lesser extent and allopurinol had no effect. In liver slices, 2-phenylethylamine is rapidly oxidized to phenylacetic acid, via phenylacetaldehyde, by aldehyde dehydrogenase and aldehyde oxidase with no contribution from xanthine oxidase.

Assessment of affective and somatic signs of ethanol withdrawal in C57BL/6J mice using a short-term ethanol treatment.

PubMed

Perez, E E; De Biasi, M

2015-05-01

Alcohol is one of the most prevalent addictive substances in the world. Withdrawal symptoms result from abrupt cessation of alcohol consumption in habitual drinkers. The emergence of both affective and physical symptoms produces a state that promotes relapse. Mice provide a preclinical model that could be used to study alcohol dependence and withdrawal while controlling for both genetic and environmental variables. The use of a liquid ethanol diet offers a reliable method for the induction of alcohol dependence in mice, but this approach is impractical when conducting high-throughput pharmacological screens or when comparing multiple strains of genetically engineered mice. The goal of this study was to compare withdrawal-associated behaviors in mice chronically treated with a liquid ethanol diet vs. mice treated with a short-term ethanol treatment that consisted of daily ethanol injections containing the alcohol dehydrogenase inhibitor, 4-methylpyrazole. Twenty-four hours after ethanol treatment, mice were tested in the open field arena, the elevated plus maze, the marble burying test, or for changes in somatic signs during spontaneous ethanol withdrawal. Anxiety-like and compulsive-like behaviors, as well as physical signs, were all significantly elevated in mice undergoing withdrawal, regardless of the route of ethanol administration. Therefore, a short-term ethanol treatment can be utilized as a screening tool for testing genetic and pharmacological agents before investing in a more time-consuming ethanol treatment. Copyright © 2015 Elsevier Inc. All rights reserved.

Genetic polymorphisms of alcohol dehydrogense-1B and aldehyde dehydrogenase-2, alcohol flushing, mean corpuscular volume, and aerodigestive tract neoplasia in Japanese drinkers.

PubMed

Yokoyama, Akira; Mizukami, Takeshi; Yokoyama, Tetsuji

2015-01-01

Genetic polymorphisms of alcohol dehydrogenase-1B (ADH1B) and aldehyde dehydrogenase-2 (ALDH2) modulate exposure levels to ethanol/acetaldehyde. Endoscopic screening of 6,014 Japanese alcoholics yielded high detection rates of esophageal squamous cell carcinoma (SCC; 4.1%) and head and neck SCC (1.0%). The risks of upper aerodigestive tract SCC/dysplasia, especially of multiple SCC/dysplasia, were increased in a multiplicative fashion by the presence of a combination of slow-metabolizing ADH1B*1/*1 and inactive heterozygous ALDH2*1/*2 because of prolonged exposure to higher concentrations of ethanol/acetaldehyde. A questionnaire asking about current and past facial flushing after drinking a glass (≈180 mL) of beer is a reliable tool for detecting the presence of inactive ALDH2. We invented a health-risk appraisal (HRA) model including the flushing questionnaire and drinking, smoking, and dietary habits. Esophageal SCC was detected at a high rate by endoscopic mass-screening in high HRA score persons. A total of 5.0% of 4,879 alcoholics had a history of (4.0%) or newly diagnosed (1.0%) gastric cancer. Their high frequency of a history of gastric cancer is partly explained by gastrectomy being a risk factor for alcoholism because of altered ethanol metabolism, e.g., by blood ethanol level overshooting. The combination of H. pylori-associated atrophic gastritis and ALDH2*1/*2 showed the greatest risk of gastric cancer in alcoholics. High detection rates of advanced colorectal adenoma/carcinoma were found in alcoholics, 15.7% of 744 immunochemical fecal occult blood test (IFOBT)-negative alcoholics and 31.5% of the 393 IFOBT-positive alcoholics. Macrocytosis with an MCV≥106 fl increased the risk of neoplasia in the entire aerodigestive tract of alcoholics, suggesting that poor nutrition as well as ethanol/acetaldehyde exposure plays an important role in neoplasia.

Cloning, expression and characterization of an aryl-alcohol dehydrogenase from the white-rot fungus Phanerochaete chrysosporium strain BKM-F-1767

PubMed Central

2012-01-01

Background The white-rot fungus Phanerochaete chrysosporium is among the small group of fungi that can degrade lignin to carbon dioxide while leaving the crystalline cellulose untouched. The efficient lignin oxidation system of this fungus requires cyclic redox reactions involving the reduction of aryl-aldehydes to the corresponding alcohols by aryl-alcohol dehydrogenase. However, the biochemical properties of this enzyme have not been extensively studied. These are of most interest for the design of metabolic engineering/synthetic biology strategies in the field of biotechnological applications of this enzyme. Results We report here the cloning of an aryl-alcohol dehydrogenase cDNA from the white-rot fungus Phanerochaete chrysosporium, its expression in Escherichia coli and the biochemical characterization of the encoded GST and His6 tagged protein. The purified recombinant enzyme showed optimal activity at 37°C and at pH 6.4 for the reduction of aryl- and linear aldehydes with NADPH as coenzyme. NADH could also be the electron donor, while having a higher Km (220 μM) compared to that of NADPH (39 μM). The purified recombinant enzyme was found to be active in the reduction of more than 20 different aryl- and linear aldehydes showing highest specificity for mono- and dimethoxylated Benzaldehyde at positions 3, 4, 3,4 and 3,5. The enzyme was also capable of oxidizing aryl-alcohols with NADP + at 30°C and an optimum pH of 10.3 but with 15 to 100-fold lower catalytic efficiency than for the reduction reaction. Conclusions In this work, we have characterized the biochemical properties of an aryl-alcohol dehydrogenase from the white-rot fungus Phanerochaete chrysosporium. We show that this enzyme functions in the reductive sense under physiological conditions and that it displays relatively large substrate specificity with highest activity towards the natural compound Veratraldehyde. PMID:22742413

In vitro activation of NAD-dependent alcohol dehydrogenases by Nudix hydrolases is more widespread than assumed.

PubMed

Ochsner, Andrea M; Müller, Jonas E N; Mora, Carlos A; Vorholt, Julia A

2014-08-25

In the Gram-positive methylotroph Bacillus methanolicus, methanol oxidation is catalyzed by an NAD-dependent methanol dehydrogenase (Mdh) that belongs to the type III alcohol dehydrogenase (Adh) family. It was previously shown that the in vitro activity of B. methanolicus Mdh is increased by the endogenous activator protein Act, a Nudix hydrolase. Here we show that this feature is not unique, but more widespread among type III Adhs in combination with Act or other Act-like Nudix hydrolases. In addition, we studied the effect of site directed mutations in the predicted active site of Mdh and two other type III Adhs with regard to activity and activation by Act. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

INACTIVATION OF E. COLI PYRUVATE FORMATE-LYASE: ROLE OF AdhE AND SMALL MOLECULES

PubMed Central

Nnyepi, Mbako R.; Peng, Yi; Broderick, Joan B.

2007-01-01

E. coli AdhE has been reported to harbor three distinct enzymatic activities: alcohol dehydrogenase, acetaldehyde-CoA dehydrogenase, and pyruvate formate-lyase (PFL) deactivase. Herein we report on the cloning, expression, and purification of E. coli AdhE, and the re-investigation of its purported enzymatic activities. While both the alcohol dehydrogenase and acetaldehyde-CoA dehydrogenase activities were readily detectible, we were unable to obtain any evidence for catalytic deactivation of PFL by AdhE, regardless of whether the reported cofactors for deactivation (Fe(II), NAD, and CoA) were present. Our results demonstrate that AdhE is not a PFL deactivating enzyme. We have also examined the potential for deactivation of active PFL by small-molecule thiols. Both β-mercaptoethanol and dithiothreitol deactivate PFL efficiently, with the former providing quite rapid deactivation. PFL deactivated by these thiols can be reactivated, suggesting that this deactivation is non-destructive transfer of an H atom equivalent to quench the glycyl radical. PMID:17280641

Cloning of the Arabidopsis and Rice Formaldehyde Dehydrogenase Genes: Implications for the Origin of Plant Adh Enzymes

PubMed Central

Dolferus, R.; Osterman, J. C.; Peacock, W. J.; Dennis, E. S.

1997-01-01

This article reports the cloning of the genes encoding the Arabidopsis and rice class III ADH enzymes, members of the alcohol dehydrogenase or medium chain reductase/dehydrogenase superfamily of proteins with glutathione-dependent formaldehyde dehydrogenase activity (GSH-FDH). Both genes contain eight introns in exactly the same positions, and these positions are conserved in plant ethanol-active Adh genes (class P). These data provide further evidence that plant class P genes have evolved from class III genes by gene duplication and acquisition of new substrate specificities. The position of introns and similarities in the nucleic acid and amino acid sequences of the different classes of ADH enzymes in plants and humans suggest that plant and animal class III enzymes diverged before they duplicated to give rise to plant and animal ethanol-active ADH enzymes. Plant class P ADH enzymes have gained substrate specificities and evolved promoters with different expression properties, in keeping with their metabolic function as part of the alcohol fermentation pathway. PMID:9215914

Endogenous Methanol Regulates Mammalian Gene Activity

PubMed Central

Komarova, Tatiana V.; Petrunia, Igor V.; Shindyapina, Anastasia V.; Silachev, Denis N.; Sheshukova, Ekaterina V.; Kiryanov, Gleb I.; Dorokhov, Yuri L.

2014-01-01

We recently showed that methanol emitted by wounded plants might function as a signaling molecule for plant-to-plant and plant-to-animal communications. In mammals, methanol is considered a poison because the enzyme alcohol dehydrogenase (ADH) converts methanol into toxic formaldehyde. However, the detection of methanol in the blood and exhaled air of healthy volunteers suggests that methanol may be a chemical with specific functions rather than a metabolic waste product. Using a genome-wide analysis of the mouse brain, we demonstrated that an increase in blood methanol concentration led to a change in the accumulation of mRNAs from genes primarily involved in detoxification processes and regulation of the alcohol/aldehyde dehydrogenases gene cluster. To test the role of ADH in the maintenance of low methanol concentration in the plasma, we used the specific ADH inhibitor 4-methylpyrazole (4-MP) and showed that intraperitoneal administration of 4-MP resulted in a significant increase in the plasma methanol, ethanol and formaldehyde concentrations. Removal of the intestine significantly decreased the rate of methanol addition to the plasma and suggested that the gut flora may be involved in the endogenous production of methanol. ADH in the liver was identified as the main enzyme for metabolizing methanol because an increase in the methanol and ethanol contents in the liver homogenate was observed after 4-MP administration into the portal vein. Liver mRNA quantification showed changes in the accumulation of mRNAs from genes involved in cell signalling and detoxification processes. We hypothesized that endogenous methanol acts as a regulator of homeostasis by controlling the mRNA synthesis. PMID:24587296

Molecular, biochemical, and functional characterization of a Nudix hydrolase protein that stimulates the activity of a nicotinoprotein alcohol dehydrogenase.

PubMed

Kloosterman, Harm; Vrijbloed, Jan W; Dijkhuizen, Lubbert

2002-09-20

The cytoplasmic coenzyme NAD(+)-dependent alcohol (methanol) dehydrogenase (MDH) employed by Bacillus methanolicus during growth on C(1)-C(4) primary alcohols is a decameric protein with 1 Zn(2+)-ion and 1-2 Mg(2+)-ions plus a tightly bound NAD(H) cofactor per subunit (a nicotinoprotein). Mg(2+)-ions are essential for binding of NAD(H) cofactor in MDH protein expressed in Escherichia coli. The low coenzyme NAD(+)-dependent activity of MDH with C(1)-C(4) primary alcohols is strongly stimulated by a second B. methanolicus protein (ACT), provided that MDH contains NAD(H) cofactor and Mg(2+)-ions are present in the assay mixture. Characterization of the act gene revealed the presence of the highly conserved amino acid sequence motif typical of Nudix hydrolase proteins in the deduced ACT amino acid sequence. The act gene was successfully expressed in E. coli allowing purification and characterization of active ACT protein. MDH activation by ACT involved hydrolytic removal of the nicotinamide mononucleotide NMN(H) moiety of the NAD(H) cofactor of MDH, changing its Ping-Pong type of reaction mechanism into a ternary complex reaction mechanism. Increased cellular NADH/NAD(+) ratios may reduce the ACT-mediated activation of MDH, thus preventing accumulation of toxic aldehydes. This represents a novel mechanism for alcohol dehydrogenase activity regulation.

Inhibition of human alcohol and aldehyde dehydrogenases by acetaminophen: Assessment of the effects on first-pass metabolism of ethanol.

PubMed

Lee, Yung-Pin; Liao, Jian-Tong; Cheng, Ya-Wen; Wu, Ting-Lun; Lee, Shou-Lun; Liu, Jong-Kang; Yin, Shih-Jiun

2013-11-01

Acetaminophen is one of the most widely used over-the-counter analgesic, antipyretic medications. Use of acetaminophen and alcohol are commonly associated. Previous studies showed that acetaminophen might affect bioavailability of ethanol by inhibiting gastric alcohol dehydrogenase (ADH). However, potential inhibitions by acetaminophen of first-pass metabolism (FPM) of ethanol, catalyzed by the human ADH family and by relevant aldehyde dehydrogenase (ALDH) isozymes, remain undefined. ADH and ALDH both exhibit racially distinct allozymes and tissue-specific distribution of isozymes, and are principal enzymes responsible for ethanol metabolism in humans. In this study, we investigated acetaminophen inhibition of ethanol oxidation with recombinant human ADH1A, ADH1B1, ADH1B2, ADH1B3, ADH1C1, ADH1C2, ADH2, and ADH4, and inhibition of acetaldehyde oxidation with recombinant human ALDH1A1 and ALDH2. The investigations were done at near physiological pH 7.5 and with a cytoplasmic coenzyme concentration of 0.5 mM NAD(+). Acetaminophen acted as a noncompetitive inhibitor for ADH enzymes, with the slope inhibition constants (Kis) ranging from 0.90 mM (ADH2) to 20 mM (ADH1A), and the intercept inhibition constants (Kii) ranging from 1.4 mM (ADH1C allozymes) to 19 mM (ADH1A). Acetaminophen exhibited noncompetitive inhibition for ALDH2 (Kis = 3.0 mM and Kii = 2.2 mM), but competitive inhibition for ALDH1A1 (Kis = 0.96 mM). The metabolic interactions between acetaminophen and ethanol/acetaldehyde were assessed by computer simulation using inhibition equations and the determined kinetic constants. At therapeutic to subtoxic plasma levels of acetaminophen (i.e., 0.2-0.5 mM) and physiologically relevant concentrations of ethanol (10 mM) and acetaldehyde (10 μm) in target tissues, acetaminophen could inhibit ADH1C allozymes (12-26%) and ADH2 (14-28%) in the liver and small intestine, ADH4 (15-31%) in the stomach, and ALDH1A1 (16-33%) and ALDH2 (8.3-19%) in all 3 tissues. The results suggest that inhibition by acetaminophen of hepatic and gastrointestinal FPM of ethanol through ADH and ALDH pathways might become significant at higher, subtoxic levels of acetaminophen. Copyright © 2013 Elsevier Inc. All rights reserved.

Mono-carbonyl curcumin analogues as 11β-hydroxysteroid dehydrogenase 1 inhibitors.

PubMed

Lin, Han; Hu, Guo-Xin; Guo, Jingjing; Ge, Yufei; Liang, Guang; Lian, Qing-Quan; Chu, Yanhui; Yuan, Xiaohuan; Huang, Ping; Ge, Ren-Shan

2013-08-01

A series of structurally novel mono-carbonyl curcumin analogues have been synthesized and biologically evaluated to test their inhibitory potencies and the structure-activity relationship (SAR) on human and rat 11β-hydroxysteroid dehydrogenase isoform (11β-HSD1) activities. 11β-HSD1 selective inhibitors have been discovered and compound A10 is discovered as a very potent with an IC50 value of 97 nM without inhibiting 11β-HSD2. Copyright © 2013 Elsevier Ltd. All rights reserved.

GS 455534 selectively suppresses binge eating of palatable food and attenuates dopamine release in the accumbens of sugar-bingeing rats.

PubMed

Bocarsly, Miriam E; Hoebel, Bartley G; Paredes, Daniel; von Loga, Isabell; Murray, Susan M; Wang, Miaoyuan; Arolfo, Maria P; Yao, Lina; Diamond, Ivan; Avena, Nicole M

2014-04-01

Binge eating palatable foods has been shown to have behavioral and neurochemical similarities to drug addiction. GS 455534 is a highly selective reversible aldehyde dehydrogenase 2 inhibitor that has been shown to reduce alcohol and cocaine intake in rats. Given the overlaps between binge eating and drug abuse, we examined the effects of GS 455534 on binge eating and subsequent dopamine release. Sprague-Dawley rats were maintained on a sugar (experiment 1) or fat (experiment 2) binge eating diet. After 25 days, GS 455534 was administered at 7.5 and 15 mg/kg by an intraperitoneal injection, and food intake was monitored. In experiment 3, rats with cannulae aimed at the nucleus accumbens shell were maintained on the binge sugar diet for 25 days. Microdialysis was performed, during which GS 455534 15 mg/kg was administered, and sugar was available. Dialysate samples were analyzed to determine extracellular levels of dopamine. In experiment 1, GS 455534 selectively decreased sugar intake food was made available in the Binge Sugar group but not the Ad libitum Sugar group, with no effect on chow intake. In experiment 2, GS 455534 decreased fat intake in the Binge Fat group, but not the Ad libitum Fat group, however, it also reduced chow intake. In experiment 3, GS 455534 attenuated accumbens dopamine release by almost 50% in binge eating rats compared with the vehicle injection. The findings suggest that selective reversible aldehyde dehydrogenase 2 inhibitors may have the therapeutic potential to reduce binge eating of palatable foods in clinical populations.

Saccharomyces cerevisiae KNU5377 stress response during high-temperature ethanol fermentation.

PubMed

Kim, Il-Sup; Kim, Young-Saeng; Kim, Hyun; Jin, Ingnyol; Yoon, Ho-Sung

2013-03-01

Fuel ethanol production is far more costly to produce than fossil fuels. There are a number of approaches to cost-effective fuel ethanol production from biomass. We characterized stress response of thermotolerant Saccharomyces cerevisiae KNU5377 during glucose-based batch fermentation at high temperature (40°C). S. cerevisiae KNU5377 (KNU5377) transcription factors (Hsf1, Msn2/4, and Yap1), metabolic enzymes (hexokinase, glyceraldehyde-3-phosphate dehydrogenase, glucose-6-phosphate dehydrogenase, isocitrate dehydrogenase, and alcohol dehydrogenase), antioxidant enzymes (thioredoxin 3, thioredoxin reductase, and porin), and molecular chaperones and its cofactors (Hsp104, Hsp82, Hsp60, Hsp42, Hsp30, Hsp26, Cpr1, Sti1, and Zpr1) are upregulated during fermentation, in comparison to S. cerevisiae S288C (S288C). Expression of glyceraldehyde-3-phosphate dehydrogenase increased significantly in KNU5377 cells. In addition, cellular hydroperoxide and protein oxidation, particularly lipid peroxidation of triosephosphate isomerase, was lower in KNU5377 than in S288C. Thus, KNU5377 activates various cell rescue proteins through transcription activators, improving tolerance and increasing alcohol yield by rapidly responding to fermentation stress through redox homeostasis and proteostasis.

Novel diamide-based inhibitors of IMPDH.

PubMed

Gu, Henry H; Iwanowicz, Edwin J; Guo, Junqing; Watterson, Scott H; Shen, Zhongqi; Pitts, William J; Dhar, T G Murali; Fleener, Catherine A; Rouleau, Katherine; Sherbina, N Z; Witmer, Mark; Tredup, Jeffrey; Hollenbaugh, Diane

2002-05-06

A series of novel amide-based small molecule inhibitors of inosine monophosphate dehydrogenase is described. The synthesis and the structure-activity relationships (SARs) derived from in vitro studies are presented.

Adaptive response due to changes in gene regulation: a study with Drosophila.

PubMed Central

McDonald, J F; Chambers, G K; David, J; Ayala, F J

1977-01-01

In spite of the critical role of the process of adaptation in evolution, there are few detailed studies of the genotypic and molecular basis of the process. Drosophila melanogaster flies selected for increased tolerance to ethanol exhibited higher levels of alcohol dehydrogenase (alcohol:NAD+ oxidoreductase; EC 1.1.1.1) activity than unselected controls. A series of tests (electrophoresis, product inhibition, temperature stability, pH optima, substrate specificity, and Michaelis constants) gave no evidence of structural differences in the enzyme of the selected and the control flies. However, quantitative immunological assays showed that the selected flies contained significantly higher amounts of alcohol dehydrogenase. Adaptation of the selected flies to higher alcohol tolerance has most likely taken place by changes not in the structural gene locus coding for the enzyme, but by regulatory changes affecting the amount of gene product. Images PMID:412190

Exploring the reversal of enantioselectivity on a zinc-dependent alcohol dehydrogenase† †Electronic supplementary information (ESI) available. See DOI: 10.1039/C7OB00482F Click here for additional data file.

PubMed Central

Maria-Solano, Miguel A.; Romero-Rivera, Adrian

2017-01-01

Alcohol Dehydrogenase (ADH) enzymes catalyse the reversible reduction of prochiral ketones to the corresponding alcohols. These enzymes present two differently shaped active site pockets, which dictate their substrate scope and selectivity. In this study, we computationally evaluate the effect of two commonly reported active site mutations (I86A, and W110T) on a secondary alcohol dehydrogenase from Thermoanaerobacter brockii (TbSADH) through Molecular Dynamics simulations. Our results indicate that the introduced mutations induce dramatic changes in the shape of the active site, but most importantly they impact the substrate–enzyme interactions. We demonstrate that the combination of Molecular Dynamics simulations with the tools POVME and NCIplot corresponds to a powerful strategy for rationalising and engineering the stereoselectivity of ADH variants. PMID:28436515

Evidence for the role of oxidative stress in the acetylation of histone H3 by ethanol in rat hepatocytes

PubMed Central

Choudhury, Mahua; Park, Pil-Hoon; Jackson, Daniel; Shukla, Shivendra D.

2010-01-01

The relationship between ethanol induced oxidative stress and acetylation of histone H3 at lysine 9 (H3AcK9) remains unknown and was therefore investigated in primary cultures of rat hepatocytes. Cells were treated with ethanol and a select group of pharmacological agents and the status of H3AcK9 and reactive oxygen species (ROS) were monitored. When hepatocytes were exposed to ethanol (50 mM, 24 hr) in the presence of N-acetyl cystein (ROS reducer) or dietary antioxidants (quercetin, resveratrol), or NADPH oxidase inhibitor apocynin, ethanol induced increases in ROS and H3AcK9, both were significantly reduced. On the other hand, l-buthionine-sulfoximine (ROS inducer) and inhibitor of mitochondrial complex I (rotenone) and III (antimycin) increased ethanol induced H3AcK9 (p<0.01). Oxidative stress also affected ethanol induced alcohol dehydrogenase 1 (ADH1) mRNA expression. These results demonstrate for the first time that oxidative stress is involved in the ethanol induced histone H3 acetylation in hepatocytes. PMID:20705415

Evidence for the role of oxidative stress in the acetylation of histone H3 by ethanol in rat hepatocytes.

PubMed

Choudhury, Mahua; Park, Pil-Hoon; Jackson, Daniel; Shukla, Shivendra D

2010-09-01

The relationship between ethanol-induced oxidative stress and acetylation of histone H3 at lysine 9 (H3AcK9) remains unknown and was therefore investigated in primary cultures of rat hepatocytes. Cells were treated with ethanol, and a select group of pharmacological agents and the status of H3AcK9 and reactive oxygen species (ROS) were monitored. Pretreatment of hepatocytes with N-acetyl cystein (ROS reducer), or dietary antioxidants (quercetin, reserveratrol), or NADPH (reduced nicotinamide adenine dinucleotide phosphate) oxidase inhibitor apocynin, significantly reduced ethanol (50 mM, 24 h) induced increases in ROS and H3AcK9. In contrast, l-buthionine sulfoximine (ROS inducer) and inhibitor of mitochondrial complexes I (rotenone) and III (antimycin) increased ethanol-induced H3AcK9 (P<.01). Oxidative stress also affected ethanol-induced alcohol dehydrogenase 1 mRNA expression. These results demonstrate for the first time that oxidative stress is involved in the ethanol-induced histone H3 acetylation in hepatocytes. Copyright © 2010 Elsevier Inc. All rights reserved.

Development of an alcohol dehydrogenase biosensor for ethanol determination with toluidine blue O covalently attached to a cellulose acetate modified electrode.

PubMed

Alpat, Senol; Telefoncu, Azmi

2010-01-01

In this work, a novel voltammetric ethanol biosensor was constructed using alcohol dehydrogenase (ADH). Firstly, alcohol dehydrogenase was immobilized on the surface of a glassy carbon electrode modified by cellulose acetate (CA) bonded to toluidine blue O (TBO). Secondly, the surface was covered by a glutaraldehyde/bovine serum albumin (BSA) cross-linking procedure to provide a new voltammetric sensor for the ethanol determination. In order to fabricate the biosensor, a new electrode matrix containing insoluble Toluidine Blue O (TBO) was obtained from the process, and enzyme/coenzyme was combined on the biosensor surface. The influence of various experimental conditions was examined for the characterization of the optimum analytical performance. The developed biosensor exhibited sensitive and selective determination of ethanol and showed a linear response between 1 × 10(-5) M and 4 × 10(-4) M ethanol. A detection limit calculated as three times the signal-to-noise ratio was 5.0 × 10(-6) M. At the end of the 20(th) day, the biosensor still retained 50% of its initial activity.

Genetic improvement of Escherichia coli for ethanol production: Chromosomal integration of Zymomonas mobilis genes encoding pyruvate decarboxylase and alcohol dehydrogenase II

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

Ohta, Kazuyoshi; Beall, D.S.; Mejia, J.P.

1991-04-01

Zymomonas mobilis genes for pyruvate decarboxylase (pdc) and alcohol dehydrogenase II (adhB) were integrated into the Escherichia coli chromosome within or near the pyruvate formate-lyase gene (pfl). Integration improved the stability of the Z. mobilis genes in E. coli, but further selection was required to increase expression. Spontaneous mutants were selected for resistance to high levels of chloramphenicol that also expressed high levels of the Z. mobilis genes. Analogous mutants were selected for increased expression of alcohol dehydrogenase on aldehyde indicator plates. These mutants were functionally equivalent to the previous plasmid-based strains for the fermentation of xylose and glucose tomore » ethanol. Ethanol concentrations of 54.4 and 41.6 g/liter were obtained from 10% glucose and 8% xylose, respectively. The efficiency of conversion exceeded theoretical limits (0.51 g of ethanol/g of sugar) on the basis of added sugars because of the additional production of ethanol from the catabolism of complex nutrients. Further mutations were introduced to inactivate succinate production (frd) and to block homologous recombination (recA).« less

Cloning, expression, and characterization of a novel (S)-specific alcohol dehydrogenase from Lactobacillus kefir.

PubMed

Chen, Qilei; Hu, Youjia; Zhao, Wenjie; Zhu, Chunbao; Zhu, Baoquan

2010-01-01

A gene encoding a novel (S)-specific NADH-dependent alcohol dehydrogenase (LK-ADH) was isolated from the genomic DNA of Lactobacillus kefir DSM 20587 by thermal asymmetric interlaced-polymerase chain reaction. The nucleotide sequence of (S)-LK-ADH gene (adhS) was determined, which consists of an open reading frame of 1,044 bp, coding for 347 amino acids with a molecular mass of 37.065 kDa. After a BLAST similarity search in GenBank database, the amino acid sequence of (S)-LK-ADH showed some homologies to several zinc containing medium-chain alcohol dehydrogenases. This novel gene was deposited into GenBank with the accession number of EU877965. adhS gene was subcloned into plasmid pET-28a(+), and recombinant (S)-LK-ADH was successfully expressed in E. coli BL21(DE3) by isopropyl-beta-D-1-thiogalactopyranoside induction. Purified enzyme showed a high enantioselectivity in the reduction of acetophenone to (S)-phenylethanol with an ee value of 99.4%. The substrate specificity and cofactor preference of recombinant (S)-LK-ADH were also tested.

Development of an Alcohol Dehydrogenase Biosensor for Ethanol Determination with Toluidine Blue O Covalently Attached to a Cellulose Acetate Modified Electrode

PubMed Central

Alpat, Şenol; Telefoncu, Azmi

2010-01-01

In this work, a novel voltammetric ethanol biosensor was constructed using alcohol dehydrogenase (ADH). Firstly, alcohol dehydrogenase was immobilized on the surface of a glassy carbon electrode modified by cellulose acetate (CA) bonded to toluidine blue O (TBO). Secondly, the surface was covered by a glutaraldehyde/bovine serum albumin (BSA) cross-linking procedure to provide a new voltammetric sensor for the ethanol determination. In order to fabricate the biosensor, a new electrode matrix containing insoluble Toluidine Blue O (TBO) was obtained from the process, and enzyme/coenzyme was combined on the biosensor surface. The influence of various experimental conditions was examined for the characterization of the optimum analytical performance. The developed biosensor exhibited sensitive and selective determination of ethanol and showed a linear response between 1 × 10−5 M and 4 × 10−4 M ethanol. A detection limit calculated as three times the signal-to-noise ratio was 5.0 × 10−6 M. At the end of the 20th day, the biosensor still retained 50% of its initial activity. PMID:22315566

Syringyl lignin is unaltered by severe sinapyl alcohol dehydrogenase suppression in tobacco.

PubMed

Barakate, Abdellah; Stephens, Jennifer; Goldie, Alison; Hunter, William N; Marshall, David; Hancock, Robert D; Lapierre, Catherine; Morreel, Kris; Boerjan, Wout; Halpin, Claire

2011-12-01

The manipulation of lignin could, in principle, facilitate efficient biofuel production from plant biomass. Despite intensive study of the lignin pathway, uncertainty exists about the enzyme catalyzing the last step in syringyl (S) monolignol biosynthesis, the reduction of sinapaldehyde to sinapyl alcohol. Traditional schemes of the pathway suggested that both guaiacyl (G) and S monolignols are produced by a single substrate-versatile enzyme, cinnamyl alcohol dehydrogenase (CAD). This was challenged by the discovery of a novel sinapyl alcohol dehydrogenase (SAD) that preferentially uses sinapaldehyde as a substrate and that was claimed to regulate S lignin biosynthesis in angiosperms. Consequently, most pathway schemes now show SAD (or SAD and CAD) at the sinapaldehyde reduction step, although functional evidence is lacking. We cloned SAD from tobacco (Nicotiana tabacum) and suppressed it in transgenic plants using RNA interference-inducing vectors. Characterization of lignin in the woody stems shows no change to content, composition, or structure, and S lignin is normal. By contrast, plants additionally suppressed in CAD have changes to lignin structure and S:G ratio and have increased sinapaldehyde in lignin, similar to plants suppressed in CAD alone. These data demonstrate that CAD, not SAD, is the enzyme responsible for S lignin biosynthesis in woody angiosperm xylem.

Syringyl Lignin Is Unaltered by Severe Sinapyl Alcohol Dehydrogenase Suppression in Tobacco[W

PubMed Central

Barakate, Abdellah; Stephens, Jennifer; Goldie, Alison; Hunter, William N.; Marshall, David; Hancock, Robert D.; Lapierre, Catherine; Morreel, Kris; Boerjan, Wout; Halpin, Claire

2011-01-01

The manipulation of lignin could, in principle, facilitate efficient biofuel production from plant biomass. Despite intensive study of the lignin pathway, uncertainty exists about the enzyme catalyzing the last step in syringyl (S) monolignol biosynthesis, the reduction of sinapaldehyde to sinapyl alcohol. Traditional schemes of the pathway suggested that both guaiacyl (G) and S monolignols are produced by a single substrate-versatile enzyme, cinnamyl alcohol dehydrogenase (CAD). This was challenged by the discovery of a novel sinapyl alcohol dehydrogenase (SAD) that preferentially uses sinapaldehyde as a substrate and that was claimed to regulate S lignin biosynthesis in angiosperms. Consequently, most pathway schemes now show SAD (or SAD and CAD) at the sinapaldehyde reduction step, although functional evidence is lacking. We cloned SAD from tobacco (Nicotiana tabacum) and suppressed it in transgenic plants using RNA interference–inducing vectors. Characterization of lignin in the woody stems shows no change to content, composition, or structure, and S lignin is normal. By contrast, plants additionally suppressed in CAD have changes to lignin structure and S:G ratio and have increased sinapaldehyde in lignin, similar to plants suppressed in CAD alone. These data demonstrate that CAD, not SAD, is the enzyme responsible for S lignin biosynthesis in woody angiosperm xylem. PMID:22158465

Estimates of alcohol-related oesophageal cancer burden in Japan: systematic review and meta-analyses

PubMed Central

Shield, Kevin D; Higuchi, Susumu; Yoshimura, Atsushi; Larsen, Elisabeth; Rehm, Maximilien X; Rehm, Jürgen

2015-01-01

Abstract Objective To refine estimates of the burden of alcohol-related oesophageal cancer in Japan. Methods We searched PubMed for published reviews and original studies on alcohol intake, aldehyde dehydrogenase polymorphisms, and risk for oesophageal cancer in Japan, published before 2014. We conducted random-effects meta-analyses, including subgroup analyses by aldehyde dehydrogenase variants. We estimated deaths and loss of disability-adjusted life years (DALYs) from oesophageal cancer using exposure distributions for alcohol based on age, sex and relative risks per unit of exposure. Findings We identified 14 relevant studies. Three cohort studies and four case-control studies had dose–response data. Evidence from cohort studies showed that people who consumed the equivalent of 100 g/day of pure alcohol had an 11.71 fold, (95% confidence interval, CI: 2.67–51.32) risk of oesophageal cancer compared to those who never consumed alcohol. Evidence from case-control studies showed that the increase in risk was 33.11 fold (95% CI: 8.15–134.43) in the population at large. The difference by study design is explained by the 159 fold (95% CI: 27.2–938.2) risk among those with an inactive aldehyde dehydrogenase enzyme variant. Applying these dose–response estimates to the national profile of alcohol intake yielded 5279 oesophageal cancer deaths and 102 988 DALYs lost – almost double the estimates produced by the most recent global burden of disease exercise. Conclusion Use of global dose–response data results in an underestimate of the burden of disease from oesophageal cancer in Japan. Where possible, national burden of disease studies should use results from the population concerned. PMID:26229204

Structure of Escherichia coli AdhP (ethanol-inducible dehydrogenase) with bound NAD.

PubMed

Thomas, Leonard M; Harper, Angelica R; Miner, Whitney A; Ajufo, Helen O; Branscum, Katie M; Kao, Lydia; Sims, Paul A

2013-07-01

The crystal structure of AdhP, a recombinantly expressed alcohol dehydrogenase from Escherichia coli K-12 (substrain MG1655), was determined to 2.01 Å resolution. The structure, which was solved using molecular replacement, also included the structural and catalytic zinc ions and the cofactor nicotinamide adenine dinucleotide (NAD). The crystals belonged to space group P21, with unit-cell parameters a = 68.18, b = 118.92, c = 97.87 Å, β = 106.41°. The final R factor and Rfree were 0.138 and 0.184, respectively. The structure of the active site of AdhP suggested a number of residues that may participate in a proton relay, and the overall structure of AdhP, including the coordination to structural and active-site zinc ions, is similar to those of other tetrameric alcohol dehydrogenase enzymes.

Discovery of 8-Membered Ring Sulfonamides as Inhibitors of Oncogenic Mutant Isocitrate Dehydrogenase 1.

PubMed

Law, Jason M; Stark, Sebastian C; Liu, Ke; Liang, Norah E; Hussain, Mahmud M; Leiendecker, Matthias; Ito, Daisuke; Verho, Oscar; Stern, Andrew M; Johnston, Stephen E; Zhang, Yan-Ling; Dunn, Gavin P; Shamji, Alykhan F; Schreiber, Stuart L

2016-10-13

Evidence suggests that specific mutations of isocitrate dehydrogenases 1 and 2 (IDH1/2) are critical for the initiation and maintenance of certain tumor types and that inhibiting these mutant enzymes with small molecules may be therapeutically beneficial. In order to discover mutant allele-selective IDH1 inhibitors with chemical features distinct from existing probes, we screened a collection of small molecules derived from diversity-oriented synthesis. The assay identified compounds that inhibit the IDH1-R132H mutant allele commonly found in glioma. Here, we report the discovery of a potent (IC 50 = 50 nM) series of IDH1-R132H inhibitors having 8-membered ring sulfonamides as exemplified by the compound BRD2879. The inhibitors suppress ( R )-2-hydroxyglutarate production in cells without apparent toxicity. Although the solubility and pharmacokinetic properties of the specific inhibitor BRD2879 prevent its use in vivo , the scaffold presents a validated starting point for the synthesis of future IDH1-R132H inhibitors having improved pharmacological properties.

Asian flushing: genetic and sociocultural factors of alcoholism among East asians.

PubMed

Lee, Haeok; Kim, Sun S; You, Kwang Soo; Park, Wanju; Yang, Jin Hyang; Kim, Minjin; Hayman, Laura L

2014-01-01

Alcohol use can lead to a cascade of problems such as increased chances of risky behavior and negative health consequences, including alcoholic liver disease and upper gastric and liver cancer. Ethanol is metabolized mainly by 2 major enzymes: alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH). Genetic variations of genes encoding the 2 enzymes are very common among East Asians but relatively rare for most other populations. Facial flushing and other physical discomforts after alcohol drinking triggered by accumulation of acetaldehyde through defective genes for ADH and ALDH have been reported. Approximately 40% of East Asians (Chinese, Japanese, and Korean) show facial flushing after drinking alcohol, known as "Asian flush," which is characterized by adverse reactions on alcohol drinking in individuals possessing the fasting metabolizing alleles for ADH, ADH1B*2, and ADH1C*1, and the null allele for ALDH and ALDH2*2. Alcoholism is determined not only by the genetic deficiency but also by behaviors that involve complex interactions between genetic and sociocultural factors. The purpose of this article was to provide nurses with the most current information about genetic and sociocultural influences on alcoholism and alcohol-related health problems specifically for East Asians and implications of this knowledge to nursing practice. The physiological phenomenon of genes and genetics in relation to alcohol metabolism in this special population is emphasized.

Activity of select dehydrogenases with sepharose-immobilized N(6)-carboxymethyl-NAD.

PubMed

Beauchamp, Justin; Vieille, Claire

2015-01-01

N(6)-carboxymethyl-NAD (N(6)-CM-NAD) can be used to immobilize NAD onto a substrate containing terminal primary amines. We previously immobilized N(6)-CM-NAD onto sepharose beads and showed that Thermotoga maritima glycerol dehydrogenase could use the immobilized cofactor with cofactor recycling. We now show that Saccharomyces cerevisiae alcohol dehydrogenase, rabbit muscle L-lactate dehydrogenase (type XI), bovine liver L-glutamic dehydrogenase (type III), Leuconostoc mesenteroides glucose-6-phosphate dehydro-genase, and Thermotoga maritima mannitol dehydrogenase are active with soluble N(6)-CM-NAD. The products of all enzymes but 6-phospho-D-glucono-1,5-lactone were formed when sepharose-immobilized N(6)-CM-NAD was recycled by T. maritima glycerol dehydrogenase, indicating that N(6)-immobilized NAD is suitable for use by a variety of different dehydrogenases. Observations of the enzyme active sites suggest that steric hindrance plays a greater role in limiting or allowing activity with the modified cofactor than do polarity and charge of the residues surrounding the N(6)-amine group on NAD.

Biochemical characterization of ethanol-dependent reduction of furfural by alcohol dehydrogenases.

PubMed

Li, Qunrui; Metthew Lam, L K; Xun, Luying

2011-11-01

Lignocellulosic biomass is usually converted to hydrolysates, which consist of sugars and sugar derivatives, such as furfural. Before yeast ferments sugars to ethanol, it reduces toxic furfural to non-inhibitory furfuryl alcohol in a prolonged lag phase. Bioreduction of furfural may shorten the lag phase. Cupriavidus necator JMP134 rapidly reduces furfural with a Zn-dependent alcohol dehydrogenase (FurX) at the expense of ethanol (Li et al. 2011). The mechanism of the ethanol-dependent reduction of furfural by FurX and three homologous alcohol dehydrogenases was investigated. The reduction consisted of two individual reactions: ethanol-dependent reduction of NAD(+) to NADH and then NADH-dependent reduction of furfural to furfuryl alcohol. The kinetic parameters of the coupled reaction and the individual reactions were determined for the four enzymes. The data indicated that limited NADH was released in the coupled reaction. The enzymes had high affinities for NADH (e.g., K ( d ) of 0.043 μM for the FurX-NADH complex) and relatively low affinities for NAD(+) (e.g., K ( d ) of 87 μM for FurX-NAD(+)). The kinetic data suggest that the four enzymes are efficient "furfural reductases" with either ethanol or NADH as the reducing power. The standard free energy change (ΔG°') for ethanol-dependent reduction of furfural was determined to be -1.1 kJ mol(-1). The physiological benefit for ethanol-dependent reduction of furfural is likely to replace toxic and recalcitrant furfural with less toxic and more biodegradable acetaldehyde.

Method To Identify Specific Inhibiutors Of Imp Dehydrogenase

DOEpatents

Collart, Frank R.; Huberman, Eliezer

2000-11-28

This invention relates to methods to identify specific inhibitors of the purine nucleotide synthesis enzyme, IMP dehydrogenase (IMPDH). IMPDH is an essential enzyme found in all free-living organisms from humans to bacteria and is an important therapeutic target. The invention allows the identification of specific inhibitors of any IMPDH enzyme which can be expressed in a functional form in a recombinant host cell. A variety of eukaryotic or prokaryotic host systems commonly used for the expression of recombinant proteins are suitable for the practice of the invention. The methods are amenable to high throughput systems for the screening of inhibitors generated by combinatorial chemistry or other methods such as antisense molecule production. Utilization of exogenous guanosine as a control component of the methods allows for the identification of inhibitors specific for IMPDH rather than other causes of decreased cell proliferation.

Alcoholism and liver disease in Mexico: Genetic and environmental factors

PubMed Central

Roman, Sonia; Zepeda-Carrillo, Eloy Alfonso; Moreno-Luna, Laura Eugenia; Panduro, Arturo

2013-01-01

Alcoholism and cirrhosis, which are two of the most serious health problems worldwide, have a broad spectrum of clinical outcomes. Both diseases are influenced by genetic susceptibility and cultural traits that differ globally but are specific for each population. In contrast to other regions around the world, Mexicans present the highest drinking score and a high mortality rate for alcoholic liver disease with an intermediate category level of per capita alcohol consumption. Mexico has a unique history of alcohol consumption that is linked to profound anthropological and social aspects. The Mexican population has an admixture genome inherited from different races, Caucasian, Amerindian and African, with a heterogeneous distribution within the country. Thus, genes related to alcohol addiction, such as dopamine receptor D2 in the brain, or liver alcohol-metabolizing enzymes, such as alcohol dehydrogenase class I polypeptide B, cytochrome P450 2E1 and aldehyde dehydrogenase class 2, may vary from one individual to another. Furthermore, they may be inherited as risk or non-risk haplogroups that confer susceptibility or resistance either to alcohol addiction or abusive alcohol consumption and possibly liver disease. Thus, in this era of genomics, personalized medicine will benefit patients if it is directed according to individual or population-based data. Additional association studies will be required to establish novel strategies for the prevention, care and treatment of liver disease in Mexico and worldwide. PMID:24307790

Alcoholism and liver disease in Mexico: genetic and environmental factors.

PubMed

Roman, Sonia; Zepeda-Carrillo, Eloy Alfonso; Moreno-Luna, Laura Eugenia; Panduro, Arturo

2013-11-28

Alcoholism and cirrhosis, which are two of the most serious health problems worldwide, have a broad spectrum of clinical outcomes. Both diseases are influenced by genetic susceptibility and cultural traits that differ globally but are specific for each population. In contrast to other regions around the world, Mexicans present the highest drinking score and a high mortality rate for alcoholic liver disease with an intermediate category level of per capita alcohol consumption. Mexico has a unique history of alcohol consumption that is linked to profound anthropological and social aspects. The Mexican population has an admixture genome inherited from different races, Caucasian, Amerindian and African, with a heterogeneous distribution within the country. Thus, genes related to alcohol addiction, such as dopamine receptor D2 in the brain, or liver alcohol-metabolizing enzymes, such as alcohol dehydrogenase class I polypeptide B, cytochrome P450 2E1 and aldehyde dehydrogenase class 2, may vary from one individual to another. Furthermore, they may be inherited as risk or non-risk haplogroups that confer susceptibility or resistance either to alcohol addiction or abusive alcohol consumption and possibly liver disease. Thus, in this era of genomics, personalized medicine will benefit patients if it is directed according to individual or population-based data. Additional association studies will be required to establish novel strategies for the prevention, care and treatment of liver disease in Mexico and worldwide.

Primary deuterium and tritium isotope effects upon V/K in the liver alcohol dehydrogenase reaction with ethanol.

PubMed

Damgaard, S E

1981-09-29

The primary isotope effect upon V/K when ethanol stereospecifically labeled with deuterium or tritium is oxidized by liver alcohol dehydrogenase has been measured between pH 6 and 9. The deuterium isotope effect was obtained with high reproducibility by the use of two different radioactive tracers, viz. 14C and 3H, to follow the rate of acetaldehyde formation from deuterium-labeled ethanol and normal ethanol, respectively. Synthesis of the necessary labeled compounds is described in this and earlier work referred to. V/K isotope effects for both tritium and deuterium have been measured with three different coenzymes, NAD+, thio-NAD+, and acetyl-NAD+. With NAD+ at pH 7, D(V/K) was 3.0 and T(V/K) was 6.5. With increasing pH, these values decreased to 1.5 and 2.5 at pH 9. The intrinsic isotope effect evaluated by the method of Northrop [Northrop, D.B. (1977) in Isotope Effects on Enzyme-Catalyzed Reactions (Cleland, W. W., O'Leary, M, H., & Northrop, D. B., Eds.) pp 112-152, University Park Press, Baltimore] varies little with pH. It amounts to about 10 with NAD+ and about 5 with the coenzyme analogues. Commitment functions and their dependence upon pH calculated in this connection appear to be in agreement with known kinetic parameters of liver alcohol dehydrogenase. This assay method was also applied in vivo in the rat. Being a noninvasive method because only trace amounts of isotopes are needed, it may yield information about alternative routes of ethanol oxidation in vivo. In naive rats at low concentrations of ethanol, it confirms the discrete role of the non alcohol dehydrogenase systems.

Two types of alcohol dehydrogenase from Perilla can form citral and perillaldehyde.

PubMed

Sato-Masumoto, Naoko; Ito, Michiho

2014-08-01

Studies on the biosynthesis of oil compounds in Perilla will help in understanding regulatory systems of secondary metabolites and in elucidating reaction mechanisms for natural product synthesis. In this study, two types of alcohol dehydrogenases, an aldo-keto reductase (AKR) and a geraniol dehydrogenase (GeDH), which are thought to participate in the biosynthesis of perilla essential oil components, such as citral and perillaldehyde, were isolated from three pure lines of perilla. These enzymes shared high amino acid sequence identity within the genus Perilla, and were expressed regardless of oil type. The overall reaction from geranyl diphosphate to citral was performed in vitro using geraniol synthase and GeDH to form a large proportion of citral and relatively little geraniol as reaction products. The biosynthetic pathway from geranyl diphosphate to citral, the main compound of citral-type perilla essential oil, was established in this study. Copyright © 2014 Elsevier Ltd. All rights reserved.

A novel alcohol dehydrogenase biosensor based on solid-state electrogenerated chemiluminescence by assembling dehydrogenase to Ru(bpy)(3)2+-Au nanoparticles aggregates.

PubMed

Zhang, Lihua; Xu, Zhiai; Sun, Xuping; Dong, Shaojun

2007-01-15

Based on electrogenerated chemiluminescence (ECL), a novel method for fabrication of alcohol dehydrogenase (ADH) biosensor by self-assembling ADH to Ru(bpy)(3)(2+)-AuNPs aggregates (Ru-AuNPs) on indium tin oxide (ITO) electrode surface has been developed. Positively charged Ru(bpy)(3)(2+) could be immobilized stably on the electrode surface with negatively charged AuNPs in the form of aggregate via electrostatic interaction. On the other hand, AuNPs are favourable candidates for the immobilization of enzymes because amine groups and cysteine residues in the enzymes are known to bind strongly with AuNPs. Moreover, AuNPs can act as tiny conduction centers to facilitate the transfer of electrons. Such biosensor combined enzymatic selectivity with the sensitivity of ECL detection for quantification of enzyme substrate, and it displayed wide linear range, high sensitivity and good stability.

Proteomic changes in Corbicula fluminea exposed to wastewater from a psychiatric hospital.

PubMed

Bebianno, M J; Sroda, S; Gomes, T; Chan, P; Bonnafe, E; Budzinski, H; Geret, F

2016-03-01

The increase use of pharmaceutical compounds in veterinary practice and human population results in the ubiquitous presence of these compounds in aquatic ecosystems. Because pharmaceuticals are highly bioactive, there is concern about their toxicological effects in aquatic organisms. Therefore, the aim of this study was to assess the effects of an effluent from a psychiatric hospital (containing a complex mixture of 25 pharmaceutical compounds from eleven therapeutic classes) on the freshwater clam Corbicula fluminea using a proteomic approach. The exposure of C. fluminea to this complex effluent containing anxiolytics, analgesics, lipid regulators, beta blockers, antidepressants, antiepileptics, antihistamines, antihypertensives, antiplatelets and antiarrhythmics induced protein changes after 1 day of exposure in clam gills and digestive gland more evident in the digestive gland. These changes included increase in the abundance of proteins associated with structural (actin and tubulin), cellular functions (calreticulin, proliferating cell nuclear antigen (PCNA), T complex protein 1 (TCP1)) and metabolism (aldehyde dehydrogenase (ALDH), alcohol dehydrogenase, 6 phosphogluconate dehydrogenase). Results from this study indicate that calreticulin, PCNA, ALDH and alcohol dehydrogenase in the digestive gland and T complex protein 1 (TCP1)) and 6 phosphogluconate dehydrogenase in the gills represent useful biomarkers for the ecotoxicological characterization of psychiatric hospital effluents in this species.

Novel inhibitors of IMPDH: a highly potent and selective quinolone-based series.

PubMed

Watterson, Scott H; Carlsen, Marianne; Dhar, T G Murali; Shen, Zhongqi; Pitts, William J; Guo, Junqing; Gu, Henry H; Norris, Derek; Chorba, John; Chen, Ping; Cheney, Daniel; Witmer, Mark; Fleener, Catherine A; Rouleau, Katherine; Townsend, Robert; Hollenbaugh, Diane L; Iwanowicz, Edwin J

2003-02-10

A series of novel quinolone-based small molecule inhibitors of inosine monophosphate dehydrogenase (IMPDH) was explored. The synthesis and the structure-activity relationships (SARs) derived from in vitro studies are described.

Alcohol and aldehyde dehydrogenase polymorphisms and a new strategy for prevention and screening for cancer in the upper aerodigestive tract in East Asians.

PubMed

Yokoyama, Akira; Omori, Tai; Yokoyama, Tetsuji

2010-01-01

The ethanol in alcoholic beverages and the acetaldehyde associated with alcohol consumption are Group 1 human carcinogens (WHO, International Agency for Research on Cancer). The combination of alcohol consumption, tobacco smoking, the inactive heterozygous aldehyde dehydrogenase-2 genotype (ALDH2*1/*2) and the less-active homozygous alcohol dehydrogenase-1B genotype (ADH1B*1/*1) increases the risk of squamous cell carcinoma (SCC) in the upper aerodigestive tract (UADT) in a multiplicative fashion in East Asians. In addition to being exposed to locally high levels of ethanol, the UADT is exposed to a very high concentration of acetaldehyde from a variety of sources, including that as an ingredient of alcoholic beverages per se and that found in tobacco smoke; acetaldehyde is also produced by salivary microorganisms and mucosal enzymes and is present as blood acetaldehyde. The inefficient degradation of acetaldehyde by weakly expressed ALDH2 in the UADT may be cri! tical to the local accumulation of acetaldehyde, especially in ALDH2*1/*2 carriers. ADH1B*1/*1 carriers tend to experience less intense alcohol flushing and are highly susceptible to heavy drinking and alcoholism. Heavy drinking by persons with the less-active ADH1B*1/*1 leads to longer exposure of the UADT to salivary ethanol and acetaldehyde. The ALDH2*1/*2 genotype is a very strong predictor of synchronous and metachronous multiple SCCs in the UADT. High red cell mean corpuscular volume (MCV), esophageal dysplasia, and melanosis in the UADT, all of which are frequently found in ALDH2*1/*2 drinkers, are useful for identifying high-risk individuals. We invented a simple flushing questionnaire that enables prediction of the ALDH2 phenotype. New health appraisal models that include ALDH2 genotype, the simple flushing questionnaire, or MCV are powerful tools for devising a new strategy for prevention and screening for UADT cancer in East Asians.

Enhancement of the infectivity of SARS-CoV in BALB/c mice by IMP dehydrogenase inhibitors, including ribavirin.

PubMed

Barnard, Dale L; Day, Craig W; Bailey, Kevin; Heiner, Matthew; Montgomery, Robert; Lauridsen, Larry; Winslow, Scott; Hoopes, Justin; Li, Joseph K-K; Lee, Jongdae; Carson, Dennis A; Cottam, Howard B; Sidwell, Robert W

2006-08-01

Because of the conflicting data concerning the SARS-CoV inhibitory efficacy of ribavirin, an inosine monophosphate (IMP) dehydrogenase inhibitor, studies were done to evaluate the efficacy of ribavirin and other IMP dehydrogenase inhibitors (5-ethynyl-1-beta-D-ribofuranosylimidazole-4-carboxamide (EICAR), mizoribine, and mycophenolic acid) in preventing viral replication in the lungs of BALB/c mice, a replication model for severe acute respiratory syndrome (SARS) infections (Subbarao, K., McAuliffe, J., Vogel, L., Fahle, G., Fischer, S., Tatti, K., Packard, M., Shieh, W.J., Zaki, S., Murphy, B., 2004. Prior infection and passive transfer of neutralizing antibody prevent replication of severe acute respiratory syndrome coronavirus (SARS-CoV) in the respiratory tract of mice. J. Virol. 78, 3572-3577). Ribavirin given at 75 mg/kg 4 h prior to virus exposure and then given twice daily for 3 days beginning at day 0 was found to increase virus lung titers and extend the length of time that virus could be detected in the lungs of mice. Other IMP dehydrogenase inhibitors administered near maximum tolerated doses using the same dosing regimen as for ribavirin were found to slightly enhance virus replication in the lungs. In addition, ribavirin treatment seemed also to promote the production of pro-inflammatory cytokines 4 days after cessation of treatment, although after 3 days of treatment ribavirin inhibited pro-inflammatory cytokine production in infected mice, significantly reducing the levels of the cytokines IL-1alpha, interleukin-5 (IL-5), monocyte chemotactic protein-1 (MCP-1), and granulocyte-macrophage colony stimulating factor (GM-CSF). These findings suggest that ribavirin may actually contribute to the pathogenesis of SARS-CoV by prolonging and/or enhancing viral replication in the lungs. By not inhibiting viral replication in the lungs of infected mice, ribavirin treatment may have provided a continual source of stimulation for the inflammatory response thought to contribute to the pathogenesis of the infection. Our data do not support the use of ribavirin or other IMP dehydrogenase inhibitors for treating SARS infections in humans.

Metabolic engineering of Clostridium autoethanogenum for selective alcohol production.

PubMed

Liew, Fungmin; Henstra, Anne M; Kӧpke, Michael; Winzer, Klaus; Simpson, Sean D; Minton, Nigel P

2017-03-01

Gas fermentation using acetogenic bacteria such as Clostridium autoethanogenum offers an attractive route for production of fuel ethanol from industrial waste gases. Acetate reduction to acetaldehyde and further to ethanol via an aldehyde: ferredoxin oxidoreductase (AOR) and alcohol dehydrogenase has been postulated alongside the classic pathway of ethanol formation via a bi-functional aldehyde/alcohol dehydrogenase (AdhE). Here we demonstrate that AOR is critical to ethanol formation in acetogens and inactivation of AdhE led to consistently enhanced autotrophic ethanol production (up to 180%). Using ClosTron and allelic exchange mutagenesis, which was demonstrated for the first time in an acetogen, we generated single mutants as well as double mutants for both aor and adhE isoforms to confirm the role of each gene. The aor1+2 double knockout strain lost the ability to convert exogenous acetate, propionate and butyrate into the corresponding alcohols, further highlighting the role of these enzymes in catalyzing the thermodynamically unfavourable reduction of carboxylic acids into alcohols. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Bioelectrochemistry of non-covalent immobilized alcohol dehydrogenase on oxidized diamond nanoparticles.

PubMed

Nicolau, Eduardo; Méndez, Jessica; Fonseca, José J; Griebenow, Kai; Cabrera, Carlos R

2012-06-01

Diamond nanoparticles are considered a biocompatible material mainly due to their non-cytotoxicity and remarkable cellular uptake. Model proteins such as cytochrome c and lysozyme have been physically adsorbed onto diamond nanoparticles, proving it to be a suitable surface for high protein loading. Herein, we explore the non-covalent immobilization of the redox enzyme alcohol dehydrogenase (ADH) from Saccharomyces cerevisiae (E.C.1.1.1.1) onto oxidized diamond nanoparticles for bioelectrochemical applications. Diamond nanoparticles were first oxidized and physically characterized by X-ray diffraction (XRD), FT-IR and TEM. Langmuir isotherms were constructed to investigate the ADH adsorption onto the diamond nanoparticles as a function of pH. It was found that a higher packing density is achieved at the isoelectric point of the enzyme. Moreover, the relative activity of the immobilized enzyme on diamond nanoparticles was addressed under optimum pH conditions able to retain up to 70% of its initial activity. Thereafter, an ethanol bioelectrochemical cell was constructed by employing the immobilized alcohol dehydrogenase onto diamond nanoparticles, this being able to provide a current increment of 72% when compared to the blank solution. The results of this investigation suggest that this technology may be useful for the construction of alcohol biosensors or biofuel cells in the near future. Copyright © 2011 Elsevier B.V. All rights reserved.

Biology, Genetics, and Environment: Underlying Factors Influencing Alcohol Metabolism.

PubMed

Wall, Tamara L; Luczak, Susan E; Hiller-Sturmhöfel, Susanne

2016-01-01

Gene variants encoding several of the alcohol-metabolizing enzymes, alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH), are among the largest genetic associations with risk for alcohol dependence. Certain genetic variants (i.e., alleles)--particularly the ADH1B*2, ADH1B*3, ADH1C*1, and ALDH2*2 alleles--have been associated with lower rates of alcohol dependence. These alleles may lead to an accumulation of acetaldehyde during alcohol metabolism, which can result in heightened subjective and objective effects. The prevalence of these alleles differs among ethnic groups; ADH1B*2 is found frequently in northeast Asians and occasionally Caucasians, ADH1B*3 is found predominantly in people of African ancestry, ADH1C*1 varies substantially across populations, and ALDH2*2 is found almost exclusively in northeast Asians. Differences in the prevalence of these alleles may account at least in part for ethnic differences in alcohol consumption and alcohol use disorder (AUD). However, these alleles do not act in isolation to influence the risk of AUD. For example, the gene effects of ALDH2*2 and ADH1B*2 seem to interact. Moreover, other factors have been found to influence the extent to which these alleles affect a person's alcohol involvement, including developmental stage, individual characteristics (e.g., ethnicity, antisocial behavior, and behavioral undercontrol), and environmental factors (e.g., culture, religion, family environment, and childhood adversity).

Discovery of nonsteroidal 17beta-hydroxysteroid dehydrogenase 1 inhibitors by pharmacophore-based screening of virtual compound libraries.

PubMed

Schuster, Daniela; Nashev, Lyubomir G; Kirchmair, Johannes; Laggner, Christian; Wolber, Gerhard; Langer, Thierry; Odermatt, Alex

2008-07-24

17Beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD1) plays a pivotal role in the local synthesis of the most potent estrogen estradiol. Its expression is a prognostic marker for the outcome of patients with breast cancer and inhibition of 17beta-HSD1 is currently under consideration for breast cancer prevention and treatment. We aimed to identify nonsteroidal 17beta-HSD1 inhibitor scaffolds by virtual screening with pharmacophore models built from crystal structures containing steroidal compounds. The most promising model was validated by comparing predicted and experimentally determined inhibitory activities of several flavonoids. Subsequently, a virtual library of nonsteroidal compounds was screened against the 3D pharmacophore. Analysis of 14 selected compounds yielded four that inhibited the activity of human 17beta-HSD1 (IC 50 below 50 microM). Specificity assessment of identified 17beta-HSD1 inhibitors emphasized the importance of including related short-chain dehydrogenase/reductase (SDR) members to analyze off-target effects. Compound 29 displayed at least 10-fold selectivity over the related SDR enzymes tested.

Signatures of cinnamyl alcohol dehydrogenase deficiency in poplar lignins.

PubMed

Lapierre, Catherine; Pilate, Gilles; Pollet, Brigitte; Mila, Isabelle; Leplé, Jean-Charles; Jouanin, Lise; Kim, Hoon; Ralph, John

2004-02-01

A series of transgenic poplars down-regulated for cinnamyl alcohol dehydrogenase (CAD) was analyzed by thioacidolysis. Among the lignin-derived monomers, the indene compounds that were recently shown to originate from sinapaldehyde incorporated into lignins through 8-O-4-cross-coupling, were found to increase as a function of CAD deficiency level. While these syringyl markers were recovered in substantial amounts in the most severely depressed lines, the markers for coniferaldehyde incorporation were recovered in only low amounts. In conjunction with these additional sinapaldehyde units and relative to the control samples, lignins in CAD-deficient poplar lines had less conventional syringyl-units and beta-O-4-bonds and more free phenolic groups. We found that almost half of the polymers in the most deficient lines could be solubilized in alkali and at room temperature. This unusual behavior suggests that lignins in CAD-deficient poplars occur as small, alkali-leachable lignin domains. That mainly sinapaldehyde incorporates into the lignins of CAD-deficient poplars suggests that the recently identified sinapyl alcohol dehydrogenase (SAD), which is structurally distinct from the CAD enzyme targeted herein, does not play any substantial role in constitutive lignification in poplar.

