Acetaldehyde coenzyme A dehydrogenase of Escherichia coli.

Mutants of Escherichia coli (adh) in which alcohol dehydrogenase is derepressed under aerobic conditions were also found to overproduce acetaldehyde coenzyme a dehydrogenase. However, acetaldehyde coenzyme A dehydrogenase was induced by ethanol or acetaldehyde and subject to strong catabolite repression, whereas alcohol dehydrogenase was little affected by these conditions. Mutants no longer able to use ethanol as carbon source were isolated from an adh strain. Some of these mutants were revertants at the adh locus and no longer produced either alcohol dehydrogenase or acetaldehyde coenzyme A dehydrogenase. Others, designated acd, were found to lack only acetaldehyde coenzyme A dehydrogenase. The acd mutation was located at min 62 of the E. coli genetic map, the gene order being thyA-lysA-acd-serA-fda. Isolation of Tn10 insertions cotransducible with acd greatly simplified the mapping procedure.     

Identification and overexpression of a bifunctional aldehyde/alcohol dehydrogenase responsible for ethanol production in Thermoanaerobacter mathranii

Thermoanaerobacter mathranii contains four genes, adhA, adhB, bdhA and adhE, predicted to code for alcohol dehydrogenases involved in ethanol metabolism. These alcohol dehydrogenases were characterized as NADP(H)-dependent primary alcohol dehydrogenase (AdhA), secondary alcohol dehydrogenase (AdhB), butanol dehydrogenase (BdhA) and NAD(H)-dependent bifunctional aldehyde/alcohol dehydrogenase (AdhE), respectively. Here we observed that AdhE is an important enzyme responsible for ethanol production in T. mathranii based on the constructed adh knockout strains. An adhE knockout strain fails to produce ethanol as a fermentation product, while other adh knockout strains showed no significant difference from the wild type. Further analysis revealed that the ΔadhE strain was defective in aldehyde dehydrogenase activity, but still maintained alcohol dehydrogenase activity. This showed that AdhE is the major aldehyde dehydrogenase in the cell and functions predominantly in the acetyl-CoA reduction to acetaldehyde in the ethanol formation pathway. Finally, AdhE was conditionally expressed from a xylose-induced promoter in a recombinant strain (BG1E1) with a concomitant deletion of a lactate dehydrogenase. Overexpressions of AdhE in strain BG1E1 with xylose as a substrate facilitate the production of ethanol at an increased yield.     

Role of NAD in regulating the adhE gene of Escherichia coli.

The fermentative alcohol dehydrogenase of Escherichia coli is encoded by the adhE gene, which is induced under anaerobic conditions but repressed in air. Previous work suggested that induction of adhE might depend on NADH levels. We therefore directly measured the NAD+ and NADH levels for cultures growing aerobically and anaerobically on a series of carbon sources whose metabolism generates different relative amounts of NADH. Expression of adhE was monitored both by assay of alcohol dehydrogenase activity and by expression of phi(adhE'-lacZ) gene fusions. The expression of the adhE gene correlated with the ratio of NADH to NAD+. The role of NADH in eliciting adhE induction was supported by a variety of treatments known to change the ratio of NADH to NAD+ or alter the total NAD+-plus-NADH pool. Blocking the electron transport chain, either by mutation or by chemical inhibitors, resulted in the artificial induction of the adhE gene under aerobic conditions. Conversely, limiting NAD synthesis, by introducing mutational blocks into the biosynthetic pathway for nicotinic acid, decreased the expression of adhE under anaerobic conditions. This, in turn, was reversed by supplementation with exogenous NAD or nicotinic acid. In merodiploid strains carrying deletion or insertion mutations abolishing the synthesis of AdhE protein, an adhE-lacZ fusion was expressed at nearly 10-fold the level observed in an adhE+ background. Introduction of mutant adhE alleles producing high levels of inactive AdhE protein gave results equivalent to those seen in absence of the AdhE protein. This finding implies that it is the buildup of NADH due to lack of enzyme activity, rather than the absence of the AdhE protein per se, which causes increased induction of the phi(adhE'-lacZ) fusion. Moreover, mutations giving elevated levels of active AdhE protein decreased the induction of the phi(adhE'-lacZ) fusion. This finding suggests that the enzymatic activity of the AdhE protein modulates the level of NADH under anaerobic conditions, thus indirectly regulating its own expression.     

Ultrastructure and pyruvate formate-lyase radical quenching property of the multienzymic AdhE protein of Escherichia coli.

The AdhE protein of Escherichia coli is a homopolymer of 96-kDa subunits harboring three Fe(2+)-dependent catalytic functions: acetaldehyde-CoA dehydrogenase, alcohol dehydrogenase, and pyruvate formatelyase (PFL) deactivase. By negative staining electron microscopy, we determined a helical assembly of 20-60 subunits into rods of 45-120 nm in length. The subunit packing is widened along the helix axis when Fe2+ and NAD are present. Chymotrypsin dissects the AdhE polypeptide between Phe762 and Ser763, thereby retaining the alcohol dehydrogenase activity on the NH2-terminal core, but destroying all other activities. PFL deactivation, i.e. quenching of the glycyl radical in PFL by the AdhE protein, was examined with respect to cofactor involvements (Fe2+, NAD, and CoA). This process is coupled to NAD reduction and requires the intact CoA sulfhydryl group. Pyruvate and NADH are inhibitors that affect the steady-state level of the radical form of PFL in a reconstituted interconversion cycle. Studies of cell cultures found that PFL deactivation in situ is initiated at redox potentials of greater than or equal to +100 mV. Our results provide insights into the structure/function organization of the AdhE multienzyme and give a rationale for how its PFL radical quenching activity may be suppressed in situ to enable effective glucose fermentation.     

The aldehyde/alcohol dehydrogenase (AdhE) in relation to the ethanol formation in Thermoanaerobacter ethanolicus JW200

A bifunctional aldehyde/alcohol dehydrogenase gene (adhE) from Thermoanaerobacter ethanolicus JW200 was identified and cloned. To unambiguously characterize the activity of AdhE, the recombinant protein was purified. The purified AdhE exhibited high enzymatic activity attributed to aldehyde dehydrogenase (11.0+/-0.3U/mg) and low alcohol dehydrogenase activity (2.6+/-0.2U/mg). Analysis of adhE homologous expression in T. ethanolicus showed that AdhE affected ethanol production.     

Ethanolamine utilization in Salmonella typhimurium: nucleotide sequence, protein expression, and mutational analysis of the cchA cchB eutE eutJ eutG eutH gene cluster.

A fragment of the Salmonella typhimurium ethanolamine utilization operon was cloned and characterized. The 6.3-kb nucleotide sequence encoded six complete open reading frames, termed cchA, cchB, eutE, eutJ, eutG, and eutH. In addition, the nucleotide sequences of two incomplete open reading frames, termed eutX and eutI, were also determined. Comparison of the deduced amino acid sequences and entries in the GenBank database indicated that eutI encodes a phosphate acetyltransferase-like enzyme. The deduced amino acid sequences of the EutE and EutG proteins revealed a significant degree of homology with the Escherichia coli alcohol dehydrogenase AdhE sequence. Mutations in eutE or eutG completely abolished the ability of mutants to utilize ethanolamine as a carbon source and reduced the ability to utilize ethanolamine as a nitrogen source. The product of eutE is most probably an acetaldehyde dehydrogenase catalyzing the conversion of acetaldehyde into acetyl coenzyme A. The product of the eutG gene, an uncommon iron-containing alcohol dehydrogenase, may protect the cell from unconverted acetaldehyde by converting it into an alcohol. The deduced amino acid sequence of cchA resembles that of carboxysome shell proteins from Thiobacillus neapolitanus and Synechococcus sp. as well as that of the PduA product from S. typhimurium. CchA and CchB proteins may be involved in the formation of an intracellular microcompartment responsible for the metabolism of ethanolamine. The hydrophobic protein encoded by the eutH gene possesses some characteristics of bacterial permeases and might therefore be involved in the transport of ethanolamine. Ethanolamine-utilization mutants were slightly attenuated in a mouse model of S. typhimurium infection, indicating that ethanolamine may be an important source of nitrogen and carbon for S. typhimurium in vivo.     

Isolation and characterization of a light-sensitive mutant of Escherichia coli K-12 with a mutation in a gene that is required for the biosynthesis of ubiquinone.

Cells with a novel mutation that is lethal when the cells are exposed to visible light were isolated from Escherichia coli K-12. The mutation was mapped at 63 min on the linkage map of the E. coli chromosome, and the gene, designated visB, was cloned and sequenced. From its map position and the evidence that the gene product VisB exhibits homology with flavin monooxygenase of Pseudomonas fluorescens, the visB gene was deduced to be identical to the ubiH gene, which is a gene required for the biosynthesis of ubiquinone and is thought to be similar to the gene for flavin monooxygenase. The photosensitive phenotype appears to be due to the accumulation of the substrate for the reaction catalyzed by the visB (ubiH) gene product because other mutations that block earlier steps in the biosynthesis of ubiquinone can reverse the photosensitivity. The accumulated intermediates may produce active species of oxygen in the mutant bacteria upon illumination by visible light, and these active oxygen species may cause the death of the cells by a mechanism similar to that associated with mutations in visA (hemH).     

Cloning and characterization of the bifunctional alcohol/acetaldehyde dehydrogenase gene (<Emphasis Type="BoldItalic">adhE</Emphasis>) in <Emphasis Type="BoldItalic">Leuconostoc mesenteroides</Emphasis> isolated from kimchi

A bifunctional alcohol/acetaldehyde dehydrogenase (AdhE) gene (adhE) was cloned from Leuconostoc mesenteroides C7 (LMC7), which is the dominant lactic acid bacterium produced during heterofermentation of kimchi. The nucleotide sequence of the DNA fragment containing putative adhE, which is 2685 bp long and encodes an 886 amino acid polypeptide, exhibits 99% homology with Leu. mesenteroides sp. cremoris. The deduced AdhE comprises two conserved domains: alcohol dehydrogenase (Adh) and acetaldehyde dehydrogenase (Aldh). Moreover, two NAD-binding sites were observed, based on the presence of the GXGXXG motif. A pADHE containing the adhE gene expressed AdhE at the translational level in Escherichia coli BL21, which was at a higher level than in E. coli DH5 and E. coli JM109. The AdhE of LMC7 showed Adh and Aldh activities that, when expressed in E. coli. BL21, were 7.5 and 5.7 U mg^-1 , respectively.     

Inhibition of the oxidation of acetaldehyde and formaldehyde by hepatocytes and mitochondria by crotonaldehyde

Crotonaldehyde was oxidized by disrupted rat liver mitochondrial fractions or by intact mitochondria at rates that were only 10 to 15% that of acetaldehyde. Although a poor substrate for oxidation, crotonaldehyde is an effective inhibitor of the oxidation of acetaldehyde by mitochondrial aldehyde dehydrogenase, by intact mitochondria, and by isolated hepatocytes. Inhibition by crotonaldehyde was competitive with respect to acetaldehyde, and the Ki for crotonaldehyde was about 5 to 20 microM. Crotonaldehyde had no effect on the oxidation of glutamate or succinate. Very low levels of acetaldehyde were detected during the metabolism of ethanol. Crotonaldehyde increased the accumulation of acetaldehyde more than 10-fold, indicating that crotonaldehyde, besides inhibiting the oxidation of added acetaldehyde, also inhibited the oxidation of acetaldehyde generated by the metabolism of ethanol. Formaldehyde was a substrate for the low-Km mitochondrial aldehyde dehydrogenase, as well as for a cytosolic, glutathione-dependent formaldehyde dehydrogenase. Crotonaldehyde was a potent inhibitor of mitochondrial oxidation of formaldehyde, but had no effect on the activity of formaldehyde dehydrogenase. In hepatocytes, crotonaldehyde produced about 30 to 40% inhibition of formaldehyde oxidation, which was similar to the inhibition produced by cyanamide. This suggested that part of the formaldehyde oxidation occurred via the mitochondrial aldehyde dehydrogenase, and part via formaldehyde dehydrogenase. The fact that inhibition by crotonaldehyde is competitive may be of value since other commonly used inhibitors of aldehyde dehydrogenase are irreversible inhibitors of the enzyme.     

Inactivation of E. coli pyruvate formate-lyase: role of AdhE and small molecules

Escherichia 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 detectable, 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 beta-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.     

