Insilico Characterization and Homology Modeling of Arabitol Dehydrogenase (ArDH) from Candida albican (original) (raw)
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Annals of the New York Academy of Sciences, 1988
The use of enzymatic reactions in biotechnological processes may be constrained by rapid inactivation of the biocatalysts involved.' The different ways to stabilize enzymes have attracted considerable attention.2 Many enzyme immobilization procedures have been proposed to avoid protein unfolding and aggregati~n.~ On the other hand, immobilization often leads to an increase in.diffusion restraint and, thus, it cannot be applied conveniently in industrial processes involving nowNewtonian reaction media. Recently, newly developed techniques such as protein engineering and directed mutagenesis have been used to limit enzyme inactivation' and to study the thermostability mechanism.' Another possible approach to increase enzyme longevity is to use additives such as sugars and polyols that preserve the biocatalyst activity by modulating its microenvironment.6 The exact mechanism of enzyme protection by additives is not yet perfectly known and, therefore, a better understanding of protein-solvent interactions is needed. Mainly, the influence of carbon chain length has been studied.' Uedaira et a1.* also have correlated the protective effect of a sugar to its hydroxyl radical position. The addition of these solutes has been reported to prevent the enzyme from unfolding by strengthening hydrogen bonds or by strengthening hydrophobic interactions (or both). Solute-solvent interactions may be approached through the thermodynamical water activity concept (a,)? When comparing the previously described it becomes obvious that the efficiency of an additive to preserve enzymatic activity is dependent both on its chemical character and on the enzyme itself. It is then of interest to focus on the understanding of stabilization mechanisms.
Structure and function of yeast alcohol dehydrogenase
Journal of the Serbian Chemical Society, 2000
1. Introduction 2. Isoenzymes of YADH 3. Substrate specificity 4. Kinetic mechanism 5. Primary structure 6. The active site 7. Mutations in the yeast enzyme 8. Chemical mechanism 9. Binding of coenzymes 10. Hydride transfer This article has been corrected. Link to the correction 10.2298/JSC0008609E
Journal of Molecular Biology, 2005
The R-specific alcohol dehydrogenase (RADH) from Lactobacillus brevis is an NADP-dependent, homotetrameric member of the extended enzyme family of short-chain dehydrogenases/reductases (SDR) with a high biotechnological application potential. Its preferred in vitro substrates are prochiral ketones like acetophenone with almost invariably a small methyl group as one substituent and a bulky (often aromatic) moiety as the other. On the basis of an atomic-resolution structure of wild-type RADH in complex with NADP and acetophenone, we designed the mutant RADH-G37D, which should possess an improved cosubstrate specificity profile for biotechnological purposes, namely, a preference for NAD rather than NADP. Comparative kinetic measurements with wild-type and mutant RADH showed that this aim was achieved. To characterize the successful mutant structurally, we determined several, partly atomic-resolution, crystal structures of RADH-G37D both as an apo-enzyme and as ternary complex with NAD or NADH and phenylethanol. The increased affinity of RADH-G37D for NAD(H) depends on an interaction between the adenosine ribose moiety of NAD and the inserted aspartate side-chain. A structural comparison between RADH-G37D as apo-enzyme and as a part of a ternary complex revealed significant rearrangements of Ser141, Glu144, Tyr189 and Met205 in the vicinity of the active site. This plasticity contributes to generate a small hydrophobic pocket for the methyl group typical for RADH substrates, and a hydrophobic coat for the second, more variable and often aromatic, substituent. Around Ser141 we even found alternative conformations in the backbone. A structural adaptability in this region, which we describe here for the first time for an SDR enzyme, is probably functionally important, because it concerns Ser142, a member of the highly conserved catalytic tetrad typical for SDR enzymes. Moreover, it affects an extended proton relay system that has been identified recently as a critical element for the catalytic mechanism in SDR enzymes.
Applied and Environmental Microbiology, 2007
Saccharomyces cerevisiae strains that produce the sugar alcohols xylitol and ribitol and the pentose sugar D-ribose from D-glucose in a single fermentation step are described. A transketolase-deficient S. cerevisiae strain accumulated D-xylulose 5-phosphate intracellularly and released ribitol and pentose sugars (D-ribose, Dribulose, and D-xylulose) into the growth medium. Expression of the xylitol dehydrogenase-encoding gene XYL2 of Pichia stipitis in the transketolase-deficient strain resulted in an 8.5-fold enhancement of the total amount of the excreted sugar alcohols ribitol and xylitol. The additional introduction of the 2-deoxy-glucose 6-phosphate phosphatase-encoding gene DOG1 into the transketolase-deficient strain expressing the XYL2 gene resulted in a further 1.6-fold increase in ribitol production. Finally, deletion of the endogenous xylulokinase-encoding gene XKS1 was necessary to increase the amount of xylitol to 50% of the 5-carbon sugar alcohols excreted. Xylitol is a naturally occurring 5-carbon sugar alcohol present in fruits and vegetables. Its sweetening power is comparable to that of sucrose, while the negative heat solution value gives a cool taste for this sugar alcohol. Xylitol inhibits dental caries and acute otitis media (18, 42) and is an ideal sweetener for diabetics because its metabolism is insulin independent. Xylitol is used in products such as chewing gums, sweets, and toothpaste. It is currently produced by chemical reduction, with a nickel catalyst, of the five-carbon sugar Dxylose from birch wood hydrolysates. D-Xylose can also be reduced to xylitol with high yields by various yeast species (9, 43). Both processes rely on the hydrolysis and/or purification of D-xylose from lignocellulosic materials. D-Glucose, on the other hand, is a common substrate in the food industry and is, compared to D-xylose, cheap and readily available. Thus, it would be attractive if xylitol could be produced from D-glucose. However, few if any natural microorganisms that produce xylitol from D-glucose are known. The species Bacillus, Zygosaccharomyces, Aureobasidium, Torula, and Candida convert D-glucose to other sugars and sugar alcohols such as D-ribose, D-arabitol, and erythritol (1, 4, 5, 12, 36). In D-ribose-producing Bacillus and Candida strains, the precursor is D-ribose 5-phosphate, a pentose phosphate pathway (PPP) intermediate. In these strains, transketolase activity, which catalyzes the conversion of D-xylulose 5-phosphate and D-ribose 5-phosphate or erythrose 4-phosphate to C 7 and C 3 and C 6 and C 3 products, respectively, in the nonoxidative branch of the PPP, is missing or defective (5, 13, 33). It was suggested that this deficiency results in the accumulation of D-ribose 5-phosphate and its dephosphorylation and excretion as D-ribose. Similarly, the PPP intermediate D-ribulose 5-phos
European Journal of Biochemistry, 1995
The lack of crystal structure for tetrameric yeast alcohol dehydrogenases (ADHs) has precluded, until now, the identification of the residues involved in subunit contacts. In order to address this question, we have characterized the thermal stability and dissociation propensity of native ADH I and ADH I1 isozymes as well as of several chimeric (ADH I-ADH 11) enzymes. Three groups of substitutions affecting the thermostability have been identified among the 24 substitutions observed between isozymes I and 11. The first group contains a Cys277+Ser substitution, located at the interface between subunits in a threedimensional model of ADH I, based on the crystallographic structure of the dimeric horse liver ADH. In the second group, the Asp236-Asn substitution is located in the same interaction zone on the model. The stabilizing effect of this substitution can result from the removal of a charge repulsion between subunits. It is shown that the effect of these two groups of substitutions correlates with changes in dissociation propensities. The third group contains the Metl68+Arg substitution that increases the thermal stability, probably by the formation of an additional salt bridge between subunits through the putative interface. These data suggest that at least part of the subunit contacts observed in horse liver ADH are located at homologous positions in yeast ADHs.
Enzyme and Microbial Technology, 2007
The present communication reports on changes in the secondary and tertiary structures of native and apo-yeast alcohol dehydrogenase upon heating at 50 • C, as evident from circular dichroism (CD) studies. The presence of sugars provided significant protection with trehalose being the most effective. Exposure of hydrophobic clusters in the protein molecule upon heat denaturation was confirmed by fluorescence studies using 1-anilinonaphthalene-8-sulfonate (ANS) as a hydrophobic reporter probe. All sugars, and especially trehalose, reduced the affinity of both forms of the enzyme for this probe. The effectiveness of sugars in diminishing ANS fluorescence enhancement is in accordance with their ability to protect aggregation of the proteins, reported earlier [Miroliaei M, Nemat-Gorgani M. Sugars protect native and apo yeast alcohol dehydrogenase against irreversible thermoinactivation. Enzyme Microb Technol 2001;29:554-9]. It is concluded that prevention of the mechanisms of irreversible thermoinactivation may occur with retention of the secondary and tertiary structural properties of the proteins.
FEBS Letters, 1984
The recently determined primary structure of glucose dehydrogenase from ~a~i~~as megater~~m was scanned by computerized comparisons for similarities with known polyol and alcohol dehydrogenases. The results revealed a highly significant similarity between this glucose dehydrogenase and ribitol dehydrogenase from Klebsiella aerogenes. Sixty-one positions of the 262 in glucose dehydrogenase are identical between these two proteins (23% identity), fitting into a homology alignment for the complete polypeptide chains. The extent of similarity is equivalent to that between other highly divergent but clearly related dehydrogenases (two zinc-containing alcohol dehydrogenases, 25%; sorbitol and zinc-containing alcohol dehydrogenases, 25%; ribitol and non-zinc-containing alcohol dehydrogenases, 20%), and suggests an ancestral relationship between glucose and ribitol dehydrogenases from different bactera. The similarities fit into a previously suggested evolutionary scheme comprjsing short and long aIcohol and polyol dehydrogenases, and greatly extend the former group to one composed of non-zinc-containing alcoholpolyol-glucose dehydrogenases.