Ethylene and 1-MCP regulate major volatile biosynthetic pathways in apple fruit.

PubMed

Yang, Xiaotang; Song, Jun; Du, Lina; Forney, Charles; Campbell-Palmer, Leslie; Fillmore, Sherry; Wismer, Paul; Zhang, Zhaoqi

2016-03-01

The effects of ethylene and 1-methylcyclopropene (1-MCP) on apple fruit volatile biosynthesis and gene expression were investigated. Statistical analysis identified 17 genes that changed significantly in response to ethylene and 1-MCP treatments. Genes encoding branched-chain amino acid aminotransferase (BCAT), aromatic amino acid aminotransferase (ArAT) and amino acid decarboxylases (AADC) were up-regulated during ripening and further enhanced by ethylene treatment. Genes related to fatty acid synthesis and metabolism, including acyl-carrier-proteins (ACPs), malonyl-CoA:ACP transacylase (MCAT), acyl-ACP-desaturase (ACPD), lipoxygenase (LOX), hydroperoxide lyase (HPL), alcohol dehydrogenase (ADH), pyruvate decarboxylase (PDC2), β-oxidation, acyl-CoA synthetase (ACS), enoyl-CoA hydratase (ECHD), acyl-CoA dehydrogenase (ACAD), and alcohol acyltransferases (AATs) also increased during ripening and in response to ethylene treatment. Allene oxide synthase (AOS), alcohol dehydrogenase 1 (ADH1), 3-ketoacyl-CoA thiolase and branched-chain amino acid aminotransferase 2 (BCAT2) decreased in ethylene-treated fruit. Treatment with 1-MCP and ethylene generally produced opposite effects on related genes, which provides evidence that regulation of these genes is ethylene dependent. Crown Copyright © 2015. Published by Elsevier Ltd. All rights reserved.

Hydrogen-driven asymmetric reduction of hydroxyacetone to (R)-1,2-propanediol by Ralstonia eutropha transformant expressing alcohol dehydrogenase from Kluyveromyces lactis.

PubMed

Oda, Takahiro; Oda, Koji; Yamamoto, Hiroaki; Matsuyama, Akinobu; Ishii, Masaharu; Igarashi, Yasuo; Nishihara, Hirofumi

2013-01-10

Conversion of industrial processes to more nature-friendly modes is a crucial subject for achieving sustainable development. Utilization of hydrogen-oxidation reactions by hydrogenase as a driving force of bioprocess reaction can be an environmentally ideal method because the reaction creates no pollutants. We expressed NAD-dependent alcohol dehydrogenase from Kluyveromyces lactis in a hydrogen-oxidizing bacterium: Ralstonia eutropha. This is the first report of hydrogen-driven in vivo coupling reaction of the alcohol dehydrogenase and indigenous soluble NAD-reducing hydrogenase. Asymmetric reduction of hydroxyacetone to (R)-1,2-propanediol, which is a commercial building block for antibacterial agents, was performed using the transformant as the microbial cell catalyst. The two enzymes coupled in vitro in vials without a marked decrease of reactivity during the 20 hr reaction because of the hydrogenase reaction, which generates no by-product that affects enzymes. Alcohol dehydrogenase was expressed functionally in R. eutropha in an activity level equivalent to that of indigenous NAD-reducing hydrogenase under the hydrogenase promoter. The hydrogen-driven in vivo coupling reaction proceeded only by the transformant cell without exogenous addition of a cofactor. The decrease of reaction velocity at higher concentration of hydroxyacetone was markedly reduced by application of an in vivo coupling system. Production of (R)-1,2-propanediol (99.8% e.e.) reached 67.7 g/l in 76 hr with almost a constant rate using a jar fermenter. The reaction velocity under 10% PH2 was almost equivalent to that under 100% hydrogen, indicating the availability of crude hydrogen gas from various sources. The in vivo coupling system enabled cell-recycling as catalysts. Asymmetric reduction of hydroxyacetone by a coupling reaction of the two enzymes continued in both in vitro and in vivo systems in the presence of hydrogen. The in vivo reaction system using R. eutropha transformant expressing heterologous alcohol dehydrogenase showed advantages for practical usage relative to the in vitro coupling system. The results suggest a hopeful perspective of the hydrogen-driven bioprocess as an environmentally outstanding method to achieve industrial green innovation. Hydrogen-oxidizing bacteria can be useful hosts for the development of hydrogen-driven microbial cell factories.

Nutritional Status, DNA Damage, and Tumor Pathology

DTIC Science & Technology

2006-08-01

tetrazolium bromide (MTT), 4.8 mM EDTA, 1 mg/ml bovine serum albumin (BSA), 50 µg/ml (U) alcohol dehydrogenase (yeast; 507 U/mg prot), and 1.9 mM phenazine ...NADP-specific isocitrate dehydrogenase (porcine heart; 26-29 U/mg prot), and 1.9 mM phenazine ethosulfate. After incubation of assays for NAD or

Inhibition effects of furfural on alcohol dehydrogenase, aldehyde dehydrogenase and pyruvate dehydrogenase.

PubMed Central

Modig, Tobias; Lidén, Gunnar; Taherzadeh, Mohammad J

2002-01-01

The kinetics of furfural inhibition of the enzymes alcohol dehydrogenase (ADH; EC 1.1.1.1), aldehyde dehydrogenase (AlDH; EC 1.2.1.5) and the pyruvate dehydrogenase (PDH) complex were studied in vitro. At a concentration of less than 2 mM furfural was found to decrease the activity of both PDH and AlDH by more than 90%, whereas the ADH activity decreased by less than 20% at the same concentration. Furfural inhibition of ADH and AlDH activities could be described well by a competitive inhibition model, whereas the inhibition of PDH was best described as non-competitive. The estimated K(m) value of AlDH for furfural was found to be about 5 microM, which was lower than that for acetaldehyde (10 microM). For ADH, however, the estimated K(m) value for furfural (1.2 mM) was higher than that for acetaldehyde (0.4 mM). The inhibition of the three enzymes by 5-hydroxymethylfurfural (HMF) was also measured. The inhibition caused by HMF of ADH was very similar to that caused by furfural. However, HMF did not inhibit either AlDH or PDH as severely as furfural. The inhibition effects on the three enzymes could well explain previously reported in vivo effects caused by furfural and HMF on the overall metabolism of Saccharomyces cerevisiae, suggesting a critical role of these enzymes in the observed inhibition. PMID:11964178

Development of Novel Therapeutics Targeting Isocitrate Dehydrogenase Mutations in Cancer.

PubMed

Sharma, Horrick

2018-05-17

Isocitrate dehydrogenases 1 and 2 (IDH1 and IDH2) are key metabolic enzymes that catalyze the conversion of isocitrate to α-ketoglutarate (αKG). IDH 1 and IDH2 regulate several cellular processes, including oxidative respiration, glutamine metabolism, lipogenesis, and cellular defense against oxidative damage. Mutations in IDH1 and IDH2 have recently been observed in multiple tumor types, including gliomas, acute myeloid leukemia, myelodysplastic syndromes, and chondrosarcoma. IDH1 and IDH2 mutations involve a gain in neomorphic activity that catalyze αKG conversion to (R)-2-hydroxyglutarate ((R)-2HG). IDH mutation-mediated accumulation of (R)-2HG result in epigenetic dysregulation, altered gene expression, and a block in cellular differentiation. Targeting mutant IDH by development of small molecule inhibitors is a rapidly emerging therapeutic approach as evidenced by the recent approval of the first selective mutant IDH2 inhibitor AG-221 (Enasidenib) for the treatment of IDH2-mutated AML. This review will focus on mutant isocitrate dehydrogenase as a therapeutic drug target and provides an update on selective and pan-mutant IDH 1/2 inhibitors in clinical trials and other mutant IDH inhibitors that are under development. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Research on alcohol metabolism among Asians and its implications for understanding causes of alcoholism.

PubMed Central

Suddendorf, R F

1989-01-01

Research into the causes of alcoholism is a relatively recent scientific endeavor. One area of study which could lead to better understanding of the disease is the possibility of a genetic predisposition to alcoholism. Recent work has demonstrated that people have varying complements of enzymes to metabolize alcohol. Current knowledge is examined about the influence of various ethanol metabolizing enzymes on alcohol consumption by Asians and members of other ethnic groups. The two principal enzymes involved in ethanol oxidative metabolism are alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH). ADH is responsible for the metabolism of ethanol to acetaldehyde. ALDH catalyzes the conversion of acetaldehyde to acetate. The different isozymes account for the diversity of alcohol metabolism among individuals. An isozyme of ADH (beta 2 beta 2) is found more frequently in Asians than in whites, and an ALDH isozyme (ALDH2), although present in Asians, often is in an inactive form. The presence of an inactive form of ALDH2 is thought to be responsible for an increase in acetaldehyde levels in the body. Acetaldehyde is considered responsible for the facial flushing reaction often observed among Asians who have consumed alcohol. A dysphoric reaction to alcohol, producing uncomfortable sensations, is believed to be a response to deter further consumption. Although the presence of an inactive ALDH2 isozyme may serve as a deterrent to alcohol consumption, its presence does not fully explain the levels of alcohol consumption by those with the inactive isozyme. Other conditions, such as social pressure, and yet undetermined biological factors, may play a significant role in alcohol consumption. PMID:2511595

Gender differences in the effects of ADH1B and ALDH2 polymorphisms on alcoholism.

PubMed

Kimura, Mitsuru; Miyakawa, Tomohiro; Matsushita, Sachio; So, Mirai; Higuchi, Susumu

2011-11-01

Gender differences are known to exist in the prevalence, characteristics, and course of alcohol dependence. Elucidating gender differences in the characteristics of alcohol dependence is important in gender-based medicine and may improve treatment outcomes. Many studies have shown that genetic factors are associated with the risk of alcohol dependence in both genders. Polymorphisms of alcohol dehydrogenase-1B (ADH1B) and aldehyde dehydrogenase-2 (ALDH2) are strong genetic determinants of alcohol dependence. This study aimed to clarify gender differences in the effects of ADH1B and ALDH2 polymorphism on the development of alcohol dependence. Subjects were 200 female alcoholics and 415 male alcoholics hospitalized in Kurihama Alcoholism Center. Clinical information and background data were obtained by chart review. ALDH2 and ADH1B genotyping was performed by the polymerase chain reaction-restriction fragment length polymorphism method. The onset age of female alcoholics with inactive ALDH2 genotype was significantly lower than those with active ALDH2 genotype, but the onset age did not differ between the inactive and active ALDH2 group in male alcoholics. The difference in onset age between the ADH1B genotype groups did not reach significant levels. The prevalence of comorbid psychiatric disorders, including major depression, eating disorder, panic disorder, and borderline personality disorder, was significantly higher in female alcoholics with inactive ALDH2 or superactive ADH1B than in those with active ALDH2 or normal ADH1B. ALDH2 polymorphism appears to have contrasting effects on the development of alcoholism in women and men. One possible reason for this gender difference may be the high prevalence of psychiatric comorbidities in female alcoholics with inactive ALDH2. Copyright © 2011 by the Research Society on Alcoholism.

Allelic variants of ADH, ALDH and the five factor model of personality in alcohol dependence syndrome

PubMed Central

Salujha, S. K.; Chaudhury, S.; Menon, P. K.; Srivastava, K.; Gupta, A.

2014-01-01

Background: The etiology of alcohol dependence is a complex interplay of biopsychosocial factors. The genes for alcohol-metabolizing enzymes: Alcohol dehydrogenase (ADH2 and ADH3) and aldehyde dehydrogenase (ALDH2) exhibit functional polymorphisms. Vulnerability of alcohol dependence may also be in part due to heritable personality traits. Aim: To determine whether any association exists between polymorphisms of ADH2, ADH3 and ALDH2 and alcohol dependence syndrome in a group of Asian Indians. In addition, the personality of these patients was assessed to identify traits predisposing to alcoholism. Materials and Methods: In this study, 100 consecutive males with alcohol dependence syndrome attending the psychiatric outpatient department of a tertiary care service hospital and an equal number of matched healthy controls were included with their consent. Blood samples of all the study cases and controls were collected and genotyped for the ADH2, ADH3 and ALDH2 loci. Personality was evaluated using the neuroticism, extraversion, openness (NEO) personality inventory and sensation seeking scale. Results: Allele frequencies of ADH2*2 (0.50), ADH3*1 (0.67) and ALSH2*2 (0.09) were significantly low in the alcohol dependent subjects. Personality traits of NEO personality inventory and sensation seeking were significantly higher when compared to controls. Conclusions: The functional polymorphisms of genes coding for alcohol metabolizing enzymes and personality traits of NEO and sensation seeking may affect the propensity to develop dependence. PMID:25535445

Role of L-alanine for redox self-sufficient amination of alcohols.

PubMed

Klatte, Stephanie; Wendisch, Volker F

2015-01-23

In white biotechnology biocatalysis represents a key technology for chemical functionalization of non-natural compounds. The plasmid-born overproduction of an alcohol dehydrogenase, an L-alanine-dependent transaminase and an alanine dehydrogenase allows for redox self-sufficient amination of alcohols in whole cell biotransformation. Here, conditions to optimize the whole cell biocatalyst presented in (Bioorg Med Chem 22:5578-5585, 2014), and the role of L-alanine for efficient amine functionalization of 1,10-decanediol to 1,10-diaminodecane were analyzed. The enzymes of the cascade for amine functionalization of alcohols were characterized in vitro to find optimal conditions for an efficient process. Transaminase from Chromobacterium violaceum, TaCv, showed three-fold higher catalytic efficiency than transaminase from Vibrio fluvialis, TaVf, and improved production at 37°C. At 42°C, TaCv was more active, which matched thermostable alcohol dehydrogenase and alanine dehydrogenase and improved the 1,10-diaminodecane production rate four-fold. To study the role of L-alanine in the whole cell biotransformation, the L-alanine concentration was varied and 1,10.diaminodecane formation tested with constant 10 mM 1,10- decanediol and 100 mM NH4Cl. Only 5.6% diamine product were observed without added L-alanine. L-alanine concentrations equimolar to that of the alcohol enabled for 94% product formation but higher L-alanine concentrations allowed for 100% product formation. L-alanine was consumed by the E. coli biocatalyst, presumably due to pyruvate catabolism since up to 16 mM acetate accumulated. Biotransformation employing E. coli strain YYC202/pTrc99a-ald-adh-ta Cv, which is unable to catabolize pyruvate, resulted in conversion with a selectivity of 42 mol-%. Biotransformation with E. coli strains only lacking pyruvate oxidase PoxB showed similar reduced amination of 1,10-decanediol indicating that oxidative decarboxylation of pyruvate to acetate by PoxB is primarily responsible for pyruvate catabolism during redox self-sufficient amination of alcohols using this whole cell biocatalyst. The replacement of the transaminase TaVf by TaCv, which showed higher activity at 42°C, in the artificial operon ald-adh-ta improved amination of alcohols in whole cell biotransformation. The addition of L-alanine, which was consumed by E. coli via pyruvate catabolism, was required for 100% product formation possibly by providing maintenance energy. Metabolic engineering revealed that pyruvate catabolism occurred primarily via oxidative decarboxylation to acetate by PoxB under the chosen biotranformation conditions.

In-vitro Wound Healing Effect of 15-Hydroxyprostaglandin Dehydrogenase Inhibitor from Plant.

PubMed

Karna, Sandeep

2017-01-01

Prostaglandins (PGs) have short existence in vivo because they are rapidly metabolized by NAD + -dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) to 15-ketoprostaglandins. Inhibition of 15-PGDH causes elevated level of PGE 2 in cellular system. It will be valuable for the therapeutic management of diseases requiring elevated PGE 2 levels, like wound healing. Ninety-eight plant samples were screened for the discovery of potent 15-PGDH inhibitor. Among them, top five plant extracts as potent 15-PGDH inhibitor were chosen to determine PGE 2 release from HaCaT (Keratinocyte cell line) cell line. Finally, top 15-PGDH inhibitor was selected to evaluate in vitro wound healing effect on HaCaT scratch model. The inhibitory activity for 15-PGDH inhibitors was evaluated using fluorescence spectrophotometer by measuring the formation of NADH at 468 nm following excitation at 340 nm. Cell viability assay and PGE 2 release was evaluated in HaCaT cell line after treatment of 15-PGDH inhibitors. Scratches were made using sterile 200 μL on HaCaT cell and wound-healing effect was evaluated after treatment of 15-PGDH inhibitor. 15-PGDH inhibitors elevated PGE 2 levels in concentration-dependent manner. Ethanol extract of Artocarpus heterophyllus (EEAH), the most potent 15-PGDH inhibitor (IC 50 = 0.62 µg/mL) with least cytotoxicity (IC 50 = 670 µg/ml), elevated both intracellular and extracellular PGE 2 levels. EEAH facilitated in-vitro wound healing in a HaCaT (Keratinocyte cell line) scratch model. EEAH might apply to treat dermal wounds by elevating PGE 2 levels via COX-1 induction and 15-PGDH inhibition. Biological inactivation of 15-PGDH causes elevated level of PGE 2 which will be useful for the management of disease that requires elevated level of PGE 2 . Abbreviations used: 15-PGDH: 15-hydroxyprostaglandin dehydrogenase, COX: Cyclooxygenase, DTT: Dithiothreitol, DMEM: Dulbecco's modified Eagle's media, EEAH: Ethanol extract of Artocarpus heterophyllus, MRP4: Multidrug resistance 4, PGs: Prostaglandins, PGT: Prostaglandin transporter, SDS: Sodium dodecylsulfate.

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

PubMed Central

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

1975-01-01

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

QSAR models based on quantum topological molecular similarity.

PubMed

Popelier, P L A; Smith, P J

2006-07-01

A new method called quantum topological molecular similarity (QTMS) was fairly recently proposed [J. Chem. Inf. Comp. Sc., 41, 2001, 764] to construct a variety of medicinal, ecological and physical organic QSAR/QSPRs. QTMS method uses quantum chemical topology (QCT) to define electronic descriptors drawn from modern ab initio wave functions of geometry-optimised molecules. It was shown that the current abundance of computing power can be utilised to inject realistic descriptors into QSAR/QSPRs. In this article we study seven datasets of medicinal interest : the dissociation constants (pK(a)) for a set of substituted imidazolines , the pK(a) of imidazoles , the ability of a set of indole derivatives to displace [(3)H] flunitrazepam from binding to bovine cortical membranes , the influenza inhibition constants for a set of benzimidazoles , the interaction constants for a set of amides and the enzyme liver alcohol dehydrogenase , the natriuretic activity of sulphonamide carbonic anhydrase inhibitors and the toxicity of a series of benzyl alcohols. A partial least square analysis in conjunction with a genetic algorithm delivered excellent models. They are also able to highlight the active site, of the ligand or the molecule whose structure determines the activity. The advantages and limitations of QTMS are discussed.

Approach to the Treatment of Methanol Intoxication.

PubMed

Kraut, Jeffrey A

2016-07-01

Methanol intoxication is an uncommon but serious poisoning. Its adverse effects are due primarily to the impact of its major metabolite formic acid and lactic acid resulting from cellular hypoxia. Symptoms including abdominal pain and loss of vision can appear a few hours to a few days after exposure, reflecting the time necessary for accumulation of the toxic byproducts. In addition to a history of exposure, increases in serum osmolal and anion gaps can be clues to its presence. However, increments in both parameters can be absent depending on the nature of the toxic alcohol, time of exposure, and coingestion of ethanol. Definitive diagnosis requires measurement with gas or liquid chromatography, which are laborious and expensive procedures. Tests under study to detect methanol or its metabolite formate might facilitate the diagnosis of this poisoning. Treatment can include administration of ethanol or fomepizole, both inhibitors of the enzyme alcohol dehydrogenase to prevent formation of its metabolites, and hemodialysis to remove methanol and formate. In this Acid-Base and Electrolyte Teaching Case, a patient with methanol intoxication due to ingestion of model airplane fuel is described, and the value and limitations of current and new diagnostic and treatment measures are discussed. Published by Elsevier Inc.

Xylitol dehydrogenase from Candida tropicalis: molecular cloning of the gene and structural analysis of the protein.

PubMed

Lima, Luanne Helena Augusto; Pinheiro, Cristiano Guimarães do Amaral; de Moraes, Lídia Maria Pepe; de Freitas, Sonia Maria; Torres, Fernando Araripe Gonçalves

2006-12-01

Yeasts can metabolize xylose by the action of two key enzymes: xylose reductase and xylitol dehydrogenase. In this work, we present data concerning the cloning of the XYL2 gene encoding xylitol dehydrogenase from the yeast Candida tropicalis. The gene is present as a single copy in the genome and is controlled at the transcriptional level by the presence of the inducer xylose. XYL2 was functionally tested by heterologous expression in Saccharomyces cerevisiae to develop a yeast strain capable of producing ethanol from xylose. Structural analysis of C. tropicalis xylitol dehydrogenase, Xyl2, suggests that it is a member of the medium-chain dehydrogenase (MDR) family. This is supported by the presence of the amino acid signature [GHE]xx[G]xxxxx[G]xx[V] in its primary sequence and a typical alcohol dehydrogenase Rossmann fold pattern composed by NAD(+) and zinc ion binding domains.

Production of C4 and C5 branched-chain alcohols by engineered Escherichia. coli.

PubMed

Chen, Xiaoyan; Xu, Jingliang; Yang, Liu; Yuan, Zhenhong; Xiao, Shiyuan; Zhang, Yu; Liang, Cuiyi; He, Minchao; Guo, Ying

2015-11-01

Higher alcohols, longer chain alcohols, contain more than 3 carbon atoms, showed close energy advantages as gasoline, and were considered as the next generation substitution for chemical fuels. Higher alcohol biosynthesis by native microorganisms mainly needs gene expression of heterologous keto acid decarboxylase and alcohol dehydrogenases. In the present study, branched-chain α-keto acid decarboxylase gene from Lactococcus lactis subsp. lactis CICC 6246 (Kivd) and alcohol dehydrogenases gene from Zymomonas mobilis CICC 41465 (AdhB) were transformed into Escherichia coli for higher alcohol production. SDS-PAGE results showed these two proteins were expressed in the recombinant strains. The resulting strain was incubated in LB medium at 37 °C in Erlenmeyer flasks and much more 3-methyl-1-butanol (104 mg/L) than isobutanol (24 mg/L) was produced. However, in 5 g/L glucose-containing medium, the production of two alcohols was similar, 156 and 161 mg/L for C4 (isobutanol) and C5 (3-methyl-1-butanol) alcohol, respectively. Effects of fermentation factors including temperature, glucose content, and α-keto acid on alcohol production were also investigated. The increase of glucose content and the adding of α-keto acids facilitated the production of C4 and C5 alcohols. The enzyme activities of pure Kivd on α-ketoisovalerate and α-ketoisocaproate were 26.77 and 21.24 μmol min(-1) mg(-1), respectively. Due to its ability on decarboxylation of α-ketoisovalerate and α-ketoisocaproate, the recombinant E. coli strain showed potential application on isoamyl alcohol and isobutanol production.

Association between ADH1C and ALDH2 polymorphisms and alcoholism in a Turkish sample.

PubMed

Ayhan, Yavuz; Gürel, Şeref Can; Karaca, Özgür; Zoto, Teuta; Hayran, Mutlu; Babaoğlu, Melih; Yaşar, Ümit; Bozkurt, Atilla; Dilbaz, Nesrin; Uluğ, Berna Diclenur; Demir, Başaran

2015-04-01

Polymorphisms in the genes encoding alcohol metabolizing enzymes are associated with alcohol dependence. To evaluate the association between the alcohol dehydrogenase 1C (ADH1C) Ile350Val and aldehyde dehydrogenase 2 (ALDH2) Glu504Lys polymorphisms and alcohol dependence in a Turkish sample. 235 individuals (115 alcohol-dependent patients and 120 controls) were genotyped for ADH1C and ALDH2 with PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism). Association between the polymorphisms and family history, daily and maximum amount of alcohol consumed was investigated. The associations between alcohol dependence, severity of consumption and family history and the polymorphisms were analyzed by chi-square or Fisher's exact test where necessary. Relationship between genotypes and dependence related features was evaluated using analysis of variance (ANOVA). The -350Val allele for ADH1C (ADH1C*2) was increased in alcohol-dependent patients (P = 0.05). In individuals with a positive family history, the genotype distribution differed significantly (P = 0.031) and more patients carried the Val allele compared with controls (P = 0.025). Genotyping of 162 participants did not reveal the -504Lys allele in ALDH2. These findings suggest that ADH1C*2 is associated with alcohol dependence in the Turkish population displaying a dominant inheritance model. ADH1C*2 allele may contribute to the variance in heritability of alcohol dependence. The ALDH2 -504Lys/Lys or Glu/Lys genotypes were not present in alcohol-dependent patients, similar to that seen in European populations and in contrast to the findings in the Asian populations.

Biology, Genetics, and Environment

PubMed Central

Wall, Tamara L.; Luczak, Susan E.; Hiller-Sturmhöfel, Susanne

2016-01-01

Gene variants encoding several of the alcohol-metabolizing enzymes, alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH), are among the largest genetic associations with risk for alcohol dependence. Certain genetic variants (i.e., alleles)—particularly the ADH1B*2, ADH1B*3, ADH1C*1, and ALDH2*2 alleles—have been associated with lower rates of alcohol dependence. These alleles may lead to an accumulation of acetaldehyde during alcohol metabolism, which can result in heightened subjective and objective effects. The prevalence of these alleles differs among ethnic groups; ADH1B*2 is found frequently in northeast Asians and occasionally Caucasians, ADH1B*3 is found predominantly in people of African ancestry, ADH1C*1 varies substantially across populations, and ALDH2*2 is found almost exclusively in northeast Asians. Differences in the prevalence of these alleles may account at least in part for ethnic differences in alcohol consumption and alcohol use disorder (AUD). However, these alleles do not act in isolation to influence the risk of AUD. For example, the gene effects of ALDH2*2 and ADH1B*2 seem to interact. Moreover, other factors have been found to influence the extent to which these alleles affect a person’s alcohol involvement, including developmental stage, individual characteristics (e.g., ethnicity, antisocial behavior, and behavioral undercontrol), and environmental factors (e.g., culture, religion, family environment, and childhood adversity). PMID:27163368

Alcoholism: genes and mechanisms.

PubMed

Oroszi, Gabor; Goldman, David

2004-12-01

Alcoholism is a chronic relapsing/remitting disease that is frequently unrecognized and untreated, in part because of the partial efficacy of treatment. Only approximately one-third of patients remain abstinent and one-third have fully relapsed 1 year after withdrawal from alcohol, with treated patients doing substantially better than untreated [1]. The partial effectiveness of strategies for prevention and treatment, and variation in clinical course and side effects, represent a challenge and an opportunity to better understand the neurobiology of addiction. The strong heritability of alcoholism suggests the existence of inherited functional variants of genes that alter the metabolism of alcohol and variants of other genes that alter the neurobiologies of reward, executive cognitive function, anxiety/dysphoria, and neuronal plasticity. Each of these neurobiologies has been identified as a critical domain in the addictions. Functional alleles that alter alcoholism-related intermediate phenotypes include common alcohol dehydrogenase 1B and aldehyde dehydrogenase 2 variants that cause the aversive flushing reaction; catechol-O-methyltransferase (COMT) Val158Met leading to differences in three aspects of neurobiology: executive cognitive function, stress/anxiety response, and opioid function; opioid receptor micro1 (OPRM1) Asn40Asp, which may serve as a gatekeeper molecule in the action of naltrexone, a drug used in alcoholism treatment; and HTTLPR, which alters serotonin transporter function and appears to affect stress response and anxiety/dysphoria, which are factors relevant to initial vulnerability, the process of addiction, and relapse.

Structure of Cryptosporidium IMP dehydrogenase bound to an inhibitor with in vivo antiparasitic activity

DOE PAGES

Kim, Youngchang; Makowska-Grzyska, Magdalena; Gorla, Suresh Kumar; ...

2015-04-21

Inosine 5´-monophosphate dehydrogenase (IMPDH) is a promising target for the treatment of Cryptosporidium infections. Here, the structure of C. parvum IMPDH ( CpIMPDH) in complex with inosine 5´-monophosphate (IMP) and P131, an inhibitor with in vivo anticryptosporidial activity, is reported. P131 contains two aromatic groups, one of which interacts with the hypoxanthine ring of IMP, while the second interacts with the aromatic ring of a tyrosine in the adjacent subunit. In addition, the amine and NO 2 moieties bind in hydrated cavities, forming water-mediated hydrogen bonds to the protein. The design of compounds to replace these water molecules is amore » new strategy for the further optimization of C. parvum inhibitors for both antiparasitic and antibacterial applications.« less

Pharmacological characterization of the selective 11β-hydroxysteroid dehydrogenase 1 inhibitor, BI 135585, a clinical candidate for the treatment of type 2 diabetes.

PubMed

Hamilton, Bradford S; Himmelsbach, Frank; Nar, Herbert; Schuler-Metz, Annette; Krosky, Paula; Guo, Joan; Guo, Rong; Meng, Shi; Zhao, Yi; Lala, Deepak S; Zhuang, Linghang; Claremon, David A; McGeehan, Gerard M

2015-01-05

To combat the increased morbidity and mortality associated with the developing diabetes epidemic new therapeutic interventions are desirable. Inhibition of intracellular cortisol generation from cortisone by blocking 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) has been shown to ameliorate the risk factors associated with the metabolic syndrome. A challenge in developing 11β-HSD1 inhibitors has been the species selectivity of small molecules, as many compounds are primate specific. Here we describe our strategy to identify potent selective 11β-HSD1 inhibitors while ensuring target engagement in key metabolic tissues, liver and fat. This strategy enabled the identification of the clinical candidate, BI 135585. Copyright © 2014 Elsevier B.V. All rights reserved.

3D-QSAR Studies on a Series of Dihydroorotate Dehydrogenase Inhibitors: Analogues of the Active Metabolite of Leflunomide

PubMed Central

Li, Shun-Lai; He, Mao-Yu; Du, Hong-Guang

2011-01-01

The active metabolite of the novel immunosuppressive agent leflunomide has been shown to inhibit the enzyme dihydroorotate dehydrogenase (DHODH). This enzyme catalyzes the fourth step in de novo pyrimidine biosynthesis. Self-organizing molecular field analysis (SOMFA), a simple three-dimensional quantitative structure-activity relationship (3D-QSAR) method is used to study the correlation between the molecular properties and the biological activities of a series of analogues of the active metabolite. The statistical results, cross-validated rCV2 (0.664) and non cross-validated r2 (0.687), show a good predictive ability. The final SOMFA model provides a better understanding of DHODH inhibitor-enzyme interactions, and may be useful for further modification and improvement of inhibitors of this important enzyme. PMID:21686163

Evaluation of alcohol dehydrogenase and aldehyde dehydrogenase enzymes as bi-enzymatic anodes in a membraneless ethanol microfluidic fuel cell

NASA Astrophysics Data System (ADS)

Galindo-de-la-Rosa, J.; Arjona, N.; Arriaga, L. G.; Ledesma-García, J.; Guerra-Balcázar, M.

2015-12-01

Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (AldH) enzymes were immobilized by covalent binding and used as the anode in a bi-enzymatic membraneless ethanol hybrid microfluidic fuel cell. The purpose of using both enzymes was to optimize the ethanol electro-oxidation reaction (EOR) by using ADH toward its direct oxidation and AldH for the oxidation of aldehydes as by-products of the EOR. For this reason, three enzymatic bioanode configurations were evaluated according with the location of enzymes: combined, vertical and horizontally separated. In the combined configuration, a current density of 16.3 mA cm-2, a voltage of 1.14 V and a power density of 7.02 mW cm-2 were obtained. When enzymes were separately placed in a horizontal and vertical position the ocp drops to 0.94 V and to 0.68 V, respectively. The current density also falls to values of 13.63 and 5.05 mA cm-2. The decrease of cell performance of bioanodes with separated enzymes compared with the combined bioanode was of 31.7% and 86.87% for the horizontal and the vertical array.

S-nitrosation versus S-glutathionylation of protein sulfhydryl groups by S-nitrosoglutathione.

PubMed

Giustarini, Daniela; Milzani, Aldo; Aldini, Giancarlo; Carini, Marina; Rossi, Ranieri; Dalle-Donne, Isabella

2005-01-01

S-Nitrosation of protein sulfhydryl groups is an established response to oxidative/nitrosative stress. The transient nature and reversibility of S-nitrosation, as well as its specificity, render this posttranslational modification an attractive mechanism of regulation of protein function and signal transduction, in analogy to S-glutathionylation. Several feasible mechanisms for protein S-nitrosation have been proposed, including transnitrosation by S-nitrosothiols, such as S-nitrosoglutathione (GSNO), where the nitrosonium moiety is directly transferred from one thiol to another. The reaction between GSNO and protein sulfhydryls can also produce a mixed disulfide by S-glutathionylation, which involves the nucleophilic attack of the sulfur of GSNO by the protein thiolate anion. In this study, we have investigated the possible occurrence of S-glutathionylation during reaction of GSNO with papain, creatine phosphokinase, glyceraldehyde-3-phosphate dehydrogenase, alcohol dehydrogenase, bovine serum albumin, and actin. Our results show that papain, creatine phosphokinase, and glyceraldehyde-3-phosphate dehydrogenase were significantly both S-nitrosated and S-glutathionylated by GSNO, whereas alcohol dehydrogenase, bovine serum albumin, and actin appeared nearly only S-nitrosated. The susceptibility of the modified proteins to denitrosation and deglutathionylation by reduced glutathione was also investigated.

Human hydroxysteroid dehydrogenases and pre-receptor regulation: Insights into inhibitor design and evaluation

PubMed Central

Penning, Trevor M.

2011-01-01

Hydroxysteroid dehydrogenases (HSDs) represent a major class of NAD(P)(H) dependent steroid hormone oxidoreductases involved in the pre-receptor regulation of hormone action. This is achieved by HSDs working in pairs so that they can interconvert ketosteroids with hydroxysteroids resulting in a change in ligand potency for nuclear receptors. HSDs belong to two protein superfamilies the aldo-keto reductases and the short-chain dehydrogenase/reductases. In humans, many of the important enzymes have been thoroughly characterized including the elucidation of their three-dimensional structures. Because these enzymes play fundamental roles in steroid hormone action they can be considered to be drug targets for a variety of steroid driven diseases: e.g. metabolic syndrome and obesity, inflammation, and hormone dependent malignancies of the endometrium, prostate and breast. This article will review how fundamental knowledge of these enzymes can be exploited in the development of isoform specific HSD inhibitors from both protein superfamilies. PMID:21272640

Cyclic ethanol metabolism in hypophysectomized rats continuously infused alcohol-containing diets

USDA-ARS?s Scientific Manuscript database

Chronic ethanol (EtOH) intake induces hepatic alcohol dehydrogenase (ADH) expression via disruption of insulin signaling in liver (JBC 2006; 281:1126-34). Total enteral nutrition (TEN) is a method of slow and continuous (approx. 23/day) feeding patients through an intragastric tube. Rats fed EtOH-co...

[Enzymatic conversion of tetradecanol in heterogenous phase by yeast-alcohol dehydrogenase].

PubMed

Rothe, U; Schöpp, W; Aurich, H

1976-01-01

Alcohol dehydrogenase from yeast converts long-chain primary alcohols not only in the dissolved state, but also at the surface of undissolved particles. Tetradecanol beads with a defined surface can be produced and employed as model substrate. The reaction rate was determined by the proton release accomplished in the reaction. The initial reaction rate depends on the enzyme concentration. The relation is nonlinear (vi = k-[e]0,4); the numerical value of the exponent (n = 0.4) argues in favour of a reaction occurring at the interface. The Lineweaver-Burk plots become linear if the substrate concentrations are based on the molar surface concentrations of the particles. The pH optimum for the reaction at the surface is displaced by 0.25 pH units towards the alkaline region (compared with ethanol as substrate). The activation energy of the reaction with tetradecanol beads as substrate is 30% lower than that for the ethanol oxydation.

S-Nitrosoglutathione Reductase Inhibition Regulates Allergen-Induced Lung Inflammation and Airway Hyperreactivity

PubMed Central

Bassett, David J. P.; Bradley, Matthews O.; Jaffar, Zeina

2013-01-01

Allergic asthma is characterized by Th2 type inflammation, leading to airway hyperresponsivenes, mucus hypersecretion and tissue remodeling. S-Nitrosoglutathione reductase (GSNOR) is an alcohol dehydrogenase involved in the regulation of intracellular levels of S-nitrosothiols. GSNOR activity has been shown to be elevated in human asthmatic lungs, resulting in diminished S-nitrosothiols and thus contributing to increased airway hyperreactivity. Using a mouse model of allergic airway inflammation, we report that intranasal administration of a new selective inhibitor of GSNOR, SPL-334, caused a marked reduction in airway hyperreactivity, allergen-specific T cells and eosinophil accumulation, and mucus production in the lungs in response to allergen inhalation. Moreover, SPL-334 treatment resulted in a significant decrease in the production of the Th2 cytokines IL-5 and IL-13 and the level of the chemokine CCL11 (eotaxin-1) in the airways. Collectively, these observations reveal that GSNOR inhibitors are effective not only in reducing airway hyperresponsiveness but also in limiting lung inflammatory responses mediated by CD4+ Th2 cells. These findings suggest that the inhibition of GSNOR may provide a novel therapeutic approach for the treatment of allergic airway inflammation. PMID:23936192

Histochemistry and cytochemistry of glucose-6-phosphate dehydrogenase.

PubMed

Van Noorden, C J

1984-01-01

Histochemistry and cytochemistry of glucose-6-phosphate dehydrogenase has found many applications in biomedical research. However, up to several years ago, the methods used often appeared to be unreliable because many artefacts occurred during processing and staining of tissue sections or cells. The development of histochemical methods preventing loss or redistribution of the enzyme by using either polyvinyl alcohol as a stabilizer or a semipermeable membrane interposed between tissue section and incubation medium, has lead to progress in the topochemical localization of glucose-6-phosphate dehydrogenase. Optimization of incubation conditions has further increased the precision of histochemical methods. Precise cytochemical methods have been developed either by the use of a polyacrylamide carrier in which individual cells have been incorporated before staining or by including polyvinyl alcohol in the incubation medium. In the present text, these methods for the histochemical and cytochemical localization of glucose-6-phosphate dehydrogenase for light microscopical and electron microscopical purposes are extensively discussed along with immunocytochemical techniques. Moreover, the validity of the staining methods is considered both for the localization of glucose-6-phosphate dehydrogenase activity in cells and tissues and for cytophotometric analysis. Finally, many applications of the methods are reviewed in the fields of functional heterogeneity of tissues, early diagnosis of carcinoma, effects of xenobiotics on cellular metabolism, diagnosis of inherited glucose-6-phosphate dehydrogenase deficiency, analysis of steroid-production in reproductive organs, and quality control of oocytes of mammals. It is concluded that the use of histochemistry and cytochemistry of glucose-6-phosphate dehydrogenase is of highly significant value in the study of diseased tissues. In many cases, the first pathological change is an increase in glucose-6-phosphate dehydrogenase activity and detection of these early changes in a few cells by histochemical means only, enables prediction of other subsequent abnormal metabolic events. Analysis of glucose-6-phosphate dehydrogenase deficiency in erythrocytes has been improved as well by the development of cytochemical tools. Heterozygous deficiency can now be detected in a reliable way. Cell biological studies of development or maturation of various tissues or cells have profited from the use of histochemistry and cytochemistry of glucose-6-phosphate dehydrogenase activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Alcohol and aldehyde dehydrogenase gene polymorphisms influence susceptibility to esophageal cancer in Japanese alcoholics.

PubMed

Yokoyama, A; Muramatsu, T; Omori, T; Matsushita, S; Yoshimizu, H; Higuchi, S; Yokoyama, T; Maruyama, K; Ishii, H

1999-11-01

Studies have consistently demonstrated that inactive aldehyde dehydrogenase-2 (ALDH2), encoded by ALDH2*1/2*2, is closely associated with alcohol-related carcinogenesis. Recently, the contributions of alcohol dehydrogenase-2 (ADH2) polymorphism to alcoholism, esophageal cancer, and the flushing response have also been described. To determine the effects of ALDH2 and ADH2 genotypes in genetically based cancer susceptibility, lymphocyte DNA samples from 668 Japanese alcoholic men more than 40 years of age (91 with and 577 without esophageal cancer) were genotyped and the results were expressed as odds ratios (ORs). This study also tested 82 of the alcoholics with esophageal cancer to determine whether cancer susceptibility is associated with patients' responses to simple questions about current or former flushing after drinking a glass of beer. The frequencies of ADH2*1/2*1 and ALDH2*1/2*2 were significantly higher in alcoholics with, than in those without, esophageal cancer (0.473 vs. 0.289 and 0.560 vs. 0.099, respectively). After adjustment for drinking and smoking, the analysis showed significantly increased cancer risk for alcoholics with either ADH2*1/2*I (OR = 2.03) or ALDH2*1/2*2 (OR = 12.76). For those having ADH2*1/2*1 combined with ALDH2*1/2*2, the esophageal cancer risk was enhanced in a multiplicative fashion (OR = 27.66). Responses to flushing questions showed that only 47.8% of the ALDH2*1/2*2 heterozygotes with ADH2*1/ 2*1, compared with 92.3% of those with ALDH2*1/2*2 and the ADH2*2 allele, reported current or former flushing. Genotyping showed that for alcoholics who reported ever flushing, the questionnaire was 71.4% correct in identifying ALDH2*1/2*2 and 87.9% correct in identifying ALDH2*1/2*1. Japanese alcoholics can be divided into cancer susceptibility groups on the basis of their combined ADH2 and ALDH2 genotypes. The flushing questionnaire can predict high risk ALDH2*1/2*2 fairly accurately in persons with ADH2*2 allele, but a reliable screening procedure for the highest risk gene combination (ADH2*1/2*1 and ALDH2*1/2*2) will require further investigation.

Electrophoretically mediated microanalysis of a nicotinamide adenine dinucleotide-dependent enzyme and its facile multiplexing using an active pixel sensor UV detector.

PubMed

Urban, Pawel L; Goodall, David M; Bergström, Edmund T; Bruce, Neil C

2007-08-31

An electrophoretically mediated microanalysis (EMMA) method has been developed for yeast alcohol dehydrogenase and quantification of reactant and product cofactors, NAD and NADH. The enzyme substrate ethanol (1% (v/v)) was added to the buffer (50 mM borate, pH 8.8). Results are presented for parallel capillary electrophoresis with a novel miniature UV area detector, with an active pixel sensor imaging an array of two or six parallel capillaries connected via a manifold to a single output capillary in a commercial CE instrument, allowing conversions with five different yeast alcohol dehydrogenase concentrations to be quantified in a single experiment.

Ethanol Extract of Pinus koraiensis Leaf Ameliorates Alcoholic Fatty Liver via the Activation of LKB1-AMPK Signaling In Vitro and In Vivo.

PubMed

Hong, Sang-Hyuk; Lee, Hyemin; Lee, Hyo-Jung; Kim, Bonglee; Nam, Min-Ho; Shim, Bum-Sang; Kim, Sung-Hoon

2017-05-01

Although Pinus koraiensis leaf (PKL) was reported for its anti-diabetes, anti-obesity and anticancer effects as a folk remedy, the inhibitory effect of PKL on alcoholic fatty liver has never been elucidated yet. This study investigated the molecular mechanisms of PKL on alcoholic fatty liver in HepG2 cells, Sprague Dawley (SD) rats and Imprinting Control Region (ICR) mice. Pinus koraiensis leaf increased phosphorylation of liver kinase B1 (LKB1)/AMP-activated protein kinase signaling, low-density lipoprotein receptor and decreased fatty acid biosynthesis-related proteins such as sterol regulatory element-binding protein 1c, fatty acid synthase, 3-hydroxy-3-methylglutaryl-CoA reductase in HepG2 cells. In SD rats with 25% alcohol-induced fatty liver, PKL suppressed the levels of aspartate aminotransferase and triglyceride and also enhanced the activities of antioxidant enzymes including superoxide dismutase, glutathione peroxidase and glutathione s-transferase compared with untreated control. Furthermore, PKL increased serum alcohol dehydrogenase and serum aldehyde dehydrogenase, but decreased serum alcohol concentration in ICR mice after alcohol administration. Consistently, histochemical analysis revealed that PKL attenuated alcohol-induced fatty liver in SD rats. Overall, these findings suggest that PKL ameliorates alcohol-induced fatty liver via activation of LKB1-AMP-activated protein kinase and modulation of proteins related to lipogenesis synthesis, cholesterol synthesis and fatty acid oxidation. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

The multifunctional isopropyl alcohol dehydrogenase of Phytomonas sp. could be the result of a horizontal gene transfer from a bacterium to the trypanosomatid lineage.

PubMed

Molinas, Sara M; Altabe, Silvia G; Opperdoes, Fred R; Rider, Mark H; Michels, Paul A M; Uttaro, Antonio D

2003-09-19

Isopropyl alcohol dehydrogenase (iPDH) is a dimeric mitochondrial alcohol dehydrogenase (ADH), so far detected within the Trypanosomatidae only in the genus Phytomonas. The cloning, sequencing, and heterologous expression of the two gene alleles of the enzyme revealed that it is a zinc-dependent medium-chain ADH. Both polypeptides have 361 amino acids. A mitochondrial targeting sequence was identified. The mature proteins each have 348 amino acids and a calculated molecular mass of 37 kDa. They differ only in one amino acid, which can explain the three isoenzymes and their respective isoelectric points previously found. A phylogenetic analysis locates iPDH within a cluster with fermentative ADHs from bacteria, sharing 74% similarity and 60% identity with Ralstonia eutropha ADH. The characterization of the two bacterially expressed Phytomonas enzymes and the comparison of their kinetic properties with those of the wild-type iPDH and of the R. eutropha ADH strongly support the idea of a horizontal gene transfer event from a bacterium to a trypanosomatid to explain the origin of the iPDH in Phytomonas. Phytomonas iPDH and R. eutropha ADH are able to use a wide range of substrates with similar Km values such as primary and secondary alcohols, diols, and aldehydes, as well as ketones such as acetone, diacetyl, and acetoin. We speculate that, as for R. eutropha ADH, Phytomonas iPDH acts as a safety valve for the release of excess reducing power.

Identification of Biomarker Genes To Predict Biodegradation of 1,4-Dioxane

PubMed Central

Gedalanga, Phillip B.; Pornwongthong, Peerapong; Mora, Rebecca; Chiang, Sheau-Yun Dora; Baldwin, Brett; Ogles, Dora

2014-01-01

Bacterial multicomponent monooxygenase gene targets in Pseudonocardia dioxanivorans CB1190 were evaluated for their use as biomarkers to identify the potential for 1,4-dioxane biodegradation in pure cultures and environmental samples. Our studies using laboratory pure cultures and industrial activated sludge samples suggest that the presence of genes associated with dioxane monooxygenase, propane monooxygenase, alcohol dehydrogenase, and aldehyde dehydrogenase are promising indicators of 1,4-dioxane biotransformation; however, gene abundance was insufficient to predict actual biodegradation. A time course gene expression analysis of dioxane and propane monooxygenases in Pseudonocardia dioxanivorans CB1190 and mixed communities in wastewater samples revealed important associations with the rates of 1,4-dioxane removal. In addition, transcripts of alcohol dehydrogenase and aldehyde dehydrogenase genes were upregulated during biodegradation, although only the aldehyde dehydrogenase was significantly correlated with 1,4-dioxane concentrations. Expression of the propane monooxygenase demonstrated a time-dependent relationship with 1,4-dioxane biodegradation in P. dioxanivorans CB1190, with increased expression occurring after over 50% of the 1,4-dioxane had been removed. While the fraction of P. dioxanivorans CB1190-like bacteria among the total bacterial population significantly increased with decrease in 1,4-dioxane concentrations in wastewater treatment samples undergoing active biodegradation, the abundance and expression of monooxygenase-based biomarkers were better predictors of 1,4-dioxane degradation than taxonomic 16S rRNA genes. This study illustrates that specific bacterial monooxygenase and dehydrogenase gene targets together can serve as effective biomarkers for 1,4-dioxane biodegradation in the environment. PMID:24632253

Pistacia lentiscus Oleoresin: Virtual Screening and Identification of Masticadienonic and Isomasticadienonic Acids as Inhibitors of 11β-Hydroxysteroid Dehydrogenase 1.

PubMed

Vuorinen, Anna; Seibert, Julia; Papageorgiou, Vassilios P; Rollinger, Judith M; Odermatt, Alex; Schuster, Daniela; Assimopoulou, Andreana N

2015-04-01

In traditional medicine, the oleoresinous gum of Pistacia lentiscus var. chia, so-called mastic gum, has been used to treat multiple conditions such as coughs, sore throats, eczema, dyslipidemia, and diabetes. Mastic gum is rich in triterpenes, which have been postulated to exert antidiabetic effects and improve lipid metabolism. In fact, there is evidence of oleanonic acid, a constituent of mastic gum, acting as a peroxisome proliferator-activated receptor γ agonist, and mastic gum being antidiabetic in mice in vivo. Despite these findings, the exact antidiabetic mechanism of mastic gum remains unknown. Glucocorticoids play a key role in regulating glucose and fatty acid metabolism, and inhibition of 11β-hydroxysteroid dehydrogenase 1 that converts inactive cortisone to active cortisol has been proposed as a promising approach to combat metabolic disturbances including diabetes. In this study, a pharmacophore-based virtual screening was applied to filter a natural product database for possible 11β-hydroxysteroid dehydrogenase 1 inhibitors. The hit list analysis was especially focused on the triterpenoids present in Pistacia species. Multiple triterpenoids, such as masticadienonic acid and isomasticadienonic acid, main constituents of mastic gum, were identified. Indeed, masticadienonic acid and isomasticadienonic acid selectively inhibited 11β-hydroxysteroid dehydrogenase 1 over 11β-hydroxysteroid dehydrogenase 2 at low micromolar concentrations. These findings suggest that inhibition of 11β-hydroxysteroid dehydrogenase 1 contributes to the antidiabetic activity of mastic gum. Georg Thieme Verlag KG Stuttgart · New York.

Association of Genetically Determined Aldehyde Dehydrogenase 2 Activity with Diabetic Complications in Relation to Alcohol Consumption in Japanese Patients with Type 2 Diabetes Mellitus: The Fukuoka Diabetes Registry

PubMed Central

Idewaki, Yasuhiro; Iwase, Masanori; Fujii, Hiroki; Ohkuma, Toshiaki; Ide, Hitoshi; Kaizu, Shinako; Jodai, Tamaki; Kikuchi, Yohei; Hirano, Atsushi; Nakamura, Udai; Kubo, Michiaki; Kitazono, Takanari

2015-01-01

Aldehyde dehydrogenase 2 (ALDH2) detoxifies aldehyde produced during ethanol metabolism and oxidative stress. A genetic defect in this enzyme is common in East Asians and determines alcohol consumption behaviors. We investigated the impact of genetically determined ALDH2 activity on diabetic microvascular and macrovascular complications in relation to drinking habits in Japanese patients with type 2 diabetes mellitus. An ALDH2 single-nucleotide polymorphism (rs671) was genotyped in 4,400 patients. Additionally, the relationship of clinical characteristics with ALDH2 activity (ALDH2 *1/*1 active enzyme activity vs. *1/*2 or *2/*2 inactive enzyme activity) and drinking habits (lifetime abstainers vs. former or current drinkers) was investigated cross-sectionally (n = 691 in *1/*1 abstainers, n = 1,315 in abstainers with *2, n = 1,711 in *1/*1 drinkers, n = 683 in drinkers with *2). The multiple logistic regression analysis for diabetic complications was adjusted for age, sex, current smoking habits, leisure-time physical activity, depressive symptoms, diabetes duration, body mass index, hemoglobin A1c, insulin use, high-density lipoprotein cholesterol, systolic blood pressure and renin-angiotensin system inhibitors use. Albuminuria prevalence was significantly lower in the drinkers with *2 than that of other groups (odds ratio [95% confidence interval (CI)]: *1/*1 abstainers as the referent, 0.94 [0.76–1.16] in abstainers with *2, 1.00 [0.80–1.26] in *1/*1 drinkers, 0.71 [0.54–0.93] in drinkers with *2). Retinal photocoagulation prevalence was also lower in drinkers with ALDH2 *2 than that of other groups. In contrast, myocardial infarction was significantly increased in ALDH2 *2 carriers compared with that in ALDH2 *1/*1 abstainers (odds ratio [95% CI]: *1/*1 abstainers as the referent, 2.63 [1.28–6.13] in abstainers with *2, 1.89 [0.89–4.51] in *1/*1 drinkers, 2.35 [1.06–5.79] in drinkers with *2). In summary, patients with type 2 diabetes and ALDH2 *2 displayed a lower microvascular complication prevalence associated with alcohol consumption but a higher macrovascular complication prevalence irrespective of alcohol consumption. PMID:26599441

Association of Genetically Determined Aldehyde Dehydrogenase 2 Activity with Diabetic Complications in Relation to Alcohol Consumption in Japanese Patients with Type 2 Diabetes Mellitus: The Fukuoka Diabetes Registry.

PubMed

Idewaki, Yasuhiro; Iwase, Masanori; Fujii, Hiroki; Ohkuma, Toshiaki; Ide, Hitoshi; Kaizu, Shinako; Jodai, Tamaki; Kikuchi, Yohei; Hirano, Atsushi; Nakamura, Udai; Kubo, Michiaki; Kitazono, Takanari

2015-01-01

Aldehyde dehydrogenase 2 (ALDH2) detoxifies aldehyde produced during ethanol metabolism and oxidative stress. A genetic defect in this enzyme is common in East Asians and determines alcohol consumption behaviors. We investigated the impact of genetically determined ALDH2 activity on diabetic microvascular and macrovascular complications in relation to drinking habits in Japanese patients with type 2 diabetes mellitus. An ALDH2 single-nucleotide polymorphism (rs671) was genotyped in 4,400 patients. Additionally, the relationship of clinical characteristics with ALDH2 activity (ALDH2 *1/*1 active enzyme activity vs. *1/*2 or *2/*2 inactive enzyme activity) and drinking habits (lifetime abstainers vs. former or current drinkers) was investigated cross-sectionally (n = 691 in *1/*1 abstainers, n = 1,315 in abstainers with *2, n = 1,711 in *1/*1 drinkers, n = 683 in drinkers with *2). The multiple logistic regression analysis for diabetic complications was adjusted for age, sex, current smoking habits, leisure-time physical activity, depressive symptoms, diabetes duration, body mass index, hemoglobin A1c, insulin use, high-density lipoprotein cholesterol, systolic blood pressure and renin-angiotensin system inhibitors use. Albuminuria prevalence was significantly lower in the drinkers with *2 than that of other groups (odds ratio [95% confidence interval (CI)]: *1/*1 abstainers as the referent, 0.94 [0.76-1.16] in abstainers with *2, 1.00 [0.80-1.26] in *1/*1 drinkers, 0.71 [0.54-0.93] in drinkers with *2). Retinal photocoagulation prevalence was also lower in drinkers with ALDH2 *2 than that of other groups. In contrast, myocardial infarction was significantly increased in ALDH2 *2 carriers compared with that in ALDH2 *1/*1 abstainers (odds ratio [95% CI]: *1/*1 abstainers as the referent, 2.63 [1.28-6.13] in abstainers with *2, 1.89 [0.89-4.51] in *1/*1 drinkers, 2.35 [1.06-5.79] in drinkers with *2). In summary, patients with type 2 diabetes and ALDH2 *2 displayed a lower microvascular complication prevalence associated with alcohol consumption but a higher macrovascular complication prevalence irrespective of alcohol consumption.

Purification and characterisation of a novel iso-propanol dehydrogenase from Phytomonas sp.

PubMed

Uttaro, A D; Opperdoes, F R

1997-04-01

An alcohol dehydrogenase with two identical subunits and a subunit molecular mass of 40,000 was purified from Phytomonas sp. isolated from the lactiferous tubes of Euphorbia characias. Digitonin titration and subcellular fractionation suggest that the enzyme is present in the mitochondrion. It utilises as substrates, primary and secondary alcohols, is specific for NAD+ as coenzyme and is inhibited by HgCl(2). The pH optimum for the oxidation of ethanol is 9.5, and for the reverse reaction 8.5. The apparent Km values for iso-propanol and ethanol are 40 and 34 microM, respectively and for the reverse reaction, with acetone as substrate, 14 microM. The respective specific activities with iso-propanol and ethanol as substrate, as measured in crude extracts are 300 and 16 mU (milligram of protein)-1. In isoelectric focusing the enzyme showed three major bands with slightly differing isoelectric points that ranged from 6.4 to 6.8. The name, iso-propanol dehydrogenase is proposed for this enzyme.

Effects of a 3β-hydroxysteroid Dehydrogenase Inhibitor, Trilostane, on the Fathead Minnow Reproductive Axis

EPA Science Inventory

A number of environmental contaminants and plant flavonoid compounds have been shown to inhibit the activity of 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase (3β-HSD). Because 3β-HSD plays a critical role in steroid hormone synthesis, inhibition of 3β-HSD represents a potentia...

Extreme halophilic alcohol dehydrogenase mediated highly efficient syntheses of enantiopure aromatic alcohols.

PubMed

Alsafadi, Diya; Alsalman, Safaa; Paradisi, Francesca

2017-11-07

Enzymatic synthesis of enantiopure aromatic secondary alcohols (including substituted, hetero-aromatic and bicyclic structures) was carried out using halophilic alcohol dehydrogenase ADH2 from Haloferax volcanii (HvADH2). This enzyme showed an unprecedented substrate scope and absolute enatioselectivity. The cofactor NADPH was used catalytically and regenerated in situ by the biocatalyst, in the presence of 5% ethanol. The efficiency of HvADH2 for the conversion of aromatic ketones was markedly influenced by the steric and electronic factors as well as the solubility of ketones in the reaction medium. Furthermore, carbonyl stretching band frequencies ν (C[double bond, length as m-dash]O) have been measured for different ketones to understand the effect of electron withdrawing or donating properties of the ketone substituents on the reaction rate catalyzed by HvADH2. Good correlation was observed between ν (C[double bond, length as m-dash]O) of methyl aryl-ketones and the reaction rate catalyzed by HvADH2. The enzyme catalyzed the reductions of ketone substrates on the preparative scale, demonstrating that HvADH2 would be a valuable biocatalyst for the preparation of chiral aromatic alcohols of pharmaceutical interest.

Synthesis of cinnamyl alcohol from cinnamaldehyde with Bacillus stearothermophilus alcohol dehydrogenase as the isolated enzyme and in recombinant E. coli cells.

PubMed

Pennacchio, Angela; Rossi, Mosè; Raia, Carlo A

2013-07-01

The synthesis of the aroma chemical cinnamyl alcohol (CMO) by means of enzymatic reduction of cinnamaldehyde (CMA) was investigated using NADH-dependent alcohol dehydrogenase from Bacillus stearothermophilus both as an isolated enzyme, and in recombinant Escherichia coli whole cells. The influence of parameters such as reaction time and cofactor, substrate, co-substrate 2-propanol and biocatalyst concentrations on the bioreduction reaction was investigated and an efficient and sustainable one-phase system developed. The reduction of CMA (0.5 g/L, 3.8 mmol/L) by the isolated enzyme occurred in 3 h at 50 °C with 97% conversion, and yielded high purity CMO (≥98%) with a yield of 88% and a productivity of 50 g/genzyme. The reduction of 12.5 g/L (94 mmol/L) CMA by whole cells in 6 h, at 37 °C and no requirement of external cofactor occurred with 97% conversion, 82% yield of 98% pure alcohol and a productivity of 34 mg/gwet cell weight. The results demonstrate the microbial system as a practical and efficient method for larger-scale synthesis of CMO.

Elucidating the contributions of multiple aldehyde/alcohol dehydrogenases to butanol and ethanol production in Clostridium acetobutylicum.

PubMed

Dai, Zongjie; Dong, Hongjun; Zhang, Yanping; Li, Yin

2016-06-20

Ethanol and butanol biosynthesis in Clostridium acetobutylicum share common aldehyde/alcohol dehydrogenases. However, little is known about the relative contributions of these multiple dehydrogenases to ethanol and butanol production respectively. The contributions of six aldehyde/alcohol dehydrogenases of C. acetobutylicum on butanol and ethanol production were evaluated through inactivation of the corresponding genes respectively. For butanol production, the relative contributions from these enzymes were: AdhE1 > BdhB > BdhA ≈ YqhD > SMB_P058 > AdhE2. For ethanol production, the contributions were: AdhE1 > BdhB > YqhD > SMB_P058 > AdhE2 > BdhA. AdhE1 and BdhB are two essential enzymes for butanol and ethanol production. AdhE1 was relatively specific for butanol production over ethanol, while BdhB, YqhD, and SMB_P058 favor ethanol production over butanol. Butanol synthesis was increased in the adhE2 mutant, which had a higher butanol/ethanol ratio (8.15:1) compared with wild type strain (6.65:1). Both the SMB_P058 mutant and yqhD mutant produced less ethanol without loss of butanol formation, which led to higher butanol/ethanol ratio, 10.12:1 and 10.17:1, respectively. To engineer a more efficient butanol-producing strain, adhE1 could be overexpressed, furthermore, adhE2, SMB_P058, yqhD are promising gene inactivation targets. This work provides useful information guiding future strain improvement for butanol production.

Comparative Study on Corrosion Protection of Reinforcing Steel by Using Amino Alcohol and Lithium Nitrite Inhibitors

PubMed Central

Lee, Han-Seung; Ryu, Hwa-Sung; Park, Won-Jun; Ismail, Mohamed A.

2015-01-01

In this study, the ability of lithium nitrite and amino alcohol inhibitors to provide corrosion protection to reinforcing steel was investigated. Two types of specimens—reinforcing steel and a reinforced concrete prism that were exposed to chloride ion levels resembling the chloride attack environment—were prepared. An autoclave accelerated corrosion test was then conducted. The variables tested included the chloride-ion concentration and molar ratios of anti-corrosion ingredients in a CaOH2-saturated aqueous solution that simulated a cement-pore solution. A concentration of 25% was used for the lithium nitrite inhibitor LiNO2, and an 80% solution of dimethyl ethanolamine ((CH3)2NCH2CH2OH, hereinafter DMEA) was used for the amino alcohol inhibitor. The test results indicated that the lithium nitrite inhibitor displayed anti-corrosion properties at a molar ratio of inhibitor of ≥0.6; the amino alcohol inhibitor also displayed anti-corrosion properties at molar ratios of inhibitor greater than approximately 0.3. PMID:28787936

Association between alcoholism and the dopamine D4 receptor gene.

