Alcohol dehydrogenase from the fruitfly Drosophila melanogaster. Inhibition studies of the alleloenzymes AdhS and AdhUF

Different metal binding inhibitors of horse liver alcohol dehydrogenase, similarly affect the Drosophila melanogaster AdhS and AdhUF alleloenzymes. However, binding is generally weaker and the experiments show that the alleloenzymes although not zinc metalloenzymes, behave to the metal binding reagents very much as if they were. The metal-directed, affinity-labelling, imidazole derivative BrImPpOH reversibly inhibits, but does not inactivate the alleolenzymes. This confirms there is no active site metal atom with cysteine as a metal ligand, as found in zinc alcohol dehydrogenases. Pyrazole is a strong ethanol-competitive inhibitor of AdhS and AdhUF alleloenzymes. Formation of the ternary enzyme-NAD-pyrazole complex gives an absorption increase between 295-330 nm. This enables an active site titration to be performed and the determination of epsilon (305 nm) of 15.8 . 10(3) M-1 . cm-1. Inhibition experiments with imidazole confirm that with secondary alcohols such as propan-2-ol, a Theorell-Chance mechanism predominates, but with ethanol and primary alcohols, interconversion of the ternary complexes is rate limiting. Salicylate is a coenzyme competitive inhibitor and KEI suggests that the coenzyme adenosine binding region is similar is Drosophila and horse liver alcohol dehydrogenase. Drosophila alcohol dehydrogenase is found not to form a ternary complex with NADH and isobutyramide. In this and other properties it is like carboxymethyl liver alcohol dehydrogenase. Both Drosophila and carboxymethyl alcohol dehydrogenase bind coenzyme in a similar manner to native horse liver alcohol dehydrogenase, but substrate binding differs between each. Inhibition by Cibacrone blue, indicates that amino acid 192 which is lysine in AdhS and threonine in AdhUF, is located in the coenzyme-binding region. Proteolytic activity present in preparations of alcohol dehydrogenase from D. melanogaster, is considered due to a metalloprotease, for which BrImPpOH is a potent inactivator.     

Complete amino acid sequence and characterization of the reaction mechanism of a glucosamine-induced novel alcohol dehydrogenase from Agrobacterium radiobacter (tumefaciens)

A glucosamine-induced novel alcohol dehydrogenase has been isolated from Agrobacterium radiobacter (tumefaciens) and its fundamental properties have been characterized. The enzyme catalyzes NAD-dependent dehydrogenation of aliphatic alcohols and amino alcohols. In this work, the complete amino acid sequence of the alcohol dehydrogenase was determined by PCR method using genomic DNA of A. radiobacter as template. The enzyme comprises 336 amino acids and has a molecular mass of 36 kDa. The primary structure of the enzyme demonstrates a high homology to structures of alcohol dehydrogenases from Shinorhizobium meliloti (83% identity, 90% positive) and Pseudomonas aeruginosa (65% identity, 76% positive). The two Zn(2+) ion binding sites, both the active site and another site that contributed to stabilization of the enzyme, are conserved in those enzymes. Sequences analysis of the NAD-dependent dehydrogenase family using a hypothetical phylogenetic tree indicates that these three enzymes form a new group distinct from other members of the Zn-containing long-chain alcohol dehydrogenase family. The physicochemical properties of alcohol dehydrogenase from A. radiobacter were characterized as follows. (1) Stereospecificity of the hydride transfer from ethanol to NADH was categorized as pro-R type by NMR spectra of NADH formed in the enzymatic reaction using ethanol-D(6) was used as substrate. (2) Optimal pH for all alcohols with no amino group examined was pH 8.5 (of the C(2)-C(6) alcohols, n-amyl alcohol demonstrated the highest activity). Conversely, glucosaminitol was optimally dehydrogenated at pH 10.0. (3) The rate-determining step of the dehydrogenase for ethanol is deprotonation of the enzyme-NAD-Zn-OHCH(2)CH(3) complex to enzyme-NAD-Zn-O(-)CH(2)CH(3) complex and that for glucosaminitol is H(2)O addition to enzyme-Zn-NADH complex.     

Chemical basis of the electrophoretic variation observed at the alcohol dehydrogenase locus of Drosophila melanogaster.

The amino acid substitution responsible for the different electrophoretic mobility of the ADHs alleloenzyme and the ADHf alleloenzyme of the alcohol dehydrogenase from a Nigerian population of Drosophila melanogaster has been established as lysine (ADHs) for threonine (ADHf). This result is discussed with reference to the charge state model of electrophoretic variation, in conjunction with other know substitutions at this locus. It is concluded that electrophoretic methods should be capable of distinguishing many alleloenzymes which have identical isoelectric points without recourse to explanations involving conformational variability.     

Primary structure of rat liver D-beta-hydroxybutyrate dehydrogenase from cDNA and protein analyses: a short-chain alcohol dehydrogenase.

The amino acid sequence of D-beta-hydroxybutyrate dehydrogenase (BDH), a phosphatidyl-choline-dependent enzyme, has been determined for the enzyme from rat liver by a combination of nucleotide sequencing of cDNA clones and amino acid sequencing of the purified protein. This represents the first report of the primary structure of this enzyme. The largest clone contained 1435 base pairs and encoded the entire amino acid sequence of mature BDH and the leader peptide of precursor BDH. Hybridization of poly(A+) rat liver mRNA revealed two bands with estimated sizes of 3.2 and 1.7 kb. A computer-based comparison of the amino acid sequence of BDH with other reported sequences reveals a homology with the superfamily of short-chain alcohol dehydrogenases, which are distinct from the classical zinc-dependent alcohol dehydrogenases. This protein family, initially discerned from Drosophila alcohol dehydrogenase and bacterial ribitol dehydrogenase, is now known to include at least 20 enzymes catalyzing oxidations of distinct substrates.     

Glucose oxidase from Aspergillus niger. Cloning, gene sequence, secretion from Saccharomyces cerevisiae and kinetic analysis of a yeast-derived enzyme

The gene for Aspergillus niger glucose oxidase (EC 1.1.3.4) has been cloned from both cDNA and genomic libraries using oligonucleotide probes derived from the amino acid sequences of peptide fragments of the enzyme. The mature enzyme consists of 583 amino acids and is preceded by a 22-amino acid presequence. No intervening sequences are found within the coding region. The enzyme contains 3 cysteine residues and 8 potential sites for N-linked glycosylation. The protein shows 26% identity with alcohol oxidase of Hansenuela polymorpha, and the N terminus has a sequence homologous with the AMP-binding region of other flavoenzymes such as p-hydroxybenzoate hydroxylase and glutathione reductase. Recombinant yeast expression plasmids have been constructed containing a hybrid yeast alcohol dehydrogenase II-glyceraldehyde-3-phosphate dehydrogenase promoter, either the yeast alpha-factor pheromone leader or the glucose oxidase presequence, and the mature glucose oxidase coding sequence. When transformed into yeast, these plasmids direct the synthesis and secretion of between 75 and 400 micrograms/ml of active glucose oxidase. Analysis of the yeast-derived enzymes shows that they are of comparable specific activity and have more extensive N-linked glycosylation than the A. niger protein.     

谷氨酸脱氢酶与醇脱氢酶的共表达及其在制备(R)-2-氯-1-苯乙醇中的应用

【背景】醇脱氢酶AdhS能催化不对称还原反应制备(R)-2-氯-1-苯乙醇,但由于自身再生辅酶NADH的能力不足,需要辅酶再生酶协助其再生NADH。谷氨酸脱氢酶能以谷氨酸为底物,再生辅酶NAD(P)H,具有辅酶再生酶的潜力。【目的】克隆表达谷氨酸脱氢酶基因gdhA,构建谷氨酸脱氢酶GdhA与醇脱氢酶AdhS的大肠杆菌共表达体系,提高AdhS制备(R)-2-氯-1-苯乙醇的转化效率。【方法】从枯草芽孢杆菌(Bacillus subtilis) 168中克隆基因gdhA,并在大肠杆菌(Escherichia coli) BL21(DE3)中表达,分析辅酶再生活力;再与醇脱氢酶AdhS共表达,优化表达条件;分析不同辅酶再生方案对制备(R)-2-氯-1-苯乙醇的转化效率的影响。【结果】谷氨酸脱氢酶GdhA再生NADH的比活力为694 U/g。经GdhA与AdhS的共表达及表达条件优化后,制备(R)-2-氯-1-苯乙醇的转化效率达465 U/L。经比较,GdhA协助再生辅酶NADH,可使AdhS制备(R)-2-氯-1-苯乙醇的转化效率提高到约3倍。【结论】谷氨酸脱氢酶GdhA为NADH高效再生酶,与醇脱氢酶AdhS共表达可显著提高AdhS制备(R)-2-氯-1-苯乙醇的转化效率。     

