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Applied and Environmental Microbiology, July 2008, p. 3949-3958, Vol. 74, No. 13
0099-2240/08/$08.00+0     doi:10.1128/AEM.00217-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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

Angela Pennacchio,1 Biagio Pucci,1,{dagger} Francesco Secundo,2 Francesco La Cara,1 Mosè Rossi,1 and Carlo A. Raia1*

Istituto di Biochimica delle Proteine, Consiglio Nazionale delle Ricerche, Via P. Castellino 111, I-80131 Naples, Italy,1 Istituto di Chimica e del Riconoscimento Molecolare, Consiglio Nazionale delle Ricerche, Via M. Bianco 9, I-20131 Milano, Italy2

Received 24 January 2008/ Accepted 27 April 2008

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 (adhTt) was heterologously overexpressed in Escherichia coli, and the protein (ADHTt) was purified to homogeneity and characterized. ADHTt 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 ~73°C and a 30-min half-inactivation temperature of ~90°C, as well as good tolerance to common organic solvents. ADHTt has a strict requirement for NAD(H) as the coenzyme, a preference for reduction of aromatic ketones and {alpha}-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, {alpha}-tetralone, and {alpha}-methyl and {alpha}-ethyl benzoylformates to (S)-(–)-1-phenylethanol (>99% enantiomeric excess [ee]), (R)-{alpha}-(trifluoromethyl)benzyl alcohol (93% ee), (S)-{alpha}-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.

* Corresponding author. Mailing address: Istituto di Biochimica delle Proteine, CNR, Via P. Castellino 111, 80131 Naples, Italy. Phone: 39-0816132560. Fax: 39-0816132277. E-mail: ca.raia{at}ibp.cnr.it

{triangledown} Published ahead of print on 2 May 2008.

{dagger} Present address: Centro Ricerche Oncologiche Mercogliano (CROM), Avellino, Italy.

Applied and Environmental Microbiology, July 2008, p. 3949-3958, Vol. 74, No. 13
0099-2240/08/$08.00+0     doi:10.1128/AEM.00217-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

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