PubMed Central

Muramatsu, T; Higuchi, S; Murayama, M; Matsushita, S; Hayashida, M

1996-01-01

A point mutation in the aldehyde dehydrogenase 2 gene (ALDH2(2) allele) is considered to be a genetic deterrent for alcoholism; however, 80 of 655 Japanese alcoholics had the mutant allele. Genotype factors that might increase susceptibility by overriding the deterrent showed a higher frequency of a five repeat allele of the dopamine D4 receptor 48 bp repeat polymorphism in alcoholics with ALDH2(2) than in 100 other alcoholics and 144 controls. Alcoholics with the five repeat allele also abused other drugs more often. These data suggest the involvement of the dopamine system in the development of alcoholism and other addictive behaviour. PMID:8929946

Identification of amino acid residues responsible for differences in substrate specificity and inhibitor sensitivity between two human liver dihydrodiol dehydrogenase isoenzymes by site-directed mutagenesis.

PubMed Central

Matsuura, K; Deyashiki, Y; Sato, K; Ishida, N; Miwa, G; Hara, A

1997-01-01

Human liver dihydrodiol dehydrogenase isoenzymes (DD1 and DD2), in which only seven amino acid residues are substituted, differ remarkably in specificity for steroidal substrates and inhibitor sensitivity: DD1 shows 20alpha-hydroxysteroid dehydrogenase activity and sensitivity to 1,10-phenanthroline, whereas DD2 oxidizes 3alpha-hydroxysteroids and is highly inhibited by bile acids. In the present study we performed site-directed mutagenesis of the seven residues (Thr-38, Arg-47, Leu-54, Cys-87, Val-151, Arg-170 and Gln-172) of DD1 to the corresponding residues (Val, His, Val, Ser, Met, His and Leu respectively) of DD2. Of the seven mutations, only the replacement of Leu-54 with Val produced an enzyme that had almost the same properties as DD2. No significant changes were observed in the other mutant enzymes. An additional site-directed mutagenesis of Tyr-55 of DD1 to Phe yielded an inactive protein, suggesting the catalytically important role of this residue. Thus a residue at a position before the catalytic Tyr residue might play a key role in determining the orientation of the substrates and inhibitors. PMID:9173902

Direct evidence that genetic variation in glycerol-3-phosphate and malate dehydrogenase genes (Gpdh and Mdh1) affects adult ethanol tolerance in Drosophila melanogaster.

PubMed

Eanes, Walter F; Merritt, Thomas J S; Flowers, Jonathan M; Kumagai, Seiji; Zhu, Chen-Tseh

2009-02-01

Many studies of alcohol adaptation in Drosophila melanogaster have focused on the Adh polymorphism, yet the metabolic elimination of alcohol should involve many enzymes and pathways. Here we evaluate the effects of glycerol-3-phosphate dehydrogenase (Gpdh) and cytosolic malate dehydrogenase (Mdh1) genotype activity on adult tolerance to ethanol. We have created a set of P-element-excision-derived Gpdh, Mdh1, and Adh alleles that generate a range of activity phenotypes from full to zero activity. Comparisons of paired Gpdh genotypes possessing 10 and 60% normal activity and 66 and 100% normal activity show significant effects where higher activity increases tolerance. Mdh1 null allele homozygotes show reductions in tolerance. We use piggyBac FLP-FRT site-specific recombination to create deletions and duplications of Gpdh. Duplications show an increase of 50% in activity and an increase of adult tolerance to ethanol exposure. These studies show that the molecular polymorphism associated with GPDH activity could be maintained in natural populations by selection related to adaptation to alcohols. Finally, we examine the interactions between activity genotypes for Gpdh, Mdh1, and Adh. We find no significant interlocus interactions. Observations on Mdh1 in both Gpdh and Adh backgrounds demonstrate significant increases in ethanol tolerance with partial reductions (50%) in cytosolic MDH activity. This observation strongly suggests the operation of pyruvate-malate and, in particular, pyruvate-citrate cycling in adaptation to alcohol exposure. We propose that an understanding of the evolution of tolerance to alcohols will require a system-level approach, rather than a focus on single enzymes.

Investigation of structure and function of mitochondrial alcohol dehydrogenase isozyme III from Komagataella phaffii GS115.

PubMed

Zhang, Huaidong; Li, Qin; Wang, Lina; Chen, Yan

2018-05-01

Alcohol dehydrogenases (ADHs) catalyze the reversible oxidation of alcohol using NAD + or NADP + as cofactor. Three ADH homologues have been identified in Komagataella phaffii GS115 (also named Pichia pastoris GS115), ADH1, ADH2 and ADH3, among which adh3 is the only gene responsible for consumption of ethanol in Komagataella phaffii GS115. However, the relationship between structure and function of mitochondrial alcohol dehydrogenase isozyme III from Komagataella phaffii GS115 (KpADH3) is still not clear yet. KpADH3 was purified, identified and characterized by multiple biophysical techniques (Nano LC-MS/MS, Enzymatic activity assay, X-ray crystallography). The crystal structure of KpADH3, which was the first ADH structure from Komagataella phaffii GS115, was solved at 1.745 Å resolution. Structural analysis indicated that KpADH3 was the sole dimeric ADH structure with face-to-face orientation quaternary structure from yeast. The major structural different conformations located on residues 100-114 (the structural zinc binding loop) and residues 337-344 (the loop between α12 and β15 which covered the catalytic domain). In addition, three channels were observed in KpADH3 crystal structure, channel 2 and channel 3 may be essential for substrate specific recognition, ingress and egress, channel 1 may be the pass-through for cofactor. KpADH3 plays an important role in the metabolism of alcohols in Komagataella phaffii GS115, and its crystal structure is the only dimeric medium-chain ADH from yeast described so far. Knowledge of the relationship between structure and function of KpADH3 is crucial for understanding the role of KpADH3 in Komagataella phaffii GS115 mitochondrial metabolism. Copyright © 2018 Elsevier B.V. All rights reserved.

Thermostable Alcohol Dehydrogenase from Thermococcus kodakarensis KOD1 for Enantioselective Bioconversion of Aromatic Secondary Alcohols

PubMed Central

Wu, Xi; Zhang, Chong; Orita, Izumi; Imanaka, Tadayuki

2013-01-01

A novel thermostable alcohol dehydrogenase (ADH) showing activity toward aromatic secondary alcohols was identified from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1 (TkADH). The gene, tk0845, which encodes an aldo-keto reductase, was heterologously expressed in Escherichia coli. The enzyme was found to be a monomer with a molecular mass of 31 kDa. It was highly thermostable with an optimal temperature of 90°C and a half-life of 4.5 h at 95°C. The apparent Km values for the cofactors NAD(P)+ and NADPH were similar within a range of 66 to 127 μM. TkADH preferred secondary alcohols and accepted various ketones and aldehydes as substrates. Interestingly, the enzyme could oxidize 1-phenylethanol and its derivatives having substituents at the meta and para positions with high enantioselectivity, yielding the corresponding (R)-alcohols with optical purities of greater than 99.8% enantiomeric excess (ee). TkADH could also reduce 2,2,2-trifluoroacetophenone to (R)-2,2,2-trifluoro-1-phenylethanol with high enantioselectivity (>99.6% ee). Furthermore, the enzyme showed high resistance to organic solvents and was particularly highly active in the presence of H2O–20% 2-propanol and H2O–50% n-hexane or n-octane. This ADH is expected to be a useful tool for the production of aromatic chiral alcohols. PMID:23354700

Synthesis and evaluation of conformationally restricted inhibitors of aspartate semialdehyde dehydrogenase.

PubMed

Evitt, Andrew S; Cox, Russell J

2011-05-01

Inhibitors of the enzyme aspartate semialdehyde dehydrogenase, a key biological target for the generation of a new class of antibiotic compounds, have been developed. To investigate improvements to binding within an inhibitor series, the lowering of the entropic barrier to binding through conformational restriction was investigated. A library of linear and cyclic substrate analogues was generated and computational docking used to aid in structure selection. The cyclic phosphonate inhibitor 18 was thus identified as complimentary to the enzyme active-site. Synthesis and in vitro inhibition assay revealed a K(i) of 3.8 mM against natural substrate, where the linear analogue of 18, compound 15, had previously shown no inhibitory activity. Two further inhibitors, phosphate analogue diastereoisomers 17a and 17b, were synthesised and also found to have low millimolar K(i) values. As a result of the computational docking investigations, a novel substrate binding interaction was discovered: hydrogen bonding between the substrate (phosphate hydroxy-group as the hydrogen bond donor) and the NADPH cofactor (2'-oxygen as the hydrogen bond acceptor).

Triazole inhibitors of Cryptosporidium parvum inosine 5′-monophosphate dehydrogenase

PubMed Central

Maurya, Sushil K.; Gollapalli, Deviprasad R.; Kirubakaran, Sivapriya; Zhang, Minjia; Johnson, Corey R.; Benjamin, Nicole N.; Hedstrom, Lizbeth; Cuny, Gregory D.

2010-01-01

Cryptosporidium parvum is an important human pathogen and potential bioterrorism agent. This protozoan parasite cannot salvage guanine or guanosine and therefore relies on inosine 5′-monophosphate dehydrogenase (IMPDH) for biosynthesis of guanine nucleotides and hence for survival. Since C. parvum IMPDH is highly divergent from the host counterpart, selective inhibitors could potentially be used to treat cryptosporidiosis with minimal effects on its mammalian host. A series of 1,2,3-triazole containing ether CpIMPDH inhibitors are described. A structure-activity relationship study revealed that a small alkyl group on the alpha-position of the ether was required with the (R)-enantiomer significantly more active than the (S)-enantiomer. Electron-withdrawing groups in the 3- and/or 4-positions of the pendent phenyl ring were best and conversion of the quinoline containing inhibitors to quinoline-N-oxides retained inhibitory activity both in the presence and absence of bovine serum albumin. The 1,2,3-triazole CpIMPDH inhibitors provide new tools for elucidating the role of IMPDH in C. parvum and may serve as potential therapeutics for treating cryptosporidiosis. PMID:19624136

Lactate dehydrogenase inhibition: exploring possible applications beyond cancer treatment.

PubMed

Di Stefano, Giuseppina; Manerba, Marcella; Di Ianni, Lorenza; Fiume, Luigi

2016-04-01

Lactate dehydrogenase (LDH) inhibition is considered a worthwhile attempt in the development of innovative anticancer strategies. Unfortunately, in spite of the involvement of several research institutions and pharma-companies, the discovery of LDH inhibitors with drug-like properties seems a hardly resolvable challenge. While awaiting new advancements, in the present review we will examine other pathologic conditions characterized by increased glycolysis and LDH activity, which could potentially benefit from LDH inhibition. The rationale for targeting LDH activity in these contexts is the same justifying the LDH-based approach in anticancer therapy: because of the enzyme position at the end of glycolytic pathway, LDH inhibitors are not expected to hinder glucose metabolism of normal cells. Moreover, we will summarize the latest contributions in the discovery of enzyme inhibitors and try to glance over the reasons underlying the complexity of this research.

Acute Ethanol Causes Hepatic Mitochondrial Depolarization in Mice: Role of Ethanol Metabolism

PubMed Central

Zhong, Zhi; Ramshesh, Venkat K.; Rehman, Hasibur; Liu, Qinlong; Theruvath, Tom P.; Krishnasamy, Yasodha; Lemasters, John J.

2014-01-01

Background/Aims An increase of ethanol metabolism and hepatic mitochondrial respiration occurs in vivo after a single binge of alcohol. Here, our aim was to determine how ethanol intake affects hepatic mitochondrial polarization status in vivo in relation to ethanol metabolism and steatosis. Methods Hepatic mitochondrial polarization, permeability transition (MPT), and reduce pyridine nucleotides, and steatosis in mice were monitored by intravital confocal/multiphoton microscopy of the fluorescence of rhodamine 123 (Rh123), calcein, NAD(P)H, and BODIPY493/503, respectively, after gavage with ethanol (1–6 g/kg). Results Mitochondria depolarized in an all-or-nothing fashion in individual hepatocytes as early as 1 h after alcohol. Depolarization was dose- and time-dependent, peaked after 6 to 12 h and maximally affected 94% of hepatocytes. This mitochondrial depolarization was not due to onset of the MPT. After 24 h, mitochondria of most hepatocytes recovered normal polarization and were indistinguishable from untreated after 7 days. Cell death monitored by propidium iodide staining, histology and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was low throughout. After alcohol, mitochondrial NAD(P)H autofluorescence increased and decreased, respectively, in hepatocytes with polarized and depolarized mitochondria. Ethanol also caused steatosis mainly in hepatocytes with depolarized mitochondria. Depolarization was linked to ethanol metabolism, since deficiency of alcohol dehydrogenase and cytochrome-P450 2E1 (CYP2E1), the major ethanol-metabolizing enzymes, decreased mitochondrial depolarization by ∼70% and ∼20%, respectively. Activation of aldehyde dehydrogenase decreased depolarization, whereas inhibition of aldehyde dehydrogenase enhanced depolarization. Activation of aldehyde dehydrogenase also markedly decreased steatosis. Conclusions Acute ethanol causes reversible hepatic mitochondrial depolarization in vivo that may contribute to steatosis and increased mitochondrial respiration. Onset of this mitochondrial depolarization is linked, at least in part, to metabolism of ethanol to acetaldehyde. PMID:24618581

Establishment of the Tree Shrew as an Alcohol-Induced Fatty Liver Model for the Study of Alcoholic Liver Diseases

PubMed Central

Xing, Huijie; Jia, Kun; He, Jun; Shi, Changzheng; Fang, Meixia; Song, Linliang; Zhang, Pu; Zhao, Yue; Fu, Jiangnan; Li, Shoujun

2015-01-01

Currently, the pathogenesis of alcoholic liver diseases (ALDs) is not clear. As a result, there is no effective treatment for ALDs. One limitation is the lack of a suitable animal model for use in studying ALDs. The tree shrew is a lower primate animal, characterized by a high-alcohol diet. This work aimed to establish a fatty liver model using tree shrews and to assess the animals’ suitability for the study of ALDs. Tree shrews were treated with alcohol solutions (10% and 20%) for two weeks. Hemophysiology, blood alcohol concentrations (BACs), oxidative stress factors, alcohol metabolic enzymes and hepatic pathology were checked and assayed with an automatic biochemical analyzer, enzyme-linked immunosorbent assay (ELISA), western blot, hematoxylin-eosin (HE) staining and oil red O staining, and magnetic resonance imaging (MRI). Compared with the normal group, the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transpeptidase (GGT), total cholesterol (TC), triglyceride (TG), reactive oxygen species (ROS), and malondialdehyde (MDA) were significantly enhanced in alcohol-treated tree shrews. However, the activity of reduced glutathione hormone (GSH) and superoxide dismutase (SOD) declined. Notable changes in alcohol dehydrogenase(ADH1), aldehyde dehydrogenase(ALDH2), CYP2E1, UDP-glucuronosyl transferase 1A1 (UGT1A1) and nuclear factor erythroid-related factor 2 (Nrf2) were observed. HE and oil red O staining showed that hepatocyte swelling, hydropic degeneration, and adipohepatic syndrome occurred in the tree shrews. Alcohol can induce fatty liver-like pathological changes and result in alterations in liver function, oxidative stress factors, alcohol metabolism enzymes and Nrf2. Therefore, the established fatty liver model of tree shrews induced by alcohol should be a promising tool for the study of ALDs. PMID:26030870

Salivary alcohol dehydrogenase in non-smoking and smoking alcohol-dependent persons.

PubMed

Waszkiewicz, Napoleon; Jelski, Wojciech; Zalewska, Anna; Szulc, Agata; Szmitkowski, Maciej; Zwierz, Krzysztof; Szajda, Sławomir Dariusz

2014-09-01

Increasing attention to the importance of saliva testing is not surprising because smoking and alcohol drinking act synergistically on oral tissues, and their metabolite levels, e.g., acetaldehyde, are much higher in saliva than in blood. The activity of salivary alcohol dehydrogenase (ADH) comes from oral microbiota, mucosa, and salivary glands. The purpose of this study was to investigate the involvement of ADH in the oral health pathology of smoking (AS) and non-smoking (ANS) alcohol-dependent males. The results indicated that the AS group had a more significant and longer duration (until the 30th day of alcohol abstinence) decrease in ADH activity and output than the ANS group (until the 15th day of alcohol abstinence) compared to controls (social drinkers; C). The decreased salivary flow (SF) in alcoholics was observed longer in the ANS group (until the 30th day of alcohol abstinence), whereas in the AS group SF normalized at the 15th day, probably due to the irritating effect of tobacco smoke on the oral mucosa. Because saliva was centrifuged to remove cells and debris (including microbial cells), the detected salivary ADH activity was derived from salivary glands and/or oral mucosa. A more profound and longer decrease in ADH activity/output in smoking than non-smoking alcoholics was likely due to the damaged salivary glands and/or oral mucosa, caused by the synergistic effect of alcohol drinking and smoking. The lower values of salivary ADH in smoking than non-smoking alcoholics might also be partly due to the reversed/inhibited ADH reaction by high levels of accumulated acetaldehyde. Copyright © 2014 Elsevier Inc. All rights reserved.

Protective effect of Flos puerariae extract following acute alcohol intoxication in mice.

PubMed

Chen, Xiao; Cai, Fei; Guo, Shuang; Ding, Fang; He, Yi; Wu, Jiliang; Liu, Chao

2014-07-01

The effect of Flos Puerariae extract (FPE) on alcohol metabolism, hepatic injury, and memory impairment was assessed following acute ethanol (EtOH) intoxication in mice. The model of acute EtOH intoxication was established by intragastric administration with 8 g/kg EtOH in mice. FPE was orally administrated (gavage) once a day for 7 consecutive days. Mice were randomly divided into 4 groups: control group, model group, and FPE groups (100, 200 mg/kg). Alcohol tolerance and intoxication time, blood alcohol concentration, the activities of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) in liver, aspartate amino transferase (AST) and alanine amino transferase (ALT) in serum, superoxide dismutase (SOD), glutathione peroxidase (GSH-px), catalase and the formation of malondialdehyde (MDA) in both liver and brain, as well as memory ability were determined after acute alcohol exposure. Compared with model group, pretreatment with FPE significantly prolonged alcohol tolerance time and shortened intoxication time, which is accompanied by decreased blood alcohol concentration and elevated activities of ADH and ALDH in liver. Moreover, the index of hepatic injury, ALT, and AST activities in serum was markedly decreased by pretreatment with FPE. Additionally, decreased MDA level, enhanced GSH-px and catalase activities in liver, as well as enhanced SOD and catalase activities in brain were found in FPE pretreated mice after acute exposure to EtOH. Furthermore, FPE pretreated mice showed markedly relieved memory disruption following acute EtOH intoxication. This study suggests that FPE pretreatment could enhance alcohol metabolism, prevent hepatic injury, and relieve memory impairment after acute alcohol intoxication and that this effect is likely related to its modulation on the alcohol metabolizing and antioxidant enzymes. Copyright © 2014 by the Research Society on Alcoholism.

COMPARISON OF IN VIVO AND IN VITRO METHODS FOR ASSESSING EFFECTS OF ALLYL ALCOHOL ON THE LIVER

EPA Science Inventory

Allyl alcohol was administered to female Fischer 344 rats at doses of 0, 3, 10 and 30 mg/kg daily for 7 days. Plasma sorbitol dehydrogenase was minimally elevated. No dose related changes were observed in hexobarbital oxidation, aniline hydroxylation, or ethylmorphine demethylati...

Identification of fused 16β,17β-oxazinone-estradiol derivatives as a new family of non-estrogenic 17β-hydroxysteroid dehydrogenase type 1 inhibitors.

PubMed

Maltais, René; Trottier, Alexandre; Delhomme, Audrey; Barbeau, Xavier; Lagüe, Patrick; Poirier, Donald

2015-03-26

A new family of cyclic carbamate-estradiol derivatives has been designed to remove the intrinsic estrogenic activity of a parent acyclic compound reported as a potent inhibitor of 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1). The synthesis of two series of fused 16β,17β-oxazinone-estradiol derivatives, saturated compounds 7a-d and unsaturated compounds 10a-d, led to the identification of 10b, a 17β-HSD1 inhibitor (IC50 = 1.4 μM) without estrogenic activity in estrogen-sensitive T-47D cells. Interestingly, this compound was found selective over 17β-HSD2 and 17β-HSD12. A computational analysis of inhibitors into 17β-HSD1 by molecular docking also revealed interesting structure-activity relationships that could be helpful in the design of new generation of 16β,17β-oxazinone-estradiol analogs. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Selective and potent urea inhibitors of Cryptosporidium parvum inosine 5′ monophosphate dehydrogenase

PubMed Central

Gorla, Suresh Kumar; Kavitha, Mandapati; Zhang, Minjia; Liu, Xiaoping; Sharling, Lisa; Gollapalli, Deviprasad R.; Striepen, Boris; Hedstrom, Lizbeth; Cuny, Gregory D.

2012-01-01

Cryptosporidium parvum and related species are zoonotic intracellular parasites of the intestine. Cryptosporidium is a leading cause of diarrhea in small children around the world. Infection can cause severe pathology in children and immunocompromised patients. This waterborne parasite is resistant to common methods of water treatment and therefore a prominent threat to drinking and recreation water even in countries with strong water safety systems. The drugs currently used to combat these organisms are ineffective. Genomic analysis revealed that the parasite relies solely on inosine-5′-monophosphate dehydrogenase (IMPDH) for the biosynthesis of guanine nucleotides. Herein, we report a selective urea-based inhibitor of C. parvum IMPDH (CpIMPDH) identified by high throughput screening. We performed a SAR study of these inhibitors with some analogues exhibiting high potency (IC50 < 2 nM) against CpIMPDH, excellent selectivity > 1000-fold versus human IMPDH type 2 and good stability in mouse liver microsomes. A subset of inhibitors also displayed potent antiparasitic activity in a Toxoplasma gondii model. PMID:22950983

Molecular Basis of Alcohol-Related Gastric and Colon Cancer.

PubMed

Na, Hye-Kyung; Lee, Ja Young

2017-05-24

Many meta-analysis, large cohort studies, and experimental studies suggest that chronic alcohol consumption increases the risk of gastric and colon cancer. Ethanol is metabolized by alcohol dehydrogenases (ADH), catalase or cytochrome P450 2E1 (CYP2E1) to acetaldehyde, which is then further oxidized to acetate by aldehyde dehydrogenase (ALDH). Acetaldehyde has been classified by the International Agency for Research on Cancer (IARC) as a Group 1 carcinogen to humans. The acetaldehyde level in the stomach and colon is locally influenced by gastric colonization by Helicobacter pylori or colonic microbes, as well as polymorphisms in the genes encoding tissue alcohol metabolizing enzymes, especially ALDH2. Alcohol stimulates the uptake of carcinogens and their metabolism and also changes the composition of enteric microbes in a way to enhance the aldehyde level. Alcohol also undergoes chemical coupling to membrane phospholipids and disrupts organization of tight junctions, leading to nuclear translocation of β-catenin and ZONAB, which may contributes to regulation of genes involved in proliferation, invasion and metastasis. Alcohol also generates reactive oxygen species (ROS) by suppressing the expression of antioxidant and cytoprotective enzymes and inducing expression of CYP2E1 which contribute to the metabolic activation of chemical carcinogens. Besides exerting genotoxic effects by directly damaging DNA, ROS can activates signaling molecules involved in inflammation, metastasis and angiogenesis. In addition, alcohol consumption induces folate deficiency, which may result in aberrant DNA methylation profiles, thereby influencing cancer-related gene expression.

Genetic analyses and molecular characterization of the pathways involved in the conversion of 2-phenylethylamine and 2-phenylethanol into phenylacetic acid in Pseudomonas putida U.

PubMed

Arias, Sagrario; Olivera, Elías R; Arcos, Mario; Naharro, Germán; Luengo, José M

2008-02-01

In Pseudomonas putida U two different pathways (Pea, Ped) are required for the conversion of 2-phenylethylamine and 2-phenylethanol into phenylacetic acid. The 2-phenylethylamine pathway (PeaABCDEFGHR) catalyses the transport of this amine, its deamination to phenylacetaldehyde by a quinohaemoprotein amine dehydrogenase and the oxidation of this compound through a reaction catalysed by a phenylacetaldehyde dehydrogenase. Another catabolic route (PedS(1)R(1)ABCS(2)R(2)DEFGHI) is needed for the uptake of 2-phenylethanol and for its oxidation to phenylacetic acid via phenylacetaldehyde. This implies the participation of two different two-component signal-transducing systems, two quinoprotein alcohol dehydrogenases, a cytochrome c, a periplasmic binding protein, an aldehyde dehydrogenase, a pentapeptide repeat protein and an ABC efflux system. Additionally, two accessory sets of elements (PqqABCDEF and CcmABCDEFGHI) are necessary for the operation of the main pathways (Pea and Ped). PqqABCDEF is required for the biosynthesis of pyrroloquinoline quinone (PQQ), a prosthetic group of certain alcohol dehydrogenases that transfers electrons to an independent cytochrome c; whereas CcmABCDEFGHI is required for cytochrome c maturation. Our data show that the degradation of phenylethylamine and phenylethanol in P. putida U is quite different from that reported in Escherichia coli, and they demonstrate that PeaABCDEFGHR and PedS(1)R(1)ABCS(2)R(2)DEFGHI are two upper routes belonging to the phenylacetyl-CoA catabolon.

Aldehyde dehydrogenase polymorphism in North American, South American, and Mexican Indian populations.

PubMed Central

Goedde, H W; Agarwal, D P; Harada, S; Rothhammer, F; Whittaker, J O; Lisker, R

1986-01-01

While about 40% of the South American Indian populations (Atacameños, Mapuche, Shuara) were found to be deficient in aldehyde dehydrogenase isozyme I (ALDH2 or E2), preliminary investigations showed very low incidence of isozyme deficiency among North American natives (Sioux, Navajo) and Mexican Indians (mestizo). Possible implications of such trait differences on cross-cultural behavioral response to alcohol drinking are discussed. PMID:3953578

Elucidation of the Mechanisms and Environmental Relevance of cis-Dichloroethene and Vinyl Chloride Biodegradation

DTIC Science & Technology

2012-11-01

in iTRAQ study Table 3-12: Ratio of aldehyde and alcohol dehydrogenases from iTRAQ study Table 3-13: Proteins involved in proposed glyoxal...pathway for cDCE degradation proceeds through chloro-, or dichloroacetaldehyde. Multiple aldehyde dehydrogenases are annotated in the genome of...dichloroacetaldehyde is a fortuitous reaction. However, if the initial reaction is monooxygenation to an aldehyde then the true intermediate would be

Pyruvate dehydrogenase complex and lactate dehydrogenase are targets for therapy of acute liver failure.

PubMed

Ferriero, Rosa; Nusco, Edoardo; De Cegli, Rossella; Carissimo, Annamaria; Manco, Giuseppe; Brunetti-Pierri, Nicola

2018-03-24

Acute liver failure is a rapidly progressive deterioration of hepatic function resulting in high mortality and morbidity. Metabolic enzymes can translocate to the nucleus to regulate histone acetylation and gene expression. Levels and activities of pyruvate dehydrogenase complex (PDHC) and lactate dehydrogenase (LDH) were evaluated in nuclear fractions of livers of mice exposed to various hepatotoxins including CD95-antibody, α-amanitin, and acetaminophen. Whole-genome gene expression profiling by RNA-seq was performed in livers of mice with acute liver failure and analyzed by gene ontology enrichment analysis. Cell viability was evaluated in cell lines knocked-down for PDHA1 or LDH-A and in cells incubated with the LDH inhibitor galloflavin after treatment with CD95-antibody. We evaluated whether the histone acetyltransferase inhibitor garcinol or galloflavin could reduce liver damage in mice with acute liver failure. Levels and activities of PDHC and LDH were increased in nuclear fractions of livers of mice with acute liver failure. The increase of nuclear PDHC and LDH was associated with increased concentrations of acetyl-CoA and lactate in nuclear fractions, and histone H3 hyper-acetylation. Gene expression in livers of mice with acute liver failure suggested that increased histone H3 acetylation induces the expression of genes related to damage response. Reduced histone acetylation by the histone acetyltransferase inhibitor garcinol decreased liver damage and improved survival in mice with acute liver failure. Knock-down of PDHC or LDH improved viability in cells exposed to a pro-apoptotic stimulus. Treatment with the LDH inhibitor galloflavin that was also found to inhibit PDHC, reduced hepatic necrosis, apoptosis, and expression of pro-inflammatory cytokines in mice with acute liver failure. Mice treated with galloflavin also showed a dose-response increase in survival. PDHC and LDH translocate to the nucleus, leading to increased nuclear concentrations of acetyl-CoA and lactate. This results in histone H3 hyper-acetylation and expression of damage response genes. Inhibition of PDHC and LDH reduces liver damage and improves survival in mice with acute liver failure. Thus, PDHC and LDH are targets for therapy of acute liver failure. Acute liver failure is a rapidly progressive deterioration of liver function resulting in high mortality. In experimental mouse models of acute liver failure, we found that two metabolic enzymes, namely pyruvate dehydrogenase complex and lactic dehydrogenase, translocate to the nucleus resulting in detrimental gene expression. Treatment with an inhibitor of these two enzymes was found to reduce liver damage and to improve survival. Copyright © 2018 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Polymorphism of ethanol-metabolism genes and alcoholism: correlation of allelic variations with the pharmacokinetic and pharmacodynamic consequences.

PubMed

Chen, Yi-Chyan; Peng, Giia-Sheun; Wang, Ming-Fang; Tsao, Tien-Ping; Yin, Shih-Jiun

2009-03-16

Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) are the principal enzymes responsible for metabolism of ethanol. Both ADH and ALDH exhibit genetic polymorphisms among racial populations. Functional variant alleles ADH1B*2 and ALDH2*2 have been consistently replicated to show protection against developing alcohol dependence. Multiple logistic regression analyses suggest that ADH1B*2 and ALDH2*2 may independently influence the risk for alcoholism. It has been well documented that homozygosity of ALDH2*2 almost fully protects against developing alcoholism and that the heterozygosity only affords a partial protection to varying degrees. Correlations of blood ethanol and acetaldehyde concentrations, cardiovascular hemodynamic responses, and subjective perceptions have been investigated in men with different combinatorial ADH1B and ALDH2 genotypes following challenge with ethanol for a period of 130 min. The pharmacokinetic and pharmacodynamic consequences indicate that acetaldehyde, rather than ethanol, is primarily responsible for the observed alcohol sensitivity reactions, suggesting that the full protection by ALDH2*2/*2 can be ascribed to the intense unpleasant physiological and psychological reactions caused by persistently elevated blood acetaldehyde after ingesting a small amount of alcohol and that the partial protection by ALDH2*1/*2 can be attributed to a faster elimination of acetaldehyde and the lower accumulation in circulation. ADH1B polymorphism does not significantly contribute to buildup of the blood acetaldehyde. Physiological tolerance or innate insensitivity to acetaldehyde may be crucial for development of alcohol dependence in alcoholics carrying ALDH2*2.

Formate generated by cellular oxidation of formaldehyde accelerates the glycolytic flux in cultured astrocytes.

PubMed

Tulpule, Ketki; Dringen, Ralf

2012-04-01

Formaldehyde is a neurotoxic compound that can be endogenously generated in the brain. Because astrocytes play a key role in metabolism and detoxification processes in brain, we have investigated the capacity of these cells to metabolize formaldehyde using primary astrocyte-rich cultures as a model system. Application of formaldehyde to these cultures resulted in the appearance of formate in cells and in a time-, concentration- and temperature-dependent disappearance of formaldehyde from the medium that was accompanied by a matching extracellular accumulation of formate. This formaldehyde-oxidizing capacity of astrocyte cultures is likely to be catalyzed by alcohol dehydrogenase 3 and aldehyde dehydrogenase 2, because the cells of the cultures contain the mRNAs of these formaldehyde-oxidizing enzymes. In addition, exposure to formaldehyde increased both glucose consumption and lactate production by the cells. Both the strong increase in the cellular formate content and the increase in glycolytic flux were only observed after application of formaldehyde to the cells, but not after treatment with exogenous methanol or formate. The accelerated lactate production was not additive to that obtained for azide, a known inhibitor of complex IV of the respiratory chain, and persisted after removal of formaldehyde after a formaldehyde exposure for 1.5 h. These data demonstrate that cultured astrocytes efficiently oxidize formaldehyde to formate, which subsequently enhances glycolytic flux, most likely by inhibition of mitochondrial respiration. Copyright © 2012 Wiley Periodicals, Inc.

Calcium Channels and Oxidative Stress Mediate a Synergistic Disruption of Tight Junctions by Ethanol and Acetaldehyde in Caco-2 Cell Monolayers.

PubMed

Samak, Geetha; Gangwar, Ruchika; Meena, Avtar S; Rao, Roshan G; Shukla, Pradeep K; Manda, Bhargavi; Narayanan, Damodaran; Jaggar, Jonathan H; Rao, RadhaKrishna

2016-12-13

Ethanol is metabolized into acetaldehyde in most tissues. In this study, we investigated the synergistic effect of ethanol and acetaldehyde on the tight junction integrity in Caco-2 cell monolayers. Expression of alcohol dehydrogenase sensitized Caco-2 cells to ethanol-induced tight junction disruption and barrier dysfunction, whereas aldehyde dehydrogenase attenuated acetaldehyde-induced tight junction disruption. Ethanol up to 150 mM did not affect tight junction integrity or barrier function, but it dose-dependently increased acetaldehyde-mediated tight junction disruption and barrier dysfunction. Src kinase and MLCK inhibitors blocked this synergistic effect of ethanol and acetaldehyde on tight junction. Ethanol and acetaldehyde caused a rapid and synergistic elevation of intracellular calcium. Calcium depletion by BAPTA or Ca 2+ -free medium blocked ethanol and acetaldehyde-induced barrier dysfunction and tight junction disruption. Diltiazem and selective knockdown of TRPV6 or Ca V 1.3 channels, by shRNA blocked ethanol and acetaldehyde-induced tight junction disruption and barrier dysfunction. Ethanol and acetaldehyde induced a rapid and synergistic increase in reactive oxygen species by a calcium-dependent mechanism. N-acetyl-L-cysteine and cyclosporine A, blocked ethanol and acetaldehyde-induced barrier dysfunction and tight junction disruption. These results demonstrate that ethanol and acetaldehyde synergistically disrupt tight junctions by a mechanism involving calcium, oxidative stress, Src kinase and MLCK.

The TosMIC approach to 3-(oxazol-5-yl) indoles: application to the synthesis of indole-based IMPDH inhibitors.

PubMed

Dhar, T G Murali; Shen, Zhongqi; Fleener, Catherine A; Rouleau, Katherine A; Barrish, Joel C; Hollenbaugh, Diane L; Iwanowicz, Edwin J

2002-11-18

A modified approach to the synthesis of 3-(oxazolyl-5-yl) indoles is reported. This method was applied to the synthesis of series of novel indole based inhibitors of inosine monophosphate dehydrogenase (IMPDH). The synthesis and the structure-activity relationships (SARs), derived from in vitro studies, for this new series of inhibitors is given.

In-vitro Wound Healing Effect of 15-Hydroxyprostaglandin Dehydrogenase Inhibitor from Plant

PubMed Central

Karna, Sandeep

2017-01-01

Background: Prostaglandins (PGs) have short existence in vivo because they are rapidly metabolized by NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) to 15-ketoprostaglandins. Inhibition of 15-PGDH causes elevated level of PGE2 in cellular system. It will be valuable for the therapeutic management of diseases requiring elevated PGE2 levels, like wound healing. Objective: Ninety-eight plant samples were screened for the discovery of potent 15-PGDH inhibitor. Among them, top five plant extracts as potent 15-PGDH inhibitor were chosen to determine PGE2 release from HaCaT (Keratinocyte cell line) cell line. Finally, top 15-PGDH inhibitor was selected to evaluate in vitro wound healing effect on HaCaT scratch model. Method: The inhibitory activity for 15-PGDH inhibitors was evaluated using fluorescence spectrophotometer by measuring the formation of NADH at 468 nm following excitation at 340 nm. Cell viability assay and PGE2 release was evaluated in HaCaT cell line after treatment of 15-PGDH inhibitors. Scratches were made using sterile 200 μL on HaCaT cell and wound-healing effect was evaluated after treatment of 15-PGDH inhibitor. Results: 15-PGDH inhibitors elevated PGE2 levels in concentration-dependent manner. Ethanol extract of Artocarpus heterophyllus (EEAH), the most potent 15-PGDH inhibitor (IC50 = 0.62 µg/mL) with least cytotoxicity (IC50 = 670 µg/ml), elevated both intracellular and extracellular PGE2 levels. EEAH facilitated in-vitro wound healing in a HaCaT (Keratinocyte cell line) scratch model. Conclusion: EEAH might apply to treat dermal wounds by elevating PGE2 levels via COX-1 induction and 15-PGDH inhibition. SUMMARY Biological inactivation of 15-PGDH causes elevated level of PGE2 which will be useful for the management of disease that requires elevated level of PGE2. Abbreviations used: 15-PGDH: 15-hydroxyprostaglandin dehydrogenase, COX: Cyclooxygenase, DTT: Dithiothreitol, DMEM: Dulbecco's modified Eagle's media, EEAH: Ethanol extract of Artocarpus heterophyllus, MRP4: Multidrug resistance 4, PGs: Prostaglandins, PGT: Prostaglandin transporter, SDS: Sodium dodecylsulfate PMID:28479736

Purification and characterization of a novel recombinant highly enantioselective short-chain NAD(H)-dependent alcohol dehydrogenase from Thermus thermophilus.

PubMed

Pennacchio, Angela; Pucci, Biagio; Secundo, Francesco; La Cara, Francesco; Rossi, Mosè; Raia, Carlo A

2008-07-01

The gene encoding a novel alcohol dehydrogenase (ADH) that belongs to the short-chain dehydrogenase/reductase (SDR) superfamily was identified in the extremely thermophilic, halotolerant gram-negative eubacterium Thermus thermophilus HB27. The T. thermophilus ADH gene (adh(Tt)) was heterologously overexpressed in Escherichia coli, and the protein (ADH(Tt)) was purified to homogeneity and characterized. ADH(Tt) is a tetrameric enzyme consisting of identical 26,961-Da subunits composed of 256 amino acids. The enzyme has remarkable thermophilicity and thermal stability, displaying activity at temperatures up to approximately 73 degrees C and a 30-min half-inactivation temperature of approximately 90 degrees C, as well as good tolerance to common organic solvents. ADH(Tt) has a strict requirement for NAD(H) as the coenzyme, a preference for reduction of aromatic ketones and alpha-keto esters, and poor activity on aromatic alcohols and aldehydes. This thermophilic enzyme catalyzes the following reactions with Prelog specificity: the reduction of acetophenone, 2,2,2-trifluoroacetophenone, alpha-tetralone, and alpha-methyl and alpha-ethyl benzoylformates to (S)-(-)-1-phenylethanol (>99% enantiomeric excess [ee]), (R)-alpha-(trifluoromethyl)benzyl alcohol (93% ee), (S)-alpha-tetralol (>99% ee), methyl (R)-(-)-mandelate (92% ee), and ethyl (R)-(-)-mandelate (95% ee), respectively, by way of an efficient in situ NADH-recycling system involving 2-propanol and a second thermophilic ADH. This study further supports the critical role of the D37 residue in discriminating NAD(H) from NADP(H) in members of the SDR superfamily.

The Enzyme Activity and Substrate Specificity of Two Major Cinnamyl Alcohol Dehydrogenases in Sorghum (Sorghum bicolor), SbCAD2 and SbCAD4.

PubMed

Jun, Se-Young; Walker, Alexander M; Kim, Hoon; Ralph, John; Vermerris, Wilfred; Sattler, Scott E; Kang, ChulHee

2017-08-01

Cinnamyl alcohol dehydrogenase (CAD) catalyzes the final step in monolignol biosynthesis, reducing sinapaldehyde, coniferaldehyde, and p -coumaraldehyde to their corresponding alcohols in an NADPH-dependent manner. Because of its terminal location in monolignol biosynthesis, the variation in substrate specificity and activity of CAD can result in significant changes in overall composition and amount of lignin. Our in-depth characterization of two major CAD isoforms, SbCAD2 (Brown midrib 6 [bmr6]) and SbCAD4, in lignifying tissues of sorghum ( Sorghum bicolor ), a strategic plant for generating renewable chemicals and fuels, indicates their similarity in both structure and activity to Arabidopsis ( Arabidopsis thaliana ) CAD5 and Populus tremuloides sinapyl alcohol dehydrogenase, respectively. This first crystal structure of a monocot CAD combined with enzyme kinetic data and a catalytic model supported by site-directed mutagenesis allows full comparison with dicot CADs and elucidates the potential signature sequence for their substrate specificity and activity. The L119W/G301F-SbCAD4 double mutant displayed its substrate preference in the order coniferaldehyde > p -coumaraldehyde > sinapaldehyde, with higher catalytic efficiency than that of both wild-type SbCAD4 and SbCAD2. As SbCAD4 is the only major CAD isoform in bmr6 mutants, replacing SbCAD4 with L119W/G301F-SbCAD4 in bmr6 plants could produce a phenotype that is more amenable to biomass processing. © 2017 American Society of Plant Biologists. All Rights Reserved.

Genetic polymorphisms of ADH1B, ADH1C and ALDH2 in Turkish alcoholics: lack of association with alcoholism and alcoholic cirrhosis.

PubMed

Vatansever, Sezgin; Tekin, Fatih; Salman, Esin; Altintoprak, Ender; Coskunol, Hakan; Akarca, Ulus Salih

2015-05-17

No data exists regarding the alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) gene polymorphisms in Turkish alcoholic cirrhotics. We studied the polymorphisms of ADH1B, ADH1C and ALDH2 genes in alcoholic cirrhotics and compared the results with non-cirrhotic alcoholics and healthy volunteers. Overall, 237 subjects were included for the study: 156 alcoholic patients (78 cirrhotics, 78 non-cirrhotic alcoholics) and 81 healthy volunteers. Three different single-nucleotide-polymorphism genotyping methods were used. ADH1C genotyping was performed using a polymerase chain reaction-restriction fragment length polymorphism method. The identified ADH1C genotypes were named according to the presence or absence of the enzyme restriction sites. ADH1B (Arg47Hys) genotyping was performed using the allele specific primer extension method, and ALDH2 (Glu487Lys) genotyping was performed by a multiplex polymerase chain reaction using two allele-specific primer pairs. For ADH1B, the frequency of allele *1 in the cirrhotics, non-cirrhotic alcoholics and healthy volunteers was 97.4%, 94.9% and 99.4%, respectively. For ADH1C, the frequency of allele *1 in the cirrhotics, non-cirrhotic alcoholics and healthy volunteers was 47%, 36.3% and 45%, respectively. There was no statistical difference between the groups for ADH1B and ADH1C (p>0.05). All alcoholic and non-alcoholic subjects (100%) had the allele *1 for ALDH2. The obtained results for ADH1B, ADH1C, and ALDH gene polymorphisms in the present study are similar to the results of Caucasian studies. ADH1B and ADH1C genetic variations are not related to the development of alcoholism or susceptibility to alcoholic cirrhosis. ALDH2 gene has no genetic variation in the Turkish population.

Fragment growing and linking lead to novel nanomolar lactate dehydrogenase inhibitors.

PubMed

Kohlmann, Anna; Zech, Stephan G; Li, Feng; Zhou, Tianjun; Squillace, Rachel M; Commodore, Lois; Greenfield, Matthew T; Lu, Xiaohui; Miller, David P; Huang, Wei-Sheng; Qi, Jiwei; Thomas, R Mathew; Wang, Yihan; Zhang, Sen; Dodd, Rory; Liu, Shuangying; Xu, Rongsong; Xu, Yongjin; Miret, Juan J; Rivera, Victor; Clackson, Tim; Shakespeare, William C; Zhu, Xiaotian; Dalgarno, David C

2013-02-14

Lactate dehydrogenase A (LDH-A) catalyzes the interconversion of lactate and pyruvate in the glycolysis pathway. Cancer cells rely heavily on glycolysis instead of oxidative phosphorylation to generate ATP, a phenomenon known as the Warburg effect. The inhibition of LDH-A by small molecules is therefore of interest for potential cancer treatments. We describe the identification and optimization of LDH-A inhibitors by fragment-based drug discovery. We applied ligand based NMR screening to identify low affinity fragments binding to LDH-A. The dissociation constants (K(d)) and enzyme inhibition (IC(50)) of fragment hits were measured by surface plasmon resonance (SPR) and enzyme assays, respectively. The binding modes of selected fragments were investigated by X-ray crystallography. Fragment growing and linking, followed by chemical optimization, resulted in nanomolar LDH-A inhibitors that demonstrated stoichiometric binding to LDH-A. Selected molecules inhibited lactate production in cells, suggesting target-specific inhibition in cancer cell lines.

Biological evaluation and 3D-QSAR studies of curcumin analogues as aldehyde dehydrogenase 1 inhibitors.

PubMed

Wang, Hui; Du, Zhiyun; Zhang, Changyuan; Tang, Zhikai; He, Yan; Zhang, Qiuyan; Zhao, Jun; Zheng, Xi

2014-05-16

Aldehyde dehydrogenase 1 (ALDH1) is reported as a biomarker for identifying some cancer stem cells, and down-regulation or inhibition of the enzyme can be effective in anti-drug resistance and a potent therapeutic for some tumours. In this paper, the inhibitory activity, mechanism mode, molecular docking and 3D-QSAR (three-dimensional quantitative structure activity relationship) of curcumin analogues (CAs) against ALDH1 were studied. Results demonstrated that curcumin and CAs possessed potent inhibitory activity against ALDH1, and the CAs compound with ortho di-hydroxyl groups showed the most potent inhibitory activity. This study indicates that CAs may represent a new class of ALDH1 inhibitor.

Effect of various kinds of beverages on stress oxidative, F2 isoprostane, serum lipid and blood glucose of elite taekwondo players.

PubMed

Maghsoudi, Zahra; Shiranian, Ashfin; Askai, Gholamreza; Ghaisvand, Reza

2016-01-01

Athletes' recovery is important in improving their performance. Nutritional strategies can be effective in enhancing recovery rate. Choosing the best food items in appropriate intervals can play effective roles in resynthesis of fuels and recovery of muscle injury. Beverage micro and macronutrient content are helpful in fuel restoration. In this study, we assess the effects of various kinds of beverages on oxidative stress, muscle injury, and metabolic risk factors in taekwondo players. This quasi-experimental study was performed on 21 taekwondo players of Isfahan. After collecting fasting blood, they performed runningbased anaerobic sprint test (RAST). Blood lactate was tested again and participants were divided into 3 intervention groups, that is, receiving 500 cc dough, non-alcoholic beer, and chocolate milk at 4 day intervals. After a 2-h recovery period, blood sampling was repeated. Elites consumed other beverages in later phases. Dietary intake and fasting triglyceride, cholesterol, blood sugar, lactate dehydrogenase, and F 2 -isoprostane concentrations were determined. Data were analyzed with a simple repeated-measures test and post-hoc tests using the Statistical Package for the Social Sciences software. Data showed that cholesterol levels non-significantly decreased after intervention. Triglyceride level was lower after taking dough and carbohydrate replacement drink. Blood glucose concentration increased after intervention periods, however, this increase was significant only after non-alcoholic beverage consumption. Lactate dehydrogenase levels reduced after all cycles, however, F 2 -isoprostane level showed no significant change. There was not significant change in lactate dehydrogenase and F 2 -isoprostane levels. Non-alcoholic beer consumption can reduce lactate dehydrogenase concentration; however, it leads to blood sugar increase. Moreover, dough consumption significantly reduced triglyceride level in taekwondo players.

Asymmetric reduction of ketones and β-keto esters by (S)-1-phenylethanol dehydrogenase from denitrifying bacterium Aromatoleum aromaticum.

PubMed

Dudzik, A; Snoch, W; Borowiecki, P; Opalinska-Piskorz, J; Witko, M; Heider, J; Szaleniec, M

2015-06-01

Enzyme-catalyzed enantioselective reductions of ketones and keto esters have become popular for the production of homochiral building blocks which are valuable synthons for the preparation of biologically active compounds at industrial scale. Among many kinds of biocatalysts, dehydrogenases/reductases from various microorganisms have been used to prepare optically pure enantiomers from carbonyl compounds. (S)-1-phenylethanol dehydrogenase (PEDH) was found in the denitrifying bacterium Aromatoleum aromaticum (strain EbN1) and belongs to the short-chain dehydrogenase/reductase family. It catalyzes the stereospecific oxidation of (S)-1-phenylethanol to acetophenone during anaerobic ethylbenzene mineralization, but also the reverse reaction, i.e., NADH-dependent enantioselective reduction of acetophenone to (S)-1-phenylethanol. In this work, we present the application of PEDH for asymmetric reduction of 42 prochiral ketones and 11 β-keto esters to enantiopure secondary alcohols. The high enantioselectivity of the reaction is explained by docking experiments and analysis of the interaction and binding energies of the theoretical enzyme-substrate complexes leading to the respective (S)- or (R)-alcohols. The conversions were carried out in a batch reactor using Escherichia coli cells with heterologously produced PEDH as whole-cell catalysts and isopropanol as reaction solvent and cosubstrate for NADH recovery. Ketones were converted to the respective secondary alcohols with excellent enantiomeric excesses and high productivities. Moreover, the progress of product formation was studied for nine para-substituted acetophenone derivatives and described by neural network models, which allow to predict reactor behavior and provides insight on enzyme reactivity. Finally, equilibrium constants for conversion of these substrates were derived from the progress curves of the reactions. The obtained values matched very well with theoretical predictions.

Alcohol dehydrogenase-1B genotype (rs1229984) is a strong determinant of the relationship between body weight and alcohol intake in Japanese alcoholic men.

PubMed

Yokoyama, Akira; Yokoyama, Tetsuji; Matsui, Toshifumi; Mizukami, Takeshi; Matsushita, Sachio; Higuchi, Susumu; Maruyama, Katsuya

2013-07-01

The calories in alcoholic beverages consumed by alcoholics are a major energy source and a strong modifier of their body weight. Genetic polymorphisms of alcohol dehydrogenase-1B (ADH1B) and aldehyde dehydrogenase-2 (ALDH2) affect susceptibility to alcoholism and may affect body weight via gene-associated differences in fuel utilization in alcoholics. We evaluated associations between ADH1B/ALDH2 genotypes and the body weight and body mass index (BMI) of 1,301 Japanese alcoholic men at the time of their first visit to an addiction center. Median (25th to 75th) caloric intake in the form of alcoholic beverages was 864 (588 to 1,176) kcal/d. Age-adjusted caloric intake did not differ according to ADH1B/ALDH2 genotypes. The body weight and BMI values showed that the ADH1B*2/*2 and *1/*2 carriers (n = 939) were significantly leaner than the ADH1B*1/*1 carriers (n = 362) irrespective of age, drinking, smoking, and dietary habits. The age-adjusted body weight values of the ADH1B*2/*2, ADH1B*1/*2, and ADH1B*1/*1 carriers were 58.4 ± 0.4, 58.7 ± 0.5, and 63.6 ± 0.5 kg, respectively (ADH1B*2 vs. ADH1B*1/*1 carriers, p < 0.0001), and the corresponding BMI values were 21.0 ± 0.1, 21.0 ± 0.1, and 22.9 ± 0.2 kg/m(2) , respectively (ADH1B*2 vs. ADH1B*1/*1 carriers, p < 0.0001). No effects of inactive ALDH2 on body weight or BMI were observed. A multivariate analysis showed that BMI decreased by 0.35 per 10-year increase in age, by 1.73 in the presence of the ADH1B*2 allele, by 1.55 when the preferred beverage was whiskey, and by 0.19 per +10 cigarettes/d and that it increased by 0.10 per +22 g ethanol (EtOH)/d and by 0.41 per increase in category of frequency of milk intake (every day, occasionally, rarely, and never). The increase in BMI as alcohol consumption increased was significantly smaller in the ADH1B*2 group than in the ADH1B*1/*1 group (p = 0.002). ADH1B genotype was a strong determinant of body weight in the alcoholics. The more rapid EtOH elimination associated with the ADH1B*2 allele may result in less efficient utilization of EtOH as an energy source in alcoholics. Copyright © 2013 by the Research Society on Alcoholism.

3D-QSAR and docking studies on 1-hydroxypyridin-2-one compounds as mutant isocitrate dehydrogenase 1 inhibitors

NASA Astrophysics Data System (ADS)

Wang, Zhenya; Chang, Yiqun; Han, Yushui; Liu, Kangjia; Hou, Jinsong; Dai, Chengli; Zhai, Yuanhao; Guo, Jialiang; Sun, Pinghua; Lin, Jing; Chen, Weimin

2016-11-01

Mutation of isocitrate dehydrogenase 1 (IDH1) which is frequently found in certain cancers such as glioma, sarcoma and acute myeloid leukemia, has been proven to be a potent drug target for cancer therapy. In silico methodologies such as 3D-QSAR and molecular docking were performed to explore compounds with better mutant isocitrate dehydrogenase 1 (MIDH1) inhibitory activity using a series of 40 newly reported 1-hydroxypyridin-2-one compounds as MIDH1 inhibitors. The satisfactory CoMFA and CoMSIA models obtained after internal and external cross-validation gave q2 values of 0.691 and 0.535, r2 values of 0.984 and 0.936, respectively. 3D contour maps generated from CoMFA and CoMSIA along with the docking results provided information about the structural requirements for better MIDH1 inhibitory activity. Based on the structure-activity relationship, 17 new potent molecules with better predicted activity than the most active compound in the literature have been designed.

Detection of in vivo DNA damage induced by ethanol in multiple organs of pregnant mice using the alkaline single cell gel electrophoresis (Comet) assay.

PubMed

Kido, Ryoko; Sato, Itaru; Tsuda, Shuji

2006-01-01

Ethanol is principal ingredient of alcohol beverage, but considered as human carcinogen, and has neurotoxicity. Alcohol consumption during pregnancy often causes fetal alcohol syndrome. The DNA damage is one of the important factors in carcinogenicity or teratogenicity. To detect the DNA damage induced by ethanol, we used an in vivo alkaline single cell gel electrophoresis (Comet) assay in pregnant mice organs and embryos. Pregnant ICR mice on Day 7 of gestation were treated with 2, 4 or 8 g/kg ethanol, and maternal organs/tissues and embryos were subjected to the Comet assay at 4, 8, 12 and 24 hr after ethanol treatment. Four and 8 g/kg ethanol induced DNA damage in brain, lung and embryos at 4 or 8 hr after the treatment. Two g/kg ethanol did not cause any DNA damage, and 8 g/kg ethanol only increased the duration of DNA damage without distinct increase in the degree of the damage. No significant DNA damage was observed in the liver. To detect the effect of acetaldehyde, disulfiram, acetaldehyde dehydrogenase inhibitor, was administered before 4 g/kg ethanol treatment. No significant increase of DNA damage was observed in the disulfiram pre-treated group. These data indicate that ethanol induces DNA damage, which might be related to ethanol toxicity. Since pre-treatment of disulfiram did not increase DNA damage, DNA damage observed in this study might not be the effect of acetaldehyde.

Improving isopropanol tolerance and production of Clostridium beijerinckii DSM 6423 by random mutagenesis and genome shuffling.

PubMed

Gérando, H Máté de; Fayolle-Guichard, F; Rudant, L; Millah, S K; Monot, F; Ferreira, Nicolas Lopes; López-Contreras, A M

2016-06-01

Random mutagenesis and genome shuffling was applied to improve solvent tolerance and isopropanol/butanol/ethanol (IBE) production in the strictly anaerobic bacteria Clostridium beijerinckii DSM 6423. Following chemical mutagenesis with N-methyl-N-nitro-N-nitrosoguanidine (NTG), screening of putatively improved strains was done by submitting the mutants to toxic levels of inhibitory chemicals or by screening for their tolerance to isopropanol (>35 g/L). Suicide substrates, such as ethyl or methyl bromobutyrate or alcohol dehydrogenase inhibitors like allyl alcohol, were tested and, finally, 36 mutants were isolated. The fermentation profiles of these NTG mutant strains were characterized, and the best performing mutants were used for consecutive rounds of genome shuffling. Screening of strains with further enhancement in isopropanol tolerance at each recursive shuffling step was then used to spot additionally improved strains. Three highly tolerant strains were finally isolated and able to withstand up to 50 g/L isopropanol on plates. Even if increased tolerance to the desired end product was not always accompanied by higher production capabilities, some shuffled strains showed increased solvent titers compared to the parental strains and the original C. beijerinckii DSM 6423. This study confirms the efficiency of genome shuffling to generate improved strains toward a desired phenotype such as alcohol tolerance. This tool also offers the possibility of obtaining improved strains of Clostridium species for which targeted genetic engineering approaches have not been described yet.

The sap of Acer okamotoanum decreases serum alcohol levels after acute ethanol ingestion in rats.

PubMed

Yoo, Yeong-Min; Jung, Eui-Man; Kang, Ha-Young; Choi, In-Gyu; Choi, Kyung-Chul; Jeung, Eui-Bae

2011-10-01

In the present study, we examined whether Acer okamotoanum (A. okamotoanum) sap decreased the serum alcohol and acetaldehyde levels after acute ethanol treatment in a rat model. Male rats were orally administered 25, 50 or 100% A. okamotoanum sap 30 min prior to oral challenge with 3 ml of ethanol (15 ml/kg of a 20% ethanol solution in water), and the blood concentrations of alcohol and acetaldehyde were analyzed up to 7 h after the treatment. Pre-treatment with the sap significantly decreased the blood ethanol and acetaldehyde concentrations after 5 h when compared with ethanol treatment alone (a negative control). The expression levels of liver alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) mRNA were increased significantly in animals pre-treated with A. okamotoanum sap when compared with negative and positive controls. The data suggest that sap pre-treatment enhanced the alcohol metabolism rate in the rat liver. To investigate the involvement of mitochondrial regulation in the ethanol-induced hepatocyte apoptosis, we carried out an immunohistochemical analysis of Bax and Bcl-2. Pre-treatment with sap significantly decreased Bax expression and increased Bcl-2 expression 7 h after ethanol administration when compared with the negative control. The data suggest that A. okamotoanum sap pre-treatment may reduce the alcohol-induced oxidative stress in the rat liver.

Characterization of an Arxula adeninivorans alcohol dehydrogenase involved in the metabolism of ethanol and 1-butanol.

PubMed

Kasprzak, Jakub; Rauter, Marion; Riechen, Jan; Worch, Sebastian; Baronian, Kim; Bode, Rüdiger; Schauer, Frieder; Kunze, Gotthard

2016-05-01

In this study, alcohol dehydrogenase 1 from Arxula adeninivorans (Aadh1p) was identified and characterized. Aadh1p showed activity with short and medium chain length primary alcohols in the forward reaction and their aldehydes in the reverse reaction. Aadh1p has 64% identity with Saccharomyces cerevisiae Adh1p, is localized in the cytoplasm and uses NAD(+) as cofactor. Gene expression analysis showed a low level increase in AADH1 gene expression with ethanol, pyruvate or xylose as the carbon source. Deletion of the AADH1 gene affects growth of the cells with 1-butanol, ethanol and glucose as the carbon source, and a strain which overexpressed the AADH1 gene metabolized 1-butanol more rapidly. An ADH activity assay indicated that Aadh1p is a major enzyme for the synthesis of ethanol and the degradation of 1-butanol in A. adeninivorans. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Genetic susceptibility factors for alcohol-induced chronic pancreatitis.

PubMed

Aghdassi, Ali A; Weiss, F Ulrich; Mayerle, Julia; Lerch, Markus M; Simon, Peter

2015-07-01

Chronic pancreatitis is a progressive inflammatory disease of the pancreas and frequently associated with immoderate alcohol consumption. Since only a small proportion of alcoholics eventually develop chronic pancreatitis genetic susceptibility factors have long been suspected to contribute to the pathogenesis of the disease. Smaller studies in ethnically defined populations have found that not only polymorphism in proteins involved in the metabolism of ethanol, such as Alcohol Dehydrogenase and Aldehyde Dehydrogenase, can confer a risk for developing chronic pancreatitis but also mutations that had previously been reported in association with idiopathic pancreatitis, such as SPINK1 mutations. In a much broader approach employing genome wide search strategies the NAPS study found that polymorphisms in the Trypsin locus (PRSS1 rs10273639), and the Claudin 2 locus (CLDN2-RIPPLY1-MORC4 locus rs7057398 and rs12688220) confer an increased risk of developing alcohol-induced pancreatitis. These results from North America have now been confirmed by a European consortium. In another genome wide approach polymorphisms in the genes encoding Fucosyltransferase 2 (FUT2) non-secretor status and blood group B were not only found in association with higher serum lipase levels in healthy volunteers but also to more than double the risk for developing alcohol-associated chronic pancreatitis. These novel genetic associations will allow to investigate the pathophysiological and biochemical basis of alcohol-induced chronic pancreatitis on a cellular level and in much more detail than previously possible. Copyright © 2015 IAP and EPC. Published by Elsevier B.V. All rights reserved.

Ethanol-induced alcohol dehydrogenase E (AdhE) potentiates pneumolysin in Streptococcus pneumoniae.

PubMed

Luong, Truc Thanh; Kim, Eun-Hye; Bak, Jong Phil; Nguyen, Cuong Thach; Choi, Sangdun; Briles, David E; Pyo, Suhkneung; Rhee, Dong-Kwon

2015-01-01

Alcohol impairs the host immune system, rendering the host more vulnerable to infection. Therefore, alcoholics are at increased risk of acquiring serious bacterial infections caused by Streptococcus pneumoniae, including pneumonia. Nevertheless, how alcohol affects pneumococcal virulence remains unclear. Here, we showed that the S. pneumoniae type 2 D39 strain is ethanol tolerant and that alcohol upregulates alcohol dehydrogenase E (AdhE) and potentiates pneumolysin (Ply). Hemolytic activity, colonization, and virulence of S. pneumoniae, as well as host cell myeloperoxidase activity, proinflammatory cytokine secretion, and inflammation, were significantly attenuated in adhE mutant bacteria (ΔadhE strain) compared to D39 wild-type bacteria. Therefore, AdhE might act as a pneumococcal virulence factor. Moreover, in the presence of ethanol, S. pneumoniae AdhE produced acetaldehyde and NADH, which subsequently led Rex (redox-sensing transcriptional repressor) to dissociate from the adhE promoter. An increase in AdhE level under the ethanol condition conferred an increase in Ply and H2O2 levels. Consistently, S. pneumoniae D39 caused higher cytotoxicity to RAW 264.7 cells than the ΔadhE strain under the ethanol stress condition, and ethanol-fed mice (alcoholic mice) were more susceptible to infection with the D39 wild-type bacteria than with the ΔadhE strain. Taken together, these data indicate that AdhE increases Ply under the ethanol stress condition, thus potentiating pneumococcal virulence. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

[Cardiovascular repercussion of lodenafil carbonate, a new PDE5 inhibitor, with and without alcohol consumption].