Expression and kinetic characterization of variants of human beta 1 beta 1 alcohol dehydrogenase containing substitutions at amino acid 47

Arg-47 of human beta 1 beta 1 alcohol dehydrogenase has been replaced with Lys, His, Gln, and Gly by site-directed mutagenesis. The mutated enzymes were expressed in Escherichia coli and purified to homogeneity. The recombinant enzymes with Arg and His at position 47 exhibit kinetic constants and stability which are similar to beta 1 beta 1 and beta 2 beta 2, respectively. The substitution of Lys, His, or Gln for Arg-47 resulted in active enzymes with lower affinity for coenzyme and higher Vmax values than beta 1 beta 1. The substitution of Gln at position 47 resulted in an enzyme with the highest Vmax for ethanol oxidation of any mammalian alcohol dehydrogenase. In this series of enzymes, the affinity for coenzyme decreases with decreasing pKa of the substituted amino acid side chains. The substitution of Gly at position 47 resulted in an enzyme with a Vmax that was one-half that of the low activity beta 1 beta 1 and coenzyme affinities that are lower than beta 1 beta 1, but are equal to or greater than the affinities exhibited by the His-47 or Gln-47 enzymes. Product inhibition studies indicated a change in mechanism from ordered Bi Bi for beta 1 beta 1 to rapid equilibrium random Bi Bi for the Gly-47 enzyme. The kinetic properties of the Gly-47 enzyme are substantially different from human liver alpha alpha which also has Gly at position 47.     

Evidence for the identity of glutathione-dependent formaldehyde dehydrogenase and class III alcohol dehydrogenase

Formaldehyde dehydrogenase (EC 1.2.1.1) is a widely occurring enzyme which catalyzes the oxidation of S-hydroxymethylglutathione, formed from formaldehyde and glutathione, into S-formyglutathione in the presence of NAD. We determined the amino acid sequences for 5 tryptic peptides (containing altogether 57 amino acids) from electrophoretically homogeneous rat liver formaldehyde dehydrogenase and found that they all were exactly homologous to the sequence of rat liver class III alcohol dehydrogenase (ADH-2). Formaldehyde dehydrogenase was found to be able at high pH values to catalyze the NAD-dependent oxidation of long-chain aliphatic alcohols like n-octanol and 12-hydroxydodecanoate but ethanol was used only at very high substrate concentrations and pyrazole was not inhibitory. The amino acid sequence homology and identical structural and kinetic properties indicate that formaldehyde dehydrogenase and the mammalian class III alcohol dehydrogenases are identical enzymes.     

Rat liver alcohol dehydrogenase of class III. Primary structure, functional consequences and relationships to other alcohol dehydrogenases

