Heterologous Expression and Characterization of an Alcohol Dehydrogenase from the Archeon <Emphasis Type="Italic">Thermoplasma acidophilum</Emphasis>

Analysis of the Thermoplasma acidophilum DSM 1728 genome identified two putative alcohol dehydrogenase (ADH) open reading frames showing 50.4% identity against each other. The corresponding genes Ta0841 and Ta1316 encode proteins of 336 and 328 amino acids with molecular masses of 36.48 and 36.01 kDa, respectively. The genes were expressed in Escherichia coli and the recombinant enzymes were functionally assessed for activity. Throughout the study only Ta1316 ADH resulted active in the oxidative reaction in the pH range 2–8 (optimal pH 5.0) and temperatures from 25 to 90°C (optimal 75°C). This ADH catalyzes the oxidation of several alcohols such as ethanol, methanol, 2-propanol, butanol, and pentanol during the reduction of the cofactor NAD⁺. The highest activity was found in the presence of ethanol producing optically pure acetaldehyde. The specific enzyme activity of the purified Ta1316 ADH with ethanol as a substrate in the optimal conditions was 628.7 U/mg.     

Molecular characterization of the recombinant iron-containing alcohol dehydrogenase from the hyperthermophilic Archaeon, Thermococcus strain ES1

The gene encoding a thermostable iron-containing alcohol dehydrogenase from Thermococcus Strain ES1 (ES1 ADH) was cloned, sequenced and expressed in Escherichia coli. The recombinant and native ES1 ADHs were purified using multistep column chromatography under anaerobic conditions. Both enzymes appeared to be homotetramers with a subunit size of 45 ± 1 kDa as revealed by SDS-PAGE, which was close to the calculated value (44.8 kDa). The recombinant ADH contained 1.0 ± 0.1 g-atom iron per subunit. Both enzymes were sensitive to oxygen with a half-life upon exposure to air of about 4 min. The recombinant enzyme exhibited a specific activity of 105 ± 2 U mg⁻¹, which was very similar to that of the native enzyme (110 ± 3 U mg⁻¹). The optimal pH-values for both enzymes for ethanol oxidation and acetaldehyde reduction were 10.4 and 7.0, respectively. Both enzymes also showed similar temperature-dependent activities, and catalyzed the oxidation of primary alcohols, but there was no activity towards methanol and secondary alcohols. Kinetic parameters of the enzymes showed lower K _m-values for acetaldehyde and NADPH and higher K _m-values for ethanol and NADP⁺. It is concluded that the gene encoding ES1 ADH was expressed successfully in E. coli. This is the first report of a fully active recombinant version of an iron-containing ADH from a hyperthermophile.     

Developmental and environmental influences in the production of a single NAD-dependent fermentative alcohol dehydrogenase by the zygomycete Mucor rouxii

A soluble NAD-dependent alcohol dehydrogenase (ADH) activity was detected in mycelium and yeast cells of wild-type Mucor rouxii. In the mycelium of cells grown in the absence of oxygen, the enzyme activity was high, whereas in yeast cells, ADH activity was high regardless of the presence or absence of oxygen. The enzyme from aerobically or anaerobically grown mycelium or yeast cells exhibited a similar optimum pH for the oxidation of ethanol to acetaldehyde (∼pH 8.5) and for the reduction of acetaldehyde to ethanol (∼pH 7.5). Zymogram analysis conducted with cell-free extracts of the wild-type and an alcohol-dehydrogenase-deficient mutant strain indicated the existence of a single ADH enzyme that was independent of the developmental stage of dimorphism, the growth atmosphere, or the carbon source in the growth medium. Purified ADH from aerobically grown mycelium was found to be a tetramer consisting of subunits of 43 kDa. The enzyme oxidized primary and secondary alcohols, although much higher activity was displayed with primary alcohols. K _m values obtained for acetaldehyde, ethanol, NADH₂, and NAD⁺ indicated that physiologically the enzyme works mainly in the reduction of acetaldehyde to ethanol. Received: 11 March 1999 / Accepted: 14 July 1999     

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

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.     

Gene cloning,expression, and characterization of a novel acetaldehyde dehydrogenase from <Emphasis Type="Italic">Issatchenkia terricola</Emphasis> strain XJ-2

Acetaldehyde is a known mutagen and carcinogen. Active aldehyde dehydrogenase (ALDH) represents an important mechanism for acetaldehyde detoxification. A yeast strain XJ-2 isolated from grape samples was found to produce acetaldehyde dehydrogenase with a high activity of 2.28 U/mg and identified as Issatchenkia terricola. The enzyme activity was validated by oxidizing acetaldehyde to acetate with NAD⁺ as coenzyme based on the headspace gas chromatography analysis. A novel acetaldehyde dehydrogenase gene (ist-ALD) was cloned by combining SiteFinding-PCR and self-formed adaptor PCR. The ist-ALD gene comprised an open reading frame of 1,578 bp and encoded a protein of 525 amino acids. The predicted protein of ist-ALD showed the highest identity (73%) to ALDH from Pichia angusta. The ist-ALD gene was expressed in Escherichia coli, and the gene product (ist-ALDH) presented a productivity of 442.3 U/mL cells. The purified ist-ALDH was a homotetramer of 232 kDa consisting of 57 kDa-subunit according to the SDS-PAGE and native PAGE analysis. Ist-ALDH exhibited the optimal activity at pH 9.0 and 40°C, respectively. The activity of ist-ALDH was enhanced by K⁺, NH4⁺, dithiothreitol, and 2-mercaptoethanol but strongly inhibited by Ag⁺, Hg²⁺, Cu²⁺, and phenylmethyl sulfonylfluoride. In the presence of NAD⁺, ist-ALDH could oxidize many aliphatic, aromatic, and heterocyclic aldehydes, preferably acetaldehyde. Kinetic study revealed that ist-ALDH had a k _cat value of 27.71/s and a k _cat/K _m value of 26.80 × 10³/(mol s) on acetaldehyde, demonstrating ist-ALDH, a catalytically active enzyme by comparing with other ALDHs. These studies indicated that ist-ALDH was a potential enzymatic product for acetaldehyde detoxification.     

Characterization of alcohol dehydrogenase 1 and 3 from <Emphasis Type="Italic">Neurospora crassa</Emphasis> FGSC2489

Alcohol dehydrogenase (ADH) is a key enzyme in the production and utilization of alcohols. Some also catalyze the formation of carboxylate esters from alcohols and aldehydes. The ADH1 and ADH3 genes of Neurospora crassa FGSC2489 were cloned and expressed in recombinant Escherichia coli to investigate their alcohol dehydrogenation and carboxylate ester formation abilities. Homology analysis and sequence alignment of amino acid sequence indicated that ADH1 and ADH3 of N. crassa contained a zinc-binding consensus sequence and a NAD⁺-binding motif and showed 54–75% identity with fungi ADHs. N. crassa ADH1 was expressed in E. coli to give a specific activity of 289 ± 9 mU/mg using ethanol and NAD⁺ as substrate and cofactor, respectively. Corresponding experiments on the expression and activity of ADH3 gave 4 mU/mg of specific activity. N. crassa ADH1 preferred primary alcohols containing C3–C8 carbons to secondary alcohols such as 2-propanol and 2-butanol. N. crassa ADH1 possessed 5.3 mU/mg of specific carboxylate ester-forming activity accumulating 0.4 mM of ethyl acetate in 18 h. Substrate specificity of various linear alcohols and aldehydes indicated that short chain-length alcohols and aldehydes were good substrates for carboxylate ester production. N. crassa ADH1 was a primary alcohol dehydrogenase using cofactor NAD⁺ preferably and possessed carboxylate ester-forming activity with short chain alcohols and aldehydes.     

Strong protective effect of the aldehyde dehydrogenase gene (<Emphasis Type="Italic">ALDH2</Emphasis>) 504lys (*2) allele against alcoholism and alcohol-induced medical diseases in Asians

Alcohol is oxidized to acetaldehyde, which in turn is oxidized to acetate. The aldehyde dehydrogenase 2 gene (ALDH2) is the most important gene responsible for acetaldehyde metabolism. Individuals heterozygous or homozygous for the lys (A or *2) allele at the single nucleotide polymorphism (SNP) glu504lys (rs671) of ALDH2 have greatly reduced ability to metabolize acetaldehyde, which greatly decreases their risk for alcohol dependence (AD). Case–control studies have shown association between this SNP and alcohol dependence as well as alcohol-induced liver disease. However, some studies have produced insignificant results. Using cumulative data from the past 20 years predominately from Asian populations (from both English and Chinese publications), this meta-analysis sought to examine and update whether the aggregate data provide new evidence of statistical significance for the proposed association. Our results (9,678 cases and 7,331 controls from 53 studies) support a strong association of alcohol abuse and dependence, with allelic P value of 3 × 10⁻⁵⁶ and OR of 0.23 (0.2, 0.28) under the random effects model. The dominant model (lys–lys + lys–glu vs. glu–glu) also showed strong association with P value of 1 × 10⁻⁴⁴ and OR of 0.22 (0.18, 0.27). When stricter criteria and various sub-group analyses were applied, the association remained strong (for example, OR = 0.23 (0.18, 0.3) and P = 2 × 10⁻²⁸ for the alcoholic patients with alcoholic liver disease, cirrhosis, or pancreatitis). These findings provide confirmation of the involvement of the human ALDH2 gene in the pathogenesis of AD as well as alcohol-induced medical illnesses in East-Asians.     

Alcohol dehydrogenase polymorphism inDrosophila: Enzyme kinetics of product inhibition

Summary Because natural populations ofDrosophila melanogaster are polymorphic for different allozymes of alcohol dehydrogenase (ADH) and becauseD. melanogaster is more tolerant to the toxic effects of ethanol than its sibling speciesD. simulans, information regarding the sensitivities of the different forms of ADH to the products of ethanol degradation are of ecological importance. ADH-F, ADH-S, ADH-71k ofD. melanogaster and the ADH ofD. simulans were inhibited by NADH, but the inhibition was relieved by NAD⁺. The order of sensitivity of NADH was ADH-F<ADH-71k, ADH-S<ADH-simulans with ADH-F being about four times less sensitive than theD. melanogaster enzymes and 12 times less sensitive than theD. simulans enzyme. Acetaldehyde inhibited the ethanolto-acetaldehyde activity of the ADHs, but at low acetaldehyde concentrations ethanol and NAD⁺ reduced the inhibition. ADH-71k and ADH-F were more subject to the inhibitory action of acetaldehyde than ADH-S and ADH-simulans, with ADH-71k being seven times more sensitive than ADH-S. The pattern of product inhibition of ADH-71k suggests a rapid equilibrium random mechanism for ethanol oxidation. Thus, although the ADH variants only differ by a few amino acids, these differences exert a far larger impact on their intrinsic properties than previously thought. How differences in product inhibition may be of significance in the evolution of the ADHs is discussed.     

Acetaldehyde mediates growth stimulation of ethanol-stressed <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>: evidence of a redox-driven mechanism

The ability of acetaldehyde (90 mg l⁻¹) to stimulate ethanol-stressed S. cerevisiae fermentations is examined and reasons for the effect explored. Alternative metabolic electron acceptors generated similar stimulatory effects to acetaldehyde, decreasing the ethanol-induced growth lag phase from 9 h to 3 h, suggesting a redox-driven effect. The exposure to ethanol caused an instant 60% decline in intracellular NAD⁺ which was largely prevented by the addition of acetaldehyde. Furthermore, the exposure to ethanol affected glycolysis by decreasing the rate of glucose utilisation from 0.33 g glucose g⁻¹ biomass h⁻¹ to 0.11 g glucose g⁻¹ biomass h⁻¹, while the addition of acetaldehyde to an ethanol stressed culture increased this rate to 0.14 g glucose g⁻¹ biomass h⁻¹.     

Tn916-induced mutants of Clostridium acetobutylicum defective in regulation of solvent formation

Sixteen Tn916-induced mutants of Clostridium acetobutylicum were selected that were defective in the production of acetone and butanol. Formation of ethanol, however, was only partially affected. The strains differed with respect to the degree of solvent formation ability and could be assigned to three different groups. Type I mutants (2 strains) were completely defective in acetone and butanol production and contained one or three copies of Tn916 in the chromosome. Analysis of the mutants for enzymes responsible for solvent production revealed the presence of a formerly unknown, specific acetaldehyde dehydrogenase. The data obtained also strongly indicate that the NADP⁺-dependent alcohol dehydrogenase is in vivo reponsible for ethanol formation, whereas the NAD⁺-dependent alcohol dehydrogenase is probably involved in butanol production. No activity of this enzyme together with all other enzymes in the acetone and butanol pathway could be found in type I strains. All tetracycline-resistant mutants obtained did no longer sporulate.Non-standard abbreviations AADC acetoacetate decarboxylase - AcaDH acetaldehyde dehydrogenase - BuaDH butyraldehyde dehydrogenase - CoA-TF acetoacetyl coenzyme A: acetate/butyrate: coenzyme A transferase - NAD-ADH, NAD⁺ dependent alcohol dehydrogenase - NADP-ADH, NADP⁺ dependent alcohol dehydrogenase     

