Functional Characterization of an NADPH Dependent 2-Alkyl-3-ketoalkanoic Acid Reductase Involved in Olefin Biosynthesis in Stenotrophomonas maltophilia

Abstract

OleD is shown to play a key reductive role in the generation of alkenes (olefins) from acyl thioesters in Stenotrophomonas maltophilia. The gene coding for OleD clusters with three other genes, oleABC, and all appear to be transcribed in the same direction as an operon in various olefin producing bacteria. In this study, a series of substrates varying in chain length and stereochemistry were synthesized and used to elucidate the functional role and substrate specificity of OleD. We demonstrated that OleD, which is an NADP(H) dependent reductase, is a homodimer which catalyzes the reversible stereospecific reduction of 2-alkyl-3-ketoalkanoic acids. Maximal catalytic efficiency was observed with syn-2-decyl-3-hydroxytetradecanoic acid, with a k(cat)/K(m) 5- and 8-fold higher than for syn-2-octyl-3-hydroxydodecanoic acid and syn-2-hexyl-3-hydroxydecanoic acid, respectively. OleD activity was not observed with syn-2-butyl-3-hydroxyoctanoic acid and compounds lacking a 2-alkyl group such as 3-ketodecanoic and 3-hydroxydecanoic acids, suggesting the necessity of the 2-alkyl chain for enzyme recognition and catalysis. Using diastereomeric pairs of substrates and 4 enantiopure isomers of 2-hexyl-3-hydroxