Characteristics of alcohol/polyol dehydrogenases. The zinc-containing long-chain alcohol dehydrogenases

Sixteen characterized alcohol dehydrogenases and one sorbitol dehydrogenase have been aligned. The proteins represent two formally different enzyme activities (EC 1.1.1.1 and EC 1.1.1.14), three different types of molecule (dimeric alcohol dehydrogenase, tetrameric alcohol dehydrogenase, tetrameric sorbitol dehydrogenase), metalloproteins with different zinc contents (1 or 2 atoms per subunit), and polypeptide chains from different kingdoms and orders (mammals, higher plants, fungus, yeasts). Present comparisons utilizing all 17 forms reveal extensive variations in alcohol dehydrogenase, but with evolutionary changes that are of the same order in different branches and at different times. They emphasize the general importance of particular residues, suggesting related overall functional constraints in the molecules. The comparisons also define a few coincidences between intron positions in the genes and gap positions in the gene products. Only 22 residues are strictly conserved; half of these are Gly, and most of the remaining ones are Pro or acidic residues. No basic residue, no straight-chain hydrophobic residues, no aromatic residues, and essentially no branched-chain or polar neutral residues are invariable. Tentative consensus sequences were calculated, defining 13 additional residues likely to be typical of but not invariant among the alcohol dehydrogenases. These show a predominance of Val, charged residues, and Gly. Combined, the comparisons, which are particularly relevant to the data base for protein engineering, illustrate the requirements for functionally important binding interactions, and the extent of space restrictions in proteins with related overall conformations and functions.     

Selective oxidation and reduction reactions with cofactor regeneration mediated by galactitol-, lactate-, and formate dehydrogenases immobilized on magnetic nanoparticles

Rapid immobilization with the one-pot purification of galactitol dehydrogenase (GatDH) and formate dehydrogenase (FDH) is achieved by using iminodiacetic acid (IDA) with chelated Co²⁺ modified magnetic nanoparticles as a carrier. Lactate dehydrogenase (LDH) from recombinant Escherichia coli and FDH commencing Candida methylica were used as an auxiliary enzyme for the regeneration of NADH/NAD⁺ with a representative synthesis of (S)-1,2-propanediol and l-tagatose starting from hydroxyacetone and galactitol. The affinity magnetic nanoparticles were characterized by scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR), while the purity of GatDH and FDH was assayed by SDS-PAGE analysis. The immobilized two-enzyme system, reflecting the pH dependence of its constituent enzymes, showed optimal activity at pH 7 and 8 for (S)-1,2-propanediol and l-tagatose production, respectively. The immobilized enzyme system retained up to 70% of its activity after one week of repeated use. The use of affinity magnetic nanoparticles offers the advantage of a one-pot purification of His(6)-tagged GatDH and FDH followed by the production of rare sugar and chiral diol.     

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.     

Embryogeny of Phaseolus: Developmental pattern of lactate and alcohol dehydrogenases

Alcohol and lactate dehydrogenase activity and their electrophoretic isoenzymes were determined in developing Phaseolus vulgaris embryos, seed coat     

Structures of human alcohol and aldehyde dehydrogenases

Human alcohol dehydrogenase is a dimeric zinc metalloenzyme for which forms of three classes, I, II and III, have been distinguished. Subunits hybridize within but not between classes. There are three types of subunit, alpha, beta, and gamma, in class I. The primary structures of all three forms have been established, as well as the overall properties and the effects of the amino acid substitutions between the various forms. Each subunit has 374 residues, of which 35 exhibit differences among the alpha, beta and gamma chains. Corresponding cDNA structures are also known, as are the genetic organization and details of the gene structures. Allelic variants occur at the beta and gamma loci. Corresponding amino acid substitutions have been characterized, and enzymatic differences between the allelic forms are explained by defined residue exchanges. The results also illustrate recent and repeated isozyme evolution, a subject where alcohol dehydrogenases exceptionally well offer detailed examples. Human aldehyde dehydrogenase occurs of two types, a mitochondrial and a cytosolic form. The enzymes are tetramers, do not contain functional metals, and have subunits which do not form inter-type hybrids. The primary structures have been determined, revealing a positional identity of 68% (in 500 residues) between the mitochondrial and cytosolic forms. The N-terminus is heterogeneous and is not blocked in the subunit of the mitochondrial enzyme, in contrast to that of the cytosolic enzyme or those of all the alcohol dehydrogenases (also cytosolic). A reactive cysteine residue at position 302 has been ascribed functional importance at or close to the active site, is conserved in the two aldehyde dehydrogenases, and is associated with the action of disulfiram on the enzyme. In Oriental populations, a mutant allelic variant of the mitochondrial protein with impaired enzyme function has also been characterized.     

Synthesis of silica particles and their application as supports for alcohol dehydrogenases and cofactor immobilizations: Conformational changes that lead to switch in enzyme stereoselectivity

FTIR, circular dichroism (CD) and fluorescence spectroscopies were used to characterize conformational changes in horse liver alcohol dehydrogenase (HLADH) and ketoreductase (KRED 117) upon physical and covalent immobilizations on silica particles (functionalized with amino, epoxy and thiol groups) of different sizes. Conformational changes for immobilized enzymes were associated with high and low frequency shifts of the amide I and II bands. CD spectra of native HLADH and KRED 117 characterized with a negative peak at 222nm indicating a α-helical structure. The disappearance of the negative peak in the CD spectra of immobilized enzymes and appearance of a positive peak at 222nm supported these observations. These findings demonstrated unfolding of folded enzymes and exposure of the amino acid residues during denaturation with a red shift in tryptophan fluorescence. The decrease in specific activities (by 60-70% in all cases) for both immobilized enzymes was correlated to those of conformational changes. Silica-attached enzyme-NADH systems were evaluated for enantioselective reduction of 1-(p-methoxyphenyl)-propan-2-one. Conformational changes enhanced the enantioselectivity of immobilized HLADH with a switch in its stereoselectivity. In the case of immobilized KRED 117, kinetic values (V(max) and K(m)) were lower than that of the free enzyme, without enhancing enzyme enantio- and stereoselectivity.     

Purification and comparative studies of alcohol dehydrogenases

Alcohol dehydrogenases from various animal and plant sources were purified by a common procedure which employed DEAE, Sephadex-G100 and affinity chromatographies. The procedure achieves an 80-130 fold purification for animal enzymes. However, only a 5-15 fold purification for plant enzymes was attained because of the instability of these enzymes. Purified alcohol dehydrogenases from animal and plant sources differ in coenzyme and substrate specificities. The enzymes from mammalian, avian and fish livers display aldehyde oxidizing and esterolytic activities in addition to alcohol oxidizing activity. However, the enzymes from plants and yeast show only the oxidative activity toward alcohols. Chemical modifications have been performed to identify amino acid residues which are essential to the oxidative and esterolytic activities of alcohol dehydrogenases.     

Developmental expression of alcohol dehydrogenases in maize

Alcohol dehydrogenase (ADH) in Zea mays exists in five distinct electrophoretic forms (isozymes), ADH-1, ADH-2, ADH-3, ADH-4, and ADH-T. The mode of inheritance of ADH-1 and ADH-2 has been previously reported; preliminary data suggest that ADH-3 is controlled by a different locus than ADH-2; no genetic analysis has yet been made for ADH-4 and ADH-T. Analyses at different stages of ontogenesis and of different organs have shown that the ADH isozyme pattern fluctuates qualitatively and quantitatively during the course of development and differentiation of the maize plant. ADH-T is controlled spatially and temporally in a very strict manner, being present only in extracts from the pericarp of 19- to 40-day-old kernels. ADH-3 and ADH-4 are present in the scutella of mature kernels and during early sporophytic development. ADH-1 and ADH-2 are the most common isozymes in all tissues examined, but ADH-1 is not found in endosperm of mature kernels or during germination. None of the isozymes have been found to be associated with any particulate cellular component at any stage of development. These findings are discussed with respect to differential gene expression, physiology, and cellular metabolism.     

