Characterization of a Zinc-Containing Alcohol Dehydrogenase with Stereoselectivity from the Hyperthermophilic Archaeon Thermococcus guaymasensis

Abstract

An alcohol dehydrogenase (ADH) from hyperthermophilic archaeon Thermococcus guaymasensis was purified to homogeneity and was found to be a homotetramer with a subunit size of 40 ؎ 1 kDa. The gene encoding the enzyme was cloned and sequenced; this gene had 1,095 bp, corresponding to 365 amino acids, and showed high sequence homology to zinc-containing ADHs and L-threonine dehydrogenases with binding motifs of catalytic zinc and NADP ؉ . Metal analyses revealed that this NADP ؉ -dependent enzyme contained 0.9 ؎ 0.03 g-atoms of zinc per subunit. It was a primary-secondary ADH and exhibited a substrate preference for secondary alcohols and corresponding ketones. Particularly, the enzyme with unusual stereoselectivity catalyzed an anti-Prelog reduction of racemic (R/S)-acetoin to (2R,3R)-2,3-butanediol and meso-2,3-butanediol. The optimal pH values for the oxidation and formation of alcohols were 10.5 and 7.5, respectively. Besides being hyperthermostable, the enzyme activity increased as the temperature was elevated up to 95°C. The enzyme was active in the presence of methanol up to 40% (vol/vol) in the assay mixture. The reduction of ketones underwent high efficiency by coupling with excess isopropanol to regenerate NADPH. The kinetic parameters of the enzyme showed that the apparent K m values and catalytic efficiency for NADPH were 40 times lower and 5 times higher than those for NADP ؉ , respectively. The physiological roles of the enzyme were proposed to be in the formation of alcohols such as ethanol or acetoin concomitant to the NADPH oxidation.

Purification and Characterization of an NAD + -Dependent XylB-Like Aryl Alcohol Dehydrogenase Identified in Acinetobacter baylyi ADP1

“…The complete inhibition of a zinc-containing enzyme by Zn 2ϩ was somehow unexpected, but was previously also reported for AlrA from Acinetobacter (31) and ADH from Thermococcus guaymasensis (37). Nevertheless, the inhibition of the enzyme activity by the mentioned metal ions suggests the importance of sulfhydryl groups for the activity of the enzyme which is also supported by the sensitivity of XylB ADP1 to N-ethylmaleimide, a thiol-blocking reagent that is known to inhibit all bacterial aromatic alcohol dehydrogenases as well as mammalian and yeast alcohol dehydrogenases (17).…”

“…In contrast to many other alcohol dehydrogenases, XylB ADP1 showed no susceptibility to iodoacetamide. Although XylB ADP1 belongs to the zinc-containing alcohol dehydrogenases, it was not or only slightly susceptible to inhibition by metal ion chelators like EDTA or 1,10-phenanthroline, respectively, a property it shares with, for example, AlrA and AreB from Acinetobacter (17,31), XylB from the P. putida TOL-plasmid (27), several other bacterial aromatic alcohol dehydrogenases (8,29), and the archaeal ADH from T. guaymasensis (37). Contrary to this, the zinccontaining yeast and horse liver alcohol dehydrogenases are sensitive to metal ion chelators (30), indicating a general difference in resistance of the zinc ions to chelation between prokaryotic and eukaryotic alcohol dehydrogenases.…”

Carbohydrate Metabolism in Archaea: Current Insights into Unusual Enzymes and Pathways and Their Regulation

“…However, in HfxGDH, none of these coordinating residues is conserved, and accordingly, the structural zinc has not been detected in the crystal structures (270). Thus, the structural zinc identified in the other archaeal GDHs seems not to be functionally essential, as also described for other MDR family members (273,(275)(276)(277).…”

Molecular Characterization of an NADPH-Dependent Acetoin Reductase/2,3-Butanediol Dehydrogenase from Clostridium beijerinckii NCIMB 8052

“…However, all known ACRs contain several cysteine residues (ranging from 3 to 11), while a sensitivity to oxygen has never been reported, except for the ACR/BDH from the archaeon Thermococcus guaymasensis (36). Moreover, as discussed above, several of the cysteine residues are proposed to be involved in binding of the zinc ions.…”

“…3) and may therefore be responsible for the observed oxygen sensitivity. Also, the ACR/BDH from T. guaymasensis contains a cysteine in an unusual position, possibly explaining its oxygen sensitivity (36).…”