PubMed

Silva, Adauto Carvalho; Toffoletto, Odaly; Lucio, Luiz Antonio Galvão; Santos, Paula Ferreira Dos; Afiune, Jorge Barros; Massud Filho, João; Tufik, Sergio

2010-02-01

Millions of men around the world suffer from erectile dysfunction, for which phosphodiesterase 5 inhibitors (PDE-5 inhibitors) are currently the first treatment option. Sexual activity and alcohol consumption are closely related, and the simultaneous use of alcohol and PDE-5 inhibitors can happen. Lodenafil carbonate is a new PDE-5 inhibitor, developed by a Brazilian pharmaceutical company. This work aimed at evaluating the cardiovascular safety of lodenafil carbonate, with and without simultaneous alcohol consumption. Fifteen male volunteers received 160 mg lodenafil carbonate (LC), in three different moments. Participants were assigned to three groups, treated with LC in fasting condition, with alcohol or receiving only placebo. The volunteers were continuously monitored during 24 hours for physical impairment, blood pressure, heart rate, QT interval and lodenafil's pharmacokinetic parameters. Lodenafil carbonate alone or with alcohol did not induce clinically relevant modifications in arterial blood pressure or heart rate. A statistically significant decrease in blood pressure was seen four hours after LC and alcohol intake, and an increase in heart rate six hours after intake of lodenafil carbonate alone. The QTc interval was not significantly modified. Lodenafil carbonate bioavailability was increased in 74% when drug intake was associated with alcohol. These results show that the use of lodenafil carbonate did not have clinically relevant effects on blood pressure or heart rate, and was not associated with QT interval prolongation. The association of lodenafil carbonate and alcohol affected its pharmacokinetic properties, increasing the bioavailability of the drug.

Molecular and physiological aspects of alcohol dehydrogenases in the ethanol metabolism of Saccharomyces cerevisiae.

PubMed

de Smidt, Olga; du Preez, James C; Albertyn, Jacobus

2012-02-01

The physiological role and possible functional substitution of each of the five alcohol dehydrogenase (Adh) isozymes in Saccharomyces cerevisiae were investigated in five quadruple deletion mutants designated strains Q1-Q5, with the number indicating the sole intact ADH gene. Their growth in aerobic batch cultures was characterised in terms of kinetic and stoichiometric parameters. Cultivation with glucose or ethanol as carbon substrate revealed that Adh1 was the only alcohol dehydrogenase capable of efficiently catalysing the reduction of acetaldehyde to ethanol. The oxidation of produced or added ethanol could also be attributed to Adh1. Growth of strains lacking the ADH1 gene resulted in the production of glycerol as a major fermentation product, concomitant with the production of a significant amount of acetaldehyde. Strains Q2 and Q3, expressing only ADH2 or ADH3, respectively, produced ethanol from glucose, albeit less than strain Q1, and were also able to oxidise added ethanol. Strains Q4 and Q5 grew poorly on glucose and produced ethanol, but were neither able to utilise the produced ethanol nor grow on added ethanol. Transcription profiles of the ADH4 and ADH5 genes suggested that participation of these gene products in ethanol production from glucose was unlikely. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Contribution of aldehyde oxidizing enzymes on the metabolism of 3,4-dimethoxy-2-phenylethylamine to 3,4-dimethoxyphenylacetic acid by guinea pig liver slices.

PubMed

Panoutsopoulos, Georgios I

2006-01-01

3,4-Dimethoxy-2-phenylethylamine is catalyzed to its aldehyde derivative by monoamine oxidase B, but the subsequent oxidation into the corresponding acid has not yet been studied. Oxidation of aromatic aldehydes is catalyzed mainly by aldehyde dehydrogenase and aldehyde oxidase. The present study examines the metabolism of 3,4-dimethoxy-2-phenylethylamine in vitro and in freshly prepared and cryopreserved guinea pig liver slices and the relative contribution of different aldehyde-oxidizing enzymes was estimated by pharmacological means. 3,4-Dimethoxy-2- phenylethylamine was converted into the corresponding aldehyde when incubated with monoamine oxidase and further oxidized into the acid when incubated with both, monoamine oxidase and aldehyde oxidase. In freshly prepared and cryopreserved liver slices, 3,4-dimethoxyphenylacetic acid was the main metabolite of 3,4-dimethoxy-2- phenylethylamine. 3,4-Dimethoxyphenylacetic acid formation was inhibited by 85% from disulfiram (aldehyde dehydrogenase inhibitor) and by 75-80% from isovanillin (aldehyde oxidase inhibitor), whereas allopurinol (xanthine oxidase inhibitor) inhibited acid formation by only 25-30%. 3,4- Dimethoxy-2-phenylethylamine is oxidized mainly to its acid, via 3,4-dimethoxyphenylacetaldehyde, by aldehyde dehydrogenase and aldehyde oxidase with a lower contribution from xanthine oxidase.

Elucidation of the Mechanisms and Environmental Relevance of cis-Dichloroethene and Vinyl Chloride Biodegradation

DTIC Science & Technology

2012-11-01

iTRAQ study Table 3-12: Ratio of aldehyde and alcohol dehydrogenases from iTRAQ study Table 3-13: Proteins involved in proposed glyoxal transformation...pathway for cDCE degradation proceeds through chloro-, or dichloroacetaldehyde. Multiple aldehyde dehydrogenases are annotated in the genome of JS666...is a fortuitous reaction. However, if the initial reaction is monooxygenation to an aldehyde then the true intermediate would be

Inhibition of Mutated Isocitrate Dehydrogenase 1 in Cancer.

PubMed

Wu, Fangrui; Cheng, Gang; Yao, Yuan; Kogiso, Mari; Jiang, Hong; Li, Xiao-Nan; Song, Yongcheng

2018-05-23

R132H mutation of isocitrate dehydrogenase 1 (IDH1) are found in ~75% of low-grade gliomas and secondary glioblastomas as well as in several other types of cancer. More chemotypes of inhibitors of IDH1(R132H) are therefore needed. To develop a new class of IDH1(R132H) inhibitors as potent antitumor agents. A biochemical assay was developed to find inhibitors of IDH1(R132H) mutant enzyme. Chemical synthesis and structure activity relationship studies were used to find compounds with improved potency. Antitumor activities of selected compounds were evaluated. A series of aromatic sulfonamide compounds were found to be novel, potent inhibitors of IDH1(R132H) with Ki values as low as 0.6 µM. Structure activity relationships of these compounds are discussed. Enzyme kinetics studies showed that one compound is a competitive inhibitor against the substrate α-KG and a non-competitive inhibitor against the cofactor NADPH. Several inhibitors were found to have no activity against wild-type IDH1, showing a high selectivity. Two potent inhibitors exhibited strong activity against proliferation of BT142 glioma cells with IDH1 R132H mutation, while these compounds did not significantly affect growth of glioma cells without IDH1 mutation. This novel series of IDH1(R132H) inhibitors have potential to be further developed for the treatment of glioma with IDH1 mutation. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Ebselen Reversibly Inhibits Human Glutamate Dehydrogenase at the Catalytic Site.

PubMed

Jin, Yanhong; Li, Di; Lu, Shiying; Zhao, Han; Chen, Zhao; Hou, Wei; Ruan, Benfang Helen

Human glutamate dehydrogenase (GDH) plays an important role in neurological diseases, tumor metabolism, and hyperinsulinism-hyperammonemia syndrome (HHS). However, there are very few inhibitors known for human GDH. Recently, Ebselen was reported to crosslink with Escherichia coli GDH at the active site cysteine residue (Cys321), but the sequence alignment showed that the corresponding residue is Ala329 in human GDH. To investigate whether Ebselen could be an inhibitor for human GDH, we cloned and expressed an N-terminal His-tagged human GDH in E. coli. The recombinant human GDH enzyme showed expected properties such as adenosine diphosphate activation and nicotinamide adenine dinucleotide/nicotinamide adenine dinucleotide phosphate dual recognition. Further, we developed a 2-(3-(2-methoxy-4-nitrophenyl)-2-(4-nitrophenyl)-2H-tetrazol-3-ium-5-yl) benzenesulfonate sodium salt (EZMTT)-based assay for human GDH, which was highly sensitive and is suitable for high-throughput screening for potent GDH inhibitors. In addition, ForteBio binding assays demonstrated that Ebselen is a reversible active site inhibitor for human GDH. Since Ebselen is a multifunctional organoselenium compound in Phase III clinical trials for inflammation, an Ebselen-based GDH inhibitor might be valuable for future drug discovery for HHS patients.

Characterization of Two Distinct Structural Classes of Selective Aldehyde Dehydrogenase 1A1 Inhibitors

DOE PAGES

Morgan, Cynthia A.; Hurley, Thomas D.

2015-01-29

Aldehyde dehydrogenases (ALDH) catalyze the irreversible oxidation of aldehydes to their corresponding carboxylic acid. Alterations in ALDH1A1 activity are associated with such diverse diseases as cancer, Parkinson’s disease, obesity, and cataracts. Inhibitors of ALDH1A1 could aid in illuminating the role of this enzyme in disease processes. However, there are no commercially available selective inhibitors for ALDH1A1. Here we characterize two distinct chemical classes of inhibitors that are selective for human ALDH1A1 compared to eight other ALDH isoenzymes. The prototypical members of each structural class, CM026 and CM037, exhibit sub-micromolar inhibition constants, but have different mechanisms of inhibition. The crystal structuresmore » of these compounds bound to ALDH1A1 demonstrate that they bind within the aldehyde binding pocket of ALDH1A1 and exploit the presence of a unique Glycine residue to achieve their selectivity. Lastly, these two novel and selective ALDH1A1 inhibitors may serve as chemical tools to better understand the contributions of ALDH1A1 to normal biology and to disease states.« less

QSAR study on the antimalarial activity of Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) inhibitors.

PubMed

Hou, X; Chen, X; Zhang, M; Yan, A

2016-01-01

Plasmodium falciparum, the most fatal parasite that causes malaria, is responsible for over one million deaths per year. P. falciparum dihydroorotate dehydrogenase (PfDHODH) has been validated as a promising drug development target for antimalarial therapy since it catalyzes the rate-limiting step for DNA and RNA biosynthesis. In this study, we investigated the quantitative structure-activity relationships (QSAR) of the antimalarial activity of PfDHODH inhibitors by generating four computational models using a multilinear regression (MLR) and a support vector machine (SVM) based on a dataset of 255 PfDHODH inhibitors. All the models display good prediction quality with a leave-one-out q(2) >0.66, a correlation coefficient (r) >0.85 on both training sets and test sets, and a mean square error (MSE) <0.32 on training sets and <0.37 on test sets, respectively. The study indicated that the hydrogen bonding ability, atom polarizabilities and ring complexity are predominant factors for inhibitors' antimalarial activity. The models are capable of predicting inhibitors' antimalarial activity and the molecular descriptors for building the models could be helpful in the development of new antimalarial drugs.

Mechanism of protection against alcoholism by an alcohol dehydrogenase polymorphism: development of an animal model.

PubMed

Rivera-Meza, Mario; Quintanilla, María Elena; Tampier, Lutske; Mura, Casilda V; Sapag, Amalia; Israel, Yedy

2010-01-01

Humans who carry a point mutation in the gene coding for alcohol dehydrogenase-1B (ADH1B*2; Arg47His) are markedly protected against alcoholism. Although this mutation results in a 100-fold increase in enzyme activity, it has not been reported to cause higher levels of acetaldehyde, a metabolite of ethanol known to deter alcohol intake. Hence, the mechanism by which this mutation confers protection against alcoholism is unknown. To study this protective effect, the wild-type rat cDNA encoding rADH-47Arg was mutated to encode rADH-47His, mimicking the human mutation. The mutated cDNA was incorporated into an adenoviral vector and administered to genetically selected alcohol-preferring rats. The V(max) of rADH-47His was 6-fold higher (P<0.001) than that of the wild-type rADH-47Arg. Animals transduced with rAdh-47His showed a 90% (P<0.01) increase in liver ADH activity and a 50% reduction (P<0.001) in voluntary ethanol intake. In animals transduced with rAdh-47His, administration of ethanol (1g/kg) produced a short-lived increase of arterial blood acetaldehyde concentration to levels that were 3.5- to 5-fold greater than those in animals transduced with the wild-type rAdh-47Arg vector or with a noncoding vector. This brief increase (burst) in arterial acetaldehyde concentration after ethanol ingestion may constitute the mechanism by which humans carrying the ADH1B*2 allele are protected against alcoholism.

Elaboration of a fragment library hit produces potent and selective aspartate semialdehyde dehydrogenase inhibitors.

PubMed

Thangavelu, Bharani; Bhansali, Pravin; Viola, Ronald E

2015-10-15

Aspartate-β-semialdehyde dehydrogenase (ASADH) lies at the first branch point in the aspartate metabolic pathway which leads to the biosynthesis of several essential amino acids and some important metabolites. This pathway is crucial for many metabolic processes in plants and microbes like bacteria and fungi, but is absent in mammals. Therefore, the key microbial enzymes involved in this pathway are attractive potential targets for development of new antibiotics with novel modes of action. The ASADH enzyme family shares the same substrate binding and active site catalytic groups; however, the enzymes from representative bacterial and fungal species show different inhibition patterns when previously screened against low molecular weight inhibitors identified from fragment library screening. In the present study several approaches, including fragment based drug discovery (FBDD), inhibitor docking, kinetic, and structure-activity relationship (SAR) studies have been used to guide ASADH inhibitor development. Elaboration of a core structure identified by FBDD has led to the synthesis of low micromolar inhibitors of the target enzyme, with high selectivity introduced between the Gram-negative and Gram-positive orthologs of ASADH. This new set of structures open a novel direction for the development of inhibitors against this validated drug-target enzyme. Copyright © 2015 Elsevier Ltd. All rights reserved.

Identification and functional evaluation of the reductases and dehydrogenases from Saccharomyces cerevisiae involved in vanillin resistance.

PubMed

Wang, Xinning; Liang, Zhenzhen; Hou, Jin; Bao, Xiaoming; Shen, Yu

2016-04-01

Vanillin, a type of phenolic released during the pre-treatment of lignocellulosic materials, is toxic to microorganisms and therefore its presence inhibits the fermentation. The vanillin can be reduced to vanillyl alcohol, which is much less toxic, by the ethanol producer Saccharomyces cerevisiae. The reducing capacity of S. cerevisiae and its vanillin resistance are strongly correlated. However, the specific enzymes and their contribution to the vanillin reduction are not extensively studied. In our previous work, an evolved vanillin-resistant strain showed an increased vanillin reduction capacity compared with its parent strain. The transcriptome analysis suggested the reductases and dehydrogenases of this vanillin resistant strain were up-regulated. Using this as a starting point, 11 significantly regulated reductases and dehydrogenases were selected in the present work for further study. The roles of these reductases and dehydrogenases in the vanillin tolerance and detoxification abilities of S. cerevisiae are described. Among the candidate genes, the overexpression of the alcohol dehydrogenase gene ADH6, acetaldehyde dehydrogenase gene ALD6, glucose-6-phosphate 1-dehydrogenase gene ZWF1, NADH-dependent aldehyde reductase gene YNL134C, and aldo-keto reductase gene YJR096W increased 177, 25, 6, 15, and 18 % of the strain μmax in the medium containing 1 g L(-1) vanillin. The in vitro detected vanillin reductase activities of strain overexpressing ADH6, YNL134C and YJR096W were notably higher than control. The vanillin specific reduction rate increased by 8 times in ADH6 overexpressed strain but not in YNL134C and YJR096W overexpressed strain. This suggested that the enzymes encoded by YNL134C and YJR096W might prefer other substrate and/or could not show their effects on vanillin on the high background of Adh6p in vivo. Overexpressing ALD6 and ZWF1 mainly increased the [NADPH]/[NADP(+)] and [GSH]/[GSSG] ratios but not the vanillin reductase activities. Their contribution to strain growth and vanillin reduction were balancing the redox state of strain when vanillin was presented. Beside the reported Adh6p, the enzymes encoded by YNL134C and YJR096W were proved to have vanillin reduction activity in present study. While ALD6 and ZWF1 did not directly reduce vanillin to vanillyl alcohol, their contribution to vanillin resistance primarily depended on the enhancement of the reducing equivalent supply.

Molecular Dissection of a Major Gene Effect on a Quantitative Trait: The Level of Alcohol Dehydrogenase Expression in Drosophila Melanogaster

PubMed Central

Stam, L. F.; Laurie, C. C.

1996-01-01

A molecular mapping experiment shows that a major gene effect on a quantitative trait, the level of alcohol dehydrogenase expression in Drosophila melanogaster, is due to multiple polymorphisms within the Adh gene. These polymorphisms are located in an intron, the coding sequence, and the 3' untranslated region. Because of nonrandom associations among polymorphisms at different sites, the individual effects combine (in some cases epistatically) to produce ``superalleles'' with large effect. These results have implications for the interpretation of major gene effects detected by quantitative trait locus mapping methods. They show that large effects due to a single locus may be due to multiple associated polymorphisms (or sequential fixations in isolated populations) rather than individual mutations of large effect. PMID:8978044

Alcohol Dehydrogenase-1B (rs1229984) and Aldehyde Dehydrogenase-2 (rs671) Genotypes Are Strong Determinants of the Serum Triglyceride and Cholesterol Levels of Japanese Alcoholic Men.

PubMed

Yokoyama, Akira; Yokoyama, Tetsuji; Matsui, Toshifumi; Mizukami, Takeshi; Kimura, Mitsuru; Matsushita, Sachio; Higuchi, Susumu; Maruyama, Katsuya

2015-01-01

Elevated serum triglyceride (TG) and high-density-lipoprotein cholesterol (HDL-C) levels are common in drinkers. The fast-metabolizing alcohol dehydrogenase-1B encoded by the ADH1B*2 allele (vs. ADH1B*1/*1 genotype) and inactive aldehyde dehydrogenase-2 encoded by the ALDH2*2 allele (vs. ALDH2*1/*1 genotype) modify ethanol metabolism and are prevalent (≈90% and ≈40%, respectively) in East Asians. We attempted to evaluate the associations between the ADH1B and ALDH2 genotypes and lipid levels in alcoholics. The population consisted of 1806 Japanese alcoholic men (≥40 years) who had undergone ADH1B and ALDH2 genotyping and whose serum TG, total cholesterol, and HDL-C levels in the fasting state had been measured within 3 days after admission. High serum levels of TG (≥150 mg/dl), HDL-C (>80 mg/dl), and low-density-lipoprotein cholesterol (LDL-C calculated by the Friedewald formula ≥140 mg/dl) were observed in 24.3%, 16.8%, and 15.6%, respectively, of the subjects. Diabetes, cirrhosis, smoking, and body mass index (BMI) affected the serum lipid levels. Multivariate analysis revealed that the presence of the ADH1B*2 allele and the active ALDH2*1/*1 genotype increased the odds ratio (OR; 95% confidence interval) for a high TG level (2.22 [1.67-2.94] and 1.39 [0.99-1.96], respectively), and decreased the OR for a high HDL-C level (0.37 [0.28-0.49] and 0.51 [0.37-0.69], respectively). The presence of the ADH1B*2 allele decreased the OR for a high LDL-C level (0.60 [0.45-0.80]). The ADH1B*2 plus ALDH2*1/*1 combination yielded the highest ORs for high TG levels and lowest OR for a high HDL-C level. The genotype effects were more prominent in relation to the higher levels of TG (≥220 mg/dl) and HDL-C (≥100 mg/dl). The fast-metabolizing ADH1B and active ALDH2, and especially a combination of the two were strongly associated with higher serum TG levels and lower serum HDL-C levels of alcoholics. The fast-metabolizing ADH1B was associated with lower serum LDL-C levels.

Disrupting androgen production of Leydig cells by resveratrol via direct inhibition of human and rat 3β-hydroxysteroid dehydrogenase.

PubMed

Li, Ling; Chen, Xiaomin; Zhu, Qiqi; Chen, Dongxin; Guo, Jingjing; Yao, Wenwen; Dong, Yaoyao; Wei, Jia; Lian, Qingquan; Ge, Ren-Shan; Yuan, Bo

2014-04-07

Resveratrol is a polyphenol produced by several plants. It has been demonstrated that it has anti-inflammatory, antitumor, and anti-diabetic effects in animal models. However, its side effects are generally unclear. In the present study, we reported that resveratrol inhibited luteinizing hormone-stimulated androgen production in rat immature Leydig cells. Further analysis demonstrated that it was a competitive inhibitor of rat and human 3β-hydroxysteroid dehydrogenase with IC₆₀ values of 3.87 ± 0.06 and 8.48 ± 0.04 μM, respectively. The inhibition on 3β-hydroxysteroid dehydrogenase was specific since it did not inhibit another hydroxysteroid dehydrogenase 17β-hydroxysteroid dehydrogenase 3 at the highest concentration (100 μM) tested. In conclusion, resveratrol potentially interferes with androgen biosynthesis of rat Leydig cells. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Effects of 20 Selected Fruits on Ethanol Metabolism: Potential Health Benefits and Harmful Impacts

PubMed Central

Zhang, Yu-Jie; Wang, Fang; Zhou, Yue; Li, Ya; Zhou, Tong; Zheng, Jie; Zhang, Jiao-Jiao; Li, Sha; Xu, Dong-Ping; Li, Hua-Bin

2016-01-01

The consumption of alcohol is often accompanied by other foods, such as fruits and vegetables. This study is aimed to investigate the effects of 20 selected fruits on ethanol metabolism to find out their potential health benefits and harmful impacts. The effects of the fruits on ethanol metabolism were characterized by the concentrations of ethanol and acetaldehyde in blood, as well as activities of alcohol dehydrogenase and acetaldehyde dehydrogenase in liver of mice. Furthermore, potential health benefits and harmful impacts of the fruits were evaluated by biochemical parameters including aspartate transaminase (AST), alanine transferase (ALT), malondialdehyde, and superoxide dismutase. Generally, effects of these fruits on ethanol metabolism were very different. Some fruits (such as Citrus limon (yellow), Averrhoa carambola, Pyrus spp., and Syzygium samarangense) could decrease the concentration of ethanol in blood. In addition, several fruits (such as Cucumis melo) showed hepatoprotective effects by significantly decreasing AST or ALT level in blood, while some fruits (such as Averrhoa carambola) showed adverse effects. The results suggested that the consumption of alcohol should not be accompanied by some fruits, and several fruits could be developed as functional foods for the prevention and treatment of hangover and alcohol use disorder. PMID:27043608

Electrolyzed-reduced water inhibits acute ethanol-induced hangovers in Sprague-Dawley rats.

PubMed

Park, Seung-Kyu; Qi, Xu-Feng; Song, Soon-Bong; Kim, Dong-Heui; Teng, Yung-Chien; Yoon, Yang-Suk; Kim, Kwang-Yong; Li, Jian-Hong; Jin, Dan; Lee, Kyu-Jae

2009-10-01

Ethanol consumption disturbs the balance between the pro- and anti-oxidant systems of the organism, leading to oxidative stress. Electrolyzed-reduced water (ERW) is widely used by people in East Asia for drinking purposes because of its therapeutic properties including scavenging effect of reactive oxygen species. This study was performed to investigate the effect of ERW on acute ethanol-induced hangovers in Sprague-Dawley rats. Alcohol concentration in serum of ERW-treated rats showed significant difference at 1 h, 3 h and 5 h respectively as compared with the rats treated with distilled water. Both alcohol dehydrogenase type 1 and acetaldehyde dehydrogenase related with oxidation of alcohol were significantly increased in liver tissue while the level of aspartate aminotransferase and alanine aminotransferase in serum was markedly decreased 24 h after pre-oral administration of ERW. Moreover, oral administration of ERW significantly activated non-ezymatic (glutathione) and enzymatic (glutathione peroxidase, glutathione-S-transferase, Cu/Zn-superoxide dismutase and catalase) antioxidants in liver tissues compared with the control group. These results suggest that drinking ERW has an effect of alcohol detoxification by antioxidant mechanism and has potentiality for relief of ethanol-induced hangover symptoms.

Effects of 20 Selected Fruits on Ethanol Metabolism: Potential Health Benefits and Harmful Impacts.

PubMed

Zhang, Yu-Jie; Wang, Fang; Zhou, Yue; Li, Ya; Zhou, Tong; Zheng, Jie; Zhang, Jiao-Jiao; Li, Sha; Xu, Dong-Ping; Li, Hua-Bin

2016-04-01

The consumption of alcohol is often accompanied by other foods, such as fruits and vegetables. This study is aimed to investigate the effects of 20 selected fruits on ethanol metabolism to find out their potential health benefits and harmful impacts. The effects of the fruits on ethanol metabolism were characterized by the concentrations of ethanol and acetaldehyde in blood, as well as activities of alcohol dehydrogenase and acetaldehyde dehydrogenase in liver of mice. Furthermore, potential health benefits and harmful impacts of the fruits were evaluated by biochemical parameters including aspartate transaminase (AST), alanine transferase (ALT), malondialdehyde, and superoxide dismutase. Generally, effects of these fruits on ethanol metabolism were very different. Some fruits (such as Citrus limon (yellow), Averrhoa carambola, Pyrus spp., and Syzygium samarangense) could decrease the concentration of ethanol in blood. In addition, several fruits (such as Cucumis melo) showed hepatoprotective effects by significantly decreasing AST or ALT level in blood, while some fruits (such as Averrhoa carambola) showed adverse effects. The results suggested that the consumption of alcohol should not be accompanied by some fruits, and several fruits could be developed as functional foods for the prevention and treatment of hangover and alcohol use disorder.

Novel indole-based inhibitors of IMPDH: introduction of hydrogen bond acceptors at indole C-3.

PubMed

Watterson, Scott H; Dhar, T G Murali; Ballentine, Shelley K; Shen, Zhongqi; Barrish, Joel C; Cheney, Daniel; Fleener, Catherine A; Rouleau, Katherine A; Townsend, Robert; Hollenbaugh, Diane L; Iwanowicz, Edwin J

2003-04-07

The development of a series of novel indole-based inhibitors of 5'-inosine monophosphate dehydrogenase (IMPDH) is described. Various hydrogen bond acceptors at C-3 of the indole were explored. The synthesis and the structure-activity relationships (SARs) derived from in vitro studies are outlined.

Overview of the Genetics of Alcohol Use Disorder

PubMed Central

Tawa, Elisabeth A.; Hall, Samuel D.; Lohoff, Falk W.

2016-01-01

Aims Alcohol Use Disorder (AUD) is a chronic psychiatric illness characterized by harmful drinking patterns leading to negative emotional, physical, and social ramifications. While the underlying pathophysiology of AUD is poorly understood, there is substantial evidence for a genetic component; however, identification of universal genetic risk variants for AUD has been difficult. Recent efforts in the search for AUD susceptibility genes will be reviewed in this article. Methods In this review, we provide an overview of genetic studies on AUD, including twin studies, linkage studies, candidate gene studies, and genome-wide association studies (GWAS). Results Several potential genetic susceptibility factors for AUD have been identified, but the genes of alcohol metabolism, alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH), have been found to be protective against the development of AUD. GWAS have also identified a heterogeneous list of SNPs associated with AUD and alcohol-related phenotypes, emphasizing the complexity and heterogeneity of the disorder. In addition, many of these findings have small effect sizes when compared to alcohol metabolism genes, and biological relevance is often unknown. Conclusions Although studies spanning multiple approaches have suggested a genetic basis for AUD, identification of the genetic risk variants has been challenging. Some promising results are emerging from GWAS studies; however, larger sample sizes are needed to improve GWAS results and resolution. As the field of genetics is rapidly developing, whole genome sequencing could soon become the new standard of interrogation of the genes and neurobiological pathways which contribute to the complex phenotype of AUD. Short summary This review examines the genetic underpinnings of Alcohol Use Disorder (AUD), with an emphasis on GWAS approaches for identifying genetic risk variants. The most promising results associated with AUD and alcohol-related phenotypes have included SNPs of the alcohol metabolism genes ADH and ALDH. PMID:27445363

Impact of chronic low to moderate alcohol consumption on blood lipid and heart energy profile in acetaldehyde dehydrogenase 2-deficient mice.

PubMed

Fan, Fan; Cao, Quan; Wang, Cong; Ma, Xin; Shen, Cheng; Liu, Xiang-wei; Bu, Li-ping; Zou, Yun-zeng; Hu, Kai; Sun, Ai-jun; Ge, Jun-bo

2014-08-01

To investigate the roles of acetaldehyde dehydrogenase 2 (ALDH2), the key enzyme of ethanol metabolism, in chronic low to moderate alcohol consumption-induced heart protective effects in mice. Twenty-one male wild-type (WT) or ALDH2-knockout (KO) mice were used in this study. In each genotype, 14 animals received alcohol (2.5%, 5% and 10% in week 1-3, respectively, and 18% in week 4-7), and 7 received water for 7 weeks. After the treatments, survival rate and general characteristics of the animals were evaluated. Serum ethanol and acetaldehyde levels and blood lipids were measured. Metabolomics was used to characterize the heart and serum metabolism profiles. Chronic alcohol intake decreased the survival rate of KO mice by 50%, and significantly decreased their body weight, but did not affect those of WT mice. Chronic alcohol intake significantly increased the serum ethanol levels in both WT and KO mice, but KO mice had significantly higher serum acetaldehyde levels than WT mice. Chronic alcohol intake significantly increased the serum HDL cholesterol levels in WT mice, and did not change the serum HDL cholesterol levels in KO mice. After chronic alcohol intake, WT and KO mice showed differential heart and serum metabolism profiles, including the 3 main energy substrate types (lipids, glucose and amino acids) and three carboxylic acid cycles. Low to moderate alcohol consumption increases HDL cholesterol levels and improves heart energy metabolism profile in WT mice but not in ALDH2-KO mice. Thus, preserved ALDH2 function is essential for the protective effect of low to moderate alcohol on the cardiovascular system.

Body odor aldehyde reduction by acetic acid bacterial extract including enzymes: alcohol dehydrogenase and aldehyde dehydrogenase.

PubMed

Yoshioka, N; Kurata, K; Takahashi, T; Ariizumi, M; Mori, T; Fujisawa, H; Kameyama, N; Okuyama, Y

2018-06-13

Body odor is mainly caused by secreted sweat. Although sweat is almost odorless immediately after secretion, decomposition or denaturation of components contained in sweat by bacteria on the skin surface contributes to unpleasant body odor. Body odor is due to various substances and aldehydes are primarily detected in body odor [1-4]. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Catalytic Mechanism of Short Ethoxy Chain Nonylphenol Dehydrogenase Belonging to a Polyethylene Glycol Dehydrogenase Group in the GMC Oxidoreductase Family

PubMed Central

Liu, Xin; Ohta, Takeshi; Kawabata, Takeshi; Kawai, Fusako

2013-01-01

Ethoxy (EO) chain nonylphenol dehydrogenase (NPEO-DH) from Ensifer sp. AS08 and EO chain octylphenol dehydrogenase from Pseudomonas putida share common molecular characteristics with polyethylene glycol (PEG) dehydrogenases (PEG-DH) and comprise a PEG-DH subgroup in the family of glucose-methanol-choline (GMC) oxidoreductases that includes glucose/alcohol oxidase and glucose/choline dehydrogenase. Three-dimensional (3D) molecular modeling suggested that differences in the size, secondary structure and hydropathy in the active site caused differences in their substrate specificities toward EO chain alkylphenols and free PEGs. Based on 3D molecular modeling, site-directed mutagenesis was utilized to introduce mutations into potential catalytic residues of NPEO-DH. From steady state and rapid kinetic characterization of wild type and mutant NPEO-DHs, we can conclude that His465 and Asn507 are directly involved in the catalysis. Asn507 mediates the transfer of proton from a substrate to FAD and His465 transfers the same proton from the reduced flavin to an electron acceptor. PMID:23306149

Catalytic mechanism of short ethoxy chain nonylphenol dehydrogenase belonging to a polyethylene glycol dehydrogenase group in the GMC oxidoreductase family.

PubMed

Liu, Xin; Ohta, Takeshi; Kawabata, Takeshi; Kawai, Fusako

2013-01-10

Ethoxy (EO) chain nonylphenol dehydrogenase (NPEO-DH) from Ensifer sp. AS08 and EO chain octylphenol dehydrogenase from Pseudomonas putida share common molecular characteristics with polyethylene glycol (PEG) dehydrogenases (PEG-DH) and comprise a PEG-DH subgroup in the family of glucose-methanol-choline (GMC) oxidoreductases that includes glucose/alcohol oxidase and glucose/choline dehydrogenase. Three-dimensional (3D) molecular modeling suggested that differences in the size, secondary structure and hydropathy in the active site caused differences in their substrate specificities toward EO chain alkylphenols and free PEGs. Based on 3D molecular modeling, site-directed mutagenesis was utilized to introduce mutations into potential catalytic residues of NPEO-DH. From steady state and rapid kinetic characterization of wild type and mutant NPEO-DHs, we can conclude that His465 and Asn507 are directly involved in the catalysis. Asn507 mediates the transfer of proton from a substrate to FAD and His465 transfers the same proton from the reduced flavin to an electron acceptor.

Expression and characterization of a class III alcohol dehydrogenase gene from Gluconobacter frateurii in the presence of methanol during glyceric acid production from glycerol.

PubMed

Sato, Shun; Morita, Naoki; Kitamoto, Dai; Habe, Hiroshi

2013-01-01

Some acetic acid bacteria have been shown to produce large amounts of glyceric acid (GA) from glycerol, which is a by-product of biodiesel fuel (BDF) production. Previously, a Gluconobacter strain was found that produced decreased amounts of GA from glycerol in the presence of methanol, a major ingredient of raw glycerol derived from the BDF industry. Thus, a comparative transcriptome analysis of Gluconobacter frateurii NBRC103465 was performed to investigate changes in gene expression during GA production from glycerol in the presence of methanol. Cells grown with methanol showed upregulated expression of a class III alcohol dehydrogenase homolog (adhC(Gf)) and decreased GA production. adhC(Gf) was cloned and expressed heterologously in Escherichia coli, and the presence of an additional protein with an approximate molecular mass of 39 kDa in the cytosol of the recombinant E. coli cells was identified by SDS-PAGE. Activity measurements of the cytosol revealed that the translational product of adhC(Gf) exhibited formaldehyde dehydrogenase activity in the presence of nicotinamide adenine dinucleotide and glutathione. Gluconobacter frateurii cells grown in 1% methanol-containing glycerol were found to have fivefold higher formaldehyde dehydrogenase activity than cells grown without methanol, suggesting that adhC(Gf) in G. frateurii cells functions in the dissimilation of methanol-derived formaldehyde.

A novel aromatic alcohol dehydrogenase in higher plants: molecular cloning and expression.

PubMed

Goffner, D; Van Doorsselaere, J; Yahiaoui, N; Samaj, J; Grima-Pettenati, J; Boudet, A M

1998-03-01

Cinnamyl alcohol dehydrogenase (CAD; EC 1.1.195) catalyses the conversion of p-hydroxy-cinnamaldehydes to the corresponding alcohols and is considered a key enzyme in lignin biosynthesis. In a previous study, an atypical form of CAD (CAD 1) was identified in Eucalyptus gunnii [12]. We report here the molecular cloning and characterization of the corresponding cDNA, CAD 1-5, which encodes this novel aromatic alcohol dehydrogenase. The identity of CAD 1-5 was unambiguously confirmed by sequence comparison of the cDNA with peptide sequences derived from purified CAD 1 protein and by functional expression of CAD 1 recombinant protein in Escherichia coli. Both native and recombinant CAD 1 exhibit high affinity towards lignin precursors including 4-coumaraldehyde and coniferaldehyde, but they do not accept sinapaldehyde. Moreover, recombinant CAD 1 can also utilize a wide range of aromatic substrates including unsubstituted and substituted benzaldehydes. The open reading frame of CAD 1-5 encodes a protein with a calculated molecular mass of 35,790 Da and an isoelectric point of 8.1. Although sequence comparisons with proteins in databases revealed significant similarities with dihydroflavonol-4-reductases (DFR; EC 1.1.1.219) from a wide range of plant species, the most striking similarity was found with cinnamoyl-CoA reductase (CCR; EC 1.2.1.44), the enzyme which directly precedes CAD in the lignin biosynthetic pathway. RNA blot analysis and immunolocalization experiments indicated that CAD 1 is expressed in both lignified and unlignified tissues/cells. Based on the catalytic activity of CAD 1 in vitro and its localization in planta, CAD 1 may function as an 'alternative' enzyme in the lignin biosynthetic pathway. However, additional roles in phenolic metabolism are not excluded.

Mitochondria-targeted ubiquinone (MitoQ) enhances acetaldehyde clearance by reversing alcohol-induced posttranslational modification of aldehyde dehydrogenase 2: A molecular mechanism of protection against alcoholic liver disease.

PubMed

Hao, Liuyi; Sun, Qian; Zhong, Wei; Zhang, Wenliang; Sun, Xinguo; Zhou, Zhanxiang

2018-04-01

Alcohol metabolism in the liver generates highly toxic acetaldehyde. Breakdown of acetaldehyde by aldehyde dehydrogenase 2 (ALDH2) in the mitochondria consumes NAD + and generates reactive oxygen/nitrogen species, which represents a fundamental mechanism in the pathogenesis of alcoholic liver disease (ALD). A mitochondria-targeted lipophilic ubiquinone (MitoQ) has been shown to confer greater protection against oxidative damage in the mitochondria compared to untargeted antioxidants. The present study aimed to investigate if MitoQ could preserve mitochondrial ALDH2 activity and speed up acetaldehyde clearance, thereby protects against ALD. Male C57BL/6J mice were exposed to alcohol for 8 weeks with MitoQ supplementation (5mg/kg/d) for the last 4 weeks. MitoQ ameliorated alcohol-induced oxidative/nitrosative stress and glutathione deficiency. It also reversed alcohol-reduced hepatic ALDH activity and accelerated acetaldehyde clearance through modulating ALDH2 cysteine S-nitrosylation, tyrosine nitration and 4-hydroxynonenol adducts formation. MitoQ ameliorated nitric oxide (NO) donor-mediated ADLH2 S-nitrosylation and nitration in Hepa-1c1c7 cells under glutathion depletion condition. In addition, alcohol-increased circulating acetaldehyde levels were accompanied by reduced intestinal ALDH activity and impaired intestinal barrier. In accordance, MitoQ reversed alcohol-increased plasma endotoxin levels and hepatic toll-like receptor 4 (TLR4)-NF-κB signaling along with subsequent inhibition of inflammatory cell infiltration. MitoQ also reversed alcohol-induced hepatic lipid accumulation through enhancing fatty acid β-oxidation. Alcohol-induced ER stress and apoptotic cell death signaling were reversed by MitoQ. This study demonstrated that speeding up acetaldehyde clearance by preserving ALDH2 activity critically mediates the beneficial effect of MitoQ on alcohol-induced pathogenesis at the gut-liver axis. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Alcoholism and alcohol drinking habits predicted from alcohol dehydrogenase genes.

PubMed

Tolstrup, Janne Schurmann; Nordestgaard, Børge Grønne; Rasmussen, Søren; Tybjaerg-Hansen, Anne; Grønbaek, Morten

2008-06-01

Alcohol drinking habits and alcoholism are partly genetically determined. Alcohol is degraded primarily by alcohol dehydrogenase (ADH) wherein genetic variation that affects the rate of alcohol degradation is found in ADH1B and ADH1C. It is biologically plausible that these variations may be associated with alcohol drinking habits and alcoholism. By genotyping 9080 white men and women from the general population, we found that men and women with ADH1B slow vs fast alcohol degradation drank more alcohol and had a higher risk of everyday drinking, heavy drinking, excessive drinking and of alcoholism. For example, the weekly alcohol intake was 9.8 drinks (95% confidence interval (CI): 9.1-11) among men with the ADH1B.1/1 genotype compared to 7.5 drinks (95% CI: 6.4-8.7) among men with the ADH1B.1/2 genotype, and the odds ratio (OR) for heavy drinking was 3.1 (95% CI: 1.7-5.7) among men with the ADH1B.1/1 genotype compared to men with the ADH1B.1/2 genotype. Furthermore, individuals with ADH1C slow vs fast alcohol degradation had a higher risk of heavy and excessive drinking. For example, the OR for heavy drinking was 1.4 (95% CI: 1.1-1.8) among men with the ADH1C.1/2 genotype and 1.4 (95% CI: 1.0-1.9) among men with the ADH1B.2/2 genotype, compared with men with the ADH1C.1/1 genotype. Results for ADH1B and ADH1C genotypes among men and women were similar. Finally, because slow ADH1B alcohol degradation is found in more than 90% of the white population compared to less than 10% of East Asians, the population attributable risk of heavy drinking and alcoholism by ADH1B.1/1 genotype was 67 and 62% among the white population compared with 9 and 24% among the East Asian population.

Three alcohol dehydrogenase genes and one acetyl-CoA synthetase gene are responsible for ethanol utilization in Yarrowia lipolytica.

PubMed

Gatter, Michael; Ottlik, Stephanie; Kövesi, Zsolt; Bauer, Benjamin; Matthäus, Falk; Barth, Gerold

2016-10-01

The non-conventional yeast Yarrowia lipolytica is able to utilize a wide range of different substrates like glucose, glycerol, ethanol, acetate, proteins and various hydrophobic molecules. Although most metabolic pathways for the utilization of these substrates have been clarified by now, it was not clear whether ethanol is oxidized by alcohol dehydrogenases or by an alternative oxidation system inside the cell. In order to detect the genes that are required for ethanol utilization in Y. lipolytica, eight alcohol dehydrogenase (ADH) genes and one alcohol oxidase gene (FAO1) have been identified and respective deletion strains were tested for their ability to metabolize ethanol. As a result of this, we found that the availability of ADH1, ADH2 or ADH3 is required for ethanol utilization in Y. lipolytica. A strain with deletions in all three genes is lacking the ability to utilize ethanol as sole carbon source. Although Adh2p showed by far the highest enzyme activity in an in vitro assay, the availability of any of the three genes was sufficient to enable a decent growth. In addition to ADH1, ADH2 and ADH3, an acetyl-CoA synthetase encoding gene (ACS1) was found to be essential for ethanol utilization. As Y. lipolytica is a non-fermenting yeast, it is neither able to grow under anaerobic conditions nor to produce ethanol. To investigate whether Y. lipolytica may produce ethanol, the key genes of alcoholic fermentation in S. cerevisiae, ScADH1 and ScPDC1, were overexpressed in an ADH and an ACS1 deletion strain. However, instead of producing ethanol, the respective strains regained the ability to use ethanol as single carbon source and were still not able to grow under anaerobic conditions. Copyright © 2016 Elsevier Inc. All rights reserved.

Alcohol and aldehyde dehydrogenase gene polymorphisms and oropharyngolaryngeal, esophageal and stomach cancers in Japanese alcoholics.

PubMed

Yokoyama, A; Muramatsu, T; Omori, T; Yokoyama, T; Matsushita, S; Higuchi, S; Maruyama, K; Ishii, H

2001-03-01

Alcohol dehydrogenase-2 (ADH2) and aldehyde dehydrogenase-2 (ALDH2) gene polymorphisms play roles in ethanol metabolism, drinking behavior and esophageal carcinogenesis in Japanese; however, the combined influence of ADH2 and ALDH2 genotypes on other aerodigestive tract cancers have not been investigated. ADH2/ALDH2 genotyping was performed on lymphocyte DNA samples from Japanese alcoholic men (526 cancer-free; 159 with solitary or multiple aerodigestive tract cancers, including 33 oropharyngolaryngeal, 112 esophageal, 38 stomach and 22 multiple primary cancers in two or three organs). After adjustment for age, drinking and smoking habits, and ADH2/ALDH2 genotypes, the presence of either ADH2*1/2*1 or ALDH2*1/2*2 significantly increased the risk for oropharyngolaryngeal cancer [odds ratios (ORs), 6.68 with ADH2*1/2*1 and 18.52 with ALDH2*1/2*2] and esophageal cancer (ORs, 2.64 and 13.50, respectively). For patients with both ADH2*1/2*1 and ALDH2*1/2*2, the risks for oropharyngolaryngeal and esophageal cancers were enhanced in a multiplicative fashion (OR = 121.77 and 40.40, respectively). A positive association with ALDH2*1/2*2 alone was observed for stomach cancer patients who also had oropharyngolaryngeal and/or esophageal cancer (OR = 110.58), but it was not observed for those with stomach cancer alone. Furthermore, in the presence of ALDH2*1/2*2, the risks for multiple intra-esophageal cancers (OR = 3.43) and for esophageal cancer with oropharyngolaryngeal and/or stomach cancer (OR = 3.95) were higher than the risks for solitary intra-esophageal cancer and for esophageal cancer alone, but these tendencies were not observed for ADH2*1/2*1 genotype. Alcoholics' population attributable risks due to ADH2/ALDH2 polymorphisms were estimated to be 82.0% for oropharyngolaryngeal cancer and 63.9% for esophageal cancer.

Aldehyde dehydrogenase 2 polymorphism for development to hepatocellular carcinoma in East Asian alcoholic liver cirrhosis.

PubMed

Abe, Hiroshi; Aida, Yuta; Seki, Nobuyoshi; Sugita, Tomonori; Tomita, Yoichi; Nagano, Tomohisa; Itagaki, Munenori; Sutoh, Satoshi; Nagatsuma, Keisuke; Itoh, Kyoko; Matsuura, Tomokazu; Aizawa, Yoshio

2015-09-01

We aimed to clarify the influences of aldehyde dehydrogenase 2 (ALDH2), alcohol dehydrogenase 1B (ADH1B) polymorphisms, and ethanol consumption profile to hepatocellular carcinoma (HCC) development in alcoholic liver cirrhosis without chronic hepatitis B and C virus infection (non-B non-C). Of 236 freshly diagnosed non-B non-C alcoholic liver cirrhosis patients, 67 were diagnosed as HCC and the remaining 169 as not having HCC. The relationship between the genetic polymorphisms and development to HCC were evaluated in well-matched patients with HCC (HCC group, n = 67) and without HCC (non-HCC group, n = 67) using propensity scores in age, sex, and prevalence of diabetes mellitus. Daily amount of ethanol consumption was significantly lower (P = 0.005), and consumptive period was significantly longer (P = 0.003) in HCC group than non-HCC group. Of 134 well-matched patients, 113 (84.3%) had ALDH2*1/*1 genotype and 21 (15.7%) had ALDH2*1/*2 genotype. In HCC development, consumptive long period (P = 0.007) and carrying ALDH2*1/*2 genotype (P = 0.026) were identified as significant factors independently participated, while there was no relation to ADH1B polymorphism. In addition, consumptive period was significantly longer in HCC group than non-HCC group in ALDH2*1/*1 genotype patients (P = 0.0005), while there was no difference in profile of ethanol consumption in ALDH2*1/*2 genotype patients. Among HCC group, daily (P = 3.78 × 10(-6) ) and cumulative amount (P = 4.89 × 10(-6) ) of ethanol consumption were significantly higher in ALDH2*1/*1 genotype patients than ALDH2*1/*2 genotype patients. In alcoholic liver cirrhosis, investigations of ALDH2 polymorphism and ethanol consumption profile are useful for prediction of HCC development. © 2015 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd.

Crystallographic analysis and structure-guided engineering of NADPH-dependent Ralstonia sp. alcohol dehydrogenase toward NADH cosubstrate specificity.

PubMed

Lerchner, Alexandra; Jarasch, Alexander; Meining, Winfried; Schiefner, André; Skerra, Arne

2013-11-01

The NADP⁺-dependent alcohol dehydrogenase from Ralstonia sp. (RasADH) belongs to the protein superfamily of short-chain dehydrogenases/reductases (SDRs). As an enzyme that accepts different types of substrates--including bulky-bulky as well as small-bulky secondary alcohols or ketones--with high stereoselectivity, it offers potential as a biocatalyst for industrial biotechnology. To understand substrate and cosubstrate specificities of RasADH we determined the crystal structure of the apo-enzyme as well as its NADP⁺-bound state with resolutions down to 2.8 Å. RasADH displays a homotetrameric quaternary structure that can be described as a dimer of homodimers while in each subunit a seven-stranded parallel β-sheet, flanked by three α-helices on each side, forms a Rossmann fold-type dinucleotide binding domain. Docking of the well-known substrate (S)-1-phenylethanol clearly revealed the structural determinants of stereospecificity. To favor practical RasADH application in the context of established cofactor recycling systems, for example, those involving an NADH-dependent amino acid dehydrogenase, we attempted to rationally change its cosubstrate specificity from NADP⁺ to NAD⁺ utilizing the structural information that NADP⁺ specificity is largely governed by the residues Asn15, Gly37, Arg38, and Arg39. Furthermore, an extensive sequence alignment with homologous dehydrogenases that have different cosubstrate specificities revealed a modified general SDR motif ASNG (instead of NNAG) at positions 86-89 of RasADH. Consequently, we constructed mutant enzymes with one (G37D), four (N15G/G37D/R38V/R39S), and six (N15G/G37D/R38V/R39S/A86N/S88A) amino acid exchanges. RasADH (N15G/G37D/R38V/R39S) was better able to accept NAD⁺ while showing much reduced catalytic efficiency with NADP⁺, leading to a change in NADH/NADPH specificity by a factor of ∼3.6 million. © 2013 Wiley Periodicals, Inc.

Haloacetic Acid Water Disinfection Byproducts Affect Pyruvate Dehydrogenase Activity and Disrupt Cellular Metabolism.

PubMed

Dad, Azra; Jeong, Clara H; Wagner, Elizabeth D; Plewa, Michael J

2018-02-06

The disinfection of drinking water has been a major public health achievement. However, haloacetic acids (HAAs), generated as byproducts of water disinfection, are cytotoxic, genotoxic, mutagenic, carcinogenic, and teratogenic. Previous studies of monoHAA-induced genotoxicity and cell stress demonstrated that the toxicity was due to inhibition of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), leading to disruption of cellular metabolism and energy homeostasis. DiHAAs and triHAAs are also produced during water disinfection, and whether they share mechanisms of action with monoHAAs is unknown. In this study, we evaluated the effects of mono-, di-, and tri-HAAs on cellular GAPDH enzyme kinetics, cellular ATP levels, and pyruvate dehydrogenase complex (PDC) activity. Here, treatments conducted in Chinese hamster ovary (CHO) cells revealed differences among mono-, di-, and triHAAs in their molecular targets. The monoHAAs, iodoacetic acid and bromoacetic acid, were the strongest inhibitors of GAPDH and greatly reduced cellular ATP levels. Chloroacetic acid, diHAAs, and triHAAs were weaker inhibitors of GAPDH and some increased the levels of cellular ATP. HAAs also affected PDC activity, with most HAAs activating PDC. The primary finding of this work is that mono- versus multi-HAAs address different molecular targets, and the results are generally consistent with a model in which monoHAAs activate the PDC through GAPDH inhibition-mediated disruption in cellular metabolites, including altering ATP-to-ADP and NADH-to-NAD ratios. The monoHAA-mediated reduction in cellular metabolites results in accelerated PDC activity by way of metabolite-ratio-dependent PDC regulation. DiHAAs and triHAAs are weaker inhibitors of GAPDH, but many also increase cellular ATP levels, and we suggest that they increase PDC activity by inhibiting pyruvate dehydrogenase kinase.

Evaluation of the influence of alcohol dehydrogenase polymorphisms on alcohol elimination rates in African Americans.

PubMed

Marshall, Vanessa J; Ramchandani, Vijay A; Kalu, Nnenna; Kwagyan, John; Scott, Denise M; Ferguson, Clifford L; Taylor, Robert E

2014-01-01

The relationship between alcohol dehydrogenase (ADH) polymorphisms and alcohol use disorders in populations of African descent has not been clearly established. This study examined the effect of ADH1B polymorphisms on alcohol metabolism and subjective response, following intravenous (IV) alcohol administration, and the influence of gender, recent drinking history, and family history of alcoholism (FHA), in nondependent African American drinkers. The sample included eighty-seven 21- to 35-year-old, light social drinkers of African descent. Participants included 39 sib pairs, 2 sibships with 3 siblings each, and 3 individuals who were not part of a sibship. Participants received infusions via the use of the clamp method that refers to the goal of controlling breath alcohol concentration in 2 randomized sessions at 0.06 g% ethanol and 0 mg% (placebo), and a battery of subjective scales at predefined time points. Dependent measures included alcohol elimination rates (AERs), alcohol disappearance rates (ADRs), subjective measures peak scores, and area under the curve. General linear model and mixed models were performed to examine the relationship between ADH1B genotype, dependent measures, and influence of covariates. Participants with ADH1B1/1 genotypes showed higher number of drinks (p = 0.023) and drinks per drinking day (p = 0.009) compared with the persons with ADH1B1/3 genotype. AER (adjusted for body weight) was higher in ADH1B*1 homozygotes (p = 0.045) compared with ADH1B1/3 heterozygotes. ADR differed significantly between males and females (p = 0.002), regardless of body weight (p = 0.004) and lean body mass (p < 0.001) adjustments. Although a few subjective measures differed across genotype, all measures were higher in alcohol sessions compared with placebo sessions (p < 0.001). These observations were mediated by drinks per drinking day, gender, and FHA. ADH1B polymorphism had a marginal effect on alcohol pharmacokinetics following IV alcohol administration in nondependent drinkers of African descent. Session (alcohol vs. placebo) and ADH1B genotype did, however, influence subjective response to alcohol with some variation by gender, FHA, and drinks per drinking day. Copyright © 2013 by the Research Society on Alcoholism.

The two-step electrochemical oxidation of alcohols using a novel recombinant PQQ alcohol dehydrogenase as a catalyst for a bioanode.

PubMed

Takeda, Kouta; Matsumura, Hirotoshi; Ishida, Takuya; Samejima, Masahiro; Igarashi, Kiyohiko; Nakamura, Nobuhumi; Ohno, Hiroyuki

2013-12-01

A bioanode has been developed based on the oxidation of ethanol by the recombinant pyrroloquinoline quinone (PQQ) dependent alcohol dehydrogenase from Pseudomonas putidaKT2440 heterologously expressed in Pichia pastoris. The apo form of the recombinant protein (PpADH) was purified and displayed catalytic activity for binding PQQ in the presence of Ca(2+). PpADH exhibited broad substrate specificity towards various alcohols and aldehydes. The Km values for the aldehydes of PpADH were increased compared to those for the alcohols, whereas the kcat values were unaltered. For instance, the Km values at T=298.15K (25 °C) for ethanol and acetaldehyde were 0.21 (± 0.02)mM and 5.8 (± 0.60)mM, respectively. The kcat values for ethanol and acetaldehyde were 24.8 (± 1.2) s(-1) and 31.1 (± 1.2) s(-1), respectively. The aminoferrocene was used as an electron transfer mediator between PpADH and the electrode during electrochemical experiments. The catalytic currents for the oxidation of alcohol and acetaldehyde by PpADH were also observed in this system. The electric charge for the oxidation of ethanol (Q = 2.09 × 10(-3) · C) was increased two-fold compared to that for the oxidation of acetaldehyde (Q = 0.95 × 10(-3) · C), as determined by chronoamperometric measurements. Thus, we have electrochemically demonstrated the two-step oxidation of ethanol to acetate using only PpADH. © 2013.

Hangover symptoms in Asian Americans with variations in the aldehyde dehydrogenase (ALDH2) gene.

PubMed

Wall, T L; Horn, S M; Johnson, M L; Smith, T L; Carr, L G

2000-01-01

Hangovers are not experienced by all people and whether they contribute to the development of alcoholism is unclear. One population that might provide some insight into the role of hangover in the etiology of alcohol use disorders is that of individuals of Asian heritage. Certain Asians have lower rates of alcohol use and alcoholism, findings associated with a mutation in the aldehyde dehydrogenase (ALDH2) gene. Asians with ALDH2*2 alleles drink less and are less likely to be alcoholic than Asians without this mutation. Following alcohol ingestion, they exhibit more intense reactions to alcohol and generate higher levels of the metabolite acetaldehyde. This study evaluated hangover symptoms in Asian Americans with variations in the ALDH2 gene. Men and women of Chinese, Japanese and Korean heritage (N = 140) were asked about their drinking history and a blood sample was collected for genotyping at the ALDH2 locus. Subjects used a Likert-type scale to estimate their severity of hangover and completed a 13-item hangover scale assessing the frequency of hangover symptoms during the previous 6 months. With abstainers (n = 17) excluded and with the effects of gender and recent drinking history controlled, ALDH2 genotype accounted for a significant amount of additional variability in the estimated severity of hangover score with a similar, but nonsignificant, trend for a five-item subscale score derived from the hangover scale. These results suggest that Asian Americans with ALDH2*2 alleles may experience more severe hangovers that may contribute, in part, to protection against the development of excessive or problematic drinking in this population.

Pyruvate Decarboxylase Catalyzes Decarboxylation of Branched-Chain 2-Oxo Acids but Is Not Essential for Fusel Alcohol Production by Saccharomyces cerevisiae

PubMed Central

ter Schure, Eelko G.; Flikweert, Marcel T.; van Dijken, Johannes P.; Pronk, Jack T.; Verrips, C. Theo

1998-01-01

The fusel alcohols 3-methyl-1-butanol, 2-methyl-1-butanol, and 2-methyl-propanol are important flavor compounds in yeast-derived food products and beverages. The formation of these compounds from branched-chain amino acids is generally assumed to occur via the Ehrlich pathway, which involves the concerted action of a branched-chain transaminase, a decarboxylase, and an alcohol dehydrogenase. Partially purified preparations of pyruvate decarboxylase (EC 4.1.1.1) have been reported to catalyze the decarboxylation of the branched-chain 2-oxo acids formed upon transamination of leucine, isoleucine, and valine. Indeed, in a coupled enzymatic assay with horse liver alcohol dehydrogenase, cell extracts of a wild-type Saccharomyces cerevisiae strain exhibited significant decarboxylation rates with these branched-chain 2-oxo acids. Decarboxylation of branched-chain 2-oxo acids was not detectable in cell extracts of an isogenic strain in which all three PDC genes had been disrupted. Experiments with cell extracts from S. cerevisiae mutants expressing a single PDC gene demonstrated that both PDC1- and PDC5-encoded isoenzymes can decarboxylate branched-chain 2-oxo acids. To investigate whether pyruvate decarboxylase is essential for fusel alcohol production by whole cells, wild-type S. cerevisiae and an isogenic pyruvate decarboxylase-negative strain were grown on ethanol with a mixture of leucine, isoleucine, and valine as the nitrogen source. Surprisingly, the three corresponding fusel alcohols were produced in both strains. This result proves that decarboxylation of branched-chain 2-oxo acids via pyruvate decarboxylase is not an essential step in fusel alcohol production. PMID:9546164

Metabolite profiling reveals a role for atypical cinnamyl alcohol dehydrogenase CAD1 in the synthesis of coniferyl alcohol in tobacco xylem.

PubMed

Damiani, Isabelle; Morreel, Kris; Danoun, Saïda; Goeminne, Geert; Yahiaoui, Nabila; Marque, Christiane; Kopka, Joachim; Messens, Eric; Goffner, Deborah; Boerjan, Wout; Boudet, Alain-Michel; Rochange, Soizic

2005-11-01

In angiosperms, lignin is built from two main monomers, coniferyl and sinapyl alcohol, which are incorporated respectively as G and S units in the polymer. The last step of their synthesis has so far been considered to be performed by a family of dimeric cinnamyl alcohol dehydrogenases (CAD2). However, previous studies on Eucalyptus gunnii xylem showed the presence of an additional, structurally unrelated, monomeric CAD form named CAD1. This form reduces coniferaldehyde to coniferyl alcohol, but is inactive on sinapaldehyde. In this paper, we report the functional characterization of CAD1 in tobacco (Nicotiana tabacum L.). Transgenic tobacco plants with reduced CAD1 expression were obtained through an RNAi strategy. These plants displayed normal growth and development, and detailed biochemical studies were needed to reveal a role for CAD1. Lignin analyses showed that CAD1 down-regulation does not affect Klason lignin content, and has a moderate impact on G unit content of the non-condensed lignin fraction. However, comparative metabolic profiling of the methanol-soluble phenolic fraction from basal xylem revealed significant differences between CAD1 down-regulated and wild-type plants. Eight compounds were less abundant in CAD1 down-regulated lines, five of which were identified as dimers or trimers of monolignols, each containing at least one moiety derived from coniferyl alcohol. In addition, 3-trans-caffeoyl quinic acid accumulated in the transgenic plants. Together, our results support a significant contribution of CAD1 to the synthesis of coniferyl alcohol in planta, along with the previously characterized CAD2 enzymes.

Problem-Based Test: The Effect of Fibroblast Growth Factor on Gene Expression

ERIC Educational Resources Information Center

Szeberenyi, Jozsef

2011-01-01

This paper shows the results of an experiment in which the effects of fibroblast growth factor (FGF), actinomycin D (Act D; an inhibitor of transcription), and cycloheximide (CHX; an inhibitor of translation) were studied on the expression of two genes: a gene called "Fnk" and the gene coding for glyceraldehyde-3-phosphate dehydrogenase (GAPDH).…

Targeting of Cancer Stem Cells and Their Microenvironment in Early-Stage Mutant K-ras Lung Cancer

DTIC Science & Technology

2016-12-01

Aldefluor reagent. (B) A549 control lung cancer cells were incubated with Alde- fluor regent and DEAB, an inhibitor of aldehyde dehydro- genase. (C...increase in liquid colony formation or in cell proliferation compared to SHH- cells. Therefore, we turned to identify aldehyde dehydrogenase (ALDH...in which a green fluorescent BODIPY moiety is linked to aminoacetaldehyde, an aldehyde dehydrogenase substrate, and thus, cells expressing ALDH

Association between ALDH2 and ADH1B polymorphisms, alcohol drinking and gastric cancer: a replication and mediation analysis.