Theoretical calculations of the catalytic triad in short-chain alcohol dehydrogenases/reductases

Three highly conserved active site residues (Ser, Tyr, and Lys) of the family of short-chain alcohol dehydrogenases/reductases (SDRs) were demonstrated to be essential for catalytic activity and have been denoted the catalytic triad of SDRs. In this study computational methods were adopted to study the ionization properties of these amino acids in SDRs from Drosophila melanogaster and Drosophila lebanonensis. Three enzyme models, with different ionization scenarios of the catalytic triad that might be possible when inhibitors bind to the enzyme cofactor complex, were constructed. The binding of the two alcohol competitive inhibitors were studied using automatic docking by the Internal Coordinate Mechanics program, molecular dynamic (MD) simulations with the AMBER program package, calculation of the free energy of ligand binding by the linear interaction energy method, and the hydropathic interactions force field. The calculations indicated that deprotonated Tyr acts as a strong base in the binary enzyme-NAD⁺ complex. Molecular dynamic simulations for 5 ns confirmed that deprotonated Tyr is essential for anchoring and orientating the inhibitors at the active site, which might be a general trend for the family of SDRs. The findings here have implications for the development of therapeutically important SDR inhibitors.     

Aerobic and anaerobic alcohol dehydrogenases in Escherichia coli

Abstract Mutants unable to use ethanol for carbon and energy were counterselected from an ethanolutilizing mutant of Escherichia coli K12 derepressed for alcohol dehydrogenase (ADH). Mutants of one class were devoid of ADH activity under anaerobic conditions but exhibited aerobic activities comparable to those of wild-type E. coli. Mutants of a second class exhibited ADH activity levels intermediate between those of the wild-type and derepressed parent. Immunological studies showed that mutants of the former class synthesized far less ADH protein than did the derepressed parent while mutants of the latter class synthesized about the same amount. The ADH mutations in both classes were located within the previously described adh region which contains the structural gene for the activity that is derepressed in the parent. An Eth⁻ adh-lac fusion mutant with an insertion in the structural gene was also isolated and characterized. It exhibited no ADH activity under anaerobic conditions and wild-type levels under aerobic conditions. These data are consistent with the existence in E. coli of distinct aerobic and anaerobic ADH enzymes and a derepression of the anaerobic but not the aerobic enzyme in the ethanol utilizing strain.     

Quinohemoprotein alcohol dehydrogenases: structure, function, and physiology

Quino(hemo)protein alcohol dehydrogenases (ADH) that have pyrroloquinoline quinone (PQQ) as the prosthetic group are classified into 3 groups, types I, II, and III. Type I ADH is a simple quinoprotein having PQQ as the only prosthetic group, while type II and type III ADHs are quinohemoprotein having heme c as well as PQQ in the catalytic polypeptide. Type II ADH is a soluble periplasmic enzyme and is widely distributed in Proteobacteria such as Pseudomonas, Ralstonia, Comamonas, etc. In contrast, type III ADH is a membrane-bound enzyme working on the periplasmic surface solely in acetic acid bacteria. It consists of three subunits that comprise a quinohemoprotein catalytic subunit, a triheme cytochrome c subunit, and a third subunit of unknown function. The catalytic subunits of all the quino(hemo)protein ADHs have a common structural motif, a quinoprotein-specific superbarrel domain, where PQQ is deeply embedded in the center. In addition, in the type II and type III ADHs this subunit contains a unique heme c domain. Various type II ADHs each have a unique substrate specificity, accepting a wide variety of alcohols, as is discussed on the basis of recent X-ray crystallographic analyses. Electron transfer within both type II and III ADHs is discussed in terms of the intramolecular reaction from PQQ to heme c and also from heme to heme, and in terms of the intermolecular reaction with azurin and ubiquinone, respectively. Unique physiological functions of both types of quinohemoprotein ADHs are also discussed.     

