Covalently anchoring of a ligand/metal via polar amino acid side chain(s) is often observed in metalloenzyme, while the substitutability of metal-binding sites remains elusive. In this study, we utilized a zinc-dependent alcohol dehydrogenase from Thermoanaerobacter brockii (TbSADH) as a model enzyme, analyzed the sequence conservation of the three residues Cys37, His59, and Asp150 that bind the zinc ion, and constructed the mutant library. After experimental validation, three out of 224 clones, which showed comparative conversion and ee values as the wild-type enzyme in the asymmetric reduction of the model substrate tetrahydrofuran-3-one, were screened out. The results reveal that the metal-binding sites in TbSADH are substitutable without tradeoff in activity and stereoselectivity, which lay a foundation for designing ADH-catalyzed new reactions via metal ion replacement.
Alcohol Dehydrogenase/metabolism* ; Catalytic Domain ; Ligands ; Protein Domains ; Zinc/metabolism*

Bacillus sp. TSH1 is a butanol-producing microorganism newly isolated in our laboratory; it can grow and ferment under facultative anaerobic conditions, while sharing similar fermentation pathways and products with Clostridium acetobutylicum. To illustrate the relationships between the products and the enzyme activities in Bacillus sp. TSH1, key butanol- and ethanol-forming enzymes were studied, including butyraldehyde dehydrogenase, butanol dehydrogenase and alcohol dehydrogenase. The activities of the three enzymes increased rapidly after the initiation of fermentation. Activities of three enzymes peaked before 21 h, and simultaneously, product concentrations also began to increase gradually. The maximum activity of alcohol dehydrogenase was 0.054 U/mg at 12 h, butyraldehyde dehydrogenase 0.035 U/mg at 21 h and butanol dehydrogenase 0.055 U/mg at 15 h. The enzyme activities then decreased, but remained constant at a low level after 24 h, while the concentrations of butanol, acetone, and ethanol continued increasing until the end of the fermentation. The results will attribute to the understanding of the butanol metabolic mechanism, and provide a reference for further study of a facultative Bacillus metabolic pathway.
Alcohol Dehydrogenase ; metabolism ; Alcohol Oxidoreductases ; metabolism ; Aldehyde Oxidoreductases ; metabolism ; Anaerobiosis ; Bacillus ; classification ; genetics ; metabolism ; Butanols ; metabolism ; Fermentation ; Metabolic Networks and Pathways

The protective effect of EtOAc fraction of Limonium tetragonum extract (EALT) against alcoholinduced hepatotoxicity was assessed following acute ethanol intoxication in Spraque-Dawley rats. EALT (200 mg/kg p.o.) was administrated once before alcohol intake (8 g/kg, p.o.). Blood ethanol concentration, and the activities of alcohol metabolic enzymes, alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) in the liver were measured. Also, the formation of malondialdehyde (MDA) and the activities of antioxidant enzymes, superoxide dismutase (SOD), glutathione peroxidase (GSH-px), catalase were determined after acute alcohol exposure. Pretreatment of rats received ethanol with EALT significantly decreased blood ethanol concentration and elevated the activities of ADH and ALDH in liver. The increased MDA level was decreased, and the reduced activities of SOD, GSH-px and catalase were markedly preserved by the treatment with EALT. This study suggests that EALT prevent hepatic injury induced by acute alcohol which is likely related to its modulation on the alcohol metabolism and antioxidant enzymes activities.
Alcohol Dehydrogenase ; Aldehyde Dehydrogenase ; Animals ; Catalase ; Ethanol* ; Glutathione Peroxidase ; Liver* ; Malondialdehyde ; Metabolism* ; Plumbaginaceae* ; Rats ; Salt-Tolerant Plants ; Superoxide Dismutase