The amino acid sequence of alcohol dehydrogenase of class III from rat liver (the enzyme ADH-2) has been determined. This type of structure is quite different from those of both the class I and the class II alcohol dehydrogenases. The rat class III structure differs from the rat and human class I structures by 133-138 residues (exact value depending on species and isozyme type); and from that of human class II by 132 residues. In contrast, the rat/human species difference within the class III enzymes is only 21 residues. The protein was carboxymethylated with iodo[2(14)C]acetate, and cleaved with CNBr and proteolytic enzymes. Peptides purified by exclusion chromatography and reverse-phase high-performance liquid chromatography were analyzed by degradation with a gas-phase sequencer and with the manual 4-N,N-dimethylaminoazobenzene-4'-isothiocyanate double-coupling method. The protein chain has 373 residues with a blocked N terminus. No evidence was obtained for heterogeneity. The rat ADH-2 enzyme of class III contains an insertion of Cys at position 60 in relation to the class I enzymes, while the latter alcohol dehydrogenase in rat (ADH-3) has another Cys insertion (at position 111) relative to ADH-2. The structure deduced explains the characteristic differences of the class III alcohol dehydrogenase in relation to the other classes of alcohol dehydrogenase, including a high absorbance, an anodic electrophoretic mobility and special kinetic properties. The main amino acid substitutions are found in the catalytic domain and in the subunit interacting segments of the coenzyme-binding domain, the latter explaining the lack of hybrid dimers between subunits of different classes. Several substitutions provide an enlarged and more hydrophilic substrate-binding pocket, which appears compatible with a higher water content in the pocket and hence could possibly explain the higher Km for all substrates as compared with the corresponding values for the class I enzymes. Finally the class III structure supports evolutionary relationships suggesting that the three classes constitute clearly separate enzymes within the group of mammalian zinc-containing alcohol dehydrogenases.     

Molecular cloning and characterization of (R)-3-hydroxybutyrate dehydrogenase from human heart.

The complete amino acid sequence of human heart (R)-3-hydroxybutyrate dehydrogenase (EC 1.1.1.30) has been deduced from the nucleotide sequence of cDNA clones. This mitochondrial enzyme has an absolute and specific requirement of phosphatidylcholine for enzymic activity (allosteric activator) and is an important prototype of lipid-requiring enzymes. Despite extensive studies, the primary sequence has not been available and is now reported. The mature form of the enzyme consists of 297 amino acids (predicted M(r) of 33,117), does not appear to contain any transmembrane helices, and is homologous with the family of short-chain alcohol dehydrogenases (SC-ADH) (Persson, B., Krook, M., and J?rnvall, H. (1991) Eur. J. Biochem. 200, 537-543) (30% residue identity with human 17 beta-hydroxysteroid dehydrogenase). The first two-thirds of the enzyme includes both putative coenzyme binding and active site conserved residues and exhibits a predicted secondary structure motif (alternating alpha-helices and beta-sheet) characteristic of SC-ADH. Bovine heart peptide sequences (174 residues in nine sequences determined by microsequencing) have extensive homology (89% identical residues) with the deduced human heart sequence. The C-terminal third (Asn-194 to Arg-297) shows little sequence homology with the SC-ADH and likely contains elements that determine the substrate specificity for the enzyme including the phospholipid (phosphatidylcholine) binding site(s). Northern blot analysis identifies a 1.3-kilobase mRNA encoding the enzyme in heart tissue.     

A vertebrate aldo-keto reductase active with retinoids and ethanol

Enzymes of the short chain and medium chain dehydrogenase/reductase families have been demonstrated to participate in the oxidoreduction of ethanol and retinoids. Mammals and amphibians contain, in the upper digestive tract mucosa, alcohol dehydrogenases of the medium chain dehydrogenase/reductase family, active with ethanol and retinol. In the present work, we searched for a similar enzyme in an avian species (Gallus domesticus). We found that chicken does not contain the homologous enzyme from the medium chain dehydrogenase/reductase family but an oxidoreductase from the aldo-keto reductase family, with retinal reductase and alcohol dehydrogenase activities. The amino acid sequence shows 66-69% residue identity with the aldose reductase and aldose reductase-like enzymes. Chicken aldo-keto reductase is a monomer of M(r) 36,000 expressed in eye, tongue, and esophagus. The enzyme can oxidize aliphatic alcohols, such as ethanol, and it is very efficient in all-trans- and 9-cis-retinal reduction (k(cat)/K(m) = 5,300 and 32,000 mm(-1).min(-1), respectively). This finding represents the inclusion of the aldo-keto reductase family, with the (alpha/beta)(8) barrel structure, into the scenario of retinoid metabolism and, therefore, of the regulation of vertebrate development and tissue differentiation.     

Malate dehydrogenase: a model for structure, evolution, and catalysis.