NADH-ubiquinone oxidoreductases of the Escherichia coli aerobic respiratory chain

Deamino-NADH/ubiquinone 1 oxidoreductase activity in membrane preparations from Escherichia coli GR19N is 20-50% of NADH/ubiquinone 1 oxidoreductase activity. In comparison, membranes from E. coli IY91, which contain amplified levels of NADH dehydrogenase, exhibit about 100-fold higher NADH/ubiquinone 1 reductase activity but about 20-fold less deamino-NADH/ubiquinone 1 reductase activity. Deamino-NADH/ubiquinone 1 reductase is more sensitive than NADH/ubiquinone 1 reductase activity to inhibition by 3-undecyl-2-hydroxyl-1,4-naphthoquinone, piericidin A, or myxothiazol. Furthermore, GR19N membranes exhibit two apparent Kms for NADH but only one for deamino-NADH. Inside-out membrane vesicles from E. coli GR19N generate a H+ electrochemical gradient (interior positive and acid) during electron transfer from deamino-NADH to ubiquinone 1 that is large and stable relative to that observed with NADH as substrate. Generation of the H+ electrochemical gradient in the presence of deamino-NADH is inhibited by 3-undecyl-2-hydroxy-1,4-naphthoquinone and is not observed in IY91 membrane vesicles or in vesicles from GR19N that are deficient in deamino-NADH/ubiquinone 1 reductase activity. The data provide a strong indication that the E. coli aerobic respiratory chain contains two species of NADH dehydrogenases: (i) an enzyme (NADH dh I) that reacts with deamino-NADH or NADH whose turnover leads to generation of a H+ electrochemical gradient at a site between the primary dehydrogenase and ubiquinone and (ii) an enzyme (NADH dh II) that reacts with NADH exclusively whose turnover does not lead to generation of a H+ electrochemical gradient between the primary dehydrogenase and ubiquinone 1.     

Inhibition of mitochondrial aldehyde dehydrogenase and acetaldehyde oxidation by the glutathione-depleting agents diethylmaleate and phorone

Experiments were carried out to study the effect of two commonly used glutathione-depleting agents, diethylmaleate and phorone, on the oxidation of acetaldehyde and the activity of aldehyde dehydrogenase. The oxidation of acetaldehyde by intact hepatocytes was inhibited when the cells were incubated with diethylmaleate. Washing and resuspending the cells in diethylmaleate-free medium afforded protection against the inhibition of acetaldehyde oxidation. The oxidation of acetaldehyde by isolated rat liver mitochondria as well as by disrupted mitochondria in the presence of excess NAD+ was inhibited by diethylmaleate or phorone, indicating inhibition of the low-Km aldehyde dehydrogenase. In addition, diethylmaleate inhibited oxidation of acetaldehyde by the high-Km cytosolic aldehyde dehydrogenase. Significant accumulation of acetaldehyde occurred when ethanol was oxidized by hepatocytes in the presence, but not in the absence, of diethylmaleate. Thus, diethylmaleate blocks the oxidation of added or metabolically generated acetaldehyde, analogous to results with other inhibitors of the low-Km aldehyde dehydrogenase such as cyanamide. These results suggest that caution should be used in interpreting the effects of diethylmaleate or phorone on metabolic reactions, especially those involving metabolism of aldehydes such as formaldehyde, because, in addition to depleting glutathione, these agents inhibit the low-Km aldehyde dehydrogenase.     

Ferulenol specifically inhibits succinate ubiquinone reductase at the level of the ubiquinone cycle

The natural compound ferulenol, a sesquiterpene prenylated coumarin derivative, was purified from Ferula vesceritensis and its mitochondrial effects were studied. Ferulenol caused inhibition of oxidative phoshorylation. At low concentrations, ferulenol inhibited ATP synthesis by inhibition of the adenine nucleotide translocase without limitation of mitochondrial respiration. At higher concentrations, ferulenol inhibited oxygen consumption. Ferulenol caused specific inhibition of succinate ubiquinone reductase without altering succinate dehydrogenase activity of the complex II. This inhibition results from a limitation of electron transfers initiated by the reduction of ubiquinone to ubiquinol in the ubiquinone cycle. This original mechanism of action makes ferulenol a useful tool to study the physiological role and the mechanism of electron transfer in the complex II. In addition, these data provide an additional mechanism by which ferulenol may alter cell function and demonstrate that mitochondrial dysfunction is an important determinant in Ferula plant toxicity.     

