Purification and characterization of a novel recombinant highly enantioselective short-chain NAD(H)-dependent alcohol dehydrogenase from Thermus thermophilus.
Pennacchio, Angela ; Pucci, Biagio ; Secundo, Francesco ; et al. ; - ASI Sponsor
Jul - 2008
ISSN : 1098-5336 ;
journal : Applied and environmental microbiology

Issue : 13
type: Article Journal

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 approximately 73 degrees C and a 30-min half-inactivation temperature of approximately 90 degrees 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 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.

keywords : Alcohol Dehydrogenase,Alcohol Dehydrogenase: chemistry,Alcohol Dehydrogenase: genetics,Alcohol Dehydrogenase: isolation \& purification,Alcohol Dehydrogenase: metabolism,Amino Acid Sequence,Enzyme Stability,Escherichia coli,Escherichia coli: enzymology,Escherichia coli: genetics,Hot Temperature,Kinetics,Molecular Sequence Data,NAD,NAD: metabolism,Recombinant Proteins,Recombinant Proteins: chemistry,Recombinant Proteins: genetics,Recombinant Proteins: isolation \& purification,Recombinant Proteins: metabolism,Sequence Alignment,Stereoisomerism,Substrate Specificity,Thermus thermophilus,Thermus thermophilus: enzymology,Thermus thermophilus: genetics