Journal of Molecular Catalysis B-enzymatic, 2002
A NAD-dependent secondary alcohol dehydrogenase (SAD) has been extracted from cells of the sterol-degrading bacterium, Rhodococcus sp. GK1 (CIP 105335). The dehydrogenase was partially purified by means of ammonium sulfate fractionation (60% saturation) and filtration on a Sepharose CL-6B column. The obtained enzyme sample was active with aliphatic secondary alcohols, such as 2-hexanol, and as reductase with aliphatic monoketones and diketones, such as 2-hexanone and 2,3-hexanedione. A hydrophobic environment was required for catalysis: methyl on one side and either methyl, ethyl, propyl, butyl, pentyl or hexyl on the other side of the function being transformed. The K m value for NAD or NADH with, respectively 2-propanol or acetone was around 1.60×10 −4 M at pH 7.0 and 30 • C. The enzyme affinity (1/K m ) for the examined 2-alcohols and 2-ketones (three to eight C atoms) increased with increasing the chain length. Its activity with 2-octanone was somewhat higher than that with 3-octanone, reflecting a better enzyme affinity for a function positioned at C-2. The K m values for the 2-alcohols (pH 7.0, 30 • C) ranged from 6.0 × 10 −2 M for 2-propanol to 1.8 × 10 −3 M for 2-octanol. Reciprocally, the K m values for the 2-ketones ranged from 6.5 × 10 −2 M for acetone to 2.1 × 10 −3 M for 2-octanone. With 2-hexanol as the substrate, the optimal temperature was around 55 • C and the activation energy of the system was 9.49 kcal/mol. The SAD was specific for the (S)-(+)-stereoisomers of 2-butanol.
Canadian Journal of Microbiology, 1992
. Purification and properties of alcohol dehydrogenase from a mutant strain of Candida guilliermondii deficient in one form of the enzyme. Can. J. Microbiol. 38: 953-957. Alcohol dehydrogenase (ADH 1) was purified from Candida guilliermondii strain BlO-05 to homogeneity, using affinity chromatography on triazine dyes and gel filtration. The enzyme was tetrameric, with a subunit molecular weight of 38 000. The purified enzyme oxidized primary and secondary alcohols, although it preferred primary alcohols. Its activity toward secondary alcohols was better than those of other yeast ADH; however, the enzyme was less sensitive toward inhibitors. Kinetic studies indicated that C. guilliermondii ADHl oxidized ethanol by an ordered bi-bi mechanism, with NAD as the first substrate fixed. Key words: Candida guilliermondii, alcohol dehydrogenase, ADH 1, tetrameric. INDRATI, R., et OHTA, Y. 1992. Purification and properties of alcohol dehydrogenase from a mutant strain of Candida guilliermondii deficient in one form of the enzyme. Can. J. Microbiol. 38 : 953-957. L'alcool deshydrogenase (ADHI) a ete purifiee jusqu'a homogeneite a partir de la souche B10-05 de Candida guilliermondii par chromatographie d'affinite sur des colorants triazines et par filtration sur gel. L'enzyme est un tetramke et posskde une sous-unite de 38 000 de poids moleculaire. L'enzyme oxyde les alcools primaires et secondaires mais a une activite preferentielle vis-a-vis des alcools primaires. L'activite sur les alcools secondaires est meilleure que celle d'autres ADH de levures, mais l'enzyme est moins sensible aux inhibiteurs. Les mesures cinetiques indiquent que l'ADH1 de C. guilliermondii oxyde l'ethanol selon un mecanisme bi-bi ordonne oh le NAD est le permier substrat fixe.
American Journal of Bioscience and Bioengineering, 2016
Allitol is an alcohol monosaccharide, is a reduction of D-psicose. It is functions as a cross linking of D-and Lhexoses. It existed in too small quantities in commercial sugars and is difficult to synthesize by using chemical methods. It has a hypoglycemic function, and can use as Laxative in treating of constipation, which can exploit in production of diabetes drugs. The present report investigates about the production of allitol by ribitol dehydrogenase (RDH), its action of the enzyme through homology and molecular docking studies. We have investigated ribitol dehydrogenase (RDH) from providencia alcalifaciens RIMD 1656011. The protein sequence of RDH was conducted for homology modeling through Swiss model. 3D structure revealed was docked with NAD + and D-psicose using AutoDock Vina software version 5.6. The results of homology modeling and docking studies revealed that the conserved residues of RDH were Tyr 153, Tyr 92, Ser 17 and Lys157 with NAD + , while conserved residues with D-psicose were GLN67 and ASP61. NAD + has good interaction with RDH showing grid score of-49.84, which is a good score for binding.