OBJECTIVE: To explore alcohol pharmacokinetics as well as acetaldehyde level in peripheral blood in human subjects with different ALDH2 genotypes after drinking. METHODS: Venous blood samples of 14 unrelated volunteers were collected. Polymerase chain reaction-restriction fragment length polymorphism technology was adopted for DNA extraction and ALDH2 genotyping. The volunteers were asked to drink beer at certain doses. The concentration of alcohol and acetaldehyde were assayed by headspace gas chromatography method at different time. The pharmacokinetic parameters were calculated. RESULTS: According to the results of electrophoresis, 5 people carried ALDH2*1/*1 as wild group and 9 people carried ALDH2*1/*2 as mutation group. The good linear range of alcohol and acetaldehyde were 0-1 570.7 microg/mL and 0-5.1772 microg/mL, respectively. The AUC values of alcohol and acetaldehyde and the t1/2Z value of alcohol were higher in the mutation group than that in the wild group. But the CL/F value of alcohol was lower in the mutation group than that in the wild group (P<0.05). CONCLUSION After the consumption of alcohol, alcohol and acetaldehyde metabolism in blood slow down in ALDH2*1/*2 mutation group influenced by the inhibition of enzyme activity, leading to the accumulation of acetaldehyde in peripheral blood, thus reinforcing their effects in the body.
Alcohol Drinking ; Aldehyde Dehydrogenase/genetics* ; Aldehyde Dehydrogenase, Mitochondrial ; Ethanol/metabolism* ; Genotype ; Humans ; Polymerase Chain Reaction ; Polymorphism, Genetic

The kinetic mechanisms of two key enzymes in the biotransformation of glycerol to 1,3-propanediol (1,3-PD) by Klebsiella pneumoniae, glycerol dehydrogenase (GDH) and 1,3-propanediol oxidoreductase (PDOR), was characterized. Kinetics on initial velocity and product inhibition revealed that GDH and PDOR follow an ordered Bi-Bi sequential mechanism. Kinetic models for GDH and PDOR showed that the oxidation reaction catalyzed by GDH was the rate-limiting step in coupled enzymatic reaction when the GDH/PDOR was 1:1, and the NAD+ was the main form of coenzyme in the reaction. Knowledge about the kinetic mechanisms will be helpful to understand how these enzymes is regulated, which will be useful for further enzyme catalysis and metabolic engineering studies.
Alcohol Dehydrogenase ; metabolism ; Bacterial Proteins ; metabolism ; Glycerol ; metabolism ; Kinetics ; Klebsiella pneumoniae ; enzymology ; Models, Theoretical ; Propylene Glycols ; metabolism ; Substrate Specificity ; Sugar Alcohol Dehydrogenases ; metabolism

Through studying the process of glycerol fermentation to 1, 3-propanediol(1, 3-PD) by Klebsiella pneumoniae, it was found that the cell growth and product (or by-product) production were under salt stress. Cell growth and product formation kept high rate at low salt concentration. High salt concentration led to low growth of cells, final concentration of 1, 3-PD and conversion from glycerol to 1, 3-PD, and, 1, 3-propanediol oxidoreductase activity decreased. When the salt concentration in 5 m3 bioreactor was controlled under appropriate manner, the concentration of 1, 3-PD production was markedly enhanced. The final 1, 3-PD concentration ,the conversion of glycerol to 1, 3-PD and productivity were 64 g/L, 61% and 2.1 g/(L x h).
Alcohol Dehydrogenase ; Alcohol Oxidoreductases ; metabolism ; Culture Media ; Culture Techniques ; Fermentation ; Glycerol ; metabolism ; Klebsiella pneumoniae ; growth & development ; metabolism ; physiology ; Propylene Glycols ; metabolism ; Sodium Chloride ; pharmacology ; Stress, Physiological

