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

Istituto di Biochimica delle Proteine, Consiglio Nazionale delle Ricerche, Via P. Castellino 111, I-80131 Naples, Italy; Istituto di Chimica e del Riconoscimento Molecolare, Consiglio Nazionale delle Ricerche, Via M. Bianco 9, I-20131 Milano, Italy

^* To whom correspondence should be addressed. Email: ca.raia{at}ibp.cnr.it.

	Abstract

The gene encoding a novel alcohol dehydrogenase (ADH) that belongs to the short-chain dehydrogenases/reductases (SDRs) superfamily has been identified in the extremely thermophilic, halotolerant gram-negative eubacterium Thermus thermophilus HB27. The ttadh gene was heterologously overexpressed in Escherichia coli and the protein (TtADH) was purified to homogeneity and characterized. TtADH is a tetrameric enzyme of identical 26,961 Da subunits composed of 256 amino acids. The enzyme has remarkable thermophilicity and thermal stability, displaying activity up to 73°C, a 30 min half-inactivation temperature of 90°C, and good tolerance towards common organic solvents. TtADH shows a strict requirement for NAD(H) as the coenzyme, preference for reduction of aromatic ketones and -keto esters, and poor activity on aromatic alcohols and aldehydes. The thermophilic enzyme catalyses the following reactions with Prelog specificity: the reduction of acetophenone, 2,2,2-trifluoroacetophenone, -tetralone, -methyl and -ethyl benzoylformate to (S)-(-)-1-phenylethanol (>99% enantiomeric excess, ee), (R)--(trifluoromethyl)benzyl alcohol (93% ee), (S)-1-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 D37 in discriminating NAD(H) from NADP(H) in members of the SDR superfamily.