Targeting Dinitrophenol to Mitochondria: Limitations to the Development of a Self-limiting Mitochondrial Protonophore

The protonmotive force (Δp) across the mitochondrial inner membrane drives ATP synthesis. In addition, the energy stored in Δp can be dissipated by proton leak through the inner membrane, contributing to basal metabolic rate and thermogenesis. Increasing mitochondrial proton leak pharmacologically should decrease the efficiency of oxidative phosphorylation and counteract obesity by enabling fatty acids to be oxidised with decreased ATP production. While protonophores such as 2,4-dinitrophenol (DNP) increase mitochondrial proton leak and have been used to treat obesity, a slight increase in DNP concentration above the therapeutically effective dose disrupts mitochondrial function and leads to toxicity. Therefore we set out to develop a less toxic protonophore that would increase proton leak significantly at high Δp but not at low Δp. Our design concept for a potential self-limiting protonophore was to couple the DNP moiety to the lipophilic triphenylphosphonium (TPP) cation and this was achieved by the preparation of 3-(3,5-dinitro-4-hydroxyphenyl)propyltriphenylphosphonium methanesulfonate (MitoDNP). TPP cations accumulate within mitochondria driven by the membrane potential (Δψ), the predominant component of Δp. Our hypothesis was that MitoDNP would accumulate in mitochondria at high Δψ where it would act as a protonophore, but that at lower Δψ the accumulation and uncoupling would be far less. We found that MitoDNP was extensively taken into mitochondria driven by Δψ. However MitoDNP did not uncouple mitochondria as judged by its inability to either increase respiration rate or decrease Δψ. Therefore MitoDNP did not act as a protonophore, probably because the efflux of deprotonated MitoDNP was inhibited.     

Integrating Horizontal Gene Transfer and Common Descent to Depict Evolution and Contrast It with “Common Design”1

ABSTRACT. 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.     

Formation of acetaldehyde-derived DNA adducts due to alcohol exposure

Epidemiological studies have identified chronic alcohol consumption as a significant risk factor for cancers of the upper aerodigestive tract, including the oral cavity, pharynx, larynx and esophagus, and for cancer of the liver. Ingested ethanol is mainly oxidized by the enzymes alcohol dehydrogenase (ADH), cytochrome P-450 2E1 (CYP2E1), and catalase to form acetaldehyde, which is subsequently oxidized by aldehyde dehydrogenase 2 (ALDH2) to produce acetate. Polymorphisms of the genes which encode enzymes for ethanol metabolism affect the ethanol/acetaldehyde oxidizing capacity. ADH1B*2 allele (ADH1B, one of the enzyme in ADH family) is commonly observed in Asian population, has much higher enzymatic activity than ADH1B*1 allele. Otherwise, approximately 40% of Japanese have single nucleotide polymorphisms (SNPs) of the ALDH2 gene. The ALDH2 *2 allele encodes a protein with an amino acid change from glutamate to lysine (derived from the ALDH2*1 allele) and devoid of enzymatic activity. Neither the homozygote (ALDH2*2/*2) nor heterozygote (ALDH2*1/*2) is able to metabolize acetaldehyde promptly. Acetaldehyde is a genotoxic compound that reacts with DNA to form primarily a Schiff base N²-ethylidene-2′-deoxyguanosine (N²-ethylidene-dG) adduct, which may be converted by reducing agents to N²-ethyl-2′-deoxyguanosine (N²-ethyl-dG) in vivo, and strongly blocked translesion DNA synthesis. Several studies have demonstrated a relationship between ALDH2 genotypes and the development of certain types of cancer. On the other hand, the drinking of alcohol induces the expression of CYP2E1, resulting in an increase in reactive oxygen species (ROS) and oxidative DNA damage. This review covers the combined effects of alcohol and ALDH2 polymorphisms on cancer risk. Studies show that ALDH2*1/*2 heterozygotes who habitually consume alcohol have higher rates of cancer than ALDH2*1/*1 homozygotes. Moreover, they support that chronic alcohol consumption contributes to formation of various DNA adducts. Although some DNA adducts formation is demonstrated to be an initiation step of carcinogenesis, it is still unclear that whether these alcohol-related DNA adducts are true factors or initiators of cancer. Future studies are needed to better characterize and to validate the roles of these DNA adducts in human study.     