Objective To explore the change rules of blood ethanol and blood acetaldehyde concentration, the impairment of psychomotor functions of different acetaldehyde dehydrogenase （ALDH） 2 genotype individuals after alcohol consumption and the relationship among them. Methods The ALDH2 genotypes in seventy-nine healthy volunteers were obtained by SNaPshot^TM method, then divided into ALDH2*1/*1 （wild type） and ALDH2*1/*2 （mutant type） group. After volunteers consumed 1.0 g/kg of alcohol, blood ethanol concentration and blood acetaldehyde concentration at a series of time points before and after alcohol consumption and psychomotor functions, such as, visual selective response time, auditory simple response time and tracking experiment were detected. Biphasic alcohol response questionnaires were collected. Results After alcohol consumption, ALDH2*1/*2 group's blood ethanol and blood acetaldehyde concentration reached the peak earlier than ALDH2*1/*1 group. Its blood acetaldehyde concentration was higher than that of ALDH2*1/*1 group, 1-6 h after alcohol consumption. The psychomotor functions, such as visual selective response time and auditory simple response time in ALDH2*1/*2 group were more significantly impaired than those in ALDH2*1/*1 group after alcohol consumption. There was no statistical significance between the two groups in excitement or sedation reactions （P>0.05）. Pearson correlation coefficient test showed that blood acetaldehyde concentration was related with psychomotor function. Conclusion There are significant differences between the psychomotor function of ALDH2 wild type and mutant type individuals after alcohol consumption estimated to be related to the difference in blood acetaldehyde concentration after alcohol consumption.
Acetaldehyde/metabolism* ; Alcohol Drinking/blood* ; Aldehyde Dehydrogenase/genetics* ; Aldehyde Dehydrogenase, Mitochondrial ; Aldehyde Oxidoreductases ; Ethanol/metabolism* ; Genotype ; Humans ; Polymorphism, Genetic/genetics* ; Psychomotor Performance/physiology*

PURPOSE: This study tried to identify novel gastric autoimmune antigens that might be involved in aggravating the atrophic gastritis among patients with Helicobacter pylori infection using two-dimensional immunoblotting analysis. MATERIALS AND METHODS: Proteins from gastric mucosal antrectomy specimens and AGS cells (gastric adenocarcinoma cell lines derived from a Caucasian patient who had received no prior therapy) were 2-dimensionally immunoblotted separately with a pool of 300 sera from H. pylroi-infected patients at Gyeongsang National University Hospital. RESULTS: Thirty-eight autoantigenic proteins including alcohol dehydrogenase [NADP+], alpha enolase, gastrokine-1, gastric triacylglycerol lipase, heat shock 70 kDa protein 1, and peroxiredoxin-2 were identified in the gastric mucosal tissue. Fourteen autoantigenic proteins including programmed cell death 6-interacting protein, serum albumin and T-complex protein 1 subunit gamma were identified in the AGS cells. Albumin, alpha-enolase, annexin A3, cytoplasmic actin 1, heat shock cognate 71 kDa protein and leukocyte elastase inhibitor were commonly observed autoantigenic proteins in both gastric mucosal tissue and AGS cells. Alpha-enolase, glutathione S-transferase P, heat shock cognate 71 kDa protein, heat shock 70 kDa protein 1, human mitochondrial adenosine triphosphate synthase (ATP) subunit beta, mitochondrial 60 kDa heat shock protein, peroxiredoxin-2, 78 kDa glucose-regulated protein precursor, tyrosine-protein phosphatase non-receptor type 11 and Tryptophan-Aspartic acid (WD) repeat-containing protein 1 showed 60% or higher amino acid positivity. CONCLUSION: These newly identified gastric autoimmune antigens might be useful in the control and prevention of gastroduodenal disorders, and might be valuable in breaking the vicious circle that exists in gastroduodenal disorders if their pathophysiological roles could be understood in the progress of chronic atrophic gastritis, gastroduodenal ulcers, intestinal metaplasia, and gastric carcinogenesis.
Alcohol Dehydrogenase/metabolism ; Autoantigens/*metabolism ; Electrophoresis, Gel, Two-Dimensional ; Gastric Mucosa/metabolism/microbiology ; Helicobacter Infections/*metabolism ; Humans ; Peptide Hormones/metabolism ; Phosphopyruvate Hydratase/metabolism

OBJECTIVETo study the activities of the key enzymes in reteplase production by Pichia pastoris.