Characteristics of short-chain alcohol dehydrogenases and related enzymes

Different short-chain dehydrogenases are distantly related, constituting a protein family now known from at least 20 separate enzymes characterized, but with extensive differences, especially in the C-terminal third of their sequences. Many of the first known members were prokaryotic, but recent additions include mammalian enzymes from placenta, liver and other tissues, including 15-hydroxyprostaglandin, 17 beta-hydroxysteroid and 11 beta-hydroxysteroid dehydrogenases. In addition, species variants, isozyme-like multiplicities and mutants have been reported for several of the structures. Alignments of the different enzymes reveal large homologous parts, with clustered similarities indicating regions of special functional/structural importance. Several of these derive from relationships within a common type of coenzyme-binding domain, but central-chain patterns of similarity go beyond this domain. Total residue identities between enzyme pairs are typically around 25%, but single forms deviate more or less (14-58%). Only six of the 250-odd residues are strictly conserved and seven more are conserved in all but single cases. Over one third of the conserved residues are glycine, showing the importance of conformational and spatial restrictions. Secondary structure predictions, residue distributions and hydrophilicity profiles outline a common, N-terminal coenzyme-binding domain similar to that of other dehydrogenases, and a C-terminal domain with unique segments and presumably individual functions in each case. Strictly conserved residues of possible functional interest are limited, essentially only three polar residues. Asp64, Tyr152 and Lys156 (in the numbering of Drosophila alcohol dehydrogenase), but no histidine or cysteine residue like in the completely different, classical medium-chain alcohol dehydrogenase family. Asp64 is in the suggested coenzyme-binding domain, whereas Tyr152 and Lys156 are close to the center of the protein chain, at a putative inter-domain, active-site segment. Consequently, the overall comparisons suggest the possibility of related mechanisms and domain properties for different members of the short-chain family.     

Enzymatic reduction of complex redox dyes using NADH-dependent reductase from Bacillus subtilis coupled with cofactor regeneration

Conventional vat dyeing involves chemical reduction of dyes into their water-soluble leuco form generating considerable amounts of toxic chemicals in effluents. In the present study, a new β-nicotinamide adenine dinucleotide disodium salt (NADH)-dependent reductase isolated from Bacillus subtilis was used to reduce the redox dyes CI Acid Blue 74, CI Natural Orange 6, and CI Vat Blue 1 into their water-soluble leuco form. Enzymatic reduction was optimized in relation to pH and temperature conditions. The reductase was able to reduce Acid Blue 74 and Natural Orange 6 in the presence of the stoichiometrically consumed cofactor NADH; meanwhile, Vat Blue 1 required the presence of mediator 1,8-dihydroxyanthraquinone. Oxygen from air was used to reoxidize the dyes into their initial forms. The enzymatic reduction of the dyes was studied and the kinetic constants determined, and these were compared to the chemically-reduced leuco form. The enzyme responsible for the reduction showed homology to a NADH-dependent reductase from B. subtilis based on results from the MS/MS peptide mass mapping of the tryptically digested protein. Additionally, the reduction of Acid Blue 74 to its leuco form by reductase from B. subtilis was confirmed using NADH regenerated by the oxidation of formic acid with formate dehydrogenase from Candida boidinii in the same solution.     

Enzyme engineering aspects of biocatalysis: cofactor regeneration as example

Reaction engineering is an important tool in the case of cofactor depending enzyme-catalyzed reactions. It allows the establishment of conditions resulting in lower product specific cofactor costs as compared with product-specific enzyme costs. This is shown for the stereospecific reduction of carbonyl compounds yielding chiral amino acids and alcohols. In continuous processes, cofactor costs can be reduced if the cofactor can be retained within the bioreactor or recycled into it after separation of the product. In case of readily water-soluble substrates it is even possible to recycle the cofactor during a single pass through a continuously operated reactor more than 4000 times because normally very low cofactor concentrations are sufficient to saturate the enzymes involved. L-tert-Leucine has been produced by reductive amination with a space-time yield of up to 366 g L(-1) d(-1) in a single continuously operated enzyme membrane reactor and a two-stage cascade. Total turnover number of the cofactor NAD(+) increased to 4230. (S)-1-Phenyl-2-propanol was obtained by reduction of the corresponding ketone in an membrane reactor with integrated extraction of the product. A new alcohol dehydrogenase from Rhodococcus erythropolis was used. A space-time yield of 63 g L(-1) d(-1) and a total turnover number of 1350 have been reached. L-Leucine has been produced using polymer-enlarged NADH. The total turnover number was 80,000 at a space-time yield of 214 g L(-1) d(-1). (c) 1996 John Wiley & Sons, Inc.     

Recent advances in biotechnological applications of alcohol dehydrogenases

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