Neuroprotective Effect of KB-R7943 Against Glutamate Excitotoxicity is Related to Mild Mitochondrial Depolarization

KB-R7943, an inhibitor of a reversed Na⁺/Ca²⁺ exchanger, exhibits neuroprotection against glutamate excitotoxicity. Taking into consideration that prolonged exposure of neurons to glutamate induces delayed calcium deregulation (DCD) and irreversible decrease of mitochondrial membrane potential (Δψ_mit), we examined the effect of KB-R7943 on glutamate and kainate-induced [Ca²⁺]_i and on Δψ_mit changes in rat cultured cerebellar granule neurons. 15 μmol/l KB-R7943 significantly delayed the onset of DCD in response to kainate but not in response to glutamate. In spite of [Ca²⁺]_i overload, KB-R7943 considerably improved the [Ca²⁺]_i recovery and restoration of Δψ_mit after glutamate and kainate washout and increased cell viability after glutamate exposure. In resting neurons, KB-R7943 induced a statistically significant decrease in Δψ_mit. KB-R7943 also depolarized isolated brain mitochondria and slightly inhibited mitochondrial Ca²⁺ uptake. These findings suggest that mild mitochondrial depolarization and diminution of Ca²⁺ accumulation in the organelles might contribute to neuroprotective effect of KB-R7943.     

Gene Cloning and Biochemical Characterization of a NAD(P)<Superscript>+</Superscript>-Dependent Aldehyde Dehydrogenase from <Emphasis Type="Italic">Bacillus licheniformis</Emphasis>

A putative aldehyde dehydrogenase (ALDH) gene, ybcD (gene locus b1467), was identified in the genome sequence of Bacillus licheniformis ATCC 14580. B. licheniformis ALDH (BlALDH) encoded by ybcD consists of 488 amino acid residues with a molecular mass of approximately 52.7 kDa. The coding sequence of ybcD gene was cloned in pQE-31, and functionally expressed in recombinant Escherichia coli M15. BlALDH had a subunit molecular mass of approximately 53 kDa and the molecular mass of the native enzyme was determined to be 220 kDa by FPLC, reflecting that the oilgomeric state of this enzyme is tetrameric. The temperature and pH optima for BlALDH were 37°C and 7.0, respectively. In the presence of either NAD⁺ or NADP⁺, the enzyme could oxidize a number of aliphatic aldehydes, particularly C3- and C5-aliphatic aldehyde. Steady-state kinetic study revealed that BlALDH had a K _M value of 0.46 mM and a k _cat value of 49.38/s when propionaldehyde was used as the substrate. BlALDH did not require metal ions for its enzymatic reaction, whereas the dehydrogenase activity was enhanced by the addition of disulfide reductants, 2-mercaptoethanol and dithiothreitol. Taken together, this study lays a foundation for future structure–function studies with BlALDH, a typical member of NAD(P)⁺-dependent aldehyde dehydrogenases.     

Mouse alcohol dehydrogenase 4: kinetic mechanism,substrate specificity and simulation of effects of ethanol on retinoid metabolism

Mouse ADH4 (purified, recombinant) has a low catalytic efficiency for ethanol and acetaldehyde, but very high activity with longer chain alcohols and aldehydes, at pH 7.3 and temperature 37°C. The observed turnover numbers and catalytic efficiencies for the oxidation of all-trans-retinol and the reduction of all-trans-retinal and 9-cis-retinal are low relative to other substrates; 9-cis-retinal is more reactive than all-trans-retinal. The reduction of all-trans- or 9-cis-retinals coupled to the oxidation of ethanol by NAD⁺ is as efficient as the reduction with NADH. However, the Michaelis constant for ethanol is about 100 mM, which indicates that the activity would be lower at physiologically relevant concentrations of ethanol. Simulations of the oxidation of retinol to retinoic acid with mouse ADH4 and human aldehyde dehydrogenase (ALDH1), using rate constants estimated for all steps in the mechanism, suggest that ethanol (50 mM) would modestly decrease production of retinoic acid. However, if the K_m for ethanol were smaller, as for human ADH4, the rate of retinol oxidation and formation of retinoic acid would be significantly decreased during metabolism of 50 mM ethanol. These studies begin to describe quantitatively the roles of enzymes involved in the metabolism of alcohols and carbonyl compounds.     