Malate dehydrogenases are widely distributed and alignment of the amino acid sequences show that the enzyme has diverged into 2 main phylogenetic groups. Multiple amino acid sequence alignments of malate dehydrogenases also show that there is a low degree of primary structural similarity, apart from in several positions crucial for nucleotide binding, catalysis, and the subunit interface. The 3-dimensional structures of several malate dehydrogenases are similar, despite their low amino acid sequence identity. The coenzyme specificity of malate dehydrogenase may be modulated by substitution of a single residue, as can the substrate specificity. The mechanism of catalysis of malate dehydrogenase is similar to that of lactate dehydrogenase, an enzyme with which it shares a similar 3-dimensional structure. Substitution of a single amino acid residue of a lactate dehydrogenase changes the enzyme specificity to that of a malate dehydrogenase, but a similar substitution in a malate dehydrogenase resulted in relaxation of the high degree of specificity for oxaloacetate. Knowledge of the 3-dimensional structures of malate and lactate dehydrogenases allows the redesign of enzymes by rational rather than random mutation and may have important commercial implications.     

UDP-glucose dehydrogenase from bovine liver: primary structure and relationship to other dehydrogenases.

The primary structure of bovine liver UDP-glucose dehydrogenase (UDPGDH), a hexameric, NAD(+)-linked enzyme, has been determined at the protein level. The 52-kDa subunits are composed of 468 amino acid residues, with a free N-terminus and a Ser/Asn microhetergeneity at one position. The sequence shares 29.6% positional identity with GDP-mannose dehydrogenase from Pseudomonas, confirming a similarity earlier noted between active site peptides. This degree of similarity is comparable to the 31.1% identity vs. the UDPGDH from type A Streptococcus. Database searching also revealed similarities to a hypothetical sequence from Salmonella typhimurium and to "UDP-N-acetyl-mannosaminuronic acid dehydrogenase" from Escherichia coli. Pairwise identities between bovine UDPGDH and each of these sequences were all in the range of approximately 26-34%. Multiple alignment of all 5 sequences indicates common ancestry for these 4-electron-transferring enzymes. There are 27 strictly conserved residues, including a cysteine residue at position 275, earlier identified by chemical modification as the expected catalytic residue of the second half-reaction (conversion of UDP-aldehydoglucose to UDP-glucuronic acid), and 2 lysine residues, at positions 219 and 338, one of which may be the expected catalytic residue for the first half-reaction (conversion of UDP-glucose to UDP-aldehydoglucose). A GXGXXG pattern characteristic of the coenzyme-binding fold is found at positions 11-16, close to the N-terminus as with "short-chain" alcohol dehydrogenases.(ABSTRACT TRUNCATED AT 250 WORDS)     

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

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.     

Cloning, sequence analysis, and expression in Escherichia coli of the gene encoding monovalent cation-activated levodione reductase from Corynebacterium aquaticum M-13

The gene encoding (6R)-2,2,6-trimethyl-1,4-cyclohexanedione (levodione) reductase was cloned from the genomic DNA of the soil isolate bacterium Corynebacterium aquaticum M-13. The gene contained an open reading frame consisting of 801 nucleotides corresponding to 267 amino acid residues. The deduced amino acid sequence showed approximately 35% identity with other short chain alcohol dehydrogenase/reductase (SDR) superfamily enzymes. The probable NADH-binding site and three catalytic residues (Ser-Tyr-Lys) were conserved. The enzyme was sufficiently produced in recombinant Escherichia coli cells using an expression vector pKK223-3, and purified to homogeneity by two-column chromatography steps. The enzyme purified from E. coli catalyzed stereo- and regio-selective reduction of levodione, and was strongly activated by monovalent cations, such as K+, Na+, and NH4+, as was the case of that from C. aquaticum M-13. To our knowledge, this is the first sequencing report of a monovalent cation-activated SDR enzyme.     