Inhibition of hepatic gluconeogenesis by ethanol

1. Gluconeogenesis from 10mm-lactate in the perfused liver of starved rats is inhibited by ethanol. The degree of inhibition reached a maximum of 66% at 10mm-ethanol under the test conditions and decreased at higher ethanol concentrations. The concentration-dependence of the inhibition is paralleled by the concentration-dependence of the activity of alcohol dehydrogenase. The enzyme is also inhibited by ethanol concentrations above 10mm. 2. Gluconeogenesis from pyruvate is not inhibited by ethanol. 3. The degree of the inhibition of gluconeogenesis from lactate by ethanol depends on the concentration of lactate and other oxidizable substances, e.g. oleate, in the perfusion medium. 4. Ethanol also inhibits, to different degrees, gluconeogenesis from glycerol, dihydroxyacetone, proline, serine, alanine, fructose and galactose. 5. The inhibition of gluconeogenesis from lactate by ethanol is reversed by acetaldehyde. 6. Pyrazole, a specific inhibitor of alcohol dehydrogenase, also reverses the inhibition of gluconeogenesis by ethanol. 7. Gluconeogenesis in kidney cortex, where the activity of alcohol dehydrogenase is very low, is not inhibited by ethanol. 8. Kidney cortex, testis, ovary, uterus and certain tissues of the alimentary tract were the only rat tissues, apart from the liver, that showed measurable alcohol dehydrogenase activity. 9. The concentrations of pyruvate in the liver were decreased to about one-fifth by ethanol. 10. The concentration of lactate in the perfused liver was about 3mm below that of the perfusion medium 30min. after the addition of 10mm-lactate. 11. The great majority of the findings support the view that the inhibition of gluconeogensis by ethanol is caused by the alcohol dehydrogenase reaction, which decreases the [free NAD(+)]/[free NADH] ratio. The decrease lowers the concentration of pyruvate and this is the immediate cause of the inhibition of gluconeogenesis from lactate, alanine and serine: the fall in the concentration of pyruvate lowers the rate of the pyruvate carboxylase reaction, one of the rate-limiting reactions of gluconeogenesis. The cause of the inhibition of gluconeogenesis from other substrates is discussed.     

The phytotoxic effect of exogenous ethanol on Euphorbia heterophylla L.

This study investigated the effects of exogenously applied ethanol on Euphorbia heterophylla L., a troublesome weed in field and plantation crops. Ethanol at concentrations ranging from 0.25 to 1.5% caused a dose-dependent inhibition of germination and growth of E. heterophylla. Measurements of respiratory activity and alcohol dehydrogenase (E.C. 1.1.1.1) activity during seed imbibition and initial seedling growth revealed that ethanol induces a prolongation of hypoxic conditions in the growing tissues. In isolated mitochondria, ethanol inhibited the respiration coupled to ADP phosphorylation, an action that probably contributed to modifications observed in the respiratory activity of embryos. A comparison of the effects of methanol, ethanol, propanol and acetaldehyde on germination and growth of E. heterophylla indicates that alcohol dehydrogenase activity is required for the observed effects, with the conversion of ethanol to acetaldehyde playing a role in the ethanol-induced injuries.     

Metabolic engineering of Clostridium autoethanogenum for selective alcohol production

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.     

Inhibition of mitochondrial NADH:ubiquinone oxidoreductase by ethoxyformic anhydride

The NADH:ubiquinone, but not the NADH:ferricyanide, reductase activity of mitochondrial complex I (NADH:ubiquinone oxidoreductase) is inhibited by incubation of the enzyme at pH 6.0 and 0 degree C with ethoxyformic anhydride (EFA), and the inhibition is partially reversed by subsequent incubation of EFA-treated complex I with hydroxylamine. These results and spectral changes of EFA-treated complex I in the u.v. region are consistent with modification of essential histidyl or tyrosyl residues between the primary NADH dehydrogenase and the site of ubiquinone reduction. Treatment of complex I with EFA in the presence of high concentrations of Seconal or Demerol did not protect against EFA inactivation, suggesting that the site of EFA modification may not be the same as the inhibiton sites of Seconal and Demerol. However, the presence of NADH during incubation of complex I with EFA greatly enhanced the inhibition rate, indicating that the reduced conformation of complex I is more susceptible to attack by EFA.