Biochemical properties and physiological roles of NADP-dependent malic enzyme in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Malic enzymes catalyze the reversible oxidative decarboxylation of L-malate using NAD(P)⁺ as a cofactor. NADP-dependent malic enzyme (MaeB) from Escherichia coli MG1655 was expressed and purified as a fusion protein. The molecular weight of MaeB was about 83 kDa, as determined by SDS-PAGE. The recombinant MaeB showed a maximum activity at pH 7.8 and 46°C. MaeB activity was dependent on the presence of Mn²⁺ but was strongly inhibited by Zn²⁺. In order to understand the physiological roles, recombinant E. coli strains (icd ^NADP/ΔmaeB and icd ^NAD/ΔmaeB) containing NADP-dependent isocitrate dehydrogenase (IDH), or engineered NAD-dependent IDH with the deletion of the maeB gene, were constructed using homologous recombination. During growth on acetate, icd ^NAD/ΔmaeB grew poorly, having a growth rate only 60% that of the wild-type strain (icd ^NADP). Furthermore, icd ^NADP/ΔmaeB exhibited a 2-fold greater adaptability to acetate than icd ^NAD/ΔmaeB, which may be explained by more NADPH production for biosynthesis in icd ^NADP/ΔmaeB due to its NADP-dependent IDH. These results indicated that MaeB was important for NADPH production for bacterial growth on acetate. We also observed that MaeB activity was significantly enhanced (7.83-fold) in icd ^NAD, which was about 3-fold higher than that in icd ^NADP, when switching from glucose to acetate. The marked increase of MaeB activity was probably induced by the shortage of NADPH in icd ^NAD. Evidently, MaeB contributed to the NADPH generation needed for bacterial growth on two carbon compounds.     

Dietary Ethanol Mediates Selection on Aldehyde Dehydrogenase Activity in Drosophila melanogaster

Ethanol is an important environmental variable for fruit-breedingDrosophila species, serving as a resource at low levels anda toxin at high levels. The first step of ethanol metabolism,the conversion of ethanol to acetaldehyde, is catalyzed primarilyby the enzyme alcohol dehydrogenase (ADH). The second step,the oxidation of acetaldehyde to acetate, has been a sourceof controversy, with some authors arguing that it is carriedout primarily by ADH itself, rather than a separate aldehydedehydrogenase (ALDH) as in mammals. We review recent evidencethat ALDH plays an important role in ethanol metabolism in Drosophila.In support of this view, we report that D. melanogaster populationsmaintained on ethanol-supplemented media evolved higher activityof ALDH, as well as of ADH. We have also tentatively identifiedthe structural gene responsible for the majority of ALDH activityin D. melanogaster. We hypothesize that variation in ALDH activitymay make an important contribution to the observed wide variationin ethanol tolerance within and among Drosophila species.     

Molecular Cloning and Characterization of a Malic Enzyme Gene from the Oleaginous Yeast <Emphasis Type="Italic">Lipomyces starkeyi</Emphasis>

The malic enzyme-encoding cDNA (GQ372891) from the oleaginous yeast Lipomyces starkeyi AS 2.1560 was isolated, which has an 1719-bp open reading frame flanked by a 290-bp 5′ untranslated sequence and a 92-bp 3′ untranslated sequence. The proposed gene, LsME1, encoded a protein with 572 amino acid residues. The protein presented 58% sequence identity with the malic enzymes from Yarrowia lipolytica CLIB122 and Aspergillus fumigatus Af293. The LsME1 gene was cloned into the vector pMAL-p4x to express a fusion protein (MBP-LsME1) in Escherichia coli TB1. The fusion protein was purified and then cleaved by Factor Xa to give the recombinant LsME1. This purified enzyme took either NAD⁺ or NADP⁺ as the coenzyme but preferred NAD⁺. The K _m values for malic acid, NAD⁺ and NADP⁺ were 0.85 ± 0.05 mM, 0.34 ± 0.08 mM, and 7.4 ± 0.32 mM, respectively, at pH 7.3.