Amino acid sequence of ovine 6-phosphogluconate dehydrogenase

The amino acid sequence of the NADP+-dependent enzyme ovine 6-phosphogluconate dehydrogenase has been determined by conventional direct protein sequence analysis of peptides resulting from digestion of the protein with trypsin and chemical cleavages with cyanogen bromide, hydroxylamine, and iodosobenzoic acid. The polypeptide contains 466 amino acids and its NH2 terminus is acetylated. The Candida utilis enzyme is inactivated by reaction of pyridoxal phosphate with two lysine residues (Minchiotti, L., Ronchi, S., and Rippa, M. (1981) Biochim. Biophys. Acta 657, 232-242). These residues are conserved in the ovine enzyme. In contrast to NAD+ dehydrogenases which have weakly related sequences and spatially related folds in their nucleotide-binding sites, no significant sequence homologies were detected between 6-phosphogluconate dehydrogenase and any of three other NADP+-requiring enzymes, glutamate dehydrogenase, p-hydroxybenzoate hydroxylase, and dihydrofolate reductase. This is in accord with structural data that show no spatial relationship between NADP+-binding sites in these enzymes.     

Structural studies on glyceraldehyde-phosphate dehydrogenase from rat skeletal muscle

The NH2-terminal amino acid sequence of rat skeletal muscle glyceraldehydephosphate dehydrogenase (D-glyceraldehyde-3-phosphate : NAD+ oxidoreductase(physphorylating), EC 1.2.1.12) was determined to be Val-Lys-Val-Gly-Val-Asn-Gly-Phe-Gly-Arg-Ile-Gly-Arg-Leu-Val-Thr-Arg-Ala-Ala-Phe-Ser-Ser-(-)-(-)--Val-Asx-Ile-Val-Ala-Ile. The presence of Asn instead of Asp in position 6 differentiates this enzyme from other glyceraldehyde-3-phosphate dehydrogenases so far sequenced with the exception of the enzymes isolated from liver. The location of Asn in position 6 has been considered as a specific property of liver glyceraldehyde-3-phosphate dehydrogenase (Kulbe, K.D., Jackson, K.W. and Tang, J. (1975) Biochem. Biophys. Res. Commun. 67, 35--42); this suggestion is not sustained by the results of the present investigation. The amino acid composition of the rat skeletal muscle dehydrogenase demonstrates the unusually low histidine content of this enzyme as compared to other mammalian muscle glyceraldehyde-phosphate dehydrogenases.     

Peromyscus alcohol dehydrogenase: Lack of cross-reacting material in enzyme-negative animals

Two forms of alcohol dehydrogenase (ADH), coded by allelic genes, have been purified to homogeneity from Peromyscus. Monospecific antisera to the purified enzymes have been raised in rabbits. These antisera fail to detect cross-reacting material in the liver of ADH-negative animals on Ouchterlony plates. Immuno-titration of anti-ADH antiserum with ADH in liver extracts from AdhS/AdhS and AdhS/AdhN animals results in identical equivalence points, again suggesting the absence of cross-reacting material coded by the AdhN allele. Over a wide range of anti-ADH antiserum dilutions, radiolabeled protein was not immunoprecipitable from liver extracts of AdhN/AdhN animals. These immunochemical tests, in conjunction with previous studies, suggest that the AdhN allele in Peromyscus does not produce inactive polypeptide in normal levels that bears immunological determinants similar to those of the fast and slow ADH isozymes.     

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

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.     

Determination of some biochemical and structural features of alcohol dehydrogenases from Drosophila simulans and Drosophila virilis. Comparison of their properties with the Drosophila melanogaster Adhs enzyme.

The biochemical properties of the enzyme alcohol dehydrogenase of two different Drosophila species, Drosophila simulans and Drosophila virilis, were studied and compared with those of Drosophila melanogaster Adhs enzyme. All of them consist of two identical subunits of molecular weight 27800 and share significant similarities in function. The substrate specificities of these enzymes were characterized and Km(app.) and Vmax.(app.) values were calculated. All these alcohol dehydrogenases show greater affinity for secondary rather than for primary alcohols. The amino acid compositions of the three enzymes were determined, and there is a close similarity between the D. simulans and the D. melanogaster enzymes, but there are significant differences from the alcohol dehydrogenase of D. virilis. The N-terminal amino acid is blocked and the C-terminal amino acid is the same for all three alcohol dehydrogenases. The enzymes from the three species were carboxymethylated and digested with trypsin. The peptide 'maps' reveal, as expected, more homologies between the enzymes of D. simulans and D. melanogaster than with the enzyme of D. virilis.