Purification and Characterization of a Novel Recombinant Highly Enantioselective Short-Chain NAD(H)-Dependent Alcohol Dehydrogenase from Thermus thermophilus

Abstract

The gene encoding a novel alcohol dehydrogenase (ADH) that belongs to the short-chain dehydrogenase/ reductase (SDR) superfamily was identified in the extremely thermophilic, halotolerant gram-negative eubacterium Thermus thermophilus HB27. The T. thermophilus ADH gene (adh Tt ) was heterologously overexpressed in Escherichia coli, and the protein (ADH Tt ) was purified to homogeneity and characterized. ADH Tt is a tetrameric enzyme consisting of identical 26,961-Da subunits composed of 256 amino acids. The enzyme has remarkable thermophilicity and thermal stability, displaying activity at temperatures up to ϳ73°C and a 30-min half-inactivation temperature of ϳ90°C, as well as good tolerance to common organic solvents. ADH Tt has a strict requirement for NAD(H) as the coenzyme, a preference for reduction of aromatic ketones and ␣-keto esters, and poor activity on aromatic alcohols and aldehydes. This thermophilic enzyme catalyzes the following reactions with Prelog specificity: the reduction of acetophenone, 2,2,2-trifluoroacetophenone, ␣-tetralone, and ␣-methyl and ␣-ethyl benzoylformates to (S)-(؊)-1-phenylethanol (>99% enantiomeric excess [ee]), (R)-␣-(trifluoromethyl)benzyl alcohol (93% ee), (S)-␣-tetralol (>99% ee), methyl (R)-(؊)-mandelate (92% ee), and ethyl (R)-(؊)-mandelate (95% ee), respectively, by way of an efficient in situ NADH-recycling system involving 2-propanol and a second thermophilic ADH. This study further supports the critical role of the D37 residue in discriminating NAD(H) from NADP(H) in members of the SDR superfamily.Alcohol dehydrogenases (ADHs) are of interest for the synthesis of the (S) or (R) enantiomers of alcohols from prochiral ketones (11). Since their early application in asymmetric synthesis using horse liver and yeast ADHs (13) and Thermoanaerobacter brockii ADH (ADH Tb ) (15), screening efforts have been directed at various species of microorganisms, which has resulted in new ADHs that have distinctive substrate specificity, good efficiency, and high enantioselectivity. Representative examples of enzymes from mesophilic microorganisms are the NADP-dependent (R)-specific ADH from Lactobacillus brevis (RADH Lb ), which is active on aryl ketones and whose crystal structure has recently been solved (29), the NAD-dependent (S)-specific 1-phenylethanol dehydrogenase from the denitrifying bacterium strain EbN1 (PED), which was characterized and crystallized (10), and the NAD-dependent ADH from Leifsonia sp. strain S749 (ADH Ls ), which was found to be active on (R)-sec alcohols, aryl ketones, aldehydes, and keto esters and whose gene has recently been cloned for protein expression in Escherichia coli (12). These enzymes are homotetrameric and belong to the short-chain dehydrogenase/ reductase (SDR) superfamily (14), which is characterized by ϳ250-residue subunits, a Gly motif in the coenzyme-binding regions, and a catalytic triad formed by the highly conserved residues Tyr, Lys, and Ser, to which an Asn residue has been added based on the proposal of Filling et al. (2), which was...

Identification of ketone reductase ChKRED20 from the genome of Chryseobacterium sp. CA49 for highly efficient anti-Prelog reduction of 3,5-bis(trifluoromethyl)acetophenone

“…ChKRED20, predicted ketone reductase in this work, KC342020; LSADH, alcohol dehydrogenase from Leifsonia sp. S749, BAD99642 [31]; LK-ADH, R-specific alcohol dehydrogenase from Lactobacillus kefir, AAP94029 [28]; LB-RADH, R-specific alcohol dehydrogenase from Lactobacillus brevis, AJ544275 [32]; TtADH, alcohol dehydrogenase from Thermus thermophilus, 2D1Y [36]; CPADH, (S)-1-phenyl-1,2-ethanediol dehydrogenase from Candida parapsilosis, DQ675534 [29]. Gray shading indicates residues highly conserved in the SDR family.…”

Asymmetric Reduction of α‐Keto Esters with Thermus thermophilus NADH‐Dependent Carbonyl Reductase using Glucose Dehydrogenase and Alcohol Dehydrogenase for Cofactor Regeneration

“…30 mg protein per litre of culture, and that this enzyme shows potential for applications involving bioconversions of substituted acetophenones and aromatic α-keto esters such as 1. [14] Kinetic studies showed that this latter compound was a better substrate when halogenated at the ortho position of the benzene ring. The k cat , K m and k cat /K m values determined for the reduction of 1 were 38.1 Ϯ 3.7 s -1 , 2.7 Ϯ 0.6 mm, and 14.1 s -1 mm -1 , respectively and those for 2 were 7.1 Ϯ 0.2 s -1 , 0.32 Ϯ 0.1 mm, and 22.2 s -1 mm -1 , respectively.…”

“…[13] More recently, an NADH-dependent, highly enantioselective ADH (TtADH), identified from the thermophilic, halotolerant gram-negative eubacterium Thermus thermophilus HB27, has been purified and characterized in our laboratory. [14] The thermophilic enzyme catalyses the reduction of α-methyl and α-ethyl benzoylformate to methyl (R)-mandelate (ee = 91 %) and ethyl (R)-mandelate (ee = 95 %), respectively, by way of an in situ NADH-recycling system involving 2-propanol and Bacillus stearothermophilus ADH (BsADH). [14] This paper reports the enzymatic synthesis of 1a and 2a by TtADH and the evaluation of glucose dehydrogenase as well as BsADH for achieving cofactor regeneration.…”

“…[14] The thermophilic enzyme catalyses the reduction of α-methyl and α-ethyl benzoylformate to methyl (R)-mandelate (ee = 91 %) and ethyl (R)-mandelate (ee = 95 %), respectively, by way of an in situ NADH-recycling system involving 2-propanol and Bacillus stearothermophilus ADH (BsADH). [14] This paper reports the enzymatic synthesis of 1a and 2a by TtADH and the evaluation of glucose dehydrogenase as well as BsADH for achieving cofactor regeneration. The work describes the determination of the optimal reaction time and substrate concentration in the absence and presence of organic solvents and the choice of the alcohol which is most suitable co-substrate for the TtADH/BsADH system.…”

In vitro conversion of glycerol to lactate with thermophilic enzymes

“…Particularly, employment of enzymes derived from thermophiles and hyperthermophiles enables the simple preparation of catalytic modules with excellent selectivity and thermal stability [5,12]. Furthermore, although the detailed mechanisms remain to be clarified, many thermophilic enzymes have also been reported to display higher tolerance towards denaturants such as detergents and organic solvents than their mesophilic counterparts [13,14], and activities of some thermophilic enzymes are even improved with organic solvents [15]. These excellent stabilities of thermophilic enzymes allow great flexibility in the operational conditions of in vitro bioconversion systems.…”