PubMed

Ishioka, Kuka; Masaoka, Hiroyuki; Ito, Hidemi; Oze, Isao; Ito, Seiji; Tajika, Masahiro; Shimizu, Yasuhiro; Niwa, Yasumasa; Nakamura, Shigeo; Matsuo, Keitaro

2018-04-03

Aldehyde dehydrogenase 2 (ALDH2; rs671, Glu504Lys) and alcohol dehydrogenase 1B (ADH1B; rs1229984, His47Arg) polymorphisms have a strong impact on carcinogenic acetaldehyde accumulation after alcohol drinking. To date, however, evidence for a significant ALDH2-alcohol drinking interaction and a mediation effect of ALDH2/ADH1B through alcohol drinking on gastric cancer have remained unclear. We conducted two case-control studies to validate the interaction and to estimate the mediation effect on gastric cancer. We calculated odds ratios (OR) and 95% confidence intervals (CI) for ALDH2/ADH1B genotypes and alcohol drinking using conditional logistic regression models after adjustment for potential confounding in the HERPACC-2 (697 cases and 1372 controls) and HERPACC-3 studies (678 cases and 678 controls). We also conducted a mediation analysis of the combination of the two studies to assess whether the effects of these polymorphisms operated through alcohol drinking or through other pathways. ALDH2 Lys alleles had a higher risk with increased alcohol consumption compared with ALDH2 Glu/Glu (OR for heavy drinking, 3.57; 95% CI 2.04-6.27; P for trend = 0.007), indicating a significant ALDH2-alcohol drinking interaction (P interaction  = 0.024). The mediation analysis indicated a significant positive direct effect (OR 1.67; 95% CI 1.38-2.03) and a protective indirect effect (OR 0.84; 95% CI 0.76-0.92) of the ALDH2 Lys alleles with the ALDH2-alcohol drinking interaction. No significant association of ADH1B with gastric cancer was observed. The observed ALDH2-alcohol drinking interaction and the direct effect of ALDH2 Lys alleles may suggest the involvement of acetaldehyde in the development of gastric cancer.

Trends in gastrectomy and ADH1B and ALDH2 genotypes in Japanese alcoholic men and their gene-gastrectomy, gene-gene and gene-age interactions for risk of alcoholism.

PubMed

Yokoyama, Akira; Yokoyama, Tetsuji; Matsui, Toshifumi; Mizukami, Takeshi; Kimura, Mitsuru; Matsushita, Sachio; Higuchi, Susumu; Maruyama, Katsuya

2013-01-01

The life-time drinking profiles of Japanese alcoholics have shown that gastrectomy increases susceptibility to alcoholism. We investigated the trends in gastrectomy and alcohol dehydrogenase-1B (ADH1B) and aldehyde dehydrogenase-2 (ALDH2) genotypes and their interactions in alcoholics. This survey was conducted on 4879 Japanese alcoholic men 40 years of age or older who underwent routine gastrointestinal endoscopic screening during the period 1996-2010. ADH1B/ALDH2 genotyping was performed in 3702 patients. A history of gastrectomy was found in 508 (10.4%) patients. The reason for the gastrectomy was peptic ulcer in 317 patients and gastric cancer in 187 patients. The frequency of gastrectomy had gradually decreased from 13.3% in 1996-2000 to 10.5% in 2001-2005 and to 7.8% in 2006-2010 (P < 0.0001). ADH1B*1/*1 was less frequent in the gastrectomy group than in the non-gastrectomy group (age-adjusted prevalence: 20.4 vs. 27.6%, P = 0.006). ALDH2 genotype distribution did not differ between the two groups. The frequency of inactive ALDH2*1/*2 heterozygotes increased slightly from 13.0% in 1996-2000 to 14.0% in 2001-2005 and to 15.4% in 2006-2010 (P < 0.08). Two alcoholism-susceptibility genotypes, ADH1B*1/*1 and ALDH2*1/*1, modestly but significantly tended not to occur in the same individual (P = 0.026). The frequency of ADH1B*1/*1 decreased with ascending age groups. The high frequency of history of gastrectomy suggested that gastrectomy is still a risk factor for alcoholism, although the percentage decreased during the period. The alcoholism-susceptibility genotype ADH1B*1/*1 was less frequent in the gastrectomy group, suggesting a competitive gene-gastrectomy interaction for alcoholism. A gene-gene interaction and gene-age interactions regarding the ADH1B genotype were observed.

Purification and characterization of a primary-secondary alcohol dehydrogenase from two strains of Clostridium beijerinckii.

PubMed Central

Ismaiel, A A; Zhu, C X; Colby, G D; Chen, J S

1993-01-01

Two primary alcohols (1-butanol and ethanol) are major fermentation products of several clostridial species. In addition to these two alcohols, the secondary alcohol 2-propanol is produced to a concentration of about 100 mM by some strains of Clostridium beijerinckii. An alcohol dehydrogenase (ADH) has been purified to homogeneity from two strains (NRRL B593 and NESTE 255) of 2-propanol-producing C. beijerinckii. When exposed to air, the purified ADH was stable, whereas the partially purified ADH was inactivated. The ADHs from the two strains had similar structural and kinetic properties. Each had a native M(r) of between 90,000 and 100,000 and a subunit M(r) of between 38,000 and 40,000. The ADHs were NADP(H) dependent, but a low level of NAD(+)-linked activity was detected. They were equally active in reducing aldehydes and 2-ketones, but a much lower oxidizing activity was obtained with primary alcohols than with secondary alcohols. The kcat/Km value for the alcohol-forming reaction appears to be a function of the size of the larger alkyl substituent on the carbonyl group. ADH activities measured in the presence of both acetone and butyraldehyde did not exceed activities measured with either substrate present alone, indicating a common active site for both substrates. There was no similarity in the N-terminal amino acid sequence between that of the ADH and those of fungi and several other bacteria. However, the N-terminal sequence had 67% identity with those of two other anaerobes, Thermoanaerobium brockii and Methanobacterium palustre. Furthermore, conserved glycine and tryptophan residues are present in ADHs of these three anaerobic bacteria and ADHs of mammals and green plants. Images PMID:8349550

Pancreatic injury in hepatic alcohol dehydrogenase-deficient deer mice after subchronic exposure to ethanol

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

Kaphalia, Bhupendra S., E-mail: bkaphali@utmb.ed; Bhopale, Kamlesh K.; Kondraganti, Shakuntala

2010-08-01

Pancreatitis caused by activation of digestive zymogens in the exocrine pancreas is a serious chronic health problem in alcoholic patients. However, mechanism of alcoholic pancreatitis remains obscure due to lack of a suitable animal model. Earlier, we reported pancreatic injury and substantial increases in endogenous formation of fatty acid ethyl esters (FAEEs) in the pancreas of hepatic alcohol dehydrogenase (ADH)-deficient (ADH{sup -}) deer mice fed 4% ethanol. To understand the mechanism of alcoholic pancreatitis, we evaluated dose-dependent metabolism of ethanol and related pancreatic injury in ADH{sup -} and hepatic ADH-normal (ADH{sup +}) deer mice fed 1%, 2% or 3.5% ethanolmore » via Lieber-DeCarli liquid diet daily for 2 months. Blood alcohol concentration (BAC) was remarkably increased and the concentration was {approx} 1.5-fold greater in ADH{sup -} vs. ADH{sup +} deer mice fed 3.5% ethanol. At the end of the experiment, remarkable increases in pancreatic FAEEs and significant pancreatic injury indicated by the presence of prominent perinuclear space, pyknotic nuclei, apoptotic bodies and dilation of glandular ER were found only in ADH{sup -} deer mice fed 3.5% ethanol. This pancreatic injury was further supported by increased plasma lipase and pancreatic cathepsin B (a lysosomal hydrolase capable of activating trypsinogen), trypsinogen activation peptide (by-product of trypsinogen activation process) and glucose-regulated protein 78 (endoplasmic reticulum stress marker). These findings suggest that ADH-deficiency and high alcohol levels in the body are the key factors in ethanol-induced pancreatic injury. Therefore, determining how this early stage of pancreatic injury advances to inflammation stage could be important for understanding the mechanism(s) of alcoholic pancreatitis.« less

Role of tryptophan 95 in substrate specificity and structural stability of Sulfolobus solfataricus alcohol dehydrogenase.

PubMed

Pennacchio, Angela; Esposito, Luciana; Zagari, Adriana; Rossi, Mosè; Raia, Carlo A

2009-09-01

A mutant of the thermostable NAD(+)-dependent (S)-stereospecific alcohol dehydrogenase from Sulfolobus solfataricus (SsADH) which has a single substitution, Trp95Leu, located at the substrate binding pocket, was fully characterized to ascertain the role of Trp95 in discriminating between chiral secondary alcohols suggested by the wild-type SsADH crystallographic structure. The Trp95Leu mutant displays no apparent activity with short-chain primary and secondary alcohols and poor activity with aromatic substrates and coenzyme. Moreover, the Trp --> Leu substitution affects the structural stability of the archaeal ADH, decreasing its thermal stability without relevant changes in secondary structure. The double mutant Trp95Leu/Asn249Tyr was also purified to assist in crystallographic analysis. This mutant exhibits higher activity but decreased affinity toward aliphatic alcohols, aldehydes as well as NAD(+) and NADH compared to the wild-type enzyme. The crystal structure of the Trp95Leu/Asn249Tyr mutant apo form, determined at 2.0 A resolution, reveals a large local rearrangement of the substrate site with dramatic consequences. The Leu95 side-chain conformation points away from the catalytic metal center and the widening of the substrate site is partially counteracted by a concomitant change of Trp117 side chain conformation. Structural changes at the active site are consistent with the reduced activity on substrates and decreased coenzyme binding.

NMR characterization of altered lignins extracted from tobacco plants down-regulated for lignification enzymes cinnamylalcohol dehydrogenase and cinnamoyl-CoA reductase.

PubMed

Ralph, J; Hatfield, R D; Piquemal, J; Yahiaoui, N; Pean, M; Lapierre, C; Boudet, A M

1998-10-27

Homologous antisense constructs were used to down-regulate tobacco cinnamyl-alcohol dehydrogenase (CAD; EC 1.1.1.195) and cinnamoyl-CoA reductase (CCR; EC 1.2.1.44) activities in the lignin monomer biosynthetic pathway. CCR converts activated cinnamic acids (hydroxycinnamoyl-SCoAs) to cinnamaldehydes; cinnamaldehydes are then reduced to cinnamyl alcohols by CAD. The transformations caused the incorporation of nontraditional components into the extractable tobacco lignins, as evidenced by NMR. Isolated lignin of antisense-CAD tobacco contained fewer coniferyl and sinapyl alcohol-derived units that were compensated for by elevated levels of benzaldehydes and cinnamaldehydes. Products from radical coupling of cinnamaldehydes, particularly sinapaldehyde, which were barely discernible in normal tobacco, were major components of the antisense-CAD tobacco lignin. Lignin content was reduced in antisense-CCR tobacco, which displayed a markedly reduced vigor. That lignin contained fewer coniferyl alcohol-derived units and significant levels of tyramine ferulate. Tyramine ferulate is a sink for the anticipated build-up of feruloyl-SCoA, and may be up-regulated in response to a deficit of coniferyl alcohol. Although it is not yet clear whether the modified lignins are true structural components of the cell wall, the findings provide further indications of the metabolic plasticity of plant lignification. An ability to produce lignin from alternative monomers would open new avenues for manipulation of lignin by genetic biotechnologies.

The Genetics of a Small Autosomal Region of DROSOPHILA MELANOGASTER Containing the Structural Gene for Alcohol Dehydrogenase. I. Characterization of Deficiencies and Mapping of ADH and Visible Mutations

PubMed Central

Woodruff, R. C.; Ashburner, M.

1979-01-01

The position of the structural gene coding for alcohol dehydrogenase (ADH) in Drosophila melanogaster has been shown to be within polytene chromosome bands 35B1 and 35B3, most probably within 35B2. The genetic and cytological properties of twelve deficiencies in polytene chromosome region 34–35 have been characterized, eleven of which include Adh. Also mapped cytogenetically are seven other recessive visible mutant loci. Flies heterozygous for overlapping deficiencies that include both the Adh locus and that for the outspread mutant (osp: a recessive wing phenotype) are homozygous viable and show a complete ADH negative phenotype and strong osp phenotype. These deficiencies probably include two polytene chromosome bands, 35B2 and 35B3. PMID:115743

High performance liquid chromatography method for the determination of cinnamyl alcohol dehydrogenase activity in soybean roots.

PubMed

dos Santos, W D; Ferrarese, Maria de Lourdes Lucio; Ferrarese-Filho, O

2006-01-01

This study proposes a simple, quick and reliable method for determining the cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.195) activity in soybean (Glycine max L. Merr.) roots using reversed-phase high performance liquid chromatography (RP-HPLC). The method includes a single extraction of the tissue and conduction of the enzymatic reaction at 30 degrees C with cinnamaldehydes (coniferyl or sinapyl), substrates of CAD. Disappearance of the substrates in the reaction mixture is monitored at 340 nm (for coniferaldehyde) or 345 nm (for sinapaldehyde) by isocratic elution with methanol/acetic acid through a GLC-ODS (M) column. This HPLC technique furnishes a rapid and reliable measure of cinnamaldehyde substrates, and may be used as an alternative tool to analyze CAD activity in enzyme preparation without previous purification.

The Last Step of Syringyl Monolignol Biosynthesis in Angiosperms Is Regulated by a Novel Gene Encoding Sinapyl Alcohol Dehydrogenase

PubMed Central

Li, Laigeng; Cheng, Xiao Fei; Leshkevich, Jacqueline; Umezawa, Toshiaki; Harding, Scott A.; Chiang, Vincent L.

2001-01-01

Cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.195) has been thought to mediate the reduction of both coniferaldehyde and sinapaldehyde into guaiacyl and syringyl monolignols in angiosperms. Here, we report the isolation of a novel aspen gene (PtSAD) encoding sinapyl alcohol dehydrogenase (SAD), which is phylogenetically distinct from aspen CAD (PtCAD). Liquid chromatography–mass spectrometry-based enzyme functional analysis and substrate level–controlled enzyme kinetics consistently demonstrated that PtSAD is sinapaldehyde specific and that PtCAD is coniferaldehyde specific. The enzymatic efficiency of PtSAD for sinapaldehyde was ∼60 times greater than that of PtCAD. These data suggest that in addition to CAD, discrete SAD function is essential to the biosynthesis of syringyl monolignol in angiosperms. In aspen stem primary tissues, PtCAD was immunolocalized exclusively to xylem elements in which only guaiacyl lignin was deposited, whereas PtSAD was abundant in syringyl lignin–enriched phloem fiber cells. In the developing secondary stem xylem, PtCAD was most conspicuous in guaiacyl lignin–enriched vessels, but PtSAD was nearly absent from these elements and was conspicuous in fiber cells. In the context of additional protein immunolocalization and lignin histochemistry, these results suggest that the distinct CAD and SAD functions are linked spatiotemporally to the differential biosynthesis of guaiacyl and syringyl lignins in different cell types. SAD is required for the biosynthesis of syringyl lignin in angiosperms. PMID:11449052

The last step of syringyl monolignol biosynthesis in angiosperms is regulated by a novel gene encoding sinapyl alcohol dehydrogenase.

PubMed

Li, L; Cheng, X F; Leshkevich, J; Umezawa, T; Harding, S A; Chiang, V L

2001-07-01

Cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.195) has been thought to mediate the reduction of both coniferaldehyde and sinapaldehyde into guaiacyl and syringyl monolignols in angiosperms. Here, we report the isolation of a novel aspen gene (PtSAD) encoding sinapyl alcohol dehydrogenase (SAD), which is phylogenetically distinct from aspen CAD (PtCAD). Liquid chromatography-mass spectrometry-based enzyme functional analysis and substrate level-controlled enzyme kinetics consistently demonstrated that PtSAD is sinapaldehyde specific and that PtCAD is coniferaldehyde specific. The enzymatic efficiency of PtSAD for sinapaldehyde was approximately 60 times greater than that of PtCAD. These data suggest that in addition to CAD, discrete SAD function is essential to the biosynthesis of syringyl monolignol in angiosperms. In aspen stem primary tissues, PtCAD was immunolocalized exclusively to xylem elements in which only guaiacyl lignin was deposited, whereas PtSAD was abundant in syringyl lignin-enriched phloem fiber cells. In the developing secondary stem xylem, PtCAD was most conspicuous in guaiacyl lignin-enriched vessels, but PtSAD was nearly absent from these elements and was conspicuous in fiber cells. In the context of additional protein immunolocalization and lignin histochemistry, these results suggest that the distinct CAD and SAD functions are linked spatiotemporally to the differential biosynthesis of guaiacyl and syringyl lignins in different cell types. SAD is required for the biosynthesis of syringyl lignin in angiosperms.

Ferrous glycinate regulates cell energy metabolism by restrictinghypoxia-induced factor-1α expression in human A549 cells.

PubMed

Kuo, Yung-Ting; Jheng, Jhong-Huei; Lo, Mei-Chen; Chen, Wei-Lu; Wang, Shyang-Guang; Lee, Horng-Mo

2018-06-04

Iron or oxygen regulates the stability of hypoxia inducible factor-1α (HIF-1α). We investigated whether ferrous glycinate would affect HIF-1α accumulation, aerobic glycolysis and mitochondrial energy metabolism in human A549 lung cancer cells. Incubation of A549 cells with ferrous glycinate decreased the protein levels of HIF-1α, which was abrogated by proteosome inhibitor, or prolyl hydroxylase inhibitor. The addition of ferrous glycinate decreased protein levels of glucose transporter-1, hexokinase-2, and lactate dehydrogenase A, and decreased pyruvate dehydrogenase kinase-1 (PDK-1) and pyruvate dehydrogenase (PDH) phosphorylation in A549 cells. Ferrous glycinate also increased the expression of the mitochondrial transcription factor A (TFAM), and the mitochondrial protein, cytochrome c oxidase (COX-IV). Silencing of HIF-1α expression mimicked the effects of ferrous glycinate on PDK-1, PDH, TFAM and COX-IV in A549 cells. Ferrous glycinate increased mitochondrial membrane potential and ATP production in A549 cells. These results suggest that ferrous glycinate may reverse Warburg effect through down regulating HIF-1α in A549 cells.

Glyceraldehyde-3-phosphate dehydrogenase aggregation inhibitor peptide: A potential therapeutic strategy against oxidative stress-induced cell death.

PubMed

Itakura, Masanori; Nakajima, Hidemitsu; Semi, Yuko; Higashida, Shusaku; Azuma, Yasu-Taka; Takeuchi, Tadayoshi

2015-11-13

The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has multiple functions, including mediating oxidative stress-induced neuronal cell death. This process is associated with disulfide-bonded GAPDH aggregation. Some reports suggest a link between GAPDH and the pathogenesis of several oxidative stress-related diseases. However, the pathological significance of GAPDH aggregation in disease pathogenesis remains unclear due to the lack of an effective GAPDH aggregation inhibitor. In this study, we identified a GAPDH aggregation inhibitor (GAI) peptide and evaluated its biological profile. The decapeptide GAI specifically inhibited GAPDH aggregation in a concentration-dependent manner. Additionally, the GAI peptide did not affect GAPDH glycolytic activity or cell viability. The GAI peptide also exerted a protective effect against oxidative stress-induced cell death in SH-SY5Y cells. This peptide could potentially serve as a tool to investigate GAPDH aggregation-related neurodegenerative and neuropsychiatric disorders and as a possible therapy for diseases associated with oxidative stress-induced cell death. Copyright © 2015 Elsevier Inc. All rights reserved.

Computational Study on New Natural Compound Inhibitors of Pyruvate Dehydrogenase Kinases

PubMed Central

Zhou, Xiaoli; Yu, Shanshan; Su, Jing; Sun, Liankun

2016-01-01

Pyruvate dehydrogenase kinases (PDKs) are key enzymes in glucose metabolism, negatively regulating pyruvate dehyrogenase complex (PDC) activity through phosphorylation. Inhibiting PDKs could upregulate PDC activity and drive cells into more aerobic metabolism. Therefore, PDKs are potential targets for metabolism related diseases, such as cancers and diabetes. In this study, a series of computer-aided virtual screening techniques were utilized to discover potential inhibitors of PDKs. Structure-based screening using Libdock was carried out following by ADME (adsorption, distribution, metabolism, excretion) and toxicity prediction. Molecular docking was used to analyze the binding mechanism between these compounds and PDKs. Molecular dynamic simulation was utilized to confirm the stability of potential compound binding. From the computational results, two novel natural coumarins compounds (ZINC12296427 and ZINC12389251) from the ZINC database were found binding to PDKs with favorable interaction energy and predicted to be non-toxic. Our study provide valuable information of PDK-coumarins binding mechanisms in PDK inhibitor-based drug discovery. PMID:26959013

Computational Study on New Natural Compound Inhibitors of Pyruvate Dehydrogenase Kinases.

PubMed

Zhou, Xiaoli; Yu, Shanshan; Su, Jing; Sun, Liankun

2016-03-04

Pyruvate dehydrogenase kinases (PDKs) are key enzymes in glucose metabolism, negatively regulating pyruvate dehyrogenase complex (PDC) activity through phosphorylation. Inhibiting PDKs could upregulate PDC activity and drive cells into more aerobic metabolism. Therefore, PDKs are potential targets for metabolism related diseases, such as cancers and diabetes. In this study, a series of computer-aided virtual screening techniques were utilized to discover potential inhibitors of PDKs. Structure-based screening using Libdock was carried out following by ADME (adsorption, distribution, metabolism, excretion) and toxicity prediction. Molecular docking was used to analyze the binding mechanism between these compounds and PDKs. Molecular dynamic simulation was utilized to confirm the stability of potential compound binding. From the computational results, two novel natural coumarins compounds (ZINC12296427 and ZINC12389251) from the ZINC database were found binding to PDKs with favorable interaction energy and predicted to be non-toxic. Our study provide valuable information of PDK-coumarins binding mechanisms in PDK inhibitor-based drug discovery.

Structural Plasticity of Malaria Dihydroorotate Dehydrogenase Allows Selective Binding of Diverse Chemical Scaffolds

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

Deng, Xiaoyi; Gujjar, Ramesh; El Mazouni, Farah

Malaria remains a major global health burden and current drug therapies are compromised by resistance. Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) was validated as a new drug target through the identification of potent and selective triazolopyrimidine-based DHODH inhibitors with anti-malarial activity in vivo. Here we report x-ray structure determination of PfDHODH bound to three inhibitors from this series, representing the first of the enzyme bound to malaria specific inhibitors. We demonstrate that conformational flexibility results in an unexpected binding mode identifying a new hydrophobic pocket on the enzyme. Importantly this plasticity allows PfDHODH to bind inhibitors from different chemical classes andmore » to accommodate inhibitor modifications during lead optimization, increasing the value of PfDHODH as a drug target. A second discovery, based on small molecule crystallography, is that the triazolopyrimidines populate a resonance form that promotes charge separation. These intrinsic dipoles allow formation of energetically favorable H-bond interactions with the enzyme. The importance of delocalization to binding affinity was supported by site-directed mutagenesis and the demonstration that triazolopyrimidine analogs that lack this intrinsic dipole are inactive. Finally, the PfDHODH-triazolopyrimidine bound structures provide considerable new insight into species-selective inhibitor binding in this enzyme family. Together, these studies will directly impact efforts to exploit PfDHODH for the development of anti-malarial chemotherapy.« less

Ultra-high-pressure processing improves proteolysis and release of bioactive peptides with activation activities on alcohol metabolic enzymes in vitro from mushroom foot protein.

PubMed

Zhao, Rui-Jie; Huo, Chun-Yan; Qian, Yang; Ren, Di-Feng; Lu, Jun

2017-09-15

This study was to find an effective process to extract bioactive peptides from mushroom foot and determine their effects on activation of alcohol metabolic enzymes in vitro. The optimum extraction assisted by ultra-high-pressure processing of mushroom foot peptides was obtained with a pressure of 400MPa and a processing time of 10min. After ultrafiltration, peptides with molecular weight of 0-3kDa had the highest activity to activate alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) by 70.79% and 71.35%, respectively. Following dextran gel chromatography, two peaks (p-I and p-II) appeared and the activation activities on ADH and ALDH of p-I were 72.00% and 73.43%, both higher than p-II. Nine peptides were found in p-I as determined by LC-MS/MS, and two of them (IPLH and IPIVLL) were synthesized. IPLH activated ADH and ALDH by 42.7% and 29.2% respectively, which were higher than IPIVLL. Copyright © 2017 Elsevier Ltd. All rights reserved.

Engineering substrate promiscuity in halophilic alcohol dehydrogenase (HvADH2) by in silico design.

PubMed

Cassidy, Jennifer; Bruen, Larah; Rosini, Elena; Molla, Gianluca; Pollegioni, Loredano; Paradisi, Francesca

2017-01-01

An alcohol dehydrogenase from the halophilic archaeon Haloferax volcanii (HvADH2) has been engineered by rational design to broaden its substrate scope towards the conversion of a range of aromatic substrates, including flurbiprofenol, that is an intermediate of the non-steroidal anti-inflammatory drug, flurbiprofen. Wild-type HvADH2 showed minimal activity with flurbiprofenol (11.1 mU/mg). A homology model of HvADH2 was built and docking experiments with this substrate revealed that the biphenyl rings of flurbiprofenol formed strong interactions with residues F85 and F108, preventing its optimal binding in the active site. Mutations at position 85 however did not increase activity. Site directed mutagenesis at position F108 allowed the identification of three variants showing a significant (up to 2.3-fold) enhancement of activity towards flurbiprofenol, when compared to wild-type HvADH2. Interestingly, F108G variant did not show the classic inhibition in the presence of (R)-enantiomer when tested with rac-1-phenylethanol, underling its potential in racemic resolution of secondary alcohols.

Loss of function of cinnamyl alcohol dehydrogenase 1 leads to unconventional lignin and a temperature-sensitive growth defect in Medicago truncatula.

PubMed

Zhao, Qiao; Tobimatsu, Yuki; Zhou, Rui; Pattathil, Sivakumar; Gallego-Giraldo, Lina; Fu, Chunxiang; Jackson, Lisa A; Hahn, Michael G; Kim, Hoon; Chen, Fang; Ralph, John; Dixon, Richard A

2013-08-13

There is considerable debate over the capacity of the cell wall polymer lignin to incorporate unnatural monomer units. We have identified Tnt1 retrotransposon insertion mutants of barrel medic (Medicago truncatula) that show reduced lignin autofluorescence under UV microscopy and red coloration in interfascicular fibers. The phenotype is caused by insertion of retrotransposons into a gene annotated as encoding cinnamyl alcohol dehydrogenase, here designated M. truncatula CAD1. NMR analysis indicated that the lignin is derived almost exclusively from coniferaldehyde and sinapaldehyde and is therefore strikingly different from classical lignins, which are derived mainly from coniferyl and sinapyl alcohols. Despite such a major alteration in lignin structure, the plants appear normal under standard conditions in the greenhouse or growth chamber. However, the plants are dwarfed when grown at 30 °C. Glycome profiling revealed an increased extractability of some xylan and pectin epitopes from the cell walls of the cad1-1 mutant but decreased extractability of others, suggesting that aldehyde-dominant lignin significantly alters cell wall structure.

Loss of function of cinnamyl alcohol dehydrogenase 1 leads to unconventional lignin and a temperature-sensitive growth defect in Medicago truncatula

PubMed Central

Zhao, Qiao; Tobimatsu, Yuki; Zhou, Rui; Pattathil, Sivakumar; Gallego-Giraldo, Lina; Fu, Chunxiang; Jackson, Lisa A.; Hahn, Michael G.; Kim, Hoon; Chen, Fang; Ralph, John; Dixon, Richard A.

2013-01-01

There is considerable debate over the capacity of the cell wall polymer lignin to incorporate unnatural monomer units. We have identified Tnt1 retrotransposon insertion mutants of barrel medic (Medicago truncatula) that show reduced lignin autofluorescence under UV microscopy and red coloration in interfascicular fibers. The phenotype is caused by insertion of retrotransposons into a gene annotated as encoding cinnamyl alcohol dehydrogenase, here designated M. truncatula CAD1. NMR analysis indicated that the lignin is derived almost exclusively from coniferaldehyde and sinapaldehyde and is therefore strikingly different from classical lignins, which are derived mainly from coniferyl and sinapyl alcohols. Despite such a major alteration in lignin structure, the plants appear normal under standard conditions in the greenhouse or growth chamber. However, the plants are dwarfed when grown at 30 °C. Glycome profiling revealed an increased extractability of some xylan and pectin epitopes from the cell walls of the cad1-1 mutant but decreased extractability of others, suggesting that aldehyde-dominant lignin significantly alters cell wall structure. PMID:23901113

Single gene insertion drives bioalcohol production by a thermophilic archaeon

PubMed Central

Basen, Mirko; Schut, Gerrit J.; Nguyen, Diep M.; Lipscomb, Gina L.; Benn, Robert A.; Prybol, Cameron J.; Vaccaro, Brian J.; Poole, Farris L.; Kelly, Robert M.; Adams, Michael W. W.

2014-01-01

Bioethanol production is achieved by only two metabolic pathways and only at moderate temperatures. Herein a fundamentally different synthetic pathway for bioalcohol production at 70 °C was constructed by insertion of the gene for bacterial alcohol dehydrogenase (AdhA) into the archaeon Pyrococcus furiosus. The engineered strain converted glucose to ethanol via acetate and acetaldehyde, catalyzed by the host-encoded aldehyde ferredoxin oxidoreductase (AOR) and heterologously expressed AdhA, in an energy-conserving, redox-balanced pathway. Furthermore, the AOR/AdhA pathway also converted exogenously added aliphatic and aromatic carboxylic acids to the corresponding alcohol using glucose, pyruvate, and/or hydrogen as the source of reductant. By heterologous coexpression of a membrane-bound carbon monoxide dehydrogenase, CO was used as a reductant for converting carboxylic acids to alcohols. Redirecting the fermentative metabolism of P. furiosus through strategic insertion of foreign genes creates unprecedented opportunities for thermophilic bioalcohol production. Moreover, the AOR/AdhA pathway is a potentially game-changing strategy for syngas fermentation, especially in combination with carbon chain elongation pathways. PMID:25368184

Single gene insertion drives bioalcohol production by a thermophilic archaeon

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

Basen, M; Schut, GJ; Nguyen, DM

2014-12-09

Bioethanol production is achieved by only two metabolic pathways and only at moderate temperatures. Herein a fundamentally different synthetic pathway for bioalcohol production at 70 degrees C was constructed by insertion of the gene for bacterial alcohol dehydrogenase (AdhA) into the archaeon Pyrococcus furiosus. The engineered strain converted glucose to ethanol via acetate and acetaldehyde, catalyzed by the host-encoded aldehyde ferredoxin oxidoreductase (AOR) and heterologously expressed AdhA, in an energy-conserving, redox-balanced pathway. Furthermore, the AOR/AdhA pathway also converted exogenously added aliphatic and aromatic carboxylic acids to the corresponding alcohol using glucose, pyruvate, and/or hydrogen as the source of reductant. Bymore » heterologous coexpression of a membrane-bound carbon monoxide dehydrogenase, CO was used as a reductant for converting carboxylic acids to alcohols. Redirecting the fermentative metabolism of P. furiosus through strategic insertion of foreign genes creates unprecedented opportunities for thermophilic bioalcohol production. Moreover, the AOR/AdhA pathway is a potentially game-changing strategy for syngas fermentation, especially in combination with carbon chain elongation pathways.« less

Expression and kinetic properties of a recombinant 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase isoenzyme of human liver.

PubMed

Deyashiki, Y; Tamada, Y; Miyabe, Y; Nakanishi, M; Matsuura, K; Hara, A

1995-08-01

Human liver cytosol contains multiple forms of 3 alpha-hydroxysteroid dehydrogenase and dihydrodiol dehydrogenase with hydroxysteroid dehydrogenase activity, and multiple cDNAs for the enzymes have been cloned from human liver cDNA libraries. To understand the relationship of the multiple enzyme froms to the genes, a cDNA, which has been reported to code for an isoenzyme of human liver 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase, was expressed in Escherichia coli. The recombinant enzyme showed structural and functional properties almost identical to those of the isoenzyme purified from human liver. In addition, the recombinant isoenzyme efficiently reduced 5 alpha-dihydrotestosterone and 5 beta-dihydrocortisone, the known substrates of human liver 3 alpha-hydroxysteroid dehydrogenase and chlordecone reductase previously purified, which suggests that these human liver enzymes are identical. Furthermore, the steady-state kinetic data for NADP(+)-linked (S)-1-indanol oxidation by the recombinant isoenzyme were consistent with a sequential ordered mechanism in which NADP+ binds first. Phenolphthalein inhibited this isoenzyme much more potently than it did the other human liver dihydrodiol dehydrogenases, and was a competitive inhibitor (Ki = 20 nM) that bound to the enzyme-NADP+ complex.

Limonene dehydrogenase hydroxylates the allylic methyl group of cyclic monoterpenes in the anaerobic terpene degradation by Castellaniella defragrans.

PubMed

Puentes-Cala, Edinson; Liebeke, Manuel; Markert, Stephanie; Harder, Jens

2018-05-01

The enzymatic functionalization of hydrocarbons is a central step in the global carbon cycle initiating the mineralization of methane, isoprene and monoterpenes, the most abundant biologically produced hydrocarbons. Also, terpene-modifying enzymes have found many applications in the energy-economic biotechnological production of fine chemicals. Here we describe a limonene dehydrogenase that was purified from the facultatively anaerobic betaproteobacterium Castellaniella defragrans 65Phen grown on monoterpenes under denitrifying conditions in the absence of molecular oxygen. The purified limonene:ferrocenium oxidoreductase activity hydroxylated the methyl group of limonene (1-methyl-4-(1-methylethenyl)-cyclohex-1-ene) yielding perillyl alcohol ([4-(prop-1-en-2-yl)cyclohex-1-en-1-yl]methanol). The enzyme had a dithiothreitol:perillyl alcohol oxidoreductase activity yielding limonene. Mass spectrometry and molecular size determinations revealed a heterodimeric enzyme comprising CtmA and CtmB. Recently the two proteins had been identified by transposon mutagenesis and proteomics as part of the cyclic terpene metabolism ( ctm ) in Castellaniella defragrans and were annotated as FAD-dependent oxidoreductases of the protein domain family phytoene dehydrogenases and related proteins (COG1233). CtmAB is the first heterodimeric enzyme in this protein superfamily. Flavins in the purified CtmAB are oxidized by ferrocenium and are reduced by limonene. Heterologous expression of CtmA, CtmB and CtmAB in E. coli demonstrated that limonene dehydrogenase activity required both subunits carrying each a flavin cofactor. Native CtmAB oxidized a wide range of monocyclic monoterpenes containing the allylic methyl group motif (1-methyl-cyclohex-1-ene). In conclusion, we have identified CtmAB as a hydroxylating limonene dehydrogenase and the first heteromer in a family of FAD-dependent dehydrogenases acting on allylic methylene or methyl CH-bonds. We suggest a placement in EC 1.17.99.8. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Comparison of a homology model and the crystallographic structure of human 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1) in a structure-based identification of inhibitors

NASA Astrophysics Data System (ADS)

Miguet, Laurence; Zhang, Ziding; Barbier, Maryse; Grigorov, Martin G.

2006-02-01

Human 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1) catalyzes the interconversion of cortisone into active cortisol. 11βHSD1 inhibition is a tempting target for the treatment of a host of human disorders that might benefit from blockade of glucocorticoid action, such as obesity, metabolic syndrome, and diabetes type 2. Here, we report an in silico screening study aimed at identifying new selective inhibitors of human 11βHSD1 enzyme. In the first step, homology modeling was employed to build the 3D structure of 11βHSD1. Further, molecular docking was used to validate the predicted model by showing that it was able to discriminate between known 11βHSD1 inhibitors or substrates and non-inhibitors. The homology model was found to reproduce closely the crystal structure that became publicly available in the final stages of this work. Finally, we carried out structure-based virtual screening experiments on both the homology model and the crystallographic structure with a database of 114'000 natural molecules. Among these, 15 molecules were consistently selected as inhibitors based on both the model and crystal structures of the enzyme, implying a good quality for the homology model. Among these putative 11βHSD1 inhibitors, two were flavonone derivatives that have already been shown to be potent inhibitors of the enzyme.

DFT-based prediction of reactivity of short-chain alcohol dehydrogenase

NASA Astrophysics Data System (ADS)

Stawoska, I.; Dudzik, A.; Wasylewski, M.; Jemioła-Rzemińska, M.; Skoczowski, A.; Strzałka, K.; Szaleniec, M.

2017-06-01

The reaction mechanism of ketone reduction by short chain dehydrogenase/reductase, ( S)-1-phenylethanol dehydrogenase from Aromatoleum aromaticum, was studied with DFT methods using cluster model approach. The characteristics of the hydride transfer process were investigated based on reaction of acetophenone and its eight structural analogues. The results confirmed previously suggested concomitant transfer of hydride from NADH to carbonyl C atom of the substrate with proton transfer from Tyr to carbonyl O atom. However, additional coupled motion of the next proton in the proton-relay system, between O2' ribose hydroxyl and Tyr154 was observed. The protonation of Lys158 seems not to affect the pKa of Tyr154, as the stable tyrosyl anion was observed only for a neutral Lys158 in the high pH model. The calculated reaction energies and reaction barriers were calibrated by calorimetric and kinetic methods. This allowed an excellent prediction of the reaction enthalpies (R2 = 0.93) and a good prediction of the reaction kinetics (R2 = 0.89). The observed relations were validated in prediction of log K eq obtained for real whole-cell reactor systems that modelled industrial synthesis of S-alcohols.

Structure of Cryptosporidium IMP dehydrogenase bound to an inhibitor with in vivo antiparasitic activity

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

Kim, Youngchang; Makowska-Grzyska, Magdalena; Gorla, Suresh Kumar

2015-04-21

Inosine 5'-monophosphate dehydrogenase (IMPDH) is a promising target for the treatment ofCryptosporidiuminfections. Here, the structure ofC. parvumIMPDH (CpIMPDH) in complex with inosine 5'-monophosphate (IMP) and P131, an inhibitor within vivoanticryptosporidial activity, is reported. P131 contains two aromatic groups, one of which interacts with the hypoxanthine ring of IMP, while the second interacts with the aromatic ring of a tyrosine in the adjacent subunit. In addition, the amine and NO 2moieties bind in hydrated cavities, forming water-mediated hydrogen bonds to the protein. The design of compounds to replace these water molecules is a new strategy for the further optimization ofC. parvuminhibitorsmore » for both antiparasitic and antibacterial applications.« less

The 2',4'-dihydroxychalcone could be explored to develop new inhibitors against the glycerol-3-phosphate dehydrogenase from Leishmania species.

PubMed

Passalacqua, Thais G; Torres, Fábio A E; Nogueira, Camila T; de Almeida, Leticia; Del Cistia, Mayara L; dos Santos, Mariana B; Dutra, Luis A; Bolzani, Vanderlan da Silva; Regasini, Luis O; Graminha, Márcia A S; Marchetto, Reinaldo; Zottis, Aderson

2015-09-01

The enzyme glycerol-3-phosphate dehydrogenase (G3PDH) from Leishmania species is considered as an attractive target to design new antileishmanial drugs and a previous in silico study reported on the importance of chalcones to achieve its inhibition. Here, we report the identification of a synthetic chalcone in our in vitro assays with promastigote cells from Leishmania amazonensis, its biological activity in animal models, and docking followed by molecular dynamics simulation to investigate the molecular interactions and structural patterns that are crucial to achieve the inhibition complex between this compound and G3PDH. A molecular fragment of this natural product derivative can provide new inhibitors with increased potency and selectivity. Copyright © 2015 Elsevier Ltd. All rights reserved.

Alcohol consumption and type 2 diabetes: influence of genetic variation in alcohol dehydrogenase.

PubMed

Beulens, Joline W J; Rimm, Eric B; Hendriks, Henk F J; Hu, Frank B; Manson, JoAnn E; Hunter, David J; Mukamal, Kenneth J

2007-09-01

We sought to investigate whether a polymorphism in the alcohol dehydrogenase 1c (ADH1C) gene modifies the association between alcohol consumption and type 2 diabetes. In nested case-control studies of 640 women with incident diabetes and 1,000 control subjects from the Nurses' Health Study and 383 men with incident diabetes and 382 control subjects from the Health Professionals Follow-Up Study, we determined associations between the ADH1C polymorphism, alcohol consumption, and diabetes risk. Moderate to heavy alcohol consumption (>5 g/day for women and >10 g/day for men) was associated with a decreased risk of diabetes among women (odds ratio [OR] 0.45 [95% CI 0.33-0.63]) but not men (1.08 [0.67-1.75]). ADH1C genotype modified the relation between alcohol consumption and diabetes for women (P(interaction) = 0.02). The number of ADH1C*2 alleles, related to a slower rate of ethanol oxidation, attenuated the lower risk of diabetes among women consuming >/=5 g alcohol/day (P(trend) = 0.002). These results were not significant among men. Results were similar in pooled analyses (P(interaction) = 0.02) with ORs for diabetes among moderate drinkers of 0.44 (95% CI 0.21-0.94) in ADH1C*1 homozygotes, 0.65 (0.39-1.06) for heterozygotes, and 0.78 (0.50-1.22) for ADH1C*2 homozygotes compared with those for ADH1C*1 homozygote abstainers (P(trend) = 0.02). ADH1C genotype modifies the association between alcohol consumption and diabetes. The ADH1C*2 allele, related to a slower oxidation rate, attenuates the lower diabetes risk among moderate to heavy drinkers. This suggests that the association between alcohol consumption and diabetes may be causal but mediated by downstream metabolites such as acetate rather than ethanol itself.

Ginsenoside-free molecules from steam-dried ginseng berry promote ethanol metabolism: an alternative choice for an alcohol hangover.

PubMed

Lee, Do Ik; Kim, Seung Tae; Lee, Dong Hoon; Yu, Jung Min; Jang, Su Kil; Joo, Seong Soo

2014-07-01

Ethanol metabolism produces harmful compounds that contribute to liver damage and cause an alcohol hangover. The intermediate metabolite acetaldehyde is responsible for alcohol hangover and CYP2E1-induced reactive oxygen species damage liver tissues. In this study, we examined whether ginsenoside-free molecules (GFMs) from steam-dried ginseng berries promote ethanol metabolism and scavenge free radicals by stimulating primary enzymes (alcohol dehydrogenase, aldehyde dehydrogenase, CYP2E1, and catalase) and antioxidant effects using in vitro and in vivo models. The results revealed that GFM effectively scavenged 2,2-diphenyl-1-picrylhydrazyl hydrate radicals and hydroxyl radicals. Notably, GFM significantly enhanced the expression of primary enzymes within 2 h in HepG2 cells. GFM clearly removed the consumed ethanol and significantly reduced the level of acetaldehyde as well as enhancement of primary gene expression in BALB/c mice. Moreover, GFM successfully protected HepG2 cells from ethanol attack. Of the major components identified in GFM, it was believed that linoleic acid was the most active ingredient. Based on these findings, we conclude that GFM holds promise for use as a new candidate for ethanol metabolism and as an antihangover agent. © 2014 Institute of Food Technologists®

Hederagenin Supplementation Alleviates the Pro-Inflammatory and Apoptotic Response to Alcohol in Rats.

PubMed

Kim, Gyeong-Ji; Song, Da Hye; Yoo, Han Seok; Chung, Kang-Hyun; Lee, Kwon Jai; An, Jeung Hee

2017-01-06

In this study, we determined the effects of hederagenin isolated from Akebia quinata fruit on alcohol-induced hepatotoxicity in rats. Specifically, we investigated the hepatoprotective, anti-inflammatory, and anti-apoptotic effects of hederagenin, as well as the role of AKT and mitogen-activated protein kinase (MAPK) signaling pathways in ethanol-induced liver injury. Experimental animals were randomly divided into three groups: normal (sham), 25% ethanol, and 25% ethanol + hederagenin (50 mg/kg/day). Each group was orally administered the respective treatments once per day for 21 days. Acetaldehyde dehydrogenase-2 mRNA expression was higher and alcohol dehydrogenase mRNA expression was lower in the ethanol + hederagenin group than those in the ethanol group. Pro-inflammatory cytokines, including TNF-α, IL-6, and cyclooxygenase-2, significantly increased in the ethanol group, but these increases were attenuated by hederagenin. Moreover, Western blot analysis showed increased expression of the apoptosis-associated protein, Bcl-2, and decreased expression of Bax and p53 after treatment with hederagenin. Hederagenin treatment attenuated ethanol-induced increases in activated p38 MAPK and increased the levels of phosphorylated AKT and ERK. Hederagenin alleviated ethanol-induced liver damage through anti-inflammatory and anti-apoptotic activities. These results suggest that hederagenin is a potential candidate for preventing alcoholic liver injury.

Coupled reactions on bioparticles: Stereoselective reduction with cofactor regeneration on PhaC inclusion bodies.

PubMed

Spieler, Valerie; Valldorf, Bernhard; Maaß, Franziska; Kleinschek, Alexander; Hüttenhain, Stefan H; Kolmar, Harald

2016-07-01

Chiral alcohols are important building blocks for specialty chemicals and pharmaceuticals. The production of chiral alcohols from ketones can be carried out stereo selectively with alcohol dehydrogenases (ADHs). To establish a process for cost-effective enzyme immobilization on solid phase for application in ketone reduction, we used an established enzyme pair consisting of ADH from Rhodococcus erythropolis and formate dehydrogenase (FDH) from Candida boidinii for NADH cofactor regeneration and co-immobilized them on modified poly-p-hydroxybutyrate synthase (PhaC)-inclusion bodies that were recombinantly produced in Escherichia coli cells. After separate production of genetically engineered and recombinantly produced enzymes and particles, cell lysates were combined and enzymes endowed with a Kcoil were captured on the surface of the Ecoil presenting particles due to coiled-coil interaction. Enzyme-loaded particles could be easily purified by centrifugation. Total conversion of 4'-chloroacetophenone to (S)-4-chloro-α-methylbenzyl alcohol could be accomplished using enzyme-loaded particles, catalytic amounts of NAD(+) and formate as substrates for FDH. Chiral GC-MS analysis revealed that immobilized ADH retained enantioselectivity with 99 % enantiomeric excess. In conclusion, this strategy may become a cost-effective alternative to coupled reactions using purified enzymes. Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Inhibition of Growth by Combined Treatment with Inhibitors of Lactate Dehydrogenase and either Phenformin or Inhibitors of 6-Phosphofructo-2-kinase/Fructose-2,6-bisphosphatase 3.

PubMed

Lea, Michael A; Guzman, Yolanda; Desbordes, Charles

2016-04-01

Enhanced glycolysis in cancer cells presents a target for chemotherapy. Previous studies have indicated that proliferation of cancer cells can be inhibited by treatment with phenformin and with an inhibitor of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB) namely 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO). In the present work, the action of two inhibitors that are effective at lower concentrations than 3PO, namely 1-(3-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one (PQP) and 1-(4-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one (PFK15) were investigated. The inhibitors of lactate dehydrogenase (LDHA) studied in order of half-maximal inhibitory concentrations were methyl 1-hydroxy-6-phenyl-4-(trifluoromethyl)-1H-indole-2-carboxylate (NHI-2) < isosafrole < oxamate. In colonic and bladder cancer cells, additive growth inhibitory effects were seen with the LDHA inhibitors, of which NHI-2 was effective at the lowest concentrations. Growth inhibition was generally greater with PFK15 than with PQP. The increased acidification of the culture medium and glucose uptake caused by phenformin was blocked by combined treatment with PFKFB3 or LDHA inhibitors. The results suggest that combined treatment with phenformin and inhibitors of glycolysis can cause additive inhibition of cell proliferation and may mitigate lactic acidosis caused by phenformin when used as a single agent. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Biochemistry of Suberization

PubMed Central

Agrawal, Vishwanath P.; Kolattukudy, P. E.

1977-01-01

A cell-free extract obtained from suberizing potato (Solanum tuberosum L.) tuber disks catalyzed the conversion of 16-hydroxy[G-3H]hexadecanoic acid to the corresponding dicarboxylic acid with NADP or NAD as the cofactor, with a slight preference for the former. This ω-hydroxyacid dehydrogenase activity, located largely in the 100,000g supernatant fraction, has a pH optimum of 9.5. It showed an apparent Km of 50 μM for 16-hydroxyhexadecanoic acid. The dehydrogenase activity was inhibited by thiol reagents, such as p-chloromercuribenzoate, N-ethylmaleimide, and iodoacetamide, and this dehydrogenase is shown to be different from alcohol dehydrogenase. That 16-oxohexadecanoic acid was an intermediate in the conversion of 16-hydroxyhexadecanoic acid to the corresponding dicarboxylic acid was suggested by the observation that the cell-free extract also catalyzed the conversion of 16-oxohexadecanoic acid to the dicarboxylic acid, with NADP as the preferred cofactor. The time course of development of the ω-hydroxyacid dehydrogenase activity in the suberizing potato disks correlated with the rate of deposition of suberin. Experiments with actinomycin D and cycloheximide suggested that the transcriptional processes, which are directly related to suberin biosynthesis and ω-hydroxyacid dehydrogenase biosynthesis, occurred between 72 and 96 hours after wounding. These results strongly suggest that a wound-induced ω-hydroxyacid dehydrogenase is involved in suberin biosynthesis in potato disks. PMID:16659915

Therapeutic Targeting of the Pyruvate Dehydrogenase Complex/Pyruvate Dehydrogenase Kinase (PDC/PDK) Axis in Cancer.

PubMed

Stacpoole, Peter W

2017-11-01

The mitochondrial pyruvate dehydrogenase complex (PDC) irreversibly decarboxylates pyruvate to acetyl coenzyme A, thereby linking glycolysis to the tricarboxylic acid cycle and defining a critical step in cellular bioenergetics. Inhibition of PDC activity by pyruvate dehydrogenase kinase (PDK)-mediated phosphorylation has been associated with the pathobiology of many disorders of metabolic integration, including cancer. Consequently, the PDC/PDK axis has long been a therapeutic target. The most common underlying mechanism accounting for PDC inhibition in these conditions is post-transcriptional upregulation of one or more PDK isoforms, leading to phosphorylation of the E1α subunit of PDC. Such perturbations of the PDC/PDK axis induce a "glycolytic shift," whereby affected cells favor adenosine triphosphate production by glycolysis over mitochondrial oxidative phosphorylation and cellular proliferation over cellular quiescence. Dichloroacetate is the prototypic xenobiotic inhibitor of PDK, thereby maintaining PDC in its unphosphorylated, catalytically active form. However, recent interest in the therapeutic targeting of the PDC/PDK axis for the treatment of cancer has yielded a new generation of small molecule PDK inhibitors. Ongoing investigations of the central role of PDC in cellular energy metabolism and its regulation by pharmacological effectors of PDKs promise to open multiple exciting vistas into the biochemical understanding and treatment of cancer and other diseases. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

77 FR 66074 - Regulation of Fuel and Fuel Additives: Modification to Octamix Waiver (TOLAD MFA-10A)

Federal Register 2010, 2011, 2012, 2013, 2014

2012-11-01

... Texas Methanol's gasoline-alcohol fuel, OCTAMIX. This correction notice explains that TOLAD MFA-10A is... inhibitor, TOLAD TM MFA-10A, at a concentration of 42 mg/l, in the OCTAMIX gasoline-alcohol fuel blend which... corrosion inhibitor in Texas Methanol's gasoline- alcohol fuel, OCTAMIX.\\2\\ \\1\\ EPA-HQ-OAR-2011-0894-0001...

Ligand-Based Pharmacophore Modeling and Virtual Screening for the Discovery of Novel 17β-Hydroxysteroid Dehydrogenase 2 Inhibitors

PubMed Central

2014-01-01

17β-Hydroxysteroid dehydrogenase 2 (17β-HSD2) catalyzes the inactivation of estradiol into estrone. This enzyme is expressed only in a few tissues, and therefore its inhibition is considered as a treatment option for osteoporosis to ameliorate estrogen deficiency. In this study, ligand-based pharmacophore models for 17β-HSD2 inhibitors were constructed and employed for virtual screening. From the virtual screening hits, 29 substances were evaluated in vitro for 17β-HSD2 inhibition. Seven compounds inhibited 17β-HSD2 with low micromolar IC50 values. To investigate structure–activity relationships (SAR), 30 more derivatives of the original hits were tested. The three most potent hits, 12, 22, and 15, had IC50 values of 240 nM, 1 μM, and 1.5 μM, respectively. All but 1 of the 13 identified inhibitors were selective over 17β-HSD1, the enzyme catalyzing conversion of estrone into estradiol. Three of the new, small, synthetic 17β-HSD2 inhibitors showed acceptable selectivity over other related HSDs, and six of them did not affect other HSDs. PMID:24960438

Multiple e-pharmacophore modelling pooled with high-throughput virtual screening, docking and molecular dynamics simulations to discover potential inhibitors of Plasmodium falciparum lactate dehydrogenase (PfLDH).

PubMed

Saxena, Shalini; Durgam, Laxman; Guruprasad, Lalitha

2018-05-14

Development of new antimalarial drugs continues to be of huge importance because of the resistance of malarial parasite towards currently used drugs. Due to the reliance of parasite on glycolysis for energy generation, glycolytic enzymes have played important role as potential targets for the development of new drugs. Plasmodium falciparum lactate dehydrogenase (PfLDH) is a key enzyme for energy generation of malarial parasites and is considered to be a potential antimalarial target. Presently, there are nearly 15 crystal structures bound with inhibitors and substrate that are available in the protein data bank (PDB). In the present work, we attempted to consider multiple crystal structures with bound inhibitors showing affinity in the range of 1.4 × 10 2 -1.3 × 10 6  nM efficacy and optimized the pharmacophore based on the energy involved in binding termed as e-pharmacophore mapping. A high throughput virtual screening (HTVS) combined with molecular docking, ADME predictions and molecular dynamics simulation led to the identification of 20 potential compounds which could be further developed as novel inhibitors for PfLDH.

Synthesis, conformational analysis, and biological activity of new analogues of thiazole-4-carboxamide adenine dinucleotide (TAD) as IMP dehydrogenase inhibitors.

PubMed

Franchetti, Palmarisa; Cappellacci, Loredana; Pasqualini, Michela; Petrelli, Riccardo; Jayaprakasan, Vetrichelvan; Jayaram, Hiremagalur N; Boyd, Donald B; Jain, Manojkumar D; Grifantini, Mario

2005-03-15

Thiazole-4-carboxamide adenine dinucleotide (TAD) analogues T-2'-MeAD (1) and T-3'-MeAD (2) containing, respectively, a methyl group at the ribose 2'-C-, and 3'-C-position of the adenosine moiety, were prepared as potential selective human inosine monophosphate dehydrogenase (IMPDH) type II inhibitors. The synthesis of heterodinucleotides was carried out by CDI-catalyzed coupling reaction of unprotected 2'-C-methyl- or 3'-C-methyl-adenosine 5'-monophosphate with 2',3'-O-isopropylidene-tiazofurin 5'-monophosphate, and then deisopropylidenation. Biological evaluation of dinucleotides 1 and 2 as inhibitors of recombinant human IMPDH type I and type II resulted in a good activity. Inhibition of both isoenzymes by T-2'-MeAD and T-3'-MeAD was noncompetitive with respect to NAD substrate. Binding of T-3'-MeAD was comparable to that of parent compound TAD, while T-2'-MeAD proved to be a weaker inhibitor. However, no significant difference was found in inhibition of the IMPDH isoenzymes. T-2'-MeAD and T-3'-MeAD were found to inhibit the growth of K562 cells (IC(50) 30.7 and 65.0muM, respectively).

Acute and chronic ethanol exposure differentially alters alcohol dehydrogenase and aldehyde dehydrogenase activity in the zebrafish liver.

PubMed

Tran, Steven; Nowicki, Magda; Chatterjee, Diptendu; Gerlai, Robert

2015-01-02

Chronic ethanol exposure paradigms have been successfully used in the past to induce behavioral and central nervous system related changes in zebrafish. However, it is currently unknown whether chronic ethanol exposure alters ethanol metabolism in adult zebrafish. In the current study we examine the effect of acute ethanol exposure on adult zebrafish behavioral responses, as well as alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) activity in the liver. We then examine how two different chronic ethanol exposure paradigms (continuous and repeated ethanol exposure) alter behavioral responses and liver enzyme activity during a subsequent acute ethanol challenge. Acute ethanol exposure increased locomotor activity in a dose-dependent manner. ADH activity was shown to exhibit an inverted U-shaped curve and ALDH activity was decreased by ethanol exposure at all doses. During the acute ethanol challenge, animals that were continuously housed in ethanol exhibited a significantly reduced locomotor response and increased ADH activity, however, ALDH activity did not change. Zebrafish that were repeatedly exposed to ethanol demonstrated a small but significant attenuation of the locomotor response during the acute ethanol challenge but ADH and ALDH activity was similar to controls. Overall, we identified two different chronic ethanol exposure paradigms that differentially alter behavioral and physiological responses in zebrafish. We speculate that these two paradigms may allow dissociation of central nervous system-related and liver enzyme-dependent ethanol induced changes in zebrafish. Copyright © 2014 Elsevier Inc. All rights reserved.

CvADH1, a member of short-chain alcohol dehydrogenase family, is inducible by gibberellin and sucrose in developing watermelon seeds.

PubMed

Kim, Joonyul; Kang, Hong-Gyu; Jun, Sung-Hoon; Lee, Jinwon; Yim, Jieun; An, Gynheung

2003-01-01

To understand the molecular mechanisms that control seed formation, we selected a seed-preferential gene (CvADH1) from the ESTs of developing watermelon seeds. RNA blot analysis and in situ localization showed that CvADH1 was preferentially expressed in the nucellar tissue. The CvADH1 protein shared about 50% homology with short-chain alcohol dehydrogenase including ABA2 in Arabidopsis thaliana, stem secoisolariciresinol dehydrogenase in Forsythia intermedia, and 3beta-hydroxysterol dehydrogenase in Digitalis lanata. We investigated gene-expression levels in seeds from both normally pollinated fruits and those made parthenocarpic via N-(2-chloro-4-pyridyl)-N'-phenylurea treatment, the latter of which lack zygotic tissues. Whereas the transcripts of CvADH1 rapidly started to accumulate from about the pre-heart stage in normal seeds, they were not detectable in the parthenocarpic seeds. Treating the parthenogenic fruit with GA(3) strongly induced gene expression, up to the level accumulated in pollinated seeds. These results suggest that the CvADH1 gene is induced in maternal tissues by signals made in the zygotic tissues, and that gibberellin might be one of those signals. We also observed that CvADH1 expression was induced by sucrose in the parthenocarpic seeds. Therefore, we propose that the CvADH1 gene is inducible by gibberellin, and that sucrose plays an important role in the maternal tissues of watermelon during early seed development.

Biochemical characterization of a recombinant short-chain NAD(H)-dependent dehydrogenase/reductase from Sulfolobus acidocaldarius.

PubMed

Pennacchio, Angela; Giordano, Assunta; Pucci, Biagio; Rossi, Mosè; Raia, Carlo A

2010-03-01

The gene encoding a novel alcohol dehydrogenase that belongs to the short-chain dehydrogenases/reductases (SDRs) superfamily was identified in the aerobic thermoacidophilic crenarchaeon Sulfolobus acidocaldarius strain DSM 639. The saadh gene was heterologously overexpressed in Escherichia coli, and the protein (SaADH) was purified to homogeneity and characterized. SaADH is a tetrameric enzyme consisting of identical 28,978-Da subunits, each composed of 264 amino acids. The enzyme has remarkable thermophilicity and thermal stability, displaying activity at temperatures up to 75 degrees C and a 30-min half-inactivation temperature of ~90 degrees C, and shows good tolerance to common organic solvents. SaADH has a strict requirement for NAD(H) as the coenzyme, and displays a preference for the reduction of alicyclic, bicyclic and aromatic ketones and alpha-keto esters, but is poorly active on aliphatic, cyclic and aromatic alcohols, and shows no activity on aldehydes. The enzyme catalyses the reduction of alpha-methyl and alpha-ethyl benzoylformate, and methyl o-chlorobenzoylformate with 100% conversion to methyl (S)-mandelate [17% enantiomeric excess (ee)], ethyl (R)-mandelate (50% ee), and methyl (R)-o-chloromandelate (72% ee), respectively, with an efficient in situ NADH-recycling system which involves glucose and a thermophilic glucose dehydrogenase. This study provides further evidence supporting the critical role of the D37 residue in discriminating NAD(H) from NAD(P)H in members of the SDR superfamily.

Alcohol drinking, mean corpuscular volume of erythrocytes, and alcohol metabolic genotypes in drunk drivers.

PubMed

Pavanello, Sofia; Snenghi, Rossella; Nalesso, Alessandro; Sartore, Daniela; Ferrara, Santo Davide; Montisci, Massimo

2012-02-01

Regular and irregular abuse of alcohol are global health priorities associated with diseases at multiple sites, including cancer. Mechanisms of diseases induced by alcohol are closely related to its metabolism. Among conventional markers of alcohol abuse, the mean corpuscular volume (MCV) of erythrocytes is prognostic of alcohol-related cancer and its predictivity increases when combined with functional polymorphisms of alcohol dehydrogenase (ADH1B [rs1229984] and ADH1C [rs698]) and the mitochondrial aldehyde dehydrogenase (ALDH2 [rs671]). Whether these genetic variants can influence abuse in alcohol drinking and MCV has never been examined in drunk-driving traffic offenders. We examined 149 drunk drivers, diagnosed as alcohol abusers according to the Diagnostic and Statistical Manual of Mental Disorders, Fourth edition Text Revision (DSM-IV-TR) and enrolled in a probation program, and 257 social drinkers (controls), all Caucasian males. Alcohol intake was assessed according to self-reported drink-units/d and MCV unadjusted and adjusted for age, smoking, and body mass index. Multivariable models were used to compute MCV adjusted means. Genotype analyses were performed by PCR on DNA from blood. The adjusted MCV mean was higher in drunk-driving abusers than in controls (92 vs. 91fL; P<.0001) and increased with the number of drink-units/d in both abusers and controls (P-trend=.0316 and .0089) already at intermediate quantities (0-1 vs. 2-4 drink-units/d: P=.054 and .024). Carriers of the common ADH1B*1/*1 (rs1229984) genotype were more likely to be drunk-driving abusers (P=.008), reported higher drink-units/d (P=.0126), and had larger MCV (P=.035). The rs698 ADH1C and rs671 ALDH2 polymorphisms were not associated with MCV. ADH1B*1/*1 polymorphism is significantly associated with being a drunk-driving abuser, higher alcohol drinking, and MCV enlargement. This suggests that drunk drivers with augmented MCV modulated by the alcohol metabolic ADH1B*1/*1 genotype may be at higher risk of driving incapability and of alcohol-related cancer. Copyright © 2012 Elsevier Inc. All rights reserved.