METHODSIn shaking flasks, a series of samples were maintained after methanol induction. The cells were sonicated to prepare cell-free suspensions, in which the activities of AOX, FAD, PDC, G-6-PD, ID, alpha-KGD and SD were measured.

RESULTSThe specific activity of AOX increased during the initial 6 h, reaching the maximum of 44.5 U/mg protein. The activity decreased quickly between 6 and 24 h, followed by increment in the following 24 h and decreased afterwards. The specific activity of FAD increased gradually in the initial 48 h and then decreased, with the peak level of 6.72 U/mg protein occurred at 48 h. The specific activity of G-6-PD increased at in 2-6 h and 24-48 h, but decreased in 6-24 h and after 48 h. The specific activity of PDC decreased during the initial 6 h and increased slowed afterwards. The specific activities of ID, alpha-KGD and SD all showed a rapid decrease in the initial 6 h and a slow decrease in 6-24 h. After 24 h, the activity of ID continued to decrease, but the other two increased in the following 24 h and then decreased, reaching the maximum at 48 h.

CONCLUSIONSAccording to the changes of these enzyme activities, the whole induction phase can be divided into 4 stages: the methanol-adaptive period in the initial 6 h, the fast growth period between 6 and 24 h, the product accumulation period in 24-48 h and the metabolism lag period in 48-72 h. In the methanol-adaptive period, complete oxidation of methanol is the dominant pathway. But in the following two stages, the metabolic pathway shifts towards glycolysis and TCA cycle.

Alcohol Oxidoreductases ; metabolism ; Fermentation ; Genetic Vectors ; Glucosephosphate Dehydrogenase ; metabolism ; Methanol ; pharmacology ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; metabolism ; Tissue Plasminogen Activator ; biosynthesis ; genetics ; metabolism

BACKGROUND/AIMS: Genetic variations of ethanol-metabolizing enzymes can affect alcohol drinking behavior. The aims of this study were to investigate and compare the distributions of these genetic polymorphisms between a healthy control group and a heavy drinker group which included an alcoholic liver cirrhosis group. METHODS: Genotypes of ADH2, ALDH2, CYP2E1, and catalase were identified by polymerase chain reaction and restriction fragment length polymorphism. Genomic DNA was extracted from peripheral leukocytes in 42 healthy controls, 12 heavy drinkers, and 30 alcoholic liver cirrhosis patients. RESULTS: 1) The genotype frequencies of ALDH2 (1*1), ADH2 (1*1), CYP2E1 (c1c1), and catalase1 (TT) were 69%, 55%, 38%, and 12%, respectively in healthy Korean males. 2) There was a significant difference in the distribution of the genetic polymorphism of ALDH2 between the control group and heavy drinker group (12 heavy drinkers and 30 alcoholic liver cirrhosis patients). The genotype frequency of ALDH2 mutant, ALDH2 (1*2) and ALDH2 (2*2) in the heavy drinker group (12%) was significantly lower than that in the control group (30%). 3) We didn't find anyone with ALDH2 homozygote mutant (DD) in the heavy drinker group. 4) There was no significant difference in the distribution of genetic polymorphisms in ADH2, CYP2E1 and catalase1 between the two groups. CONCLUSIONS: These results suggest that the absence of ALDH2 mutant genotype is strongly related to heavy drinking behavior. We can not prove, however, any evidence that the polymorphisms of other ethanol-metabolizing enzymes are associated with the determination of alcohol-drinking behavior.
Adult ; Alcohol Dehydrogenase/*genetics ; Alcohol Drinking ; Alcoholism/enzymology/*genetics ; Aldehyde Dehydrogenase/*genetics ; Cytochrome P-450 CYP2E1/*genetics ; Ethanol/metabolism ; Humans ; Liver Cirrhosis, Alcoholic/enzymology/*genetics ; Male ; Middle Aged ; *Polymorphism, Genetic