Genetic polymorphisms of alcohol dehydrogenase-1B and aldehyde dehydrogenase-2 and liver cirrhosis, chronic calcific pancreatitis, diabetes mellitus, and hypertension among Japanese alcoholic men.

PubMed

Yokoyama, Akira; Mizukami, Takeshi; Matsui, Toshifumi; Yokoyama, Tetsuji; Kimura, Mitsuru; Matsushita, Sachio; Higuchi, Susumu; Maruyama, Katsuya

2013-08-01

The presence of the less-active form of alcohol dehydrogenase-1B encoded by ADH1B*1/*1 (vs. *2 allele) and active form of aldehyde dehydrogenase-2 (ALDH2) encoded by ALDH2*1/*1 (vs. *2 allele) increases the risk of alcoholism in East Asians. The subjects in this cross-sectional survey were 1,902 Japanese alcoholic men (≥40 years) who underwent ADH1B/ALDH2 genotyping. Age-adjusted daily alcohol consumption did not differ according to the ADH1B/ALDH2 genotypes. The age-adjusted odds ratios (AORs; 95% confidence interval) for liver cirrhosis (LC; n = 359, 1.58 [1.19 to 2.09]), chronic calcific pancreatitis (CP; n = 80, 2.24 [1.20 to 4.20]), and diabetes mellitus (DM; n = 383, 1.51 [1.15 to 1.99]) were higher in the ADH1B*2 allele carriers than in the ADH1B*1/*1 carriers. The AORs for LC (1.43 [1.01 to 2.02]), CP (1.68 [0.80 to 3.53]), DM (1.63 [1.15 to 2.30]), and hypertension (HT; n = 495, 1.52 [1.11 to 2.07]) were higher in the ALDH2*1/*1 carriers than in the ALDH2*1/*2 carriers. The ADH1B*2-associated AOR for LC was 2.08 (1.46 to 2.94) among those aged 40 to 59 years, but 0.89 (0.56 to 1.43) among those aged 60 years or over, and the interaction between ADH1B genotype and age on the LC risk was significant (p = 0.009). When the group with non-LC and no/mild fibrosis was used as controls, the ADH1B*2-associated AORs increased according to the severity of their liver disease: 1.67 (1.32 to 2.11) for the group with non-LC and serum type IV collagen values ≥200 ng/ml, 1.81 (1.24 to 2.63) for the group of Child-Pugh class A LC, and 3.17 (1.98 to 5.07) for the group with Child-Pugh class B/C LC. Anti-hepatitis C virus (HCV) antibody was positive in 103 patients, and the groups with a high anti-HCV antibody titer and either the ADH1B*2/*2 genotype or the ALDH2*1/*1 genotype had the highest AORs (8.83 and 4.90, respectively). The population attributable fraction (PAF) due to the ADH1B*2 allele was 29% for LC, 47% for CP, and 27% for DM, and the PAF due to the ALDH2*1/*1 genotype was 26% for LC, 34% for DM, and 30% for HT. The ADH1B*2 allele increased the AORs for LC, CP, and DM of the alcoholics, and the ALDH2*1/*1 genotype increased their AORs for LC, DM, and HT. HCV infection and genetic susceptibility had a synergistic effect on the AOR for LC. Copyright © 2013 by the Research Society on Alcoholism.

S-adenosylmethionine decreases the peak blood alcohol levels 3 h after an acute bolus of ethanol by inducing alcohol metabolizing enzymes in the liver.

PubMed

Bardag-Gorce, Fawzia; Oliva, Joan; Wong, Wesley; Fong, Stephanie; Li, Jun; French, Barbara A; French, Samuel W

2010-12-01

An alcohol bolus causes the blood alcohol level (BAL) to peak at 1-2 h post ingestion. The ethanol elimination rate is regulated by alcohol metabolizing enzymes, primarily alcohol dehydrogenase (ADH1), acetaldehyde dehydrogenase (ALDH), and cytochrome P450 (CYP2E1). Recently, S-adenosylmethionine (SAMe) was found to reduce acute BALs 3 h after an alcohol bolus. The question, then, was: what is the mechanism involved in this reduction of BAL by feeding SAMe? To answer this question, we investigated the changes in ethanol metabolizing enzymes and the epigenetic changes that regulate the expression of these enzymes during acute binge drinking and chronic drinking. Rats were fed a bolus of ethanol with or without SAMe, and were sacrificed at 3 h or 12 h after the bolus. RT-PCR and Western blot analyses showed that SAMe significantly induced ADH1 levels in the 3 h liver samples. However, SAMe did not affect the changes in ADH1 protein levels 12 h post bolus. Since SAMe is a methyl donor, it was postulated that the ADH1 gene expression up regulation at 3 h was due to a histone modification induced by methylation from methyl transferases. Dimethylated histone 3 lysine 4 (H3K4me2), a modification responsible for gene expression activation, was found to be significantly increased by SAMe at 3 h post bolus. These results correlated with the low BAL found at 3 h post bolus, and support the concept that SAMe increased the gene expression to increase the elimination rate of ethanol in binge drinking by increasing H3K4me2. Copyright © 2010 Elsevier Inc. All rights reserved.

Fast internal dynamics in alcohol dehydrogenase

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

Monkenbusch, M.; Stadler, A., E-mail: a.stadler@fz-juelich.de; Biehl, R.

2015-08-21

Large-scale domain motions in alcohol dehydrogenase (ADH) have been observed previously by neutron spin-echo spectroscopy (NSE). We have extended the investigation on the dynamics of ADH in solution by using high-resolution neutron time-of-flight (TOF) and neutron backscattering (BS) spectroscopy in the incoherent scattering range. The observed hydrogen dynamics were interpreted in terms of three mobility classes, which allowed a simultaneous description of the measured TOF and BS spectra. In addition to the slow global protein diffusion and domain motions observed by NSE, a fast internal process could be identified. Around one third of the protons in ADH participate in themore » fast localized diffusive motion. The diffusion coefficient of the fast internal motions is around two third of the value of the surrounding D{sub 2}O solvent. It is tempting to associate the fast internal process with solvent exposed amino acid residues with dangling side chains.« less

NMR characterization of altered lignins extracted from tobacco plants down-regulated for lignification enzymes cinnamylalcohol dehydrogenase and cinnamoyl-CoA reductase

PubMed Central

Ralph, John; Hatfield, Ronald D.; Piquemal, Joël; Yahiaoui, Nabila; Pean, Michel; Lapierre, Catherine; Boudet, Alain M.

1998-01-01

Homologous antisense constructs were used to down-regulate tobacco cinnamyl-alcohol dehydrogenase (CAD; EC 1.1.1.195) and cinnamoyl-CoA reductase (CCR; EC 1.2.1.44) activities in the lignin monomer biosynthetic pathway. CCR converts activated cinnamic acids (hydroxycinnamoyl–SCoAs) to cinnamaldehydes; cinnamaldehydes are then reduced to cinnamyl alcohols by CAD. The transformations caused the incorporation of nontraditional components into the extractable tobacco lignins, as evidenced by NMR. Isolated lignin of antisense-CAD tobacco contained fewer coniferyl and sinapyl alcohol-derived units that were compensated for by elevated levels of benzaldehydes and cinnamaldehydes. Products from radical coupling of cinnamaldehydes, particularly sinapaldehyde, which were barely discernible in normal tobacco, were major components of the antisense-CAD tobacco lignin. Lignin content was reduced in antisense-CCR tobacco, which displayed a markedly reduced vigor. That lignin contained fewer coniferyl alcohol-derived units and significant levels of tyramine ferulate. Tyramine ferulate is a sink for the anticipated build-up of feruloyl–SCoA, and may be up-regulated in response to a deficit of coniferyl alcohol. Although it is not yet clear whether the modified lignins are true structural components of the cell wall, the findings provide further indications of the metabolic plasticity of plant lignification. An ability to produce lignin from alternative monomers would open new avenues for manipulation of lignin by genetic biotechnologies. PMID:9788995

Inversion of substrate stereoselectivity of horse liver alcohol dehydrogenase by substitutions of Ser-48 and Phe-93

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

Kim, Keehyuk; Plapp, Bryce V.

The substrate specificities of alcohol dehydrogenases (ADH) are of continuing interest for understanding the physiological functions of these enzymes. Ser-48 and Phe-93 have been identified as important residues in the substrate binding sites of ADHs, but more comprehensive structural and kinetic studies are required. The S48T substitution in horse ADH1E has small effects on kinetic constants and catalytic efficiency (V/Km) with ethanol, but decreases activity with benzyl alcohol and affinity for 2,2,2-trifluoroethanol (TFE) and 2,3,4,5,6-pentafluorobenzyl alcohol (PFB). Nevertheless, atomic resolution crystal structures of the S48T enzyme complexed with NAD+ and TFE or PFB are very similar to the structures formore » the wild-type enzyme. (The S48A substitution greatly diminishes catalytic activity.) The F93A substitution significantly decreases catalytic efficiency (V/Km) for ethanol and acetaldehyde while increasing activity for larger secondary alcohols and the enantioselectivity for the R-isomer relative to the S-isomer of 2-alcohols. The doubly substituted S48T/F93A enzyme has kinetic constants for primary and secondary alcohols similar to those for the F93A enzyme, but the effect of the S48T substitution is to decrease V/Km for (S)-2-alcohols without changing V/Km for (R)-2-alcohols. Thus, the S48T/F93A substitutions invert the enantioselectivity for alcohol oxidation, increasing the R/S ratio by 10, 590, and 200-fold for 2-butanol, 2-octanol, and sec-phenethyl alcohol, respectively. Transient kinetic studies and simulations of the ordered bi bi mechanism for the oxidation of the 2-butanols by the S48T/F93A ADH show that the rate of hydride transfer is increased about 7-fold for both isomers (relative to wild-type enzyme) and that the inversion of enantioselectivity is due to more productive binding for (R)-2-butanol than for (S)-2-butanol in the ternary complex. Molecular modeling suggests that both of the sec-phenethyl alcohols could bind to the enzyme and that dynamics must affect the rates of catalysis.« less

Disulfiram treatment increases plasma and red blood cell acetaldehyde in abstinent alcoholics.

PubMed

Rosman, A S; Waraich, A; Baraona, E; Lieber, C S

2000-07-01

Much of alcohol's toxicity is due to its product, acetaldehyde. The role of acetaldehyde derived from endogenous sources was assessed in alcoholic patients administered disulfiram, an inhibitor of aldehyde dehydrogenase. The first part of the study included 23 subjects without biochemical or clinical evidence of chronic liver disease who were abstinent for 2 weeks; 11 patients were started on disulfiram (250 mg/day), whereas the other 12 were not given disulfiram and served as controls. The second part of the study included 13 alcoholic patients with clinical or pathological evidence of cirrhosis who also were administered disulfiram for 2 weeks. Plasma and red blood cell (RBC) acetaldehyde as well as serum transaminases were measured at baseline and after 1 and 2 weeks of treatment. In the disulfiram-treated group of alcoholics without known cirrhosis, RBC acetaldehyde levels increased from the pretreatment value of 2.98+/-0.18 microM to 4.14+/-0.33 microM after 1 week and to 4.14+/-0.26 microM after 2 weeks of treatment (p < 0.001). Compared with the pretreatment values (2.07+/-0.24 microM), plasma acetaldehyde levels also increased after 1 week (3.18+/-0.32 microM) and 2 weeks (3.15+/-0.26 microM) of disulfiram treatment (p < 0.001). There were no significant differences in sequential levels measured in either plasma or RBC acetaldehyde levels in patients who were not administered disulfiram. In the group of cirrhotic patients, the mean baseline RBC acetaldehyde value (3.60+/-0.22 microM) was significantly higher than in noncirrhotics. Disulfiram therapy increased the RBC acetaldehyde after 1 week (4.63+/-0.27 microM, p < 0.001) and 2 weeks of treatment (4.06+/-0.28 microM, p < 0.05). Compared with baseline values, plasma acetaldehyde levels were significantly higher after 1 week but not after 2 weeks of disulfiram. There were no significant differences among serum transaminases in alcoholics administered disulfiram, although three cirrhotic patients did have clinically significant elevations. In abstaining subjects given disulfiram, acetaldehyde concentrations increase, possibly due to diminished catabolism of endogenously generated acetaldehyde. Disulfiram should be given cautiously, especially in patients with cirrhosis.

Checkpoint kinase 1 inhibition sensitises transformed cells to dihydroorotate dehydrogenase inhibition

PubMed Central

Arnould, Stéphanie; Rodier, Geneviève; Matar, Gisèle; Vincent, Charles; Pirot, Nelly; Delorme, Yoann; Berthet, Charlène; Buscail, Yoan; Noël, Jean Yohan; Lachambre, Simon; Jarlier, Marta; Bernex, Florence; Delpech, Hélène; Vidalain, Pierre Olivier; Janin, Yves L.; Theillet, Charles; Sardet, Claude

2017-01-01

Reduction in nucleotide pools through the inhibition of mitochondrial enzyme dihydroorotate dehydrogenase (DHODH) has been demonstrated to effectively reduce cancer cell proliferation and tumour growth. The current study sought to investigate whether this antiproliferative effect could be enhanced by combining Chk1 kinase inhibition. The pharmacological activity of DHODH inhibitor teriflunomide was more selective towards transformed mouse embryonic fibroblasts than their primary or immortalised counterparts, and this effect was amplified when cells were subsequently exposed to PF477736 Chk1 inhibitor. Flow cytometry analyses revealed substantial accumulations of cells in S and G2/M phases, followed by increased cytotoxicity which was characterised by caspase 3-dependent induction of cell death. Associating PF477736 with teriflunomide also significantly sensitised SUM159 and HCC1937 human triple negative breast cancer cell lines to dihydroorotate dehydrogenase inhibition. The main characteristic of this effect was the sustained accumulation of teriflunomide-induced DNA damage as cells displayed increased phospho serine 139 H2AX (γH2AX) levels and concentration-dependent phosphorylation of Chk1 on serine 345 upon exposure to the combination as compared with either inhibitor alone. Importantly a similar significant increase in cell death was observed upon dual siRNA mediated depletion of Chk1 and DHODH in both murine and human cancer cell models. Altogether these results suggest that combining DHODH and Chk1 inhibitions may be a strategy worth considering as a potential alternative to conventional chemotherapies. PMID:29221122

Original 2-(3-Alkoxy-1H-pyrazol-1-yl)azines Inhibitors of Human Dihydroorotate Dehydrogenase (DHODH)

PubMed Central

2016-01-01

Following our discovery of human dihydroorotate dehydrogenase (DHODH) inhibition by 2-(3-alkoxy-1H-pyrazol-1-yl)pyrimidine derivatives as well as 2-(4-benzyl-3-ethoxy-5-methyl-1H-pyrazol-1-yl)-5-methylpyridine, we describe here the syntheses and evaluation of an array of azine-bearing analogues. As in our previous report, the structure–activity study of this series of human DHODH inhibitors was based on a phenotypic assay measuring measles virus replication. Among other inhibitors, this round of syntheses and biological evaluation iteration led to the highly active 5-cyclopropyl-2-(4-(2,6-difluorophenoxy)-3-isopropoxy-5-methyl-1H-pyrazol-1-yl)-3-fluoropyridine. Inhibition of DHODH by this compound was confirmed in an array of in vitro assays, including enzymatic tests and cell-based assays for viral replication and cellular growth. This molecule was found to be more active than the known inhibitors of DHODH, brequinar and teriflunomide, thus opening perspectives for its use as a tool or for the design of an original series of immunosuppressive agent. Moreover, because other series of inhibitors of human DHODH have been found to also affect Plasmodium falciparum DHODH, all the compounds were assayed for their effect on P. falciparum growth. However, the modest in vitro inhibition solely observed for two compounds did not correlate with their inhibition of P. falciparum DHODH. PMID:26079043

Regulation of the Biosynthesis of Amino Acids of the Aspartate Family in Coliform Bacteria and Pseudomonads

PubMed Central

Cohen, G. N.; Stanier, R. Y.; Bras, Gisele Le

1969-01-01

The control of aspartokinase and homoserine dehydrogenase activities was compared in aerobic and fermentative pseudomonads (genera Pseudomonas and Aeromonas), and in coliform bacteria representative of the principal genera of the Enterobacteriaceae. Isofunctional aspartokinases subject to independent end-product control occur in the Enterobacteriaceae and in Aeromonas. In Pseudomonas, there appears to be a single aspartokinase, subject to concerted feedback inhibition by lysine and threonine. Within this genus, the sensitivity of aspartokinase to the single allosteric inhibitors varies considerably: the aspartokinase of the acidovorans group is little affected by the single inhibitors, whereas that of the fluorescent group is severely inhibited by either amino acid at high concentration. In all bacteria examined, homoserine dehydrogenase activity is inhibited by threonine; inhibition is more severe in aerobic pseudomonads than in the other groups. In most of the bacteria examined, either nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate can serve as a cofactor for this enzyme, though the relative activity with the two pyridine nucleotides varies widely. Aerobic pseudomonads of the acidovorans group contain a homoserine dehydrogenase that is absolutely specific for NAD. The taxonomic implications of these findings are discussed. PMID:4391829

Butylated hydroxyanisole alters rat 5α-reductase and 3α-hydroxysteroid dehydrogenase: Implications for influences of neurosteroidogenesis.

PubMed

Guo, Jingjing; Li, Lili; Zhou, Songyi; Su, Ying; Li, Xiaoheng; Sun, Jianliang; Ge, Ren-Shan

2017-07-13

Butylated hydroxyanisole is a synthetic antioxidant. It may affect the function of the nerve system. The objective of the present study is to investigate the direct effects of butylated hydroxyanisole on rat brain neurosteroidogenic 5α-reductase 1 (SRD5A1), 3α-hydroxysteroid dehydrogenase (AKR1C14), and retinol dehydrogenase 2 (RDH2). Rat SRD5A1, AKR1C14, and RDH2 were cloned and expressed in COS1 cells, and the effects of butylated hydroxyanisole on these enzyme activities were measured. Butylated hydroxyanisole inhibited SRD5A1, AKR1C14, and RDH2 with IC 50 values of 4.731±0.079μM, 5.753±0.073μM, and over 100μM, respectively. Butylated hydroxyanisole is a competitive inhibitor for both SRD5A1 and AKR1C14. Docking analysis shows that butylated hydroxyanisole binds to the dihydrotestosterone-binding site of AKR1C14. In conclusion, butylated hydroxyanisole is a potent inhibitor of SRD5A1 and AKR1C14, thus reducing the formation of active neurosteroids. Copyright © 2017 Elsevier B.V. All rights reserved.

Acridone-based inhibitors of inosine 5'-monophosphate dehydrogenase: discovery and SAR leading to the identification of N-(2-(6-(4-ethylpiperazin-1-yl)pyridin-3-yl)propan-2-yl)-2- fluoro-9-oxo-9,10-dihydroacridine-3-carboxamide (BMS-566419).

PubMed

Watterson, Scott H; Chen, Ping; Zhao, Yufen; Gu, Henry H; Dhar, T G Murali; Xiao, Zili; Ballentine, Shelley K; Shen, Zhongqi; Fleener, Catherine A; Rouleau, Katherine A; Obermeier, Mary; Yang, Zheng; McIntyre, Kim W; Shuster, David J; Witmer, Mark; Dambach, Donna; Chao, Sam; Mathur, Arvind; Chen, Bang-Chi; Barrish, Joel C; Robl, Jeffrey A; Townsend, Robert; Iwanowicz, Edwin J

2007-07-26

Inosine monophosphate dehydrogenase (IMPDH), a key enzyme in the de novo synthesis of guanosine nucleotides, catalyzes the irreversible nicotinamide-adenine dinucleotide dependent oxidation of inosine-5'-monophosphate to xanthosine-5'-monophosphate. Mycophenolate Mofetil (MMF), a prodrug of mycophenolic acid, has clinical utility for the treatment of transplant rejection based on its inhibition of IMPDH. The overall clinical benefit of MMF is limited by what is generally believed to be compound-based, dose-limiting gastrointestinal (GI) toxicity that is related to its specific pharmacokinetic characteristics. Thus, development of an IMPDH inhibitor with a novel structure and a different pharmacokinetic profile may reduce the likelihood of GI toxicity and allow for increased efficacy. This article will detail the discovery and SAR leading to a novel and potent acridone-based IMPDH inhibitor 4m and its efficacy and GI tolerability when administered orally in a rat adjuvant arthritis model.

Endocrine disruptors and other inhibitors of 11β-hydroxysteroid dehydrogenase 1 and 2: Tissue-specific consequences of enzyme inhibition.

PubMed

Vitku, Jana; Starka, Luboslav; Bicikova, Marie; Hill, Martin; Heracek, Jiri; Sosvorova, Lucie; Hampl, Richard

2016-01-01

Numerous chemicals in the environment have the ability to interact with the endocrine system. These compounds are called endocrine disruptors (EDs). Exposure to EDs represents one of the hypotheses for decreasing fertility, the increased risk of numerous cancers and obesity, metabolic syndrome and type 2 diabetes. There are various mechanisms of ED action, one of which is their interference in the action of 11β-hydroxysteroid dehydrogenase (11βHSD) that maintains a balance between active and inactive glucocorticoids on the intracellular level. This enzyme has two isoforms and is expressed in various tissues. Inhibition of 11βHSD in various tissues can have different consequences. In the case of EDs, the results of exposure are mainly adverse; on the other hand pharmaceutically developed inhibitors of 11βHSD type 1 are evaluated as an option for treating metabolic syndrome, as well as related diseases and depressive disorders. This review focuses on the effects of 11βHSD inhibitors in the testis, colon, adipose tissue, kidney, brain and placenta. Copyright © 2014 Elsevier Ltd. All rights reserved.

Novel 11β-hydroxysteroid dehydrogenase 1 inhibitors reduce cortisol levels in keratinocytes and improve dermal collagen content in human ex vivo skin after exposure to cortisone and UV.

PubMed

Boudon, Stéphanie M; Vuorinen, Anna; Geotti-Bianchini, Piero; Wandeler, Eliane; Kratschmar, Denise V; Heidl, Marc; Campiche, Remo; Jackson, Eileen; Odermatt, Alex

2017-01-01

Activity and selectivity assessment of new bi-aryl amide 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) inhibitors, prepared in a modular manner via Suzuki cross-coupling, are described. Several compounds inhibiting 11β-HSD1 at nanomolar concentrations were identified. Compounds 2b, 3e, 7b and 12e were shown to selectively inhibit 11β-HSD1 over 11β-HSD2, 17β-HSD1 and 17β-HSD2. These inhibitors also potently inhibited 11β-HSD1 activity in intact HEK-293 cells expressing the recombinant enzyme and in intact primary human keratinocytes expressing endogenous 11β-HSD1. Moreover, compounds 2b, 3e and 12e were tested for their activity in human skin biopsies. They were able to prevent, at least in part, both the cortisone- and the UV-mediated decreases in collagen content. Thus, inhibition of 11β-HSD1 by these compounds can be further investigated to delay or prevent UV-mediated skin damage and skin aging.

Structure-Guided Lead Optimization of Triazolopyrimidine-Ring Substituents Identifies Potent Plasmodium falciparum Dihydroorotate Dehydrogenase Inhibitors with Clinical Candidate Potential

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

Coteron, Jose M.; Marco, Maria; Esquivias, Jorge

2012-02-27

Drug therapy is the mainstay of antimalarial therapy, yet current drugs are threatened by the development of resistance. In an effort to identify new potential antimalarials, we have undertaken a lead optimization program around our previously identified triazolopyrimidine-based series of Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) inhibitors. The X-ray structure of PfDHODH was used to inform the medicinal chemistry program allowing the identification of a potent and selective inhibitor (DSM265) that acts through DHODH inhibition to kill both sensitive and drug resistant strains of the parasite. This compound has similar potency to chloroquine in the humanized SCID mouse P. falciparum model,more » can be synthesized by a simple route, and rodent pharmacokinetic studies demonstrated it has excellent oral bioavailability, a long half-life and low clearance. These studies have identified the first candidate in the triazolopyrimidine series to meet previously established progression criteria for efficacy and ADME properties, justifying further development of this compound toward clinical candidate status.« less

Inhibition of Cancer-Associated Mutant Isocitrate Dehydrogenases by 2-thiohydantoin compounds

PubMed Central

Kogiso, Mari; Yao, Yuan; Zhou, Chao; Li, Xiao-Nan; Song, Yongcheng

2015-01-01

Somatic mutations of isocitrate dehydrogenase 1 (IDH1) at R132 are frequently found in certain cancers such as glioma. With losing the activity of wild-type IDH1, the R132H and R132C mutant proteins can reduce α-ketoglutaric acid (α-KG) to D-2-hydroxyglutaric acid (D2HG). The resulting high concentration of D2HG inhibits many α-KG-dependent dioxygenases, including histone demethylases, to cause broad histone hypermethylation. These aberrant epigenetic changes are responsible for initiation of these cancers. We report the synthesis, structure activity relationships, enzyme kinetics and binding thermodynamics of a novel series of 2-thiohydantoin and related compounds, among which several compounds are potent inhibitors of mutant IDH1 with Ki as low as 420 nM. X-ray crystal structures of IDH1(R132H) in complex with two inhibitors are reported, showing their inhibitor-protein interactions. These compounds can decrease the cellular concentration of D2HG, reduce the levels of histone methylation, and suppress proliferation of stem-like cancer cells in BT142 glioma with IDH1 R132H mutation. PMID:26280302

Inhibition of human alcohol and aldehyde dehydrogenases by aspirin and salicylate: assessment of the effects on first-pass metabolism of ethanol.

PubMed

Lee, Shou-Lun; Lee, Yung-Pin; Wu, Min-Li; Chi, Yu-Chou; Liu, Chiu-Ming; Lai, Ching-Long; Yin, Shih-Jiun

2015-05-01

Previous studies have reported that aspirin significantly reduced the first-pass metabolism (FPM) of ethanol in humans thereby increasing adverse effects of alcohol. The underlying causes, however, remain poorly understood. Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH), principal enzymes responsible for metabolism of ethanol, are complex enzyme families that exhibit functional polymorphisms among ethnic groups and distinct tissue distributions. We investigated the inhibition profiles by aspirin and its major metabolite salicylate of ethanol oxidation by recombinant human ADH1A, ADH1B1, ADH1B2, ADH1B3, ADH1C1, ADH1C2, ADH2, and ADH4, and acetaldehyde oxidation by ALDH1A1 and ALDH2, at pH 7.5 and 0.5 mM NAD(+). Competitive inhibition pattern was found to be a predominant type among the ADHs and ALDHs studied, although noncompetitive and uncompetitive inhibitions were also detected in a few cases. The inhibition constants of salicylate for the ADHs and ALDHs were considerably lower than that of aspirin with the exception of ADH1A that can be ascribed to a substitution of Ala-93 at the bottom of substrate pocket as revealed by molecular docking experiments. Kinetic inhibition equation-based simulations show at higher therapeutic levels of blood plasma salicylate (1.5 mM) that the decrease of activities at 2-10 mM ethanol for ADH1A/ADH2 and ADH1B2/ADH1B3 are predicted to be 75-86% and 31-52%, respectively, and that the activity decline for ALDH1A1 and ALDH2 at 10-50 μM acetaldehyde to be 62-73%. Our findings suggest that salicylate may substantially inhibit hepatic FPM of alcohol at both the ADH and ALDH steps when concurrent intaking aspirin. Copyright © 2015 Elsevier Inc. All rights reserved.

Purification and characterization of a novel cytosolic NADP(H)-dependent retinol oxidoreductase from rabbit liver.

PubMed

Huang, D Y; Ichikawa, Y

1997-03-07

Rabbit liver cytosol exhibits very high retinol dehydrogenase activity. At least two retinol dehydrogenases were demonstrated to exist in rabbit liver cytosol, and the major one, a cytosolic NADP(H)-dependent retinol dehydrogenase (systematic name: retinol oxidoreductase) was purified about 1795-fold to electrophoretic and column chromatographic homogeneity by a procedure involving column chromatography on AF-Red Toyopearl twice and then hydroxyapatite. Its molecular mass was estimated to be 34 kDa by SDS-PAGE, and 144 kDa by HPLC gel filtration, suggesting that it is a homo-tetramer. The enzyme uses free retinol and retinal, and their complexes with CRBP as substrates in vitro. The optimum pH values for retinol oxidation of free retinol and CRBP-retinol were 8.8-9.2 and 8.0-9.0, respectively, and those for retinal reduction of free retinal and retinal-CRBP were the same, 7.0-7.6. Km for free retinol and Vmax for retinal formation were 2.8 microM and 2893 nmol/min per mg protein at 37 degrees C (pH 9.0) and the corresponding values with retinol-CRBP as a substrate were 2.5 microM and 2428 nmol/min per mg protein at 37 degrees C (pH 8.6); Km for free retinal and Vmax for retinol formation were 6.5 microM and 4108 nmol/min per mg protein, and the corresponding values with retinal-CRBP as a substrate were 5.1 microM and 3067 nmol/min per mg protein at 37 degrees C, pH 7.4. NAD(H) was not effective as a cofactor. 4-Methylpyrazole was a weak inhibitor (IC50 = 28 mM) of the enzyme, and ethanol was neither a substrate nor an inhibitor of the enzyme. This enzyme exhibits relatively broad aldehyde reductase activity and some ketone reductase activity, the activity for aromatic substitutive aldehydes being especially high and effective. Whereas, except in the case of retinol, oxidative activity toward the corresponding alcohols was not detected. This novel cytosolic enzyme may play an important role in vivo in maintaining the homeostasis of retinal, the substrate of retinoic acid synthesis, at least in rabbit liver, since a high concentration of retinol in liver and the lower Km of the enzyme for retinol force the oxidative reaction, while higher activity of retinal reductase at physiological pH forces the reductive reaction.

Gene ercA, encoding a putative iron-containing alcohol dehydrogenase, is involved in regulation of ethanol utilization in Pseudomonas aeruginosa.

PubMed

Hempel, Niels; Görisch, Helmut; Mern, Demissew S

2013-09-01

Several two-component regulatory systems are known to be involved in the signal transduction pathway of the ethanol oxidation system in Pseudomonas aeruginosa ATCC 17933. These sensor kinases and response regulators are organized in a hierarchical manner. In addition, a cytoplasmic putative iron-containing alcohol dehydrogenase (Fe-ADH) encoded by ercA (PA1991) has been identified to play an essential role in this regulatory network. The gene ercA (PA1991) is located next to ercS, which encodes a sensor kinase. Inactivation of ercA (PA1991) by insertion of a kanamycin resistance cassette created mutant NH1. NH1 showed poor growth on various alcohols. On ethanol, NH1 grew only with an extremely extended lag phase. During the induction period on ethanol, transcription of structural genes exa and pqqABCDEH, encoding components of initial ethanol oxidation in P. aeruginosa, was drastically reduced in NH1, which indicates the regulatory function of ercA (PA1991). However, transcription in the extremely delayed logarithmic growth phase was comparable to that in the wild type. To date, the involvement of an Fe-ADH in signal transduction processes has not been reported.

Gene ercA, Encoding a Putative Iron-Containing Alcohol Dehydrogenase, Is Involved in Regulation of Ethanol Utilization in Pseudomonas aeruginosa

PubMed Central

Hempel, Niels; Görisch, Helmut

2013-01-01

Several two-component regulatory systems are known to be involved in the signal transduction pathway of the ethanol oxidation system in Pseudomonas aeruginosa ATCC 17933. These sensor kinases and response regulators are organized in a hierarchical manner. In addition, a cytoplasmic putative iron-containing alcohol dehydrogenase (Fe-ADH) encoded by ercA (PA1991) has been identified to play an essential role in this regulatory network. The gene ercA (PA1991) is located next to ercS, which encodes a sensor kinase. Inactivation of ercA (PA1991) by insertion of a kanamycin resistance cassette created mutant NH1. NH1 showed poor growth on various alcohols. On ethanol, NH1 grew only with an extremely extended lag phase. During the induction period on ethanol, transcription of structural genes exa and pqqABCDEH, encoding components of initial ethanol oxidation in P. aeruginosa, was drastically reduced in NH1, which indicates the regulatory function of ercA (PA1991). However, transcription in the extremely delayed logarithmic growth phase was comparable to that in the wild type. To date, the involvement of an Fe-ADH in signal transduction processes has not been reported. PMID:23813731

Drosophila genetics in the classroom.

PubMed

Sofer, W; Tompkins, L

1994-01-01

Drosophila has long been useful for demonstrating the principles of classical Mendelian genetics in the classroom. In recent years, the organism has also helped students understand biochemical and behavioral genetics. In this connection, this article describes the development of a set of integrated laboratory exercises and descriptive materials--a laboratory module--in biochemical genetics for use by high-school students. The module focuses on the Adh gene and its product, the alcohol dehydrogenase enzyme. Among other activities, students using the module get to measure alcohol tolerance and to assay alcohol dehydrogenase activity in Adh-negative and -positive flies. To effectively present the module in the classroom, teachers attend a month-long Dissemination Institute in the summer. During this period, they learn about other research activities that can be adapted for classroom use. One such activity that has proved popular with teachers and students utilizes Drosophila to introduce some of the concepts of behavioral genetics to the high-school student. By establishing closer interactions between high-school educators and research scientists, the gulf between the two communities can begin to be bridged. It is anticipated that the result of a closer relationship will be that the excitement and creativity of science will be more effectively conveyed to students.

Thermostable NADP+-Dependent Medium-Chain Alcohol Dehydrogenase from Acinetobacter sp. Strain M-1: Purification and Characterization and Gene Expression in Escherichia coli

PubMed Central

Tani, Akio; Sakai, Yasuyoshi; Ishige, Takeru; Kato, Nobuo

2000-01-01

NADPH-dependent alkylaldehyde reducing enzyme, which was greatly induced by n-hexadecane, from Acinetobacter sp. strain M-1 was purified and characterized. The purified enzyme had molecular masses of 40 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 160 kDa as determined by gel filtration chromatography. The enzyme, which was shown to be highly thermostable, was most active toward n-heptanal and could use n-alkylaldehydes ranging from C2 to C14 and several substituted benzaldehydes, including the industrially important compounds cinnamyl aldehyde and anisaldehyde, as substrates. The alrA gene coding for this enzyme was cloned, and its nucleotide sequence was determined. The deduced amino acid sequence encoded by the alrA gene exhibited homology to the amino acid sequences of zinc-containing alcohol dehydrogenases from various sources. The gene could be highly expressed in Escherichia coli, and the product was purified to homogeneity by simpler procedures from the recombinant than from the original host. Our results show that this enzyme can be used for industrial bioconversion of useful alcohols and aldehydes. PMID:11097895

In-Silico molecular docking and simulation studies on novel chalcone and flavone hybrid derivatives with 1, 2, 3-triazole linkage as vital inhibitors of Plasmodium falciparum dihydroorotate dehydrogenase.

PubMed

Thillainayagam, Mahalakshmi; Malathi, Kullappan; Ramaiah, Sudha

2017-11-27

The structural motifs of chalcones, flavones, and triazoles with varied substitutions have been studied for the antimalarial activity. In this study, 25 novel derivatives of chalcone and flavone hybrid derivatives with 1, 2, 3-triazole linkage are docked with Plasmodium falciparum dihydroorotate dehydrogenase to establish their inhibitory activity against Plasmodium falciparum. The best binding conformation of the ligands at the catalytic site of dihydroorotate dehydrogenase are selected to characterize the best bound ligand using the best consensus score and the number of hydrogen bond interactions. The ligand namely (2E)-3-(4-{[1-(3-chloro-4-fluorophenyl)-1H-1, 2, 3-triazol-4-yl]methoxy}-3-methoxyphenyl-1-(2-hydroxy-4,6-dimethoxyphenyl)prop-2-en-1-one, is one the among the five best docked ligands, which interacts with the protein through nine hydrogen bonds and with a consensus score of five. To refine and confirm the docking study results, the stability of complexes is verified using Molecular Dynamics Simulations, Molecular Mechanics /Poisson-Boltzmann Surface Area free binding energy analysis, and per residue contribution for the binding energy. The study implies that the best docked Plasmodium falciparum dihydroorotate dehydrogenase-ligand complex is having high negative binding energy, most stable, compact, and rigid with nine hydrogen bonds. The study provides insight for the optimization of chalcone and flavone hybrids with 1, 2, 3-triazole linkage as potent inhibitors.

Identification of a magnesium-dependent NAD(P)(H)-binding domain in the nicotinoprotein methanol dehydrogenase from Bacillus methanolicus.

PubMed

Hektor, Harm J; Kloosterman, Harm; Dijkhuizen, Lubbert

2002-12-06

The Bacillus methanolicus methanol dehydrogenase (MDH) is a decameric nicotinoprotein alcohol dehydrogenase (family III) with one Zn(2+) ion, one or two Mg(2+) ions, and a tightly bound cofactor NAD(H) per subunit. The Mg(2+) ions are essential for binding of cofactor NAD(H) in MDH. A B. methanolicus activator protein strongly stimulates the relatively low coenzyme NAD(+)-dependent MDH activity, involving hydrolytic removal of the NMN(H) moiety of cofactor NAD(H) (Kloosterman, H., Vrijbloed, J. W., and Dijkhuizen, L. (2002) J. Biol. Chem. 277, 34785-34792). Members of family III of NAD(P)-dependent alcohol dehydrogenases contain three unique, conserved sequence motifs (domains A, B, and C). Domain C is thought to be involved in metal binding, whereas the functions of domains A and B are still unknown. This paper provides evidence that domain A constitutes (part of) a new magnesium-dependent NAD(P)(H)-binding domain. Site-directed mutants D100N and K103R lacked (most of the) bound cofactor NAD(H) and had lost all coenzyme NAD(+)-dependent MDH activity. Also mutants G95A and S97G were both impaired in cofactor NAD(H) binding but retained coenzyme NAD(+)-dependent MDH activity. Mutant G95A displayed a rather low MDH activity, whereas mutant S97G was insensitive to activator protein but displayed "fully activated" MDH reaction rates. The various roles of these amino acid residues in coenzyme and/or cofactor NAD(H) binding in MDH are discussed.

Efficient reduction of the formation of by-products and improvement of production yield of 2,3-butanediol by a combined deletion of alcohol dehydrogenase, acetate kinase-phosphotransacetylase, and lactate dehydrogenase genes in metabolically engineered Klebsiella oxytoca in mineral salts medium.

PubMed

Jantama, Kaemwich; Polyiam, Pattharasedthi; Khunnonkwao, Panwana; Chan, Sitha; Sangproo, Maytawadee; Khor, Kirin; Jantama, Sirima Suvarnakuta; Kanchanatawee, Sunthorn

2015-07-01

Klebsiella oxytoca KMS005 (∆adhE∆ackA-pta∆ldhA) was metabolically engineered to improve 2,3-butanediol (BDO) yield. Elimination of alcohol dehydrogenase E (adhE), acetate kinase A-phosphotransacetylase (ackA-pta), and lactate dehydrogenase A (ldhA) enzymes allowed BDO production as a primary pathway for NADH re-oxidation, and significantly reduced by-products. KMS005 was screened for the efficient glucose utilization by metabolic evolution. KMS005-73T improved BDO production at a concentration of 23.5±0.5 g/L with yield of 0.46±0.02 g/g in mineral salts medium containing 50 g/L glucose in a shake flask. KMS005-73T also exhibited BDO yields of about 0.40-0.42 g/g from sugarcane molasses, cassava starch, and maltodextrin. During fed-batch fermentation, KMS005-73T produced BDO at a concentration, yield, and overall and specific productivities of 117.4±4.5 g/L, 0.49±0.02 g/g, 1.20±0.05 g/Lh, and 27.2±1.1 g/gCDW, respectively. No acetoin, lactate, and formate were detected, and only trace amounts of acetate and ethanol were formed. The strain also produced the least by-products and the highest BDO yield among other Klebsiella strains previously developed. Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Association between alcohol dehydrogenase 1C gene *1/*2 polymorphism and pancreatitis risk: a meta-analysis.

PubMed

Fang, F; Pan, J; Su, G H; Xu, L X; Li, G; Li, Z H; Zhao, H; Wang, J

2015-11-30

Numerous studies have focused on the relationship be-tween alcohol dehydrogenase 1C gene (ADH1C) *1/*2 polymorphism (Ile350Val, rs698, also known as ADH1C *1/*2) and pancreatitis risk, but the results have been inconsistent. Thus, we conducted a meta-anal-ysis to more precisely estimate this association. Relevant publications were searched in several widely used databases and 9 eligible studies were included in the meta-analysis. Pooled odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to evaluate the strength of the association. Significant associations between ADH1C *1/*2 poly-morphism and pancreatitis risk were observed in both overall meta-analysis for 12 vs 22 (OR = 1.53, 95%CI = 1.12-2.10) and 11 + 12 vs 22 (OR = 1.44, 95%CI = 1.07-1.95), and the chronic alcoholic pancre-atitis subgroup for 12 vs 22 (OR = 1.64, 95%CI = 1.17-2.29) and 11 + 12 vs 22 (OR = 1.53, 95%CI = 1.11-2.11). Significant pancreatitis risk variation was also detected in Caucasians for 11 + 12 vs 22 (OR = 1.45, 95%CI = 1.07-1.98). In conclusion, the ADH1C *1/*2 polymorphism is likely associated with pancreatitis risk, particularly chronic alcoholic pancreatitis risk, with the *1 allele functioning as a risk factor.

[Discovery of the target genes inhibited by formic acid in Candida shehatae].

PubMed

Cai, Peng; Xiong, Xujie; Xu, Yong; Yong, Qiang; Zhu, Junjun; Shiyuan, Yu

2014-01-04

At transcriptional level, the inhibitory effects of formic acid was investigated on Candida shehatae, a model yeast strain capable of fermenting xylose to ethanol. Thereby, the target genes were regulated by formic acid and the transcript profiles were discovered. On the basis of the transcriptome data of C. shehatae metabolizing glucose and xylose, the genes responsible for ethanol fermentation were chosen as candidates by the combined method of yeast metabolic pathway analysis and manual gene BLAST search. These candidates were then quantitatively detected by RQ-PCR technique to find the regulating genes under gradient doses of formic acid. By quantitative analysis of 42 candidate genes, we finally identified 10 and 5 genes as markedly down-regulated and up-regulated targets by formic acid, respectively. With regard to gene transcripts regulated by formic acid in C. shehatae, the markedly down-regulated genes ranking declines as follows: xylitol dehydrogenase (XYL2), acetyl-CoA synthetase (ACS), ribose-5-phosphate isomerase (RKI), transaldolase (TAL), phosphogluconate dehydrogenase (GND1), transketolase (TKL), glucose-6-phosphate dehydrogenase (ZWF1), xylose reductase (XYL1), pyruvate dehydrogenase (PDH) and pyruvate decarboxylase (PDC); and a declining rank for up-regulated gens as follows: fructose-bisphosphate aldolase (ALD), glucokinase (GLK), malate dehydrogenase (MDH), 6-phosphofructokinase (PFK) and alcohol dehydrogenase (ADH).

Atomic-Resolution Structures of Horse Liver Alcohol Dehydrogenase with NAD[superscript +] and Fluoroalcohols Define Strained Michaelis Complexes

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

Plapp, Bryce V.; Ramaswamy, S.; Iowa)

2013-01-16

Structures of horse liver alcohol dehydrogenase complexed with NAD{sup +} and unreactive substrate analogues, 2,2,2-trifluoroethanol or 2,3,4,5,6-pentafluorobenzyl alcohol, were determined at 100 K at 1.12 or 1.14 {angstrom} resolution, providing estimates of atomic positions with overall errors of 0.02 {angstrom}, the geometry of ligand binding, descriptions of alternative conformations of amino acid residues and waters, and evidence of a strained nicotinamide ring. The four independent subunits from the two homodimeric structures differ only slightly in the peptide backbone conformation. Alternative conformations for amino acid side chains were identified for 50 of the 748 residues in each complex, and Leu-57 andmore » Leu-116 adopt different conformations to accommodate the different alcohols at the active site. Each fluoroalcohol occupies one position, and the fluorines of the alcohols are well-resolved. These structures closely resemble the expected Michaelis complexes with the pro-R hydrogens of the methylene carbons of the alcohols directed toward the re face of C4N of the nicotinamide rings with a C-C distance of 3.40 {angstrom}. The oxygens of the alcohols are ligated to the catalytic zinc at a distance expected for a zinc alkoxide (1.96 {angstrom}) and participate in a low-barrier hydrogen bond (2.52 {angstrom}) with the hydroxyl group of Ser-48 in a proton relay system. As determined by X-ray refinement with no restraints on bond distances and planarity, the nicotinamide rings in the two complexes are slightly puckered (quasi-boat conformation, with torsion angles of 5.9{sup o} for C4N and 4.8{sup o} for N1N relative to the plane of the other atoms) and have bond distances that are somewhat different compared to those found for NAD(P){sup +}. It appears that the nicotinamide ring is strained toward the transition state on the path to alcohol oxidation.« less

β-Catenin is Essential for Ethanol Metabolism and Protection Against Alcohol-mediated Liver Steatosis in Mice

PubMed Central

Liu, Shiguang; Yeh, Tzu-Hsuan; Singh, Vijay P.; Shiva, Sruti; Krauland, Lindsay; Li, Huanan; Zhang, Pili; Kharbanda, Kusum; Ritov, Vladimir; Monga, Satdarshan P. S.; Scott, Donald K.; Eagon, Patricia K.; Behari, Jaideep

2011-01-01

The liver plays a central role in ethanol metabolism and oxidative stress is implicated in alcohol-mediated liver injury. β-Catenin regulates hepatic metabolic zonation and adaptive response to oxidative stress. We hypothesized that β-catenin regulates the hepatic response to ethanol ingestion. Female liver-specific β-catenin knockout (KO) mice and wild type (WT) littermates were fed the Lieber-Decarli liquid diet (5% ethanol) in a pair-wise fashion. Liver histology, biochemistry, and gene expression studies were performed. Plasma alcohol and ammonia levels were measured using standard assays. Ethanol-fed KO mice exhibited systemic toxicity and early mortality. KO mice exhibited severe macrovesicular steatosis and five to six-fold higher serum ALT and AST levels. KO mice had modest increase in hepatic oxidative stress, lower expression of mitochondrial superoxide dismutase (SOD-2), and lower citrate synthase activity, the first step in the tricarboxylic acid cycle. N-Acetyl cysteine (NAC) did not prevent ethanol-induced mortality in KO mice. In WT livers, β-catenin was found to co-precipitate with FoxO3, the upstream regulator of SOD-2. Hepatic alcohol dehydrogenase and aldehyde dehydrogenase activities and expression were lower in KO mice. Hepatic cytochrome P450 2E1 protein levels were upregulated in ethanol-fed WT mice but were nearly undetectable in KO mice. These changes in ethanol-metabolizing enzymes were associated with 30-fold higher blood alcohol levels in KO mice. Conclusion β-catenin is essential for hepatic ethanol metabolism and plays a protective role in alcohol-mediated liver steatosis. Our results strongly suggest that integration of these functions by β-catenin is critical for adaptation to ethanol ingestion in vivo. PMID:22031168

Protective effect of Xingnaojia formulation on rats with brain and liver damage caused by chronic alcoholism.

PubMed

Li, Shuang; Wang, S U; Guo, Zhi-Gang; Huang, Ning; Zhao, Fan-Rong; Zhu, Mo-Li; Ma, Li-Juan; Liang, Jin-Ying; Zhang, Yu-Lin; Huang, Zhong-Lin; Wan, Guang-Rui

2015-11-01

The aim of this study was to observe the effect of a formulation of traditional Chinese medicine extracts known as Xingnaojia (XNJ) on the liver function, learning ability and memory of rats with chronic alcoholism and to verify the mechanism by which it protects the brain and liver. A rat model of chronic alcoholism was used in the study. The spatial learning ability and memory of the rats were tested. The rats were then sacrificed and their brains and hepatic tissues were isolated. The activity of superoxide dismutase (SOD) and levels of glutamate (Glu), N-methyl D-aspartate receptor subtype 2B (NR2B), cyclin-dependent kinase 5 (CDK5) and cannabinoid receptor 1 (CB1) in the hippocampus were analyzed. The ultrastructure of the hepatic tissue was observed by electron microscopy. In addition, the activities of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) in serum were tested and the levels of low-density lipoprotein (LDL), high-density lipoprotein (HDL), triglycerides (TG) and total cholesterol (TCHOL) were analyzed. XNJ enhanced the learning and memory of rats with chronic alcoholism. Treatment with XNJ increased the activity of SOD, and decreased the expression levels of NR2B mRNA and NR2B, CB1 and CDK5 proteins in the brain tissues compared with those in the model rats. It also increased the activity of ALDH in the serum and liver, decreased the serum levels of LDL, TG and TCHOL and increased the serum level of HDL. These results indicate that XNJ exhibited a protective effect against brain and liver damage in rats with chronic alcoholism.

Maternal folate, alcohol and energy metabolism-related gene polymorphisms and the risk of recurrent pregnancy loss.

PubMed

Sata, F; Yamada, H; Kishi, R; Minakami, H

2012-10-01

Epidemiological studies have suggested that the condition of recurrent pregnancy loss (RPL) may be multifactorial, with both genetic predisposition and environmental factors potentially involved in its pathogenesis. The aim of this study is to elucidate the associations between maternal folate, alcohol and energy metabolism-related gene polymorphisms and the risk of RPL. This case-control study, which involved 116 cases with two or more instances of RPL and 306 fertile controls, was performed in the city of Sapporo, Japan. The associations between eight single nucleotide polymorphisms of folate, alcohol and energy metabolism-related genes [methylenetetrahydrofolate reductase (MTHFR), 5-methyltetrahydrofolate-homocysteine methyltransferase (MTR), 5-methyltetrahydrofolate-homocysteine methyltransferase reductase (MTRR), alcohol dehydrogenase 1B (ADH1B), aldehyde dehydrogenase 2 (ALDH2), beta-3-adrenergic receptor (ADRB3) and peroxisome proliferator-activated receptor gamma (PPARG)], and RPL were assessed. Without consideration of cigarette smoking or alcohol use, the risk of RPL significantly decreased in women with the MTHFR rs1801133 TT, MTR rs1805087 AG or ALDH2 rs671 AA genotype (P < 0.05). The risk of RPL associated with cigarette smoking and alcohol use decreased significantly in women carrying the MTHFR rs1801133 T allele [odds ratio (OR), 0.51; 95% confidence interval (CI), 0.27-0.95]. Similarly, the risk of RPL significantly decreased in women carrying the MTR rs1805087 G allele (OR, 0.44; 95% CI, 0.23-0.85). Our findings suggest that maternal gene polymorphisms related to folate metabolism may decrease the risk of RPL. Molecular epidemiological studies are needed to unequivocally elucidate the multifactorial effects of both genetic and environmental factors on human fecundity.

Novel Scheme for Biosynthesis of Aryl Metabolites from l-Phenylalanine in the Fungus Bjerkandera adusta

PubMed Central

Lapadatescu, Carmen; Giniès, Christian; Le Quéré, Jean-Luc; Bonnarme, Pascal

2000-01-01

Aryl metabolite biosynthesis was studied in the white rot fungus Bjerkandera adusta cultivated in a liquid medium supplemented with l-phenylalanine. Aromatic compounds were analyzed by gas chromatography-mass spectrometry following addition of labelled precursors (14C- and 13C-labelled l-phenylalanine), which did not interfere with fungal metabolism. The major aromatic compounds identified were benzyl alcohol, benzaldehyde (bitter almond aroma), and benzoic acid. Hydroxy- and methoxybenzylic compounds (alcohols, aldehydes, and acids) were also found in fungal cultures. Intracellular enzymatic activities (phenylalanine ammonia lyase, aryl-alcohol oxidase, aryl-alcohol dehydrogenase, aryl-aldehyde dehydrogenase, lignin peroxidase) and extracellular enzymatic activities (aryl-alcohol oxidase, lignin peroxidase), as well as aromatic compounds, were detected in B. adusta cultures. Metabolite formation required de novo protein biosynthesis. Our results show that l-phenylalanine was deaminated to trans-cinnamic acid by a phenylalanine ammonia lyase and trans-cinnamic acid was in turn converted to aromatic acids (phenylpyruvic, phenylacetic, mandelic, and benzoylformic acids); benzaldehyde was a metabolic intermediate. These acids were transformed into benzaldehyde, benzyl alcohol, and benzoic acid. Our findings support the hypothesis that all of these compounds are intermediates in the biosynthetic pathway from l-phenylalanine to aryl metabolites. Additionally, trans-cinnamic acid can also be transformed via β-oxidation to benzoic acid. This was confirmed by the presence of acetophenone as a β-oxidation degradation intermediate. To our knowledge, this is the first time that a β-oxidation sequence leading to benzoic acid synthesis has been found in a white rot fungus. A novel metabolic scheme for biosynthesis of aryl metabolites from l-phenylalanine is proposed. PMID:10742235

Reversal of alcohol induced testicular hyperlipidemia by supplementation of ascorbic acid and its comparison with abstention in male guinea pigs.

PubMed

Radhakrishnakartha, Harikrishnan; Appu, Abhilash Puthuvelvippel; Madambath, Indira

2014-02-01

Chronic ethanol exposure causes hyperlipidemia. The present study was designed to investigate the impact of ascorbic acid supplementation on ethanol induced hyperlipidemia in testis and to compare it with that of abstinence from taking alcohol. Thirty-six male guinea pigs were divided into two groups and were maintained for 90 days as follows (1) control (C) (2) ethanol treated group (E) (4 g/kg body wt/day). Ethanol was administered for 90 days and on 90th day, alanine amino transaminase (ALT), aspartate amino transaminase (AST) and γ-glutamyltransferase (GGT) in serum was assayed. The animals in the ethanol group were further divided into an ascorbic acid supplemented group (25 mg/100 g body wt/day) (E+AA) and an ethanol abstention group (EAG) and those in the control group were divided into a control group and a control+ascorbic acid group (C+AA). There was significant increase in levels of testicular cholesterol, free fatty acid, phospholipids and triglycerides in the ethanol group. There was also a significant increase in the activity of HMG CoA reductase and decrease in activity of testicular glucose-6-phosphate dehydrogenase (G6PDH) and malic enzyme in ethanol-ingested animals that further led to decreased levels of serum testosterone. Alcohol administration also enhanced the activity of testicular alcohol dehydrogenase (ADH). Ascorbic acid supplementation and abstention altered all these parameters induced by chronic alcohol administration. Histological studies were also in line with the above results. Ascorbic acid was able to reinstate the cholesterol homeostasis in testis which could have further restored the testicular steroidogenesis. The present study demonstrated that ascorbic acid is effective in reducing the hyperlipidemia induced by chronic alcohol administration and produced a better recovery than abstention.

A multiple mediator analysis approach to quantify the effects of the ADH1B and ALDH2 genes on hepatocellular carcinoma risk.

PubMed

Shih, Stephannie; Huang, Yen-Tsung; Yang, Hwai-I

2018-06-01

Previous work suggested a genetic component affecting the risk of hepatocellular carcinoma (HCC) and mediation analyses have elucidated potential indirect pathways of these genetic effects. Specifically, the effects of alcohol dehydrogenase (ADH1B) and aldehyde dehydrogenase (ALDH2) genes on HCC risk vary based on alcohol consumption habits. However, alcohol consumption may not be the only mediator in the identified pathway: factors related to alcohol consumption may contribute to the same indirect pathway. Thus, we developed a multimediator model to quantify the genetic effects on HCC risk through sequential dichotomous mediators under the counterfactual framework. Our method provided a closed form formula for the mediation effects through different indirect paths, which requires no assumption for the rarity of outcome. In simulation studies of a finite sample, we presented the utility of the method with the variance of the effects estimated using the delta method and bootstrapping. We applied our method to data from participants in Taiwan (580 cases and 3,207 controls) and quantified the mediation effects of single nucleotide polymorphisms (SNPs) in the ADH1B and ALDH2 genes on HCC through alcohol consumption (yes/no) and high alanine transaminase (ALT) levels (greater than or equal to 45 U/L or below 45 U/L). Assuming a dominant risk model, we identified that the SNPs' effects through alcohol consumption is more significant than through ALT levels on HCC risk. This new method provides insight to the magnitude of various casual mechanisms as a closed form solution and can be readily applied in other genomic studies. © 2018 WILEY PERIODICALS, INC.

Oxalomalate, a competitive inhibitor of NADP+ -dependent isocitrate dehydrogenase, regulates lipid peroxidation-mediated apoptosis in U937 cells.

PubMed

Yang, Eun Sun; Yang, Joon-Hyuck; Park, Ji Eun; Park, Jeen-Woo

2005-01-01

Membrane lipid peroxidation processes yield products that may react with DNA and proteins to cause oxidative modifications. Recently, we demonstrated that the control of cytosolic redox balance and the cellular defense against oxidative damage is one of the primary functions of cytosolic NADP+ -dependent isocitrate dehydrogenase (IDPc) through to supply NADPH for antioxidant systems. The protective role of IDPc against lipid peroxidation-mediated apoptosis in U937 cells was investigated in control and cells pre-treated with oxlalomalate, a competitive inhibitor of IDPc. Upon exposure to 2,2'-azobis (2-amidinopropane) hydrochloride (AAPH) to U937 cells, which induces lipid peroxidation in membranes, the susceptibility to apoptosis was higher in oxalomalate-treated cells as compared to control cells. The results suggest that IDPc plays an important protective role in apoptosis of U937 cells induced by lipid peroxidation-mediated oxidative stress.

Discovering Novel Alternaria solani Succinate Dehydrogenase Inhibitors by In Silico Modeling and Virtual Screening Strategies to Combat Early Blight

NASA Astrophysics Data System (ADS)

Iftikhar, Sehrish; Shahid, Ahmad A.; Halim, Sobia A.; Wolters, Pieter J.; Vleeshouwers, Vivianne G. A. A.; Khan, Ajmal; Al-Harrasi, Ahmed; Ahmad, Shahbaz

2017-11-01

Alternaria blight is an important foliage disease caused by Alternaria solani. The enzyme Succinate dehydrogenase (SDH) is a potential drug target because of its role in tricarboxylic acid cycle. Hence targeting Alternaria solani SDH enzyme could be efficient tool to design novel fungicides against A. solani. We employed computational methodologies to design new SDH inhibitors using homology modeling; pharmacophore modeling and structure based virtual screening protocol. The three dimensional SDH model showed good stereo-chemical and structural properties. Based on virtual screening results twelve commercially available compounds were purchased and tested in vitro and in vivo. The compounds were found to inhibit mycelial growth of A. solani. Moreover in vitro trials showed that inhibitory effects were enhanced with increase in concentrations. Similarly increased disease control was observed in pre-treated potato tubers. Hence the applied in silico strategy led us to identify new and novel fungicides.

Discovering Novel Alternaria solani Succinate Dehydrogenase Inhibitors by in Silico Modeling and Virtual Screening Strategies to Combat Early Blight

PubMed Central

Iftikhar, Sehrish; Shahid, Ahmad A.; Halim, Sobia A.; Wolters, Pieter J.; Vleeshouwers, Vivianne G. A. A.; Khan, Ajmal; Al-Harrasi, Ahmed; Ahmad, Shahbaz

2017-01-01

Alternaria blight is an important foliage disease caused by Alternaria solani. The enzyme Succinate dehydrogenase (SDH) is a potential drug target because of its role in tricarboxylic acid cycle. Hence targeting Alternaria solani SDH enzyme could be efficient tool to design novel fungicides against A. solani. We employed computational methodologies to design new SDH inhibitors using homology modeling; pharmacophore modeling and structure based virtual screening. The three dimensional SDH model showed good stereo-chemical and structural properties. Based on virtual screening results twelve commercially available compounds were purchased and tested in vitro and in vivo. The compounds were found to inhibit mycelial growth of A. solani. Moreover in vitro trials showed that inhibitory effects were enhanced with increase in concentrations. Similarly increased disease control was observed in pre-treated potato tubers. Hence the applied in silico strategy led us to identify novel fungicides. PMID:29204422

Synthesis of sterically encumbered 11β-aminoprogesterone derivatives and evaluation as 11β-hydroxysteroid dehydrogenase inhibitors and mineralocorticoid receptor antagonists.

PubMed

Pandya, Keyur; Dietrich, David; Seibert, Julia; Vederas, John C; Odermatt, Alex

2013-11-01

11β-Hydroxyprogesterone is a well-known nonselective inhibitor of 11β-hydroxysteroid dehydrogenase (11βHSD) types 1 and 2. It also activates the mineralocorticoid receptor (MR). Modulation of corticosteroid action by inhibition of 11βHSDs or blocking MR is currently under consideration for treatment of electrolyte disturbances, metabolic diseases and chronic inflammatory disorders. We established conditions to synthesize sterically demanding 11β-aminoprogesterone, which following subsequent nucleophilic or reductive amination, allowed extension of the amino group to prepare amino acid derivatives. Biological testing revealed that some of the 11β-aminoprogesterone derivatives selectively inhibit 11βHSD2. Moreover, two compounds that did not significantly inhibit 11βHSDs had antagonist properties on MR. The 11β-aminoprogesterone derivatives form a basis for the further development of improved modulators of corticosteroid action. Copyright © 2013 Elsevier Ltd. All rights reserved.

In vivo antitumor activity of 4-amino 4-methyl 2-pentyne 1-al, an inhibitor of aldehyde dehydrogenase.

PubMed

Quemener, V; Quash, G; Moulinoux, J P; Penlap, V; Ripoll, H; Havouis, R; Doutheau, A; Goré, J

1989-01-01

4-amino-4-methyl-2-pentyne-1-al (AMPAL), a new irreversible inhibitor of aldehyde dehydrogenase (ALDH) has been assayed for its in vitro and in vivo antitumor activity. In vitro, AMPAL inhibits the proliferation and the ALDH activity of L1210 and RBL5 cell lines. In vivo, AMPAL significantly increases the mean survival time of mice i.p. grafted with leukemia (L1210, P815, MBL2, EL4, RBL5 cell lines) or carcinoma cells (Krebs cell line), without haematopoetic toxicity. No carcinostatic effect was observed against the P388 leukemia and the 3LL Lewis lung carcinoma. A possible relationship between the ALDH isoenzyme activity of the tumor and its sensitivity to AMPAL is discussed in the light of previous reports concerning the role of aldehydes in cell growth control.

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

Ding, Y.S.

(9, 10-/sup 3/H/sub 2/)Z9-14:Ac was synthesized at high specific activity (/sup 3/H, 58 Ci/mmole) by partial tritiation of the corresponding alkyne and was converted to the labeled Z9-14:OH and Z9-14:Al to study tissue specificity of acetate esterase (E), alcohol oxidase (OX), and aldehyde dehydrogenase (ALDH) in male and female Heliothis virescens. Soluble and membrane-associated enzyme activities were determined by radio-TLC assays. Compounds of the tritium-labeled Z11-16 series were synthesized and their in vitro fates examined as well. In order to achieve an alternative approach in which (1) pheromone receptor proteins would be stoichiometrically and irreversibly modified, or (2) pheromone-catabolizing enzymesmore » are inactivated by tight-binding or irreversible inhibitors, we have designed analogues of pheromones of lepidopterous insect pests and assayed their biological activity in vitro and in vivo. Various fluorinated molecules such as acyl fluorides, fluoroolefins, 2-fluoro aldehydes, 2,2-difluoro aldehydes and trifluoromethyl ketones were synthesized. The synthesis of some other functional groups such as cyclopropanones, cyclopropanols, cyclopropyl carbinols, cyclopropyl aldehydes and Michael acceptors will also be discussed.« less

Purification and characterization of an N alpha-acetyltransferase from Saccharomyces cerevisiae.

PubMed

Lee, F J; Lin, L W; Smith, J A

1988-10-15

N alpha-Acetyltransferase, which catalyzes the transfer of an acetyl group from acetyl coenzyme A to the alpha-NH2 group of proteins and peptides, was isolated from Saccharomyces cerevisiae and demonstrated by protein sequence analysis to be NH2-terminally blocked. The enzyme was purified 4,600-fold to apparent homogeneity by successive purification steps using DEAE-Sepharose, hydroxylapatite, DE52 cellulose, and Affi-Gel blue. The Mr of the native enzyme was estimated to be 180,000 +/- 10,000 by gel filtration chromatography, and the Mr of each subunit was estimated to be 95,000 +/- 2,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme has a pH optimum near 9.0, and its pI is 4.3 as determined by chromatofocusing on Mono-P. The enzyme catalyzed the transfer of an acetyl group to various synthetic peptides, including human adrenocorticotropic hormone (ACTH) (1-24) and its [Phe2] analogue, yeast alcohol dehydrogenase I (1-24), yeast alcohol dehydrogenase II (1-24), and human superoxide dismutase (1-24). These peptides contain either Ser or Ala as NH2-terminal residues which together with Met are the most commonly acetylated NH2-terminal residues (Persson, B., Flinta, C., von Heijne, G., and Jornvall, H. (1985) Eur. J. Biochem. 152, 523-527). Yeast enolase, containing a free NH2-terminal Ala residue, is known not to be N alpha-acetylated in vivo (Chin, C. C. Q., Brewer, J. M., and Wold, F. (1981) J. Biol. Chem. 256, 1377-1384), and enolase (1-24), a synthetic peptide mimicking the protein's NH2 terminus, was not acetylated in vitro by yeast acetyltransferase. The enzyme did not catalyze the N alpha-acetylation of other synthetic peptides including ACTH(11-24), ACTH(7-38), ACTH(18-39), human beta-endorphin, yeast superoxide dismutase (1-24). Each of these peptides has an NH2-terminal residue which is rarely acetylated in proteins (Lys, Phe, Arg, Tyr, Val, respectively). Among a series of divalent cations, Cu2+ and Zn2+ were demonstrated to be the most potent inhibitors. The enzyme was inactivated by chemical modification with diethyl pyrocarbonate and N-bromosuccinimide.

Substrate specificity of sheep liver sorbitol dehydrogenase.

PubMed Central

Lindstad, R I; Köll, P; McKinley-McKee, J S

1998-01-01

The substrate specificity of sheep liver sorbitol dehydrogenase has been studied by steady-state kinetics over the range pH 7-10. Sorbitol dehydrogenase stereo-selectively catalyses the reversible NAD-linked oxidation of various polyols and other secondary alcohols into their corresponding ketones. The kinetic constants are given for various novel polyol substrates, including L-glucitol, L-mannitol, L-altritol, D-altritol, D-iditol and eight heptitols, as well as for many aliphatic and aromatic alcohols. The maximum velocities (kcat) and the substrate specificity-constants (kcat/Km) are positively correlated with increasing pH. The enzyme-catalysed reactions occur by a compulsory ordered kinetic mechanism with the coenzyme as the first, or leading, substrate. With many substrates, the rate-limiting step for the overall reaction is the enzyme-NADH product dissociation. However, with several substrates there is a transition to a mechanism with partial rate-limitation at the ternary complex level, especially at low pH. The kinetic data enable the elucidation of new empirical rules for the substrate specificity of sorbitol dehydrogenase. The specificity-constants for polyol oxidation vary as a function of substrate configuration with D-xylo> D-ribo > L-xylo > D-lyxo approximately L-arabino > D-arabino > L-lyxo. Catalytic activity with a polyol or an aromatic substrate and various 1-deoxy derivatives thereof varies with -CH2OH > -CH2NH2 > -CH2OCH3 approximately -CH3. The presence of a hydroxyl group at each of the remaining chiral centres of a polyol, apart from the reactive C2, is also nonessential for productive ternary complex formation and catalysis. A predominantly nonpolar enzymic epitope appears to constitute an important structural determinant for the substrate specificity of sorbitol dehydrogenase. The existence of two distinct substrate binding regions in the enzyme active site, along with that of the catalytic zinc, is suggested to account for the lack of stereospecificity at C2 in some polyols. PMID:9461546

Alcohol Selectivity in a Synthetic Thermophilic n-Butanol Pathway Is Driven by Biocatalytic and Thermostability Characteristics of Constituent Enzymes.

PubMed

Loder, Andrew J; Zeldes, Benjamin M; Garrison, G Dale; Lipscomb, Gina L; Adams, Michael W W; Kelly, Robert M

2015-10-01

n-Butanol is generated as a natural product of metabolism by several microorganisms, but almost all grow at mesophilic temperatures. A synthetic pathway for n-butanol production from acetyl coenzyme A (acetyl-CoA) that functioned at 70°C was assembled in vitro from enzymes recruited from thermophilic bacteria to inform efforts for engineering butanol production into thermophilic hosts. Recombinant versions of eight thermophilic enzymes (β-ketothiolase [Thl], 3-hydroxybutyryl-CoA dehydrogenase [Hbd], and 3-hydroxybutyryl-CoA dehydratase [Crt] from Caldanaerobacter subterraneus subsp. tengcongensis; trans-2-enoyl-CoA reductase [Ter] from Spirochaeta thermophila; bifunctional acetaldehyde dehydrogenase/alcohol dehydrogenase [AdhE] from Clostridium thermocellum; and AdhE, aldehyde dehydrogenase [Bad], and butanol dehydrogenase [Bdh] from Thermoanaerobacter sp. strain X514) were utilized to examine three possible pathways for n-butanol. These pathways differed in the two steps required to convert butyryl-CoA to n-butanol: Thl-Hbd-Crt-Ter-AdhE (C. thermocellum), Thl-Hbd-Crt-Ter-AdhE (Thermoanaerobacter X514), and Thl-Hbd-Crt-Ter-Bad-Bdh. n-Butanol was produced at 70°C, but with different amounts of ethanol as a coproduct, because of the broad substrate specificities of AdhE, Bad, and Bdh. A reaction kinetics model, validated via comparison to in vitro experiments, was used to determine relative enzyme ratios needed to maximize n-butanol production. By using large relative amounts of Thl and Hbd and small amounts of Bad and Bdh, >70% conversion to n-butanol was observed in vitro, but with a 60% decrease in the predicted pathway flux. With more-selective hypothetical versions of Bad and Bdh, >70% conversion to n-butanol is predicted, with a 19% increase in pathway flux. Thus, more-selective thermophilic versions of Bad, Bdh, and AdhE are needed to fully exploit biocatalytic n-butanol production at elevated temperatures. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Alcohol Selectivity in a Synthetic Thermophilic n-Butanol Pathway Is Driven by Biocatalytic and Thermostability Characteristics of Constituent Enzymes

PubMed Central

Loder, Andrew J.; Zeldes, Benjamin M.; Garrison, G. Dale; Lipscomb, Gina L.; Adams, Michael W. W.

2015-01-01

n-Butanol is generated as a natural product of metabolism by several microorganisms, but almost all grow at mesophilic temperatures. A synthetic pathway for n-butanol production from acetyl coenzyme A (acetyl-CoA) that functioned at 70°C was assembled in vitro from enzymes recruited from thermophilic bacteria to inform efforts for engineering butanol production into thermophilic hosts. Recombinant versions of eight thermophilic enzymes (β-ketothiolase [Thl], 3-hydroxybutyryl-CoA dehydrogenase [Hbd], and 3-hydroxybutyryl-CoA dehydratase [Crt] from Caldanaerobacter subterraneus subsp. tengcongensis; trans-2-enoyl-CoA reductase [Ter] from Spirochaeta thermophila; bifunctional acetaldehyde dehydrogenase/alcohol dehydrogenase [AdhE] from Clostridium thermocellum; and AdhE, aldehyde dehydrogenase [Bad], and butanol dehydrogenase [Bdh] from Thermoanaerobacter sp. strain X514) were utilized to examine three possible pathways for n-butanol. These pathways differed in the two steps required to convert butyryl-CoA to n-butanol: Thl-Hbd-Crt-Ter-AdhE (C. thermocellum), Thl-Hbd-Crt-Ter-AdhE (Thermoanaerobacter X514), and Thl-Hbd-Crt-Ter-Bad-Bdh. n-Butanol was produced at 70°C, but with different amounts of ethanol as a coproduct, because of the broad substrate specificities of AdhE, Bad, and Bdh. A reaction kinetics model, validated via comparison to in vitro experiments, was used to determine relative enzyme ratios needed to maximize n-butanol production. By using large relative amounts of Thl and Hbd and small amounts of Bad and Bdh, >70% conversion to n-butanol was observed in vitro, but with a 60% decrease in the predicted pathway flux. With more-selective hypothetical versions of Bad and Bdh, >70% conversion to n-butanol is predicted, with a 19% increase in pathway flux. Thus, more-selective thermophilic versions of Bad, Bdh, and AdhE are needed to fully exploit biocatalytic n-butanol production at elevated temperatures. PMID:26253677

Molecular and Biochemical Basis for Stress-Induced Accumulation of Free and Bound p-Coumaraldehyde in Cucumber1[W][OA

PubMed Central

Varbanova, Marina; Porter, Katie; Lu, Fachuang; Ralph, John; Hammerschmidt, Ray; Jones, A. Daniel; Day, Brad

2011-01-01

To elucidate the genetic and biochemical regulation of elicitor-induced p-coumaraldehyde accumulation in plants, we undertook a multifaceted approach to characterize the metabolic flux through the phenylpropanoid pathway via the characterization and chemical analysis of the metabolites in the p-coumaryl, coniferyl, and sinapyl alcohol branches of this pathway. Here, we report the identification and characterization of four cinnamyl alcohol dehydrogenases (CADs) from cucumber (Cucumis sativus) with low activity toward p-coumaraldehyde yet exhibiting significant activity toward other phenylpropanoid hydroxycinnamaldehydes. As part of this analysis, we identified and characterized the activity of a hydroxycinnamoyl-coenzyme A:shikimate hydroxycinnamoyl transferase (HCT) capable of utilizing shikimate and p-coumaroyl-coenzyme A to generate p-coumaroyl shikimate. Following pectinase treatment of cucumber, we observed the rapid accumulation of p-coumaraldehyde, likely the result of low aldehyde reductase activity (i.e. alcohol dehydrogenase in the reverse reaction) of CsCAD enzymes on p-coumaraldehyde. In parallel, we noted a concomitant reduction in the activity of CsHCT. Taken together, our findings support the hypothesis that the up-regulation of the phenylpropanoid pathway upon abiotic stress greatly enhances the overall p-coumaryl alcohol branch of the pathway. The data presented here point to a role for CsHCT (as well as, presumably, p-coumarate 3-hydroxylase) as a control point in the regulation of the coniferyl and sinapyl alcohol branches of this pathway. This mechanism represents a potentially evolutionarily conserved process to efficiently and quickly respond to biotic and abiotic stresses in cucurbit plants, resulting in the rapid lignification of affected tissues. PMID:21940999

Analysis of the enzymatic formation of citral in the glands of sweet basil.

PubMed

Iijima, Yoko; Wang, Guodong; Fridman, Eyal; Pichersky, Eran

2006-04-15

Basil glands of the Sweet Dani cultivar contain high levels of citral, a mixture of geranial and its cis-isomer neral, as well as low levels of geraniol and nerol. We have previously reported the identification of a cDNA from Sweet Dani that encodes an enzyme responsible for the formation of geraniol from geranyl diphosphate in the glands, and that these glands cannot synthesize nerol directly from geranyl diphosphate. Here, we report the identification of two basil cDNAs encoding NADP+-dependent dehydrogenases that can use geraniol as the substrate. One cDNA, designated CAD1, represents a gene whose expression is highly specific to gland cells of all three basil cultivars examined, regardless of their citral content, and encodes an enzyme with high sequence similarity to known cinnamyl alcohol dehydrogenases (CADs). The enzyme encoded by CAD1 reversibly oxidizes geraniol to produce geranial (which reversibly isomerizes to neral via keto-enol tautomerization) at half the efficiency compared with its activity with cinnamyl alcohol. CAD1 does not use nerol and neral as substrates. A second cDNA, designated GEDH1, encodes an enzyme with sequence similarity to CAD1 that is capable of reversibly oxidizing geraniol and nerol in equal efficiency, and prolonged incubation of geraniol with GEDH1 in vitro produces not only geranial and neral, but also nerol. GEDH1 is also active, although at a lower efficiency, with cinnamyl alcohol. However, GEDH1 is expressed at low levels in glands of all cultivars compared with its expression in leaves. These and additional data presented indicate that basil glands may contain additional dehydrogenases capable of oxidizing geraniol.

Identification of a Long-range Protein Network That Modulates Active Site Dynamics in Extremophilic Alcohol Dehydrogenases*

PubMed Central

Nagel, Zachary D.; Cun, Shujian; Klinman, Judith P.

2013-01-01

A tetrameric thermophilic alcohol dehydrogenase from Bacillus stearothermophilus (ht-ADH) has been mutated at an aromatic side chain in the active site (Trp-87). The ht-W87A mutation results in a loss of the Arrhenius break seen at 30 °C for the wild-type enzyme and an increase in cold lability that is attributed to destabilization of the active tetrameric form. Kinetic isotope effects (KIEs) are nearly temperature-independent over the experimental temperature range, and similar in magnitude to those measured above 30 °C for the wild-type enzyme. This suggests that the rigidification in the wild-type enzyme below 30 °C does not occur for ht-W87A. A mutation at the dimer-dimer interface in a thermolabile psychrophilic homologue of ht-ADH, ps-A25Y, leads to a more thermostable enzyme and a change in the rate-determining step at low temperature. The reciprocal mutation in ht-ADH, ht-Y25A, results in kinetic behavior similar to that of W87A. Collectively, the results indicate that flexibility at the active site is intimately connected to a subunit interaction 20 Å away. The convex Arrhenius curves previously reported for ht-ADH (Kohen, A., Cannio, R., Bartolucci, S., and Klinman, J. P. (1999) Nature 399, 496–499) are proposed to arise, at least in part, from a change in subunit interactions that rigidifies the substrate-binding domain below 30 °C, and impedes the ability of the enzyme to sample the catalytically relevant conformational landscape. These results implicate an evolutionarily conserved, long-range network of dynamical communication that controls C-H activation in the prokaryotic alcohol dehydrogenases. PMID:23525111

Environmental stresses of field growth allow cinnamyl alcohol dehydrogenase-deficient Nicotiana attenuata plants to compensate for their structural deficiencies.

PubMed

Kaur, Harleen; Shaker, Kamel; Heinzel, Nicolas; Ralph, John; Gális, Ivan; Baldwin, Ian T

2012-08-01

The organized lignocellulosic assemblies of cell walls provide the structural integrity required for the large statures of terrestrial plants. Silencing two CINNAMYL ALCOHOL DEHYDROGENASE (CAD) genes in Nicotiana attenuata produced plants (ir-CAD) with thin, red-pigmented stems, low CAD and sinapyl alcohol dehydrogenase activity, low lignin contents, and rubbery, structurally unstable stems when grown in the glasshouse (GH). However, when planted into their native desert habitat, ir-CAD plants produced robust stems that survived wind storms as well as the wild-type plants. Despite efficient silencing of NaCAD transcripts and enzymatic activity, field-grown ir-CAD plants had delayed and restricted spread of red stem pigmentation, a color change reflecting blocked lignification by CAD silencing, and attained wild-type-comparable total lignin contents. The rubbery GH phenotype was largely restored when field-grown ir-CAD plants were protected from wind, herbivore attack, and ultraviolet B exposure and grown in restricted rooting volumes; conversely, it was lost when ir-CAD plants were experimentally exposed to wind, ultraviolet B, and grown in large pots in growth chambers. Transcript and liquid chromatography-electrospray ionization-time-of-flight analysis revealed that these environmental stresses enhanced the accumulation of various phenylpropanoids in stems of field-grown plants; gas chromatography-mass spectrometry and nuclear magnetic resonance analysis revealed that the lignin of field-grown ir-CAD plants had GH-grown comparable levels of sinapaldehyde and syringaldehyde cross-linked into their lignins. Additionally, field-grown ir-CAD plants had short, thick stems with normal xylem element traits, which collectively enabled field-grown ir-CAD plants to compensate for the structural deficiencies associated with CAD silencing. Environmental stresses play an essential role in regulating lignin biosynthesis in lignin-deficient plants.

Environmental Stresses of Field Growth Allow Cinnamyl Alcohol Dehydrogenase-Deficient Nicotiana attenuata Plants to Compensate for their Structural Deficiencies1[C][W][OA

PubMed Central

Kaur, Harleen; Shaker, Kamel; Heinzel, Nicolas; Ralph, John; Gális, Ivan; Baldwin, Ian T.

2012-01-01

The organized lignocellulosic assemblies of cell walls provide the structural integrity required for the large statures of terrestrial plants. Silencing two CINNAMYL ALCOHOL DEHYDROGENASE (CAD) genes in Nicotiana attenuata produced plants (ir-CAD) with thin, red-pigmented stems, low CAD and sinapyl alcohol dehydrogenase activity, low lignin contents, and rubbery, structurally unstable stems when grown in the glasshouse (GH). However, when planted into their native desert habitat, ir-CAD plants produced robust stems that survived wind storms as well as the wild-type plants. Despite efficient silencing of NaCAD transcripts and enzymatic activity, field-grown ir-CAD plants had delayed and restricted spread of red stem pigmentation, a color change reflecting blocked lignification by CAD silencing, and attained wild-type-comparable total lignin contents. The rubbery GH phenotype was largely restored when field-grown ir-CAD plants were protected from wind, herbivore attack, and ultraviolet B exposure and grown in restricted rooting volumes; conversely, it was lost when ir-CAD plants were experimentally exposed to wind, ultraviolet B, and grown in large pots in growth chambers. Transcript and liquid chromatography-electrospray ionization-time-of-flight analysis revealed that these environmental stresses enhanced the accumulation of various phenylpropanoids in stems of field-grown plants; gas chromatography-mass spectrometry and nuclear magnetic resonance analysis revealed that the lignin of field-grown ir-CAD plants had GH-grown comparable levels of sinapaldehyde and syringaldehyde cross-linked into their lignins. Additionally, field-grown ir-CAD plants had short, thick stems with normal xylem element traits, which collectively enabled field-grown ir-CAD plants to compensate for the structural deficiencies associated with CAD silencing. Environmental stresses play an essential role in regulating lignin biosynthesis in lignin-deficient plants. PMID:22645069

Mitochondrial Impairment May Increase Cellular NAD(P)H: Resazurin Oxidoreductase Activity, Perturbing the NAD(P)H-Based Viability Assays.

PubMed

Aleshin, Vasily A; Artiukhov, Artem V; Oppermann, Henry; Kazantsev, Alexey V; Lukashev, Nikolay V; Bunik, Victoria I

2015-08-21

Cellular NAD(P)H-dependent oxidoreductase activity with artificial dyes (NAD(P)H-OR) is an indicator of viability, as the cellular redox state is important for biosynthesis and antioxidant defense. However, high NAD(P)H due to impaired mitochondrial oxidation, known as reductive stress, should increase NAD(P)H-OR yet perturb viability. To better understand this complex behavior, we assayed NAD(P)H-OR with resazurin (Alamar Blue) in glioblastoma cell lines U87 and T98G, treated with inhibitors of central metabolism, oxythiamin, and phosphonate analogs of 2-oxo acids. Targeting the thiamin diphosphate (ThDP)-dependent enzymes, the inhibitors are known to decrease the NAD(P)H production in the pentose phosphate shuttle and/or upon mitochondrial oxidation of 2-oxo acids. Nevertheless, the inhibitors elevated NAD(P)H-OR with resazurin in a time- and concentration-dependent manner, suggesting impaired NAD(P)H oxidation rather than increased viability. In particular, inhibition of the ThDP-dependent enzymes affects metabolism of malate, which mediates mitochondrial oxidation of cytosolic NAD(P)H. We showed that oxythiamin not only inhibited mitochondrial 2-oxo acid dehydrogenases, but also induced cell-specific changes in glutamate and malate dehydrogenases and/or malic enzyme. As a result, inhibition of the 2-oxo acid dehydrogenases compromises mitochondrial metabolism, with the dysregulated electron fluxes leading to increases in cellular NAD(P)H-OR. Perturbed mitochondrial oxidation of NAD(P)H may thus complicate the NAD(P)H-based viability assay.

Mitochondrial Impairment May Increase Cellular NAD(P)H: Resazurin Oxidoreductase Activity, Perturbing the NAD(P)H-Based Viability Assays

PubMed Central

Aleshin, Vasily A.; Artiukhov, Artem V.; Oppermann, Henry; Kazantsev, Alexey V.; Lukashev, Nikolay V.; Bunik, Victoria I.

2015-01-01

Cellular NAD(P)H-dependent oxidoreductase activity with artificial dyes (NAD(P)H-OR) is an indicator of viability, as the cellular redox state is important for biosynthesis and antioxidant defense. However, high NAD(P)H due to impaired mitochondrial oxidation, known as reductive stress, should increase NAD(P)H-OR yet perturb viability. To better understand this complex behavior, we assayed NAD(P)H-OR with resazurin (Alamar Blue) in glioblastoma cell lines U87 and T98G, treated with inhibitors of central metabolism, oxythiamin, and phosphonate analogs of 2-oxo acids. Targeting the thiamin diphosphate (ThDP)-dependent enzymes, the inhibitors are known to decrease the NAD(P)H production in the pentose phosphate shuttle and/or upon mitochondrial oxidation of 2-oxo acids. Nevertheless, the inhibitors elevated NAD(P)H-OR with resazurin in a time- and concentration-dependent manner, suggesting impaired NAD(P)H oxidation rather than increased viability. In particular, inhibition of the ThDP-dependent enzymes affects metabolism of malate, which mediates mitochondrial oxidation of cytosolic NAD(P)H. We showed that oxythiamin not only inhibited mitochondrial 2-oxo acid dehydrogenases, but also induced cell-specific changes in glutamate and malate dehydrogenases and/or malic enzyme. As a result, inhibition of the 2-oxo acid dehydrogenases compromises mitochondrial metabolism, with the dysregulated electron fluxes leading to increases in cellular NAD(P)H-OR. Perturbed mitochondrial oxidation of NAD(P)H may thus complicate the NAD(P)H-based viability assay. PMID:26308058

Abnormal lignin in a loblolly pine mutant.

PubMed

Ralph, J; MacKay, J J; Hatfield, R D; O'Malley, D M; Whetten, R W; Sederoff, R R

1997-07-11

Novel lignin is formed in a mutant loblolly pine (Pinus taeda L.) severely depleted in cinnamyl alcohol dehydrogenase (E.C. 1.1.1.195), which converts coniferaldehyde to coniferyl alcohol, the primary lignin precursor in pines. Dihydroconiferyl alcohol, a monomer not normally associated with the lignin biosynthetic pathway, is the major component of the mutant's lignin, accounting for approximately 30 percent (versus approximately 3 percent in normal pine) of the units. The level of aldehydes, including new 2-methoxybenzaldehydes, is also increased. The mutant pines grew normally indicating that, even within a species, extensive variations in lignin composition need not disrupt the essential functions of lignin.

The yeast ADH7 promoter enables gene expression under pronounced translation repression caused by the combined stress of vanillin, furfural, and 5-hydroxymethylfurfural.

PubMed

Ishida, Yoko; Nguyen, Trinh Thi My; Izawa, Shingo

2017-06-20

Lignocellulosic biomass conversion inhibitors such as vanillin, furfural, and 5-hydroxymethylfurfural (HMF) inhibit the growth of and fermentation by Saccharomyces cerevisiae. A high concentration of each fermentation inhibitor represses translation and increases non-translated mRNAs. We previously reported that the mRNAs of ADH7 and BDH2, which encode putative NADPH- and NADH-dependent alcohol dehydrogenases, respectively, were efficiently translated even with translation repression in response to severe vanillin stress. However, the combined effects of these fermentation inhibitors on the expression of ADH7 and BDH2 remain unclear. We herein demonstrated that exposure to a combined stress of vanillin, furfural, and HMF repressed translation. The protein synthesis of Adh7, but not Bdh2 was significantly induced under combined stress conditions, even though the mRNA levels of ADH7 and BDH2 were up-regulated. Additionally, adh7Δ cells were more sensitive to the combined stress than wild-type and bdh2Δ cells. These results suggest that induction of the ADH7 expression plays a role in the tolerance to the combined stress of vanillin, furfural, and HMF. Furthermore, we succeeded in improving yeast tolerance to the combined stress by controlling the expression of ALD6 with the ADH7 promoter. Our results demonstrate that the ADH7 promoter can overcome the pronounced translation repression caused by the combined stress of vanillin, furfural, and HMF, and also suggest a new gene engineering strategy to breed robust and optimized yeasts for bioethanol production from a lignocellulosic biomass. Copyright © 2017 Elsevier B.V. All rights reserved.

Role of αA-crystallin-derived αA66-80 peptide in guinea pig lens crystallin aggregation and insolubilization

PubMed Central

Raju, Murugesan; Mooney, Brian P.; Thakkar, Kavi M.; Giblin, Frank J.; Schey, Kevin L.; Sharma, K. Krishna

2015-01-01

Earlier we reported that low molecular weight (LMW) peptides accumulate in aging human lens tissue and that among the LMW peptides, the chaperone inhibitor peptide αA66-80, derived from α-crystallin protein, is one of the predominant peptides. We showed that in vitro αA66-80 induces protein aggregation. The current study was undertaken to determine whether LMW peptides are also present in guinea pig lens tissue subjected to hyperbaric oxygen (HBO) in vivo. The nuclear opacity induced by HBO in guinea pig lens is the closest animal model for studying age-related cataract formation in humans. A LMW peptide profile by mass spectrometry showed the presence of an increased amount of LMW peptides in HBO-treated guinea pig lenses compared to age-matched controls. Interestingly, the mass spectrometric data also showed that the chaperone inhibitor peptide αA66-80 accumulates in HBO-treated guinea pig lens. Following incubation of synthetic chaperone inhibitor peptide αA66-80 with α-crystallin from guinea pig lens extracts, we observed a decreased ability of α-crystallin to inhibit the amorphous aggregation of the target protein alcohol dehydrogenase and the formation of large light scattering aggregates, similar to those we have observed with human α-crystallin and αA66-80 peptide. Further, time-lapse recordings showed that a preformed complex of α-crystallin and αA66-80 attracted additional crystallin molecules to form even larger aggregates. These results demonstrate that LMW peptide–mediated cataract development in aged human lens and in HBO-induced lens opacity in the guinea pig may have common molecular pathways. PMID:25639202

Role of αA-crystallin-derived αA66-80 peptide in guinea pig lens crystallin aggregation and insolubilization.

PubMed

Raju, Murugesan; Mooney, Brian P; Thakkar, Kavi M; Giblin, Frank J; Schey, Kevin L; Sharma, K Krishna

2015-03-01

Earlier we reported that low molecular weight (LMW) peptides accumulate in aging human lens tissue and that among the LMW peptides, the chaperone inhibitor peptide αA66-80, derived from α-crystallin protein, is one of the predominant peptides. We showed that in vitro αA66-80 induces protein aggregation. The current study was undertaken to determine whether LMW peptides are also present in guinea pig lens tissue subjected to hyperbaric oxygen (HBO) in vivo. The nuclear opacity induced by HBO in guinea pig lens is the closest animal model for studying age-related cataract formation in humans. A LMW peptide profile by mass spectrometry showed the presence of an increased amount of LMW peptides in HBO-treated guinea pig lenses compared to age-matched controls. Interestingly, the mass spectrometric data also showed that the chaperone inhibitor peptide αA66-80 accumulates in HBO-treated guinea pig lens. Following incubation of synthetic chaperone inhibitor peptide αA66-80 with α-crystallin from guinea pig lens extracts, we observed a decreased ability of α-crystallin to inhibit the amorphous aggregation of the target protein alcohol dehydrogenase and the formation of large light scattering aggregates, similar to those we have observed with human α-crystallin and αA66-80 peptide. Further, time-lapse recordings showed that a preformed complex of α-crystallin and αA66-80 attracted additional crystallin molecules to form even larger aggregates. These results demonstrate that LMW peptide-mediated cataract development in aged human lens and in HBO-induced lens opacity in the guinea pig may have common molecular pathways. Copyright © 2015 Elsevier Ltd. All rights reserved.

Crystallization and preliminary X-ray study of a (2R,3R)-2,3-butanediol dehydrogenase from Bacillus coagulans 2-6

PubMed Central

Miao, Xiangzhi; Huang, Xianhui; Zhang, Guofang; Zhao, Xiufang; Zhu, Xianming; Dong, Hui

2013-01-01

(2R,3R)-2,3-Butanediol dehydrogenase (R,R-BDH) from Bacillus coagulans 2-6 is a zinc-dependent medium-chain alcohol dehydrogenase. Recombinant R,R-BDH with a His6 tag at the C-terminus was expressed in Escherichia coli BL21 (DE3) cells and purified by Ni2+-chelating affinity and size-exclusion chromatography. Crystals were grown by the hanging-drop vapour-diffusion method at 289 K. The crystallization condition consisted of 8%(v/v) Tacsimate pH 4.6, 18%(w/v) polyethylene glycol 3350. The crystal diffracted to 2.8 Å resolution in the orthorhombic space group P212121, with unit-cell parameters a = 88.35, b = 128.73, c = 131.03 Å. PMID:24100567

Crystallization and preliminary X-ray study of a (2R,3R)-2,3-butanediol dehydrogenase from Bacillus coagulans 2-6.

PubMed

Miao, Xiangzhi; Huang, Xianhui; Zhang, Guofang; Zhao, Xiufang; Zhu, Xianming; Dong, Hui

2013-10-01

(2R,3R)-2,3-Butanediol dehydrogenase (R,R-BDH) from Bacillus coagulans 2-6 is a zinc-dependent medium-chain alcohol dehydrogenase. Recombinant R,R-BDH with a His6 tag at the C-terminus was expressed in Escherichia coli BL21 (DE3) cells and purified by Ni2+-chelating affinity and size-exclusion chromatography. Crystals were grown by the hanging-drop vapour-diffusion method at 289 K. The crystallization condition consisted of 8%(v/v) Tacsimate pH 4.6, 18%(w/v) polyethylene glycol 3350. The crystal diffracted to 2.8 Å resolution in the orthorhombic space group P2₁2₁2₁, with unit-cell parameters a=88.35, b=128.73, c=131.03 Å.

Ethanol production in recombinant hosts

DOEpatents

Ingram, Lonnie O'Neal; Barbosa-Alleyne, Maria D.

2005-02-01

The subject invention concerns the transformation of Gram-positive bacteria with heterologous genes which confer upon these microbes the ability to produce ethanol as a fermentation product. Specifically exemplified is the transformation of bacteria with genes, obtainable from Zymomonas mobilis, which encode pyruvate decarboxylase and alcohol dehydrogenase.

Ethanol production in gram-positive microbes

DOEpatents

Ingram, Lonnie O'Neal; Barbosa-Alleyne, Maria D. F.

1999-01-01

The subject invention concerns the transformation of Gram-positive bacteria with heterologous genes which confer upon these microbes the ability to produce ethanol as a fermentation product. Specifically exemplified is the transformation of bacteria with genes, obtainable from Zymomonas mobilis, which encode pyruvate decarboxylase and alcohol dehydrogenase.

Ethanol production in Gram-positive microbes

DOEpatents

Ingram, Lonnie O'Neal; Barbosa-Alleyne, Maria D. F.

1996-01-01

The subject invention concerns the transformation of Gram-positive bacteria with heterologous genes which confer upon these microbes the ability to produce ethanol as a fermentation product. Specifically exemplified is the transformation of bacteria with genes, obtainable from Zymomonas mobilis, which encode pyruvate decarboxylase and alcohol dehydrogenase.

Ameliorating effects of Mango (Mangifera indica L.) fruit on plasma ethanol level in a mouse model assessed with 1H-NMR based metabolic profiling

PubMed Central

Kim, So-Hyun; K. Cho, Somi; Min, Tae-Sun; Kim, Yujin; Yang, Seung-Ok; Kim, Hee-Su; Hyun, Sun-Hee; Kim, Hana; Kim, Young-Suk; Choi, Hyung-Kyoon

2011-01-01

The ameliorating effects of Mango (Mangifera indica L.) flesh and peel samples on plasma ethanol level were investigated using a mouse model. Mango fruit samples remarkably decreased mouse plasma ethanol levels and increased the activities of alcohol dehydrogenase and acetaldehyde dehydrogenase. The 1H-NMR-based metabolomic technique was employed to investigate the differences in metabolic profiles of mango fruits, and mouse plasma samples fed with mango fruit samples. The partial least squares-discriminate analysis of 1H-NMR spectral data of mouse plasma demonstrated that there were clear separations among plasma samples from mice fed with buffer, mango flesh and peel. A loading plot demonstrated that metabolites from mango fruit, such as fructose and aspartate, might stimulate alcohol degradation enzymes. This study suggests that mango flesh and peel could be used as resources for functional foods intended to decrease plasma ethanol level after ethanol uptake. PMID:21562641

Mutant alcohol dehydrogenase leads to improved ethanol tolerance in Clostridium thermocellum

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

Brown, Steven D; Guss, Adam M; Karpinets, Tatiana V

2011-01-01

Clostridium thermocellum is a thermophilic, obligately anaerobic, Gram-positive bacterium that is a candidate microorganism for converting cellulosic biomass into ethanol through consolidated bioprocessing. Ethanol intolerance is an important metric in terms of process economics, and tolerance has often been described as a complex and likely multigenic trait for which complex gene interactions come into play. Here, we resequence the genome of an ethanol-tolerant mutant, show that the tolerant phenotype is primarily due to a mutated bifunctional acetaldehyde-CoA/alcohol dehydrogenase gene (adhE), hypothesize based on structural analysis that cofactor specificity may be affected, and confirm this hypothesis using enzyme assays. Biochemical assaysmore » confirm a complete loss of NADH-dependent activity with concomitant acquisition of NADPH-dependent activity, which likely affects electron flow in the mutant. The simplicity of the genetic basis for the ethanol-tolerant phenotype observed here informs rational engineering of mutant microbial strains for cellulosic ethanol production.« less

Nonsense mutations in the alcohol dehydrogenase gene of Drosophila melanogaster correlate with an abnormal 3' end processing of the corresponding pre-mRNA.

PubMed Central

Brogna, S

1999-01-01

From bacteria to mammals, mutations that generate premature termination codons have been shown to result in the reduction in the abundance of the corresponding mRNA. In mammalian cells, more often than not, the reduction happens while the RNA is still associated with the nucleus. Here, it is reported that mutations in the alcohol dehydrogenase gene (Adh) of Drosophila melanogaster that generate premature termination codons lead to reduced levels of cytoplasmic and nuclear mRNA. Unexpectedly, it has been found that the poly(A) tails of Adh mRNAs and pre-mRNAs that carry a premature termination codon are longer than in the wild-type transcript. The more 5' terminal the mutation is, the longer is the poly(A) tail of the transcript. These findings suggest that the integrity of the coding region may be required for accurate mRNA 3' end processing. PMID:10199572

Aldehyde dehydrogenase 1a1 mediates a GABA synthesis pathway in midbrain dopaminergic neurons.

PubMed

Kim, Jae-Ick; Ganesan, Subhashree; Luo, Sarah X; Wu, Yu-Wei; Park, Esther; Huang, Eric J; Chen, Lu; Ding, Jun B

2015-10-02

Midbrain dopamine neurons are an essential component of the basal ganglia circuitry, playing key roles in the control of fine movement and reward. Recently, it has been demonstrated that γ-aminobutyric acid (GABA), the chief inhibitory neurotransmitter, is co-released by dopamine neurons. Here, we show that GABA co-release in dopamine neurons does not use the conventional GABA-synthesizing enzymes, glutamate decarboxylases GAD65 and GAD67. Our experiments reveal an evolutionarily conserved GABA synthesis pathway mediated by aldehyde dehydrogenase 1a1 (ALDH1a1). Moreover, GABA co-release is modulated by ethanol (EtOH) at concentrations seen in blood alcohol after binge drinking, and diminished ALDH1a1 leads to enhanced alcohol consumption and preference. These findings provide insights into the functional role of GABA co-release in midbrain dopamine neurons, which may be essential for reward-based behavior and addiction. Copyright © 2015, American Association for the Advancement of Science.

A General Tool for Engineering the NAD/NADP Cofactor Preference of Oxidoreductases.

PubMed

Cahn, Jackson K B; Werlang, Caroline A; Baumschlager, Armin; Brinkmann-Chen, Sabine; Mayo, Stephen L; Arnold, Frances H

2017-02-17

The ability to control enzymatic nicotinamide cofactor utilization is critical for engineering efficient metabolic pathways. However, the complex interactions that determine cofactor-binding preference render this engineering particularly challenging. Physics-based models have been insufficiently accurate and blind directed evolution methods too inefficient to be widely adopted. Building on a comprehensive survey of previous studies and our own prior engineering successes, we present a structure-guided, semirational strategy for reversing enzymatic nicotinamide cofactor specificity. This heuristic-based approach leverages the diversity and sensitivity of catalytically productive cofactor binding geometries to limit the problem to an experimentally tractable scale. We demonstrate the efficacy of this strategy by inverting the cofactor specificity of four structurally diverse NADP-dependent enzymes: glyoxylate reductase, cinnamyl alcohol dehydrogenase, xylose reductase, and iron-containing alcohol dehydrogenase. The analytical components of this approach have been fully automated and are available in the form of an easy-to-use web tool: Cofactor Specificity Reversal-Structural Analysis and Library Design (CSR-SALAD).

Monoterpene alcohol metabolism: identification, purification, and characterization of two geraniol dehydrogenase isoenzymes from Polygonum minus leaves.

PubMed

Hassan, Maizom; Maarof, Nur Diyana; Ali, Zainon Mohd; Noor, Normah Mohd; Othman, Roohaida; Mori, Nobuhiro

2012-01-01

NADP(+)-dependent geraniol dehydrogenase (EC 1.1.1.183) is an enzyme that catalyzes the oxidation of geraniol to geranial. Stable, highly active cell-free extract was obtained from Polygonum minus leaves using polyvinylpolypyrrolidone, Amberlite XAD-4, glycerol, 2-mercaptoethanol, thiourea, and phenylmethylsulfonylfluoride in tricine-NaOH buffer (pH 7.5). The enzyme preparation was separated into two activity peaks, geraniol-DH I and II, by DEAE-Toyopearl 650M column chromatography at pH 7.5. Both isoenzymes were purified to homogeneity in three chromatographic steps. The geraniol-DH isoenzymes were similar in molecular mass, optimal temperature, and pH, but the isoelectric point, substrate specificity, and kinetic parameters were different. The K(m) values for geraniol of geraniol-DH I and II appeared to be 0.4 mM and 0.185 mM respectively. P. minus geraniol-DHs are unusual among geraniol-DHs in view of their thermal stability and optimal temperatures, and also their high specificity for allylic alcohols and NADP(+).

Purification and characterization of cinnamyl alcohol-NADPH-dehydrogenase from the leaf tissues of a basin mangrove Lumnitzera racemosa Willd.

PubMed

Murugan, K; Arunkumar, N S; Mohankumar, C

2004-01-01

Cinnamyl alcohol-NADPH-dehydrogenase (CAD), the marker enzyme of lignin biosynthesis was purified from the leaf tissues of a basin mangrove Lumnitzera racemosa by ammonium sulphate precipitation, followed by anion-exchange, gel filtration and affinity chromatography. The molecular mass of the CAD enzyme was determined as 89 kDa, by size elution chromatography. SDS-PAGE of CAD revealed two closely associated bands of 45 kDa and 42 kDa as heterogenous subunits. The optimum pH of CAD was found to be 4.0. Km for the substrates cinnamaldehyde, coniferaldehyde and sinapaldehyde was determined. Cinnamaldehyde showed higher Km value than sinapaldehyde and coniferaldehyde. The correlation of activity of CAD with the amount of lignin was found less significant in L. racemosa, compared to plant species of other habitats viz., mesophytes, xerophytes and hydrophytes, suggesting that CAD possibly exhibits physiological suppression due to the saline habitat of the plant.

Enzyme dynamics and hydrogen tunnelling in a thermophilic alcohol dehydrogenase

NASA Astrophysics Data System (ADS)

Kohen, Amnon; Cannio, Raffaele; Bartolucci, Simonetta; Klinman, Judith P.; Klinman, Judith P.

1999-06-01

Biological catalysts (enzymes) speed up reactions by many orders of magnitude using fundamental physical processes to increase chemical reactivity. Hydrogen tunnelling has increasingly been found to contribute to enzyme reactions at room temperature. Tunnelling is the phenomenon by which a particle transfers through a reaction barrier as a result of its wave-like property. In reactions involving small molecules, the relative importance of tunnelling increases as the temperature is reduced. We have now investigated whether hydrogen tunnelling occurs at elevated temperatures in a biological system that functions physiologically under such conditions. Using a thermophilic alcohol dehydrogenase (ADH), we find that hydrogen tunnelling makes a significant contribution at 65°C this is analogous to previous findings with mesophilic ADH at 25°C ( ref. 5). Contrary to predictions for tunnelling through a rigid barrier, the tunnelling with the thermophilic ADH decreases at and below room temperature. These findings provide experimental evidence for a role of thermally excited enzyme fluctuations in modulating enzyme-catalysed bond cleavage.

Nitric oxide inhibition of alcohol dehydrogenase in fresh-cut apples ( Malus domestica Borkh).

PubMed

Amissah, Joris Gerald Niilante; Hotchkiss, Joseph H; Watkins, Chris B

2013-11-20

The effects of nitric oxide (NO) and nitrite treatment on alcohol dehydrogenase activity and the shelf life of apple tissue were investigated. Fresh-cut apple slices were stored for 2 days at 6 °C in 0.25-1% NO (v/v, balance N2) or 100% N2 atmospheres. Slices were also treated with 1% NO or 2 mM sodium nitrite (NaNO2) for 20 min, stored for 6 weeks in 100% N2 at 6 °C, and analyzed for acetaldehyde, ethanol, and ethyl acetate accumulation, firmness, and color. Compared with N2 or deionized water controls, treatment with 1% NO or 2 mM NaNO2 inhibited ethanol accumulation, whereas that of acetaldehyde increased. Ethyl acetate accumulation was inhibited only by NO. Slice firmness was not affected by NO or NaNO2 treatment, but slices were darker than the untreated controls. NO and nitrite may extend the shelf life of fresh-cut produce with low concentrations of phenolic compounds.

Expression levels of chaperones influence biotransformation activity of recombinant Escherichia coli expressing Micrococcus luteus alcohol dehydrogenase and Pseudomonas putida Baeyer-Villiger monooxygenase.

PubMed

Baek, A-Hyong; Jeon, Eun-Yeong; Lee, Sun-Mee; Park, Jin-Byung

2015-05-01

We demonstrated for the first time that the archaeal chaperones (i.e., γ-prefoldin and thermosome) can stabilize enzyme activity in vivo. Ricinoleic acid biotransformation activity of recombinant Escherichia coli expressing Micrococcus luteus alcohol dehydrogenase and the Pseudomonas putida KT2440 Baeyer-Villiger monooxygenase improved significantly with co-expression of γ-prefoldin or recombinant themosome originating from the deep-sea hyperthermophile archaea Methanocaldococcus jannaschii. Furthermore, the degree of enhanced activity was dependent on the expression levels of the chaperones. For example, whole-cell biotransformation activity was highest at 12 µmol/g dry cells/min when γ-prefoldin expression level was approximately 46% of the theoretical maximum. This value was approximately two-fold greater than that in E. coli, where the γ-prefoldin expression level was zero or set to the theoretical maximum. Therefore, it was assumed that the expression levels of chaperones must be optimized to achieve maximum biotransformation activity in whole-cell biocatalysts. © 2014 Wiley Periodicals, Inc.

Inversion of substrate stereoselectivity of horse liver alcohol dehydrogenase by substitutions of Ser-48 and Phe-93.

PubMed

Kim, Keehyuk; Plapp, Bryce V

2017-10-01

The substrate specificities of alcohol dehydrogenases (ADH) are of continuing interest for understanding the physiological functions of these enzymes. Ser-48 and Phe-93 have been identified as important residues in the substrate binding sites of ADHs, but more comprehensive structural and kinetic studies are required. The S48T substitution in horse ADH1E has small effects on kinetic constants and catalytic efficiency (V/K m ) with ethanol, but decreases activity with benzyl alcohol and affinity for 2,2,2-trifluoroethanol (TFE) and 2,3,4,5,6-pentafluorobenzyl alcohol (PFB). Nevertheless, atomic resolution crystal structures of the S48T enzyme complexed with NAD + and TFE or PFB are very similar to the structures for the wild-type enzyme. (The S48A substitution greatly diminishes catalytic activity.) The F93A substitution significantly decreases catalytic efficiency (V/K m ) for ethanol and acetaldehyde while increasing activity for larger secondary alcohols and the enantioselectivity for the R-isomer relative to the S-isomer of 2-alcohols. The doubly substituted S48T/F93A enzyme has kinetic constants for primary and secondary alcohols similar to those for the F93A enzyme, but the effect of the S48T substitution is to decrease V/K m for (S)-2-alcohols without changing V/K m for (R)-2-alcohols. Thus, the S48T/F93A substitutions invert the enantioselectivity for alcohol oxidation, increasing the R/S ratio by 10, 590, and 200-fold for 2-butanol, 2-octanol, and sec-phenethyl alcohol, respectively. Transient kinetic studies and simulations of the ordered bi bi mechanism for the oxidation of the 2-butanols by the S48T/F93A ADH show that the rate of hydride transfer is increased about 7-fold for both isomers (relative to wild-type enzyme) and that the inversion of enantioselectivity is due to more productive binding for (R)-2-butanol than for (S)-2-butanol in the ternary complex. Molecular modeling suggests that both of the sec-phenethyl alcohols could bind to the enzyme and that dynamics must affect the rates of catalysis. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Acetylcholine promotes the emergence and elongation of lateral roots of Raphanus sativus

PubMed Central

Sugiyama, Kou-ichi

2011-01-01

Radish (Raphanus sativus L.) was grown on four layers of paper towel moistened with distilled water with and without acetylcholine (ACh) for five days in the dark after sowing. ACh at 1 nM promoted the growth (emergence and elongation) of lateral roots of radish plants, but had no effect on the stems and main roots. Moreover, ACh enhanced the dry weight of roots [main (primary) + lateral roots]. Neostigmine, an inhibitor of acetylcholinesterase (AChE) also promoted the emergence and elongation of lateral roots, and atropine, a competitive inhibitor of ACh receptor, suppressed the emergence and elongation. ACh promoted the activities of glyceraldehyde-3-phosephate dehydrogenase (G-3-PD), nicotinamide adenine dinucleotide-specific isocitrate dehydrogenase (NAD-ICDH), succinate dehydrogenase (SDH) and cytochrome-c oxidase (Cyt-c OD) in seedlings. Moreover, ACh suppressed the activity of AChE and increased the amount of proteins and pyridine nucleotides (NAD and NADH) in the roots of the seedlings. It also increased the activities of NAD-forming enzymes [NAD synthetase and ATP-nicotinamide mononucleotide (ATP-NMN) adenyltransferase], and enhanced the amount of DNA in the roots of the seedlings. The relationship between ACh and the emergence and growth of lateral roots was discussed from a biochemical viewpoint. PMID:21900743

Discovery of Clinical Candidate 2-((2S,6S)-2-Phenyl-6-hydroxyadamantan-2-yl)-1-(3'-hydroxyazetidin-1-yl)ethanone [BMS-816336], an Orally Active Novel Selective 11β-Hydroxysteroid Dehydrogenase Type 1 Inhibitor.

PubMed

Ye, Xiang-Yang; Chen, Stephanie Y; Wu, Shung; Yoon, David S; Wang, Haixia; Hong, Zhenqiu; O'Connor, Stephen P; Li, Jun; Li, James J; Kennedy, Lawrence J; Walker, Steven J; Nayeem, Akbar; Sheriff, Steven; Camac, Daniel M; Ramamurthy, Vidyhashankar; Morin, Paul E; Zebo, Rachel; Taylor, Joseph R; Morgan, Nathan N; Ponticiello, Randolph P; Harrity, Thomas; Apedo, Atsu; Golla, Rajasree; Seethala, Ramakrishna; Wang, Mengmeng; Harper, Timothy W; Sleczka, Bogdan G; He, Bin; Kirby, Mark; Leahy, David K; Li, Jianqing; Hanson, Ronald L; Guo, Zhiwei; Li, Yi-Xin; DiMarco, John D; Scaringe, Raymond; Maxwell, Brad; Moulin, Frederick; Barrish, Joel C; Gordon, David A; Robl, Jeffrey A

2017-06-22

BMS-816336 (6n-2), a hydroxy-substituted adamantyl acetamide, has been identified as a novel, potent inhibitor against human 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) enzyme (IC 50 3.0 nM) with >10000-fold selectivity over human 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2). 6n-2 exhibits a robust acute pharmacodynamic effect in cynomolgus monkeys (ED 50 0.12 mg/kg) and in DIO mice. It is orally bioavailable (%F ranges from 20 to 72% in preclinical species) and has a predicted pharmacokinetic profile of a high peak to trough ratio and short half-life in humans. This ADME profile met our selection criteria for once daily administration, targeting robust inhibition of 11β-HSD1 enzyme for the first 12 h period after dosing followed by an "inhibition holiday" so that the potential for hypothalamic-pituitary-adrenal (HPA) axis activation might be mitigated. 6n-2 was found to be well-tolerated in phase 1 clinical studies and represents a potential new treatment for type 2 diabetes, metabolic syndrome, and other human diseases modulated by glucocorticoid control.

Discovery of Clinical Candidate 2-((2 S,6 S)-2-Phenyl-6-hydroxyadamantan-2-yl)-1-(3'-hydroxyazetidin-1-yl)ethanone [BMS-816336], an Orally Active Novel Selective 11β-Hydroxysteroid Dehydrogenase Type 1 Inhibitor

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

Ye, Xiang-Yang; Chen, Stephanie Y.; Wu, Shung

BMS-816336 (6n-2), a hydroxy-substituted adamantyl acetamide, has been identified as a novel, potent inhibitor against human 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) enzyme (IC 50 3.0 nM) with >10000-fold selectivity over human 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2). 6n-2 exhibits a robust acute pharmacodynamic effect in cynomolgus monkeys (ED 50 0.12 mg/kg) and in DIO mice. It is orally bioavailable (%F ranges from 20 to 72% in preclinical species) and has a predicted pharmacokinetic profile of a high peak to trough ratio and short half-life in humans. This ADME profile met our selection criteria for once daily administration, targeting robust inhibition ofmore » 11β-HSD1 enzyme for the first 12 h period after dosing followed by an “inhibition holiday” so that the potential for hypothalamic–pituitary–adrenal (HPA) axis activation might be mitigated. 6n-2 was found to be well-tolerated in phase 1 clinical studies and represents a potential new treatment for type 2 diabetes, metabolic syndrome, and other human diseases modulated by glucocorticoid control.« less

Targeting 6-phosphogluconate dehydrogenase in the oxidative PPP sensitizes leukemia cells to antimalarial agent dihydroartemisinin.

PubMed

Elf, S; Lin, R; Xia, S; Pan, Y; Shan, C; Wu, S; Lonial, S; Gaddh, M; Arellano, M L; Khoury, H J; Khuri, F R; Lee, B H; Boggon, T J; Fan, J; Chen, J

2017-01-12

The oxidative pentose phosphate pathway (PPP) is crucial for cancer cell metabolism and tumor growth. We recently reported that targeting a key oxidative PPP enzyme, 6-phosphogluconate dehydrogenase (6PGD), using our novel small-molecule 6PGD inhibitors Physcion and its derivative S3, shows anticancer effects. Notably, humans with genetic deficiency of either 6PGD or another oxidative PPP enzyme, glucose-6-phosphate dehydrogenase, exhibit non-immune hemolytic anemia upon exposure to aspirin and various antimalarial drugs. Inspired by these clinical observations, we examined the anticancer potential of combined treatment with 6PGD inhibitors and antimalarial drugs. We found that stable knockdown of 6PGD sensitizes leukemia cells to antimalarial agent dihydroartemisinin (DHA). Combined treatment with DHA and Physcion activates AMP-activated protein kinase, leading to synergistic inhibition of human leukemia cell viability. Moreover, our combined therapy synergistically attenuates tumor growth in xenograft nude mice injected with human K562 leukemia cells and cell viability of primary leukemia cells from human patients, but shows minimal toxicity to normal hematopoietic cells in mice as well as red blood cells and mononucleocytes from healthy human donors. Our findings reveal the potential for combined therapy using optimized doses of Physcion and DHA as a novel antileukemia treatment without inducing hemolysis.

Role of malate dehydrogenase in facilitating lactate dehydrogenase to support the glycolysis pathway in tumors.

PubMed

Mansouri, Siavash; Shahriari, Ali; Kalantar, Hadi; Moini Zanjani, Taraneh; Haghi Karamallah, Mojtaba

2017-04-01

High aerobic glycolysis, as one of the hallmarks of cancer cells, requires nicotinamide adenine dinucleotide (NAD + ) as a vital co-factor, to guarantee the flow of glycolysis. Malate dehydrogenase (MDH), as an important enzyme in cancer metabolism, is a source of NAD + additional to lactate dehydrogenase (LDH). The current study aimed to elucidate the kinetic parameters of MDH in human breast cancer and evaluate its supportive role in the glycolysis pathway. The Michaelis-Menten constant (K m ) and maximum velocity (V max ) of MDH were determined in the crude extracts of human breast tumors and healthy tissue samples, which were obtained directly from the operating theatre. To assess the potential role of MDH in supporting glycolysis, the MDH activity was measured when the LDH activity was inhibited by different concentrations of oxamate, an inhibitor of LDH in breast cancer cell lines. The K m of cancerous MDH (C-MDH) was the same as the healthy MDH, although the V max of C-MDH was higher relative to the healthy MDH. Notably, the MDH activity was increased in the MDA-MB-231 cell line, which was treated with the LDH inhibitor (oxamate), but not in the MCF-7 cell line (P<0.05). The higher tendency of C-MDH for NAD + and malate generation in cancer cells is an effective approach for supporting glycolysis. Increasing MDH activity in the absence of LDH demonstrates the supportive role of MDH in glycolysis. Therefore, decreasing MDH activity and expression in a forward reaction may present as a valid molecular target to abolish its potential effect on tumor metabolism.

Expression of catalase, alcohol dehydrogenase, and malate dehydrogenase in rot grains upon fungicide use on maize hybrids grown at different spacings.

PubMed

Kluge, E R; Mendes, M C; Faria, M V; Santos, H O; Santos, L A; Sandini, I E

2017-04-20

In this study, we evaluated the fungicide effect on the incidence of rot grains and expression of catalase (CAT), alcohol dehydrogenase (ADH), and malate dehydrogenase (MDH) enzymes in commercial maize hybrids grown with conventional and reduced spacing in Guarapuava, PR, Brazil. The experiment was designed in random blocks with a 3 × 8-factorial scheme, totaling 24 treatments. The first factor constituted three levels, the first with foliar fungicide application [150.0 g/L trifloxystrobin (15.0%, w/v) + 175.0 g/L prothioconazole (17.5%, w/v)] at a dose of 0.4 L/ha at V8-stage eight expanded leaves and the second with an application of 0.5 L/ha at VT-tasseling and check (no fungicide application) stage. The second factor comprised eight maize hybrids that were divided into two groups, complex (AG 9045PRO, AG 8041PRO, DKB245PRO2, and 2B707PW) and susceptible (P 32R48H, DKB390PRO, P 30F53H, and P 30R50H), according to their reaction to the causative fungus, totaling 72 plots at each site in the crop of 2013/2014. The percentage of rot grains and the expression of CAT, ADH, and MDH were evaluated for each hybrid. The percentage of rot grains was influenced by the hybrid and fungicide used. The (trifloxystrobin + prothioconazole) reduced the incidence of rot grains, with relatively higher reduction in the hybrids considered susceptible. The higher expression of CAT enzyme was related to the higher incidence of rot grains because of grain deterioration, depending on the hybrids evaluated. A higher expression of ADH and MDH enzymes was observed in the maize hybrids belonging to the group considered tolerant.

Ethanol production in Gram-positive microbes

DOEpatents

Ingram, L.O.; Barbosa-Alleyne, M.D.F.

1999-06-29

The subject invention concerns the transformation of Gram-positive bacteria with heterologous genes which confer upon these microbes the ability to produce ethanol as a fermentation product. Specifically exemplified is the transformation of bacteria with genes, obtainable from Zymomonas mobilis, which encode pyruvate decarboxylase and alcohol dehydrogenase. 2 figs.

Oxidation Numbers in the Study of Metabolism

ERIC Educational Resources Information Center

Bentley, Ronald; Franzen, James; Chasteen, Thomas G.

2002-01-01

The calculation and use of oxidation numbers in the study of metabolic reactions are discussed for normal oxidations (alcohol dehydrogenase and the NAD+/NADH couple, propanediol dehydratase) and for enzymatic reactions with a "hidden" redox component (transamination, the coupled conversion of ethylamine to ethanol, and the biomethylation of…

Ethanol production in Gram-positive microbes

DOEpatents

Ingram, L.O.; Barbosa-Alleyne, M.D.F.

1996-01-09

The subject invention concerns the transformation of Gram-positive bacteria with heterologous genes which confer upon these microbes the ability to produce ethanol as a fermentation product. Specifically exemplified is the transformation of bacteria with genes, obtainable from Zymomonas mobilis, which encode pyruvate decarboxylase and alcohol dehydrogenase. 2 figs.

Integrating Horizontal Gene Transfer and Common Descent to Depict Evolution and Contrast It with “Common Design”1

PubMed Central

GUILLERMO PAZ-Y-MIÑO-C; ESPINOSA, AVELINA

2016-01-01

Horizontal gene transfer (HGT) and common descent interact in space and time. Because events of HGT co-occur with phylogenetic evolution, it is difficult to depict evolutionary patterns graphically. Tree-like representations of life’s diversification are useful, but they ignore the significance of HGT in evolutionary history, particularly of unicellular organisms, ancestors of multicellular life. Here we integrate the reticulated-tree model, ring of life, symbiogenesis whole-organism model, and eliminative pattern pluralism to represent evolution. Using Entamoeba histolytica alcohol dehydrogenase 2 (EhADH2), a bifunctional enzyme in the glycolytic pathway of amoeba, we illustrate how EhADH2 could be the product of both horizontally acquired features from ancestral prokaryotes (i.e. aldehyde dehydrogenase [ALDH] and alcohol dehydrogenase [ADH]), and subsequent functional integration of these enzymes into EhADH2, which is now inherited by amoeba via common descent. Natural selection has driven the evolution of EhADH2 active sites, which require specific amino acids (cysteine 252 in the ALDH domain; histidine 754 in the ADH domain), iron- and NAD+ as cofactors, and the substrates acetyl-CoA for ALDH and acetaldehyde for ADH. Alternative views invoking “common design” (i.e. the non-naturalistic emergence of major taxa independent from ancestry) to explain the interaction between horizontal and vertical evolution are unfounded. PMID:20021546

Molecular evolution and functional divergence of alcohol dehydrogenases in animals, fungi and plants.

PubMed

Thompson, Claudia E; Freitas, Loreta B; Salzano, Francisco M

2018-01-01

Alcohol dehydrogenases belong to the large superfamily of medium-chain dehydrogenases/reductases, which occur throughout the biological world and are involved with many important metabolic routes. We considered the phylogeny of 190 ADH sequences of animals, fungi, and plants. Non-class III Caenorhabditis elegans ADHs were seen closely related to tetrameric fungal ADHs. ADH3 forms a sister group to amphibian, reptilian, avian and mammalian non-class III ADHs. In fishes, two main forms are identified: ADH1 and ADH3, whereas in amphibians there is a new ADH form (ADH8). ADH2 is found in Mammalia and Aves, and they formed a monophyletic group. Additionally, mammalian ADH4 seems to result from an ADH1 duplication, while in Fungi, ADH formed clusters based on types and genera. The plant ADH isoforms constitute a basal clade in relation to ADHs from animals. We identified amino acid residues responsible for functional divergence between ADH types in fungi, mammals, and fishes. In mammals, these differences occur mainly between ADH1/ADH4 and ADH3/ADH5, whereas functional divergence occurred in fungi between ADH1/ADH5, ADH5/ADH4, and ADH5/ADH3. In fishes, the forms also seem to be functionally divergent. The ADH family expansion exemplifies a neofunctionalization process where reiterative duplication events are related to new activities.

Sortase A-mediated crosslinked short-chain dehydrogenases/reductases as novel biocatalysts with improved thermostability and catalytic efficiency.

PubMed

Li, Kunpeng; Zhang, Rongzhen; Xu, Yan; Wu, Zhimeng; Li, Jing; Zhou, Xiaotian; Jiang, Jiawei; Liu, Haiyan; Xiao, Rong

2017-06-08

(S)-carbonyl reductase II (SCRII) from Candida parapsilosis is a short-chain alcohol dehydrogenase/reductase. It catalyses the conversion of 2-hydroxyacetophenone to (S)-1-phenyl-1,2-ethanediol with low efficiency. Sortase was reported as a molecular "stapler" for site-specific protein conjugation to strengthen or add protein functionality. Here, we describe Staphylococcus aureus sortase A-mediated crosslinking of SCRII to produce stable catalysts for efficient biotransformation. Via a native N-terminal glycine and an added GGGGSLPETGG peptide at C-terminus of SCRII, SCRII subunits were conjugated by sortase A to form crosslinked SCRII, mainly dimers and trimers. The crosslinked SCRII showed over 6-fold and 4-fold increases, respectively, in activity and k cat /K m values toward 2-hydroxyacetophenone compared with wild-type SCRII. Moreover, crosslinked SCRII was much more thermostable with its denaturation temperature (T m ) increased to 60 °C. Biotransformation result showed that crosslinked SCRII gave a product optical purity of 100% and a yield of >99.9% within 3 h, a 16-fold decrease in transformation duration with respect to Escherichia coli/pET-SCRII. Sortase A-catalysed ligation also obviously improved T m s and product yields of eight other short-chain alcohol dehydrogenases/reductases. This work demonstrates a generic technology to improve enzyme function and thermostability through sortase A-mediated crosslinking of oxidoreductases.

The functional divergence of short-chain dehydrogenases involved in tropinone reduction.

PubMed

Brock, Andrea; Brandt, Wolfgang; Dräger, Birgit

2008-05-01

Tropane alkaloids typically occur in the Solanaceae and are also found in Cochlearia officinalis, a member of the Brassicaceae. Tropinone reductases are key enzymes of tropane alkaloid metabolism. Two different tropinone reductases form one stereoisomeric product each, either tropine for esterified alkaloids or pseudotropine that is converted to calystegines. A cDNA sequence with similarity to known tropinone reductases (TR) was cloned from C. officinalis. The protein was expressed in Escherichia coli, and found to catalyze the reduction of tropinone. The enzyme is a member of the short-chain dehydrogenase enzyme family and shows broad substrate specificity. Several synthetic ketones were accepted as substrates, with higher affinity and faster enzymatic turnover than observed for tropinone. C. officinalis TR produced both the isomeric alcohols tropine and pseudotropine from tropinone using NADPH + H(+) as co-substrate. Tropinone reductases of the Solanaceae, in contrast, are strictly stereospecific and form one tropane alcohol only. The Arabidopsis thaliana homologue of C. officinalis TR showed high sequence similarity, but did not reduce tropinone. A tyrosine residue was identified in the active site of C. officinalis TR that appeared responsible for binding and orientation of tropinone. Mutagenesis of the tyrosine residue yielded an active reductase, but with complete loss of TR activity. Thus C. officinalis TR presents an example of an enzyme with relaxed substrate specificity, like short-chain dehydrogenases, that provides favorable preconditions for the evolution of novel functions in biosynthetic sequences.

Structural Studies of Cinnamoyl-CoA Reductase and Cinnamyl-Alcohol Dehydrogenase, Key Enzymes of Monolignol Biosynthesis[C][W

PubMed Central

Pan, Haiyun; Zhou, Rui; Louie, Gordon V.; Mühlemann, Joëlle K.; Bomati, Erin K.; Bowman, Marianne E.; Dudareva, Natalia; Dixon, Richard A.; Noel, Joseph P.; Wang, Xiaoqiang

2014-01-01

The enzymes cinnamoyl-CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) catalyze the two key reduction reactions in the conversion of cinnamic acid derivatives into monolignol building blocks for lignin polymers in plant cell walls. Here, we describe detailed functional and structural analyses of CCRs from Medicago truncatula and Petunia hybrida and of an atypical CAD (CAD2) from M. truncatula. These enzymes are closely related members of the short-chain dehydrogenase/reductase (SDR) superfamily. Our structural studies support a reaction mechanism involving a canonical SDR catalytic triad in both CCR and CAD2 and an important role for an auxiliary cysteine unique to CCR. Site-directed mutants of CAD2 (Phe226Ala and Tyr136Phe) that enlarge the phenolic binding site result in a 4- to 10-fold increase in activity with sinapaldehyde, which in comparison to the smaller coumaraldehyde and coniferaldehyde substrates is disfavored by wild-type CAD2. This finding demonstrates the potential exploitation of rationally engineered forms of CCR and CAD2 for the targeted modification of monolignol composition in transgenic plants. Thermal denaturation measurements and structural comparisons of various liganded and unliganded forms of CCR and CAD2 highlight substantial conformational flexibility of these SDR enzymes, which plays an important role in the establishment of catalytically productive complexes of the enzymes with their NADPH and phenolic substrates. PMID:25217505

Inhibition of Cancer-Associated Mutant Isocitrate Dehydrogenases: Synthesis, Structure–Activity Relationship, and Selective Antitumor Activity

PubMed Central

2015-01-01

Mutations of isocitrate dehydrogenase 1 (IDH1) are frequently found in certain cancers such as glioma. Different from the wild-type (WT) IDH1, the mutant enzymes catalyze the reduction of α-ketoglutaric acid to d-2-hydroxyglutaric acid (D2HG), leading to cancer initiation. Several 1-hydroxypyridin-2-one compounds were identified to be inhibitors of IDH1(R132H). A total of 61 derivatives were synthesized, and their structure–activity relationships were investigated. Potent IDH1(R132H) inhibitors were identified with Ki values as low as 140 nM, while they possess weak or no activity against WT IDH1. Activities of selected compounds against IDH1(R132C) were found to be correlated with their inhibitory activities against IDH1(R132H), as well as cellular production of D2HG, with R2 of 0.83 and 0.73, respectively. Several inhibitors were found to be permeable through the blood–brain barrier in a cell-based model assay and exhibit potent and selective activity (EC50 = 0.26–1.8 μM) against glioma cells with the IDH1 R132H mutation. PMID:25271760

Alcohol cues impair learning inhibitory signals in beer drinkers

PubMed Central

Laude, Jennifer R.; Fillmore, Mark T.

2015-01-01

Background Models of drug addiction emphasize the reciprocal influence of incentive-motivational properties of drug-related cues and poor impulse control resulting in drug use. Recent studies have shown that alcohol-related cues can impair response inhibition. What is unknown is whether these cues also disrupt learning of inhibitory associations. Methods Participants performed a Conditioned Inhibition (CI) task and were required to learn that a neutral image was a conditioned inhibitor when presented in the context of either an alcohol image intended to draw their attention away from the to-be-trained inhibitor, or a control condition in which the alcohol image was absent. After training, subjects in each condition rated the likelihood that the neutral image would signal the outcome. Eye tracking was used to verify that attention to the neutral image was in fact reduced when the alcohol image was present. Results Compared with controls those trained in the alcohol image condition reported a greater likelihood that the presence of the inhibitor would be followed by the outcome and thus were less able to acquire CI. Measures of eye-tracking verified that attention to the alcohol cue was associated with this maladaptive behavior. Conclusions When alcohol cues are present, there is a reduced ability to learn that such information is irrelevant to an outcome, and this impairs ones’ ability to inhibit perseveration of a response. This has implications for persistence of a drinking episode. PMID:25872597

De Novo Sequencing and Analysis of Lemongrass Transcriptome Provide First Insights into the Essential Oil Biosynthesis of Aromatic Grasses.

PubMed

Meena, Seema; Kumar, Sarma R; Venkata Rao, D K; Dwivedi, Varun; Shilpashree, H B; Rastogi, Shubhra; Shasany, Ajit K; Nagegowda, Dinesh A

2016-01-01

Aromatic grasses of the genus Cymbopogon (Poaceae family) represent unique group of plants that produce diverse composition of monoterpene rich essential oils, which have great value in flavor, fragrance, cosmetic, and aromatherapy industries. Despite the commercial importance of these natural aromatic oils, their biosynthesis at the molecular level remains unexplored. As the first step toward understanding the essential oil biosynthesis, we performed de novo transcriptome assembly and analysis of C. flexuosus (lemongrass) by employing Illumina sequencing. Mining of transcriptome data and subsequent phylogenetic analysis led to identification of terpene synthases, pyrophosphatases, alcohol dehydrogenases, aldo-keto reductases, carotenoid cleavage dioxygenases, alcohol acetyltransferases, and aldehyde dehydrogenases, which are potentially involved in essential oil biosynthesis. Comparative essential oil profiling and mRNA expression analysis in three Cymbopogon species (C. flexuosus, aldehyde type; C. martinii, alcohol type; and C. winterianus, intermediate type) with varying essential oil composition indicated the involvement of identified candidate genes in the formation of alcohols, aldehydes, and acetates. Molecular modeling and docking further supported the role of identified protein sequences in aroma formation in Cymbopogon. Also, simple sequence repeats were found in the transcriptome with many linked to terpene pathway genes including the genes potentially involved in aroma biosynthesis. This work provides the first insights into the essential oil biosynthesis of aromatic grasses, and the identified candidate genes and markers can be a great resource for biotechnological and molecular breeding approaches to modulate the essential oil composition.

De Novo Sequencing and Analysis of Lemongrass Transcriptome Provide First Insights into the Essential Oil Biosynthesis of Aromatic Grasses

PubMed Central

Meena, Seema; Kumar, Sarma R.; Venkata Rao, D. K.; Dwivedi, Varun; Shilpashree, H. B.; Rastogi, Shubhra; Shasany, Ajit K.; Nagegowda, Dinesh A.

2016-01-01

Aromatic grasses of the genus Cymbopogon (Poaceae family) represent unique group of plants that produce diverse composition of monoterpene rich essential oils, which have great value in flavor, fragrance, cosmetic, and aromatherapy industries. Despite the commercial importance of these natural aromatic oils, their biosynthesis at the molecular level remains unexplored. As the first step toward understanding the essential oil biosynthesis, we performed de novo transcriptome assembly and analysis of C. flexuosus (lemongrass) by employing Illumina sequencing. Mining of transcriptome data and subsequent phylogenetic analysis led to identification of terpene synthases, pyrophosphatases, alcohol dehydrogenases, aldo-keto reductases, carotenoid cleavage dioxygenases, alcohol acetyltransferases, and aldehyde dehydrogenases, which are potentially involved in essential oil biosynthesis. Comparative essential oil profiling and mRNA expression analysis in three Cymbopogon species (C. flexuosus, aldehyde type; C. martinii, alcohol type; and C. winterianus, intermediate type) with varying essential oil composition indicated the involvement of identified candidate genes in the formation of alcohols, aldehydes, and acetates. Molecular modeling and docking further supported the role of identified protein sequences in aroma formation in Cymbopogon. Also, simple sequence repeats were found in the transcriptome with many linked to terpene pathway genes including the genes potentially involved in aroma biosynthesis. This work provides the first insights into the essential oil biosynthesis of aromatic grasses, and the identified candidate genes and markers can be a great resource for biotechnological and molecular breeding approaches to modulate the essential oil composition. PMID:27516768

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.

Identification of a new selective chemical inhibitor of mutant isocitrate dehydrogenase-1.

PubMed

Kim, Hyo-Joon; Choi, Bu Young; Keum, Young-Sam

2015-03-01

Recent genome-wide sequencing studies have identified unexpected genetic alterations in cancer. In particular, missense mutations in isocitrate dehydrogenase-1 (IDH1) at arginine 132, mostly substituted into histidine (IDH1-R132H) were observed to frequently occur in glioma patients. We have purified recombinant IDH1 and IDH1-R132H proteins and monitored their catalytic activities. In parallel experiments, we have attempted to find new selective IDH1-R132H chemical inhibitor(s) from a fragment-based chemical library. We have found that IDH1, but not IDH1-R132H, can catalyze the conversion of isocitrate into α-ketoglutarate (α-KG). In addition, we have observed that IDH1-R132H was more efficient than IDH1 in converting α-KG into (R)-2-hydroxyglutarate (R-2HG). Moreover, we have identified a new hit molecule, e.g., 2-(3-trifluoromethylphenyl)isothioazol-3(2H)-one as a new selective IDH1-R132H inhibitor. We have observed an underlying biochemical mechanism explaining how a heterozygous IDH1 mutation contributes to the generation of R-2HG and increases cellular histone H3 trimethylation levels. We have also identified a novel selective IDH1-R132H chemical hit molecule, e.g., 2-(3-trifluoromethylphenyl)isothioazol-3(2H)-one, which could be used for a future lead development against IDH1-R132H.

Binding Energy Calculation of Patchouli Alcohol Isomer Cyclooxygenase Complexes Suggested as COX-1/COX-2 Selective Inhibitor

PubMed Central

Mahdi, Chanif; Nurdiana, Nurdiana; Kikuchi, Takheshi; Fatchiyah, Fatchiyah

2014-01-01

To understand the structural features that dictate the selectivity of the two isoforms of the prostaglandin H2 synthase (PGHS/COX), the three-dimensional (3D) structure of COX-1/COX-2 was assessed by means of binding energy calculation of virtual molecular dynamic with using ligand alpha-Patchouli alcohol isomers. Molecular interaction studies with COX-1 and COX-2 were done using the molecular docking tools by Hex 8.0. Interactions were further visualized by using Discovery Studio Client 3.5 software tool. The binding energy of molecular interaction was calculated by AMBER12 and Virtual Molecular Dynamic 1.9.1 software. The analysis of the alpha-Patchouli alcohol isomer compounds showed that all alpha-Patchouli alcohol isomers were suggested as inhibitor of COX-1 and COX-2. Collectively, the scoring binding energy calculation (with PBSA Model Solvent) of alpha-Patchouli alcohol isomer compounds (CID442384, CID6432585, CID3080622, CID10955174, and CID56928117) was suggested as candidate for a selective COX-1 inhibitor and CID521903 as nonselective COX-1/COX-2. PMID:25484897

Cloning, sequencing, and expression of the gene coding for bile acid 7 alpha-hydroxysteroid dehydrogenase from Eubacterium sp. strain VPI 12708.

PubMed Central

Baron, S F; Franklund, C V; Hylemon, P B

1991-01-01

Southern blot analysis indicated that the gene encoding the constitutive, NADP-linked bile acid 7 alpha-hydroxysteroid dehydrogenase of Eubacterium sp. strain VPI 12708 was located on a 6.5-kb EcoRI fragment of the chromosomal DNA. This fragment was cloned into bacteriophage lambda gt11, and a 2.9-kb piece of this insert was subcloned into pUC19, yielding the recombinant plasmid pBH51. DNA sequence analysis of the 7 alpha-hydroxysteroid dehydrogenase gene in pBH51 revealed a 798-bp open reading frame, coding for a protein with a calculated molecular weight of 28,500. A putative promoter sequence and ribosome binding site were identified. The 7 alpha-hydroxysteroid dehydrogenase mRNA transcript in Eubacterium sp. strain VPI 12708 was about 0.94 kb in length, suggesting that it is monocistronic. An Escherichia coli DH5 alpha transformant harboring pBH51 had approximately 30-fold greater levels of 7 alpha-hydroxysteroid dehydrogenase mRNA, immunoreactive protein, and specific activity than Eubacterium sp. strain VPI 12708. The 7 alpha-hydroxysteroid dehydrogenase purified from the pBH51 transformant was similar in subunit molecular weight, specific activity, and kinetic properties to that from Eubacterium sp. strain VPI 12708, and it reached with antiserum raised against the authentic enzyme on Western immunoblots. Alignment of the amino acid sequence of the 7 alpha-hydroxysteroid dehydrogenase with those of 10 other pyridine nucleotide-linked alcohol/polyol dehydrogenases revealed six conserved amino acid residues in the N-terminal regions thought to function in coenzyme binding. Images PMID:1856160

Discovery of N-[2-[2-[[3-methoxy-4-(5-oxazolyl)phenyl]amino]-5-oxazolyl]phenyl]-N-methyl-4- morpholineacetamide as a novel and potent inhibitor of inosine monophosphate dehydrogenase with excellent in vivo activity.

PubMed

Dhar, T G Murali; Shen, Zhongqi; Guo, Junqing; Liu, Chunjian; Watterson, Scott H; Gu, Henry H; Pitts, William J; Fleener, Catherine A; Rouleau, Katherine A; Sherbina, N Z; McIntyre, Kim W; Shuster, David J; Witmer, Mark R; Tredup, Jeffrey A; Chen, Bang-Chi; Zhao, Rulin; Bednarz, Mark S; Cheney, Daniel L; MacMaster, John F; Miller, Laura M; Berry, Karen K; Harper, Timothy W; Barrish, Joel C; Hollenbaugh, Diane L; Iwanowicz, Edwin J

2002-05-23

Inosine monophosphate dehydrogenase (IMPDH) is a key enzyme that is involved in the de novo synthesis of purine nucleotides. Novel 2-aminooxazoles were synthesized and tested for inhibition of IMPDH catalytic activity. Multiple analogues based on this chemotype were found to inhibit IMPDH with low nanomolar potency. One of the analogues (compound 23) showed excellent in vivo activity in the inhibition of antibody production in mice and in the adjuvant induced arthritis model in rats.

Binding, hydration, and decarboxylation of the reaction intermediate glutaconyl-coenzyme A by human glutaryl-CoA dehydrogenase.

PubMed

Westover, J B; Goodman, S I; Frerman, F E

2001-11-20

Glutaconyl-coenzyme A (CoA) is the presumed enzyme-bound intermediate in the oxidative decarboxylation of glutaryl-CoA that is catalyzed by glutaryl-CoA dehydrogenase. We demonstrated glutaconyl-CoA bound to glutaryl-CoA dehydrogenase after anaerobic reduction of the dehydrogenase with glutaryl-CoA. Glutaryl-CoA dehydrogenase also has intrinsic enoyl-CoA hydratase activity, a property of other members of the acyl-CoA dehydrogenase family. The enzyme rapidly hydrates glutaconyl-CoA at pH 7.6 with a k(cat) of 2.7 s(-1). The k(cat) in the overall oxidation-decarboxylation reaction at pH 7.6 is about 9 s(-1). The binding of glutaconyl-CoA was quantitatively assessed from the K(m) in the hydratase reaction, 3 microM, and the K(i), 1.0 microM, as a competitive inhibitor of the dehydrogenase. These values compare with K(m) and K(i) of 4.0 and 12.9 microM, respectively, for crotonyl-CoA. Glu370 is the general base catalyst in the dehydrogenase that abstracts an alpha-proton of the substrate to initiate the catalytic pathway. The mutant dehydrogenase, Glu370Gln, is inactive in the dehydrogenation and the hydratase reactions. However, this mutant dehydrogenase decarboxylates glutaconyl-CoA to crotonyl-CoA without oxidation-reduction reactions of the dehydrogenase flavin. Addition of glutaconyl-CoA to this mutant dehydrogenase results in a rapid, transient increase in long-wavelength absorbance (lambda(max) approximately 725 nm), and crotonyl-CoA is found as the sole product. We propose that this 725 nm-absorbing species is the delocalized crotonyl-CoA anion that follows decarboxylation and that the decay is the result of slow protonation of the anion in the absence of the general acid catalyst, Glu370(H(+)). In the absence of detectable oxidation-reduction, the data indicate that oxidation-reduction of the dehydrogenase flavin is not essential for decarboxylation of glutaconyl-CoA.

Analysis of flavin oxidation and electron-transfer inhibition in Plasmodium falciparum dihydroorotate dehydrogenase.

PubMed

Malmquist, Nicholas A; Gujjar, Ramesh; Rathod, Pradipsinh K; Phillips, Margaret A

2008-02-26

Plasmodium falciparum dihydroorotate dehydrogenase (pfDHODH) is a flavin-dependent mitochondrial enzyme that provides the only route to pyrimidine biosynthesis in the parasite. Clinically significant inhibitors of human DHODH (e.g., A77 1726) bind to a pocket on the opposite face of the flavin cofactor from dihydroorotate (DHO). This pocket demonstrates considerable sequence variability, which has allowed species-specific inhibitors of the malarial enzyme to be identified. Ubiquinone (CoQ), the physiological oxidant in the reaction, has been postulated to bind this site despite a lack of structural evidence. To more clearly define the residues involved in CoQ binding and catalysis, we undertook site-directed mutagenesis of seven residues in the structurally defined A77 1726 binding site, which we term the species-selective inhibitor site. Mutation of several of these residues (H185, F188, and F227) to Ala substantially decreased the affinity of pfDHODH-specific inhibitors (40-240-fold). In contrast, only a modest increase in the Kmapp for CoQ was observed, although mutation of Y528 in particular caused a substantial reduction in kcat (40-100-fold decrease). Pre-steady-state kinetic analysis by single wavelength stopped-flow spectroscopy showed that the mutations had no effect on the rate of the DHO-dependent reductive half-reaction, but most reduced the rate of the CoQ-dependent flavin oxidation step (3-20-fold decrease), while not significantly altering the Kdox for CoQ. As with the mutants, inhibitors that bind this site block the CoQ-dependent oxidative half-reaction without affecting the DHO-dependent step. These results identify residues involved in inhibitor binding and electron transfer to CoQ. Importantly, the data provide compelling evidence that the binding sites for CoQ and species-selective site inhibitors do not overlap, and they suggest instead that inhibitors act either by blocking the electron path between flavin and CoQ or by stabilizing a conformation that excludes CoQ binding.

Kinetics of Alcohol Dehydrogenase-Catalyzed Oxidation of Ethanol Followed by Visible Spectroscopy

ERIC Educational Resources Information Center

Bendinskas, Kestutis; DiJiacomo, Christopher; Krill, Allison; Vitz, Ed

2005-01-01

The effect of substrate concentration on the rate of enzymatic reaction was investigated and typical Michaelis-Mentin kinetics was observed during the first week. The first order reaction at relatively low concentrations of ethanol and the pseudo zero-order reaction at high concentrations of ethanol were emphasized.

Meta-analysis of ADH1B and ALDH2 polymorphisms and esophageal cancer risk in China.

PubMed

Zhang, Guo-Hong; Mai, Rui-Qin; Huang, Bo

2010-12-21

To evaluate whether alcohol dehydrogenase-1B (ADH1B) His47Arg and aldehyde dehydrogenase-2 (ALDH2) Glu487Lys polymorphism is involved in the esophageal squamous cell carcinoma (ESCC) risk in Chinese Han population. Seven studies of ADH1B and ALDH2 genotypes in Chinese Han population in 1450 cases and 2459 controls were included for meta-analysis. Stratified analyses were carried out to determine the gene-alcohol and gene-gene interaction with ESCC risk. Potential sources of heterogeneity between studies were explored, and publication bias was also evaluated. Individuals with ADH1B arginine (Arg)/Arg genotype showed 3.95-fold increased ESCC risk in the recessive genetic model [Arg/Arg vs Arg/histidine (His) + His/His: odds ratio (OR) = 3.95, 95% confidence interval (CI): 2.76-5.67]. Significant association was found in the dominant model for ALDH2 lysine (Lys) allele [glutamate (Glu)/Lys + Lys/Lys vs Glu/Glu: OR = 2.00, 95% CI: 1.54-2.61]. Compared with the non-alcoholics, Arg/Arg (OR = 25.20, 95% CI: 10.87-53.44) and Glu/Lys + Lys/Lys (OR = 21.47, 95% CI: 6.44-71.59) were found to interact with alcohol drinking to increase the ESCC risk. ADH1B Arg+ and ALDH2 Lys+ had a higher risk for ESCC (OR = 7.09, 95% CI: 2.16-23.33). The genetic variations of ADH1B His47Arg and ALDH2 Glu487Lys are susceptible loci for ESCC in Chinese Han population and interact substantially with alcohol consumption. The individuals carrying both risky genotypes have a higher baseline risk of ESCC.

Candidate genes for alcohol dependence: A genetic association study from India.

PubMed

Malhotra, Savita; Basu, Debasish; Khullar, Madhu; Ghosh, Abhishek; Chugh, Neera

2016-11-01

Search for candidate genes for alcohol dependence (AD) has been inconsistent and inconclusive. Moreover, most of the research has been confined to a few specific ethnic groups. Hence, the aim of our study was to explore specific candidate genes for AD in north Indian male population. In this clinic-based genetic association study, 210 males with AD and 200 controls matched for age, gender and ethnicity were recruited from the clinic and the general population, respectively. Cases were diagnosed with Semi-structured Assessment for Genetics of Alcoholism-II (SSAGA-II). Single-nucleotide polymorphism genotyping was done by real-time quantitative-polymerase chain reaction (PCR) using Taq Man assay (ABI 7500) fast real-time PCR system. Both at the genotypic level and at allelic frequency, Met158 variant of catechol-O-methyl transferase (COMT) showed significant increase in cases as compared to controls. The frequency of heterozygous genotype (A/G) of gamma-aminobutyric acid receptor A1 (GABRA1) was significantly lower in cases as compared to controls. Likewise, for GABRA2, the frequency of homozygous recessive genotype (G/G) was significantly higher in the control group. With respect to the 5-hydroxytryptamine (5HT) transporter long promoter region (5HTTLPR), cholinergic receptor muscarinic (CHRM2) and alcohol dehydrogenase 1B (ADH1B) genes, there was no significant difference between the cases and the controls. Aldehyde dehydrogenase (ALDH2) gene was found to be monomorphic in our study population. Our study findings showed COMT polymorphism conferring risk and GABRA polymorphism as a protective genotype for Indian male with AD. Genes for alcohol metabolism, serotonin transporter and cholinergic receptor gene polymorphism were perhaps not contributory to AD for Indian population.

Optimization of benzoxazole-based inhibitors of Cryptosporidium parvum inosine 5′-monophosphate dehydrogenase

PubMed Central

Gorla, Suresh Kumar; Kavitha, Mandapati; Zhang, Minjia; Chin, James En Wai; Liu, Xiaoping; Striepen, Boris; Makowska-Grzyska, Magdalena; Kim, Youngchang; Joachimiak, Andrzej; Hedstrom, Lizbeth; Cuny, Gregory D.

2013-01-01

Cryptosporidium parvum is an enteric protozoan parasite that has emerged as a major cause of diarrhea, malnutrition and gastroenteritis as well as posing a potential bioterrorism threat. C. parvum synthesizes guanine nucleotides from host adenosine in a streamlined pathway that relies on inosine 5′-monophosphate dehydrogenase (IMPDH). We have previously identified several parasite-selective C. parvum IMPDH (CpIMPDH) inhibitors by high-throughput screening. In this paper, we report the structure-activity relationship (SAR) for a series of benzoxazole derivatives with many compounds demonstrating CpIMPDH IC50 values in the nanomolar range and > 500-fold selectivity over human IMPDH (hIMPDH). Unlike previously reported CpIMPDH inhibitors, these compounds are competitive inhibitors versus NAD+. The SAR study reveals that pyridine and other small heteroaromatic substituents are required at the 2-position of the benzoxazole for potent inhibitory activity. In addition, several other SAR conclusions are highlighted with regard to the benzoxazole and the amide portion of the inhibitor, including preferred stereochemistry. An x-ray crystal structure of a representative E•IMP•inhibitor complex is also presented. Overall, the secondary amine derivative 15a (Q67) demonstrated excellent CpIMPDH inhibitory activity (IC50 = 0.5 ± 0.1 nM) and moderate stability (t1/2 = 44 min) in mouse liver microsomes. Compound 73, the racemic version of 15a, also displayed superb antiparasitic activity in a Toxoplasma gondii strain that relies on CpIMPDH (EC50 = 20 ± 20 nM), and selectivity versus a wild-type T. gondii strain (200-fold). No toxicity was observed (LD50 > 50 μM) against a panel of four mammalian cells lines. PMID:23668331

[Carbohydrates metabolism disturbances when simulating prenatal alcohol intoxication].

PubMed

Kurch, N M; Vysokogorskiĭ, V E

2013-01-01

The influence of prenatal alcohol intoxication on carbohydrate metabolism markers has been investigated at different terms of postnatal offspring development (15, 30 and 60 days). Plasma glucose decreased as compared with the same in control group was detected. In the liver homogenates an increase of phosphorylase activity and a decrease of glucose-6-phosphatase, aldolase and glucose-6-phosphate dehydrogenase activities were found. These changes were accompanied by the incease in the lactate/pyruvate index attributed to increased lactate content in the liver tissue. The obtained data indicate essential disturbances of carbohydrate metabolism markers in prenatal alcoholized offspring, which include stable hypoglycemia, suppression of glycolytic and pentosephosphate pathways of glucose metabolism and lactate accumulation in the liver.

NADP(+)-dependent D-xylose dehydrogenase from pig liver. Purification and properties.

PubMed

Zepeda, S; Monasterio, O; Ureta, T

1990-03-15

An NADP(+)-dependent D-xylose dehydrogenase from pig liver cytosol was purified about 2000-fold to apparent homogeneity with a yield of 15% and specific activity of 6 units/mg of protein. An Mr value of 62,000 was obtained by gel filtration. PAGE in the presence of SDS gave an Mr value of 32,000, suggesting that the native enzyme is a dimer of similar or identical subunits. D-Xylose, D-ribose, L-arabinose, 2-deoxy-D-glucose, D-glucose and D-mannose were substrates in the presence of NADP+ but the specificity constant (ratio kcat./Km(app.)) is, by far, much higher for D-xylose than for the other sugars. The enzyme is specific for NADP+; NAD+ is not reduced in the presence of D-xylose or other sugars. Initial-velocity studies for the forward direction with xylose or NADP+ concentrations varied at fixed concentrations of the nucleotide or the sugar respectively revealed a pattern of parallel lines in double-reciprocal plots. Km values for D-xylose and NADP+ were 8.8 mM and 0.99 mM respectively. Dead-end inhibition studies to confirm a ping-pong mechanism showed that NAD+ acted as an uncompetitive inhibitor versus NADP+ (Ki 5.8 mM) and as a competitive inhibitor versus xylose. D-Lyxose was a competitive inhibitor versus xylose and uncompetitive versus NADP+. These results fit better to a sequential compulsory ordered mechanism with NADP+ as the first substrate, but a ping-pong mechanism with xylose as the first substrate has not been ruled out. The presence of D-xylose dehydrogenase suggests that in mammalian liver D-xylose is utilized by a pathway other than the pentose phosphate pathway.

Functional characterization of SlscADH1, a fruit-ripening-associated short-chain alcohol dehydrogenase of tomato.

PubMed

Moummou, Hanane; Tonfack, Libert Brice; Chervin, Christian; Benichou, Mohamed; Youmbi, Emmanuel; Ginies, Christian; Latché, Alain; Pech, Jean-Claude; van der Rest, Benoît

2012-10-15

A tomato short-chain dehydrogenase-reductase (SlscADH1) is preferentially expressed in fruit with a maximum expression at the breaker stage while expression in roots, stems, leaves and flowers is very weak. It represents a potential candidate for the formation of aroma volatiles by interconverting alcohols and aldehydes. The SlscADH1 recombinant protein produced in Escherichia coli exhibited dehydrogenase-reductase activity towards several volatile compounds present in tomato flavour with a strong preference for the NAD/NADH co-factors. The strongest activity was observed for the reduction of hexanal (K(m)=0.175mM) and phenylacetaldehyde (K(m)=0.375mM) in the presence of NADH. The oxidation process of hexanol and 1-phenylethanol was much less efficient (K(m)s of 2.9 and 23.0mM, respectively), indicating that the enzyme preferentially acts as a reductase. However activity was observed only for hexanal, phenylacetaldehyde, (E)-2-hexenal and acetaldehyde and the corresponding alcohols. No activity could be detected for other aroma volatiles important for tomato flavour, such as methyl-butanol/methyl-butanal, 5-methyl-6-hepten-2-one/5-methyl-6-hepten-2-ol, citronellal/citronellol, neral/nerol, geraniol. In order to assess the function of the SlscADH1 gene, transgenic plants have been generated using the technique of RNA interference (RNAi). Constitutive down-regulation using the 35S promoter resulted in the generation of dwarf plants, indicating that the SlscADH1 gene, although weakly expressed in vegetative tissues, had a function in regulating plant development. Fruit-specific down-regulation using the 2A11 promoter had no morphogenetic effect and did not alter the aldehyde/alcohol balance of the volatiles compounds produced by the fruit. Nevertheless, SlscADH1-inhibited fruit unexpectedly accumulated higher concentrations of C5 and C6 volatile compounds of the lipoxygenase pathway, possibly as an indirect effect of the suppression of SlscADH1 on the catabolism of phospholipids and/or integrity of membranes. Copyright © 2012 Elsevier GmbH. All rights reserved.

Crystallization and preliminary X-ray analysis of binary and ternary complexes of Haloferax mediterranei glucose dehydrogenase

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

Esclapez, Julia; Britton, K. Linda; Baker, Patrick J.

2005-08-01

Single crystals of binary and ternary complexes of wild-type and D38C mutant H. mediterranei glucose dehydrogenase have been obtained by the hanging-drop vapour-diffusion method. Haloferax mediterranei glucose dehydrogenase (EC 1.1.1.47) belongs to the medium-chain alcohol dehydrogenase superfamily and requires zinc for catalysis. In the majority of these family members, the catalytic zinc is tetrahedrally coordinated by the side chains of a cysteine, a histidine, a cysteine or glutamate and a water molecule. In H. mediterranei glucose dehydrogenase, sequence analysis indicates that the zinc coordination is different, with the invariant cysteine replaced by an aspartate residue. In order to analyse themore » significance of this replacement and to contribute to an understanding of the role of the metal ion in catalysis, a range of binary and ternary complexes of the wild-type and a D38C mutant protein have been crystallized. For most of the complexes, crystals belonging to space group I222 were obtained using sodium/potassium citrate as a precipitant. However, for the binary and non-productive ternary complexes with NADPH/Zn, it was necessary to replace the citrate with 2-methyl-2,4-pentanediol. Despite the radical change in conditions, the crystals thus formed were isomorphous.« less

[How aliphatic alcohols and ph affect reactional capability of the horse blood serum cholinesterase at its interaction with organophosphorus inhibitors].

PubMed

Basova, N E; Kormilitsin, B N; Perchenok, A Iu; Rozengart, E V; Saakov, V S; Suvorov, A A

2013-01-01

There was studied action of aliphatic alcohols (ethanol, propanol, isopropanol, n-butanol, isobutanol, secbutanol, tretbetanol) and pH on various kinds of reactional capability the serum cholinesterase. At the alcohols-affected inhibition of the cholinesterase hydrolytic activity, the determining role was played not the total number carbon atoms in the alcohol molecule, but by the "effective length" of the carbohydrate chain. The fact that the presence of alcohols did not affect parameters of the reverse cholinesterase inhibition with onium ions tetramethylammonium and choline allows suggesting the absence of effect solvents on specific acetylcholine sorption in the enzyme active center. With aid of two rows of hydrophobic organophosphorus inhibitors (OPI), we have managed to estimate both the degree and the character itself of the modifying action of alcohols and pH on the process of irreversible inhibition of serum cholinesterase.

Molecular Modeling Studies of 11β-Hydroxysteroid Dehydrogenase Type 1 Inhibitors through Receptor-Based 3D-QSAR and Molecular Dynamics Simulations.

PubMed

Qian, Haiyan; Chen, Jiongjiong; Pan, Youlu; Chen, Jianzhong

2016-09-19

11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) is a potential target for the treatment of numerous human disorders, such as diabetes, obesity, and metabolic syndrome. In this work, molecular modeling studies combining molecular docking, 3D-QSAR, MESP, MD simulations and free energy calculations were performed on pyridine amides and 1,2,4-triazolopyridines as 11β-HSD1 inhibitors to explore structure-activity relationships and structural requirement for the inhibitory activity. 3D-QSAR models, including CoMFA and CoMSIA, were developed from the conformations obtained by docking strategy. The derived pharmacophoric features were further supported by MESP and Mulliken charge analyses using density functional theory. In addition, MD simulations and free energy calculations were employed to determine the detailed binding process and to compare the binding modes of inhibitors with different bioactivities. The binding free energies calculated by MM/PBSA showed a good correlation with the experimental biological activities. Free energy analyses and per-residue energy decomposition indicated the van der Waals interaction would be the major driving force for the interactions between an inhibitor and 11β-HSD1. These unified results may provide that hydrogen bond interactions with Ser170 and Tyr183 are favorable for enhancing activity. Thr124, Ser170, Tyr177, Tyr183, Val227, and Val231 are the key amino acid residues in the binding pocket. The obtained results are expected to be valuable for the rational design of novel potent 11β-HSD1 inhibitors.

Selective Inhibition of Mutant Isocitrate Dehydrogenase 1 (IDH1) via Disruption of a Metal Binding Network by an Allosteric Small Molecule

PubMed Central

Deng, Gejing; Shen, Junqing; Yin, Ming; McManus, Jessica; Mathieu, Magali; Gee, Patricia; He, Timothy; Shi, Chaomei; Bedel, Olivier; McLean, Larry R.; Le-Strat, Frank; Zhang, Ying; Marquette, Jean-Pierre; Gao, Qiang; Zhang, Bailin; Rak, Alexey; Hoffmann, Dietmar; Rooney, Eamonn; Vassort, Aurelie; Englaro, Walter; Li, Yi; Patel, Vinod; Adrian, Francisco; Gross, Stefan; Wiederschain, Dmitri; Cheng, Hong; Licht, Stuart

2015-01-01

Cancer-associated point mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) confer a neomorphic enzymatic activity: the reduction of α-ketoglutarate to d-2-hydroxyglutaric acid, which is proposed to act as an oncogenic metabolite by inducing hypermethylation of histones and DNA. Although selective inhibitors of mutant IDH1 and IDH2 have been identified and are currently under investigation as potential cancer therapeutics, the mechanistic basis for their selectivity is not yet well understood. A high throughput screen for selective inhibitors of IDH1 bearing the oncogenic mutation R132H identified compound 1, a bis-imidazole phenol that inhibits d-2-hydroxyglutaric acid production in cells. We investigated the mode of inhibition of compound 1 and a previously published IDH1 mutant inhibitor with a different chemical scaffold. Steady-state kinetics and biophysical studies show that both of these compounds selectively inhibit mutant IDH1 by binding to an allosteric site and that inhibition is competitive with respect to Mg2+. A crystal structure of compound 1 complexed with R132H IDH1 indicates that the inhibitor binds at the dimer interface and makes direct contact with a residue involved in binding of the catalytically essential divalent cation. These results show that targeting a divalent cation binding residue can enable selective inhibition of mutant IDH1 and suggest that differences in magnesium binding between wild-type and mutant enzymes may contribute to the inhibitors' selectivity for the mutant enzyme. PMID:25391653

Structure-based design and mechanisms of allosteric inhibitors for mitochondrial branched-chain α-ketoacid dehydrogenase kinase

PubMed Central

Qi, Xiangbing; Gui, Wen-Jun; Morlock, Lorraine K.; Wallace, Amy L.; Ahmed, Kamran; Laxman, Sunil; Campeau, Philippe M.; Lee, Brendan H.; Hutson, Susan M.; Tu, Benjamin P.; Williams, Noelle S.; Tambar, Uttam K.; Wynn, R. Max; Chuang, David T.

2013-01-01

The branched-chain amino acids (BCAAs) leucine, isoleucine, and valine are elevated in maple syrup urine disease, heart failure, obesity, and type 2 diabetes. BCAA homeostasis is controlled by the mitochondrial branched-chain α-ketoacid dehydrogenase complex (BCKDC), which is negatively regulated by the specific BCKD kinase (BDK). Here, we used structure-based design to develop a BDK inhibitor, (S)-α-chloro-phenylpropionic acid [(S)-CPP]. Crystal structures of the BDK-(S)-CPP complex show that (S)-CPP binds to a unique allosteric site in the N-terminal domain, triggering helix movements in BDK. These conformational changes are communicated to the lipoyl-binding pocket, which nullifies BDK activity by blocking its binding to the BCKDC core. Administration of (S)-CPP to mice leads to the full activation and dephosphorylation of BCKDC with significant reduction in plasma BCAA concentrations. The results buttress the concept of targeting mitochondrial BDK as a pharmacological approach to mitigate BCAA accumulation in metabolic diseases and heart failure. PMID:23716694

Isocitrate dehydrogenase mutations confer dasatinib hypersensitivity and SRC-dependence in intrahepatic cholangiocarcinoma

PubMed Central

Saha, Supriya K.; Gordan, John D.; Kleinstiver, Benjamin P.; Vu, Phuong; Najem, Mortada S.; Yeo, Jia-Chi; Shi, Lei; Kato, Yasutaka; Levin, Rebecca S.; Webber, James T.; Damon, Leah J.; Egan, Regina K.; Greninger, Patricia; McDermott, Ultan; Garnett, Mathew J.; Jenkins, Roger L.; Rieger-Christ, Kimberly M.; Sullivan, Travis B.; Hezel, Aram F.; Liss, Andrew S.; Mizukami, Yusuke; Goyal, Lipika; Ferrone, Cristina R.; Zhu, Andrew X.; Joung, J. Keith; Shokat, Kevan M.; Benes, Cyril H.; Bardeesy, Nabeel

2017-01-01

Intrahepatic cholangiocarcinoma (ICC) is an aggressive liver bile duct malignancy exhibiting frequent isocitrate dehydrogenase (IDH1/IDH2) mutations. Through a high-throughput drug screen of a large panel of cancer cell lines including 17 biliary tract cancers, we found that IDH mutant (IDHm) ICC cells demonstrate a striking response to the multi-kinase inhibitor dasatinib, with the highest sensitivity among 682 solid tumor cell lines. Using unbiased proteomics to capture the activated kinome and CRISPR/Cas9-based genome editing to introduce dasatinib-resistant ‘gatekeeper’ mutant kinases, we identified SRC as a critical dasatinib target in IDHm ICC. Importantly, dasatinib-treated IDHm xenografts exhibited pronounced apoptosis and tumor regression. Our results show that IDHm ICC cells have a unique dependency on SRC and suggest that dasatinib may have therapeutic benefit against IDHm ICC. Moreover, these proteomic and genome-editing strategies provide a systematic and broadly applicable approach to define targets of kinase inhibitors underlying drug responsiveness. PMID:27231123

Inhibitory effects of Aphanizomenon flos-aquae constituents on human UDP-glucose dehydrogenase activity.

PubMed

Scoglio, Stefano; Lo Curcio, Valeria; Catalani, Simona; Palma, Francesco; Battistelli, Serafina; Benedetti, Serena

2016-12-01

The purpose of this study was to investigate the in vitro inhibitory effects of the edible microalga Aphanizomenon flos-aquae (AFA) on human UDP-α-d-glucose 6-dehydrogenase (UGDH) activity, a cytosolic enzyme involved both in tumor progression and in phytochemical bioavailability. Both the hydrophilic and ethanolic AFA extracts as well as the constitutive active principles phycocyanin (PC), phycocyanobilin (PCB) and mycosporine-like amino acids (MAAs) were tested. Among AFA components, PCB presented the strongest inhibitory effect on UGDH activity, acting as a competitive inhibitor with respect to UDP-glucose and a non-competitive inhibitor with respect to NAD(+). In preliminary experiments, AFA PCB was also effective in reducing the colony formation capacity of PC-3 prostate cancer cells and FTC-133 thyroid cancer cells. Overall, these findings confirmed that AFA and its active principles are natural compounds with high biological activity. Further studies evaluating the effects of AFA PCB in reducing tumor cell growth and phytochemical glucuronidation are encouraged.

Metal organic frameworks for enzyme immobilization in biofuel cells

NASA Astrophysics Data System (ADS)

Bodell, JaDee

Interest in biofuel cells has been rapidly expanding as an ever-growing segment of the population gains access to electronic devices. The largest areas of growth for new populations using electronic devices are often in communities without electrical infrastructure. This lack of infrastructure in remote environments is one of the key driving factors behind the development of biofuel cells. Biofuel cells employ biological catalysts such as enzymes to catalyze oxidation and reduction reactions of select fuels to generate power. There are several benefits to using enzymes to catalyze reactions as compared to traditional fuel cells which use metal catalysts. First, enzymes are able to catalyze reactions at or near room temperature, whereas traditional metal catalysts are only efficient at very high temperatures. Second, biofuel cells can operate under mild pH conditions which is important for the eventual design of safe, commercially viable devices. Also, biofuel cells allow for implantable and flexible technologies. Finally, enzymes exhibit high selectivity and can be combined to fully oxidize or reduce the fuel which can generate several electrons from a single molecule of fuel, increasing the overall device efficiency. One of the main challenges which persist in biofuel cells is the instability of enzymes over time which tend to denature after hours or days. For a viable commercial biofuel cell to be produced, the stability of enzymes must be extended to months or years. Enzymes have been shown to have improved stability after being immobilized. The focus of this research was to find a metal organic framework (MOF) structure which could successfully immobilize enzymes while still allowing for electron transport to occur between the catalytic center of the enzyme and the electrode surface within a biofuel cell for power generation. Four MOF structures were successfully synthesized and were subsequently tested to determine the MOF's ability to immobilize the following enzymes: nicotinamide adenine dinucleotide (NAD)-dependent alcohol and aldehyde dehydrogenases, and pyrroloquinoline quinone (PQQ)-dependent alcohol and aldehyde dehydrogenases, as well as flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase. Tb-meso MOF was shown to immobilize PQQ-dependent enzymes through ? stacking interactions of the heme in the enzyme and the triazine molecules in the ligand of the MOF. However, the PQQ-dependent dehydrogenases did not have enough catalytic activity present to be measured electrochemically. Finally, ZIF-90 was synthesized under aqueous conditions in the presence of FAD-dependent glucose dehydrogenase (GDH) which led to size selective sheltering of FAD-GDH. FAD-GDH had activity an order of magnitude larger than any of the alcohol dehydrogenases, which provided sufficient catalytic activity to measure electrochemically. The FAD-GDH bound within ZIF-90 was used to build a full biofuel cell resulting in an open circuit voltage of 708 +/- 16 mV and a maximum power density of 2.75 +/- 0.40 microW/cm2.

[Effect of total ischemia and 3',5'-cAMP on the activity of the thermostable cytoplasmic inhibitor of Ca2+ ion transport in rat heart mitochondria].

PubMed

Turakulov, Ia Kh; Luchenko, M B; Gaĭnutdinov, M Kh; Abidov, A A

1985-01-01

Activity of cytoplasmic inhibitor of Ca2+ transport in rat heart mitochondria was studied after total ischemia and incubation of heart homogenates with cAMP. Distinct inactivation of the inhibitor occurred under these conditions. The decrease of the inhibitor activity in ischemic myocardium appears to serve as a compensatory mechanism: 1. pyruvate dehydrogenase and the enzymes of tricarboxylic acid cycle were activated due to increase in Ca2+ concentration in mitochondria, 2. as a result of Ca2+ accumulation in mitochondria the elevated concentration of Ca2+ was decreased in myoplasm, which developed after impairment of plasmatic membranes and of sarcoplasmic reticulum membranes.

Alcohol dehydrogenase ADH2-1 and ADH2-2 allelic isoforms in the Russian population correlate with type of alcoholic disease.

PubMed

Ogurtsov, Pavel P.; Garmash, Irina V.; Miandina, Galina I.; Guschin, Alexander E.; Itkes, Alexander V.; Moiseev, Valentin S.

2001-09-01

The frequency ADH2-2 allele in the Moscow urban population and a correlation between the ADH2-2 allele, alcoholic dependence without cirrhosis, symptomatic alcoholic cirrhosis and status on hepatitis B and C infection have been studied. One hundred and twenty-three inhabitants of Moscow (50 healthy donors, 36 patients with alcoholic cirrhosis (subdivided into infected and uninfected by HBV and/or HCV) and 37 patients with alcoholic dependence) of a similar age/sex and drinking pattern have been analysed. The frequency of 41% for ADH2-2 allele is characteristic for an urban Moscow population. This value is intermediate between that found for Asian peoples and for Central and Western Europe. There is a negative correlation between the ADH2-2 allele and alcohol misuse (both alcoholic dependence and alcoholic cirrhosis). This correlation is expressed more in alcoholic dependence. In spite of the possession of the ADH2-2 allele (or genotype ADH2-1/2), alcohol misuse increases the risk of cirrhosis. At the same time, positive status for active hepatitis B, C or combined infection B + C (replication markers HBV-DNA or HCV-RNA) increases the risk for symptomatic alcoholic cirrhosis in alcohol abusing patients, independently of ADH2 genotype.

The carbanion of nitroethane is an inhibitor of, and not a substrate for, flavocytochrome b2 [L-(+)-lactate dehydrogenase

PubMed Central

Genet, R; Lederer, F

1990-01-01

Although nitroethane does not bind to the active site of flavocytochrome b2, its anion, ethane nitronate, behaves as a competitive inhibitor, with a Ki of 2.2 mM. No electron transfer can be detected between the nitronate and the enzyme, in contrast with the observations of other workers on D-amino acid oxidase. Propionate is a competitive inhibitor, with a Ki of 28 mM. The significance of these results with respect to the proposed carbanion mechanism and the putative existence of a covalent enzyme-substrate intermediate is discussed. PMID:2178603

A theoretical study of the molecular mechanism of the GAPDH Trypanosoma cruzi enzyme involving iodoacetate inhibitor

NASA Astrophysics Data System (ADS)

Carneiro, Agnaldo Silva; Lameira, Jerônimo; Alves, Cláudio Nahum

2011-10-01

The glyceraldehyde-3-phosphate dehydrogenase enzyme (GAPDH) is an important biological target for the development of new chemotherapeutic agents against Chagas disease. In this Letter, the inhibition mechanism of GAPDH involving iodoacetate (IAA) inhibitor was studied using the hybrid quantum mechanical/molecular mechanical (QM/MM) approach and molecular dynamic simulations. Analysis of the potential energy surface and potential of mean force show that the covalent attachment of IAA inhibitor to the active site of the enzyme occurs as a concerted process. In addition, the energy terms decomposition shows that NAD+ plays an important role in stabilization of the reagents and transition state.

Identification of p-hydroxybenzyl alcohol, tyrosol, phloretin and its derivate phloridzin as tyrosinase substrates.

PubMed

Ortiz-Ruiz, Carmen Vanessa; Berna, Jose; Garcia-Molina, Maria Del Mar; Tudela, Jose; Tomas, Virginia; Garcia-Canovas, Francisco

2015-07-01

In recent years, the hydroxyalkylphenols p-hydroxybenzyl alcohol and tyrosol, and the compound phloretin and its derivate phloridzin have been described as inhibitors of the enzyme tyrosinase. When the monophenolase and the diphenolase activities of tyrosinase on its physiological substrates l-dopa and/or l-tyrosine are measured in the presence of these compounds, the rate of action of the enzyme decreases. These findings led to the identification of these compounds as inhibitors. However, these molecules show an unusual behavior as inhibitors of the enzyme indeed, in this study, we demonstrate that they are not true inhibitors but alternative substrates of the enzyme. Copyright © 2015 Elsevier Ltd. All rights reserved.

The Pyruvate and α-Ketoglutarate Dehydrogenase Complexes of Pseudomonas aeruginosa Catalyze Pyocyanin and Phenazine-1-carboxylic Acid Reduction via the Subunit Dihydrolipoamide Dehydrogenase*

PubMed Central

Glasser, Nathaniel R.; Wang, Benjamin X.; Hoy, Julie A.; Newman, Dianne K.

2017-01-01

Phenazines are a class of redox-active molecules produced by diverse bacteria and archaea. Many of the biological functions of phenazines, such as mediating signaling, iron acquisition, and redox homeostasis, derive from their redox activity. Although prior studies have focused on extracellular phenazine oxidation by oxygen and iron, here we report a search for reductants and catalysts of intracellular phenazine reduction in Pseudomonas aeruginosa. Enzymatic assays in cell-free lysate, together with crude fractionation and chemical inhibition, indicate that P. aeruginosa contains multiple enzymes that catalyze the reduction of the endogenous phenazines pyocyanin and phenazine-1-carboxylic acid in both cytosolic and membrane fractions. We used chemical inhibitors to target general enzyme classes and found that an inhibitor of flavoproteins and heme-containing proteins, diphenyleneiodonium, effectively inhibited phenazine reduction in vitro, suggesting that most phenazine reduction derives from these enzymes. Using natively purified proteins, we demonstrate that the pyruvate and α-ketoglutarate dehydrogenase complexes directly catalyze phenazine reduction with pyruvate or α-ketoglutarate as electron donors. Both complexes transfer electrons to phenazines through the common subunit dihydrolipoamide dehydrogenase, a flavoprotein encoded by the gene lpdG. Although we were unable to co-crystallize LpdG with an endogenous phenazine, we report its X-ray crystal structure in the apo-form (refined to 1.35 Å), bound to NAD+ (1.45 Å), and bound to NADH (1.79 Å). In contrast to the notion that phenazines support intracellular redox homeostasis by oxidizing NADH, our work suggests that phenazines may substitute for NAD+ in LpdG and other enzymes, achieving the same end by a different mechanism. PMID:28174304

The Pyruvate and α-Ketoglutarate Dehydrogenase Complexes of Pseudomonas aeruginosa Catalyze Pyocyanin and Phenazine-1-carboxylic Acid Reduction via the Subunit Dihydrolipoamide Dehydrogenase.

PubMed

Glasser, Nathaniel R; Wang, Benjamin X; Hoy, Julie A; Newman, Dianne K

2017-03-31

Phenazines are a class of redox-active molecules produced by diverse bacteria and archaea. Many of the biological functions of phenazines, such as mediating signaling, iron acquisition, and redox homeostasis, derive from their redox activity. Although prior studies have focused on extracellular phenazine oxidation by oxygen and iron, here we report a search for reductants and catalysts of intracellular phenazine reduction in Pseudomonas aeruginosa Enzymatic assays in cell-free lysate, together with crude fractionation and chemical inhibition, indicate that P. aeruginosa contains multiple enzymes that catalyze the reduction of the endogenous phenazines pyocyanin and phenazine-1-carboxylic acid in both cytosolic and membrane fractions. We used chemical inhibitors to target general enzyme classes and found that an inhibitor of flavoproteins and heme-containing proteins, diphenyleneiodonium, effectively inhibited phenazine reduction in vitro , suggesting that most phenazine reduction derives from these enzymes. Using natively purified proteins, we demonstrate that the pyruvate and α-ketoglutarate dehydrogenase complexes directly catalyze phenazine reduction with pyruvate or α-ketoglutarate as electron donors. Both complexes transfer electrons to phenazines through the common subunit dihydrolipoamide dehydrogenase, a flavoprotein encoded by the gene lpdG Although we were unable to co-crystallize LpdG with an endogenous phenazine, we report its X-ray crystal structure in the apo-form (refined to 1.35 Å), bound to NAD + (1.45 Å), and bound to NADH (1.79 Å). In contrast to the notion that phenazines support intracellular redox homeostasis by oxidizing NADH, our work suggests that phenazines may substitute for NAD + in LpdG and other enzymes, achieving the same end by a different mechanism. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Nitric oxide inhibits succinate dehydrogenase-driven oxygen consumption in potato tuber mitochondria in an oxygen tension-independent manner.

PubMed

Simonin, Vagner; Galina, Antonio

2013-01-01

NO (nitric oxide) is described as an inhibitor of plant and mammalian respiratory chains owing to its high affinity for COX (cytochrome c oxidase), which hinders the reduction of oxygen to water. In the present study we show that in plant mitochondria NO may interfere with other respiratory complexes as well. We analysed oxygen consumption supported by complex I and/or complex II and/or external NADH dehydrogenase in Percoll-isolated potato tuber (Solanum tuberosum) mitochondria. When mitochondrial respiration was stimulated by succinate, adding the NO donors SNAP (S-nitroso-N-acetyl-DL-penicillamine) or DETA-NONOate caused a 70% reduction in oxygen consumption rate in state 3 (stimulated with 1 mM of ADP). This inhibition was followed by a significant increase in the Km value of SDH (succinate dehydrogenase) for succinate (Km of 0.77±0.19 to 34.3±5.9 mM, in the presence of NO). When mitochondrial respiration was stimulated by external NADH dehydrogenase or complex I, NO had no effect on respiration. NO itself and DETA-NONOate had similar effects to SNAP. No significant inhibition of respiration was observed in the absence of ADP. More importantly, SNAP inhibited PTM (potato tuber mitochondria) respiration independently of oxygen tensions, indicating a different kinetic mechanism from that observed in mammalian mitochondria. We also observed, in an FAD reduction assay, that SNAP blocked the intrinsic SDH electron flow in much the same way as TTFA (thenoyltrifluoroacetone), a non-competitive SDH inhibitor. We suggest that NO inhibits SDH in its ubiquinone site or its Fe-S centres. These data indicate that SDH has an alternative site of NO action in plant mitochondria.

Ethanol metabolism, oxidative stress, and endoplasmic reticulum stress responses in the lungs of hepatic alcohol dehydrogenase deficient deer mice after chronic ethanol feeding.

PubMed

Kaphalia, Lata; Boroumand, Nahal; Hyunsu, Ju; Kaphalia, Bhupendra S; Calhoun, William J

2014-06-01

Consumption and over-consumption of alcoholic beverages are well-recognized contributors to a variety of pulmonary disorders, even in the absence of intoxication. The mechanisms by which alcohol (ethanol) may produce disease include oxidative stress and prolonged endoplasmic reticulum (ER) stress. Many aspects of these processes remain incompletely understood due to a lack of a suitable animal model. Chronic alcohol over-consumption reduces hepatic alcohol dehydrogenase (ADH), the principal canonical metabolic pathway of ethanol oxidation. We therefore modeled this situation using hepatic ADH-deficient deer mice fed 3.5% ethanol daily for 3 months. Blood ethanol concentration was 180 mg% in ethanol fed mice, compared to <1.0% in the controls. Acetaldehyde (oxidative metabolite of ethanol) was minimally, but significantly increased in ethanol-fed vs. pair-fed control mice. Total fatty acid ethyl esters (FAEEs, nonoxidative metabolites of ethanol) were 47.6 μg/g in the lungs of ethanol-fed mice as compared to 1.5 μg/g in pair-fed controls. Histological and immunohistological evaluation showed perivascular and peribronchiolar lymphocytic infiltration, and significant oxidative injury, in the lungs of ethanol-fed mice compared to pair-fed controls. Several fold increases for cytochrome P450 2E1, caspase 8 and caspase 3 found in the lungs of ethanol-fed mice as compared to pair-fed controls suggest role of oxidative stress in ethanol-induced lung injury. ER stress and unfolded protein response signaling were also significantly increased in the lungs of ethanol-fed mice. Surprisingly, no significant activation of inositol-requiring enzyme-1α and spliced XBP1 was observed indicating a lack of activation of corrective mechanisms to reinstate ER homeostasis. The data suggest that oxidative stress and prolonged ER stress, coupled with formation and accumulation of cytotoxic FAEEs may contribute to the pathogenesis of alcoholic lung disease. Copyright © 2014 Elsevier Inc. All rights reserved.

Ethanol metabolism, oxidative stress, and endoplasmic reticulum stress responses in the lungs of hepatic alcohol dehydrogenase deficient deer mice after chronic ethanol feeding

PubMed Central

Kaphalia, Lata; Boroumand, Nahal; Ju, Hyunsu; Kaphalia, Bhupendra S.; Calhoun, William J.

2014-01-01

Consumption and over-consumption of alcoholic beverages are well-recognized contributors to a variety of pulmonary disorders, even in the absence of intoxication. The mechanisms by which alcohol (ethanol) may produce disease include oxidative stress and prolonged endoplasmic reticulum (ER) stress. Many aspects of these processes remain incompletely understood due to a lack of a suitable animal model. Chronic alcohol over-consumption reduces hepatic alcohol dehydrogenase (ADH), the principal canonical metabolic pathway of ethanol oxidation. We therefore modeled this situation using hepatic ADH-deficient deer mice fed 3.5% ethanol daily for 3 months. Blood ethanol concentration was 180 mg% in ethanol fed mice, compared to <0.2% in the controls. Acetaldehyde (oxidative metabolite of ethanol) was minimally, but significantly increased in ethanol-fed vs. pair-fed control mice. Total fatty acid ethyl esters (FAEEs, nonoxidative metabolites of ethanol) were 47.6 μg/g in the lungs of ethanol-fed mice as compared to 1.5 μg/g in pair-fed controls. Histological and immunohistological evaluation showed perivascular and peribronchiolar lymphocytic infiltration, and significant oxidative injury, in the lungs of ethanol-fed mice compared to pair-fed controls. Several fold increases for cytochrome P450 2E1, caspase 8 and caspase 3 found in the lungs of ethanol-fed mice as compared to pair-fed controls suggest role of oxidative stress in ethanol-induced lung injury. ER stress and unfolded protein response signaling were also significantly increased in the lungs of ethanol-fed mice. Surprisingly, no significant activation of inositol-requiring enzyme-1α and spliced XBP1 were observed indicating a lack of activation of corrective mechanisms to reinstate ER homeostasis. The data suggest that oxidative stress and prolonged ER stress, coupled with formation and accumulation of cytotoxic FAEEs may contribute to the pathogenesis of alcoholic lung disease. PMID:24625836

Deep Sequencing of 71 Candidate Genes to Characterize Variation Associated with Alcohol Dependence.

PubMed

Clark, Shaunna L; McClay, Joseph L; Adkins, Daniel E; Kumar, Gaurav; Aberg, Karolina A; Nerella, Srilaxmi; Xie, Linying; Collins, Ann L; Crowley, James J; Quackenbush, Corey R; Hilliard, Christopher E; Shabalin, Andrey A; Vrieze, Scott I; Peterson, Roseann E; Copeland, William E; Silberg, Judy L; McGue, Matt; Maes, Hermine; Iacono, William G; Sullivan, Patrick F; Costello, Elizabeth J; van den Oord, Edwin J

2017-04-01

Previous genomewide association studies (GWASs) have identified a number of putative risk loci for alcohol dependence (AD). However, only a few loci have replicated and these replicated variants only explain a small proportion of AD risk. Using an innovative approach, the goal of this study was to generate hypotheses about potentially causal variants for AD that can be explored further through functional studies. We employed targeted capture of 71 candidate loci and flanking regions followed by next-generation deep sequencing (mean coverage 78X) in 806 European Americans. Regions included in our targeted capture library were genes identified through published GWAS of alcohol, all human alcohol and aldehyde dehydrogenases, reward system genes including dopaminergic and opioid receptors, prioritized candidate genes based on previous associations, and genes involved in the absorption, distribution, metabolism, and excretion of drugs. We performed single-locus tests to determine if any single variant was associated with AD symptom count. Sets of variants that overlapped with biologically meaningful annotations were tested for association in aggregate. No single, common variant was significantly associated with AD in our study. We did, however, find evidence for association with several variant sets. Two variant sets were significant at the q-value <0.10 level: a genic enhancer for ADHFE1 (p = 1.47 × 10 -5 ; q = 0.019), an alcohol dehydrogenase, and ADORA1 (p = 5.29 × 10 -5 ; q = 0.035), an adenosine receptor that belongs to a G-protein-coupled receptor gene family. To our knowledge, this is the first sequencing study of AD to examine variants in entire genes, including flanking and regulatory regions. We found that in addition to protein coding variant sets, regulatory variant sets may play a role in AD. From these findings, we have generated initial functional hypotheses about how these sets may influence AD. Copyright © 2017 by the Research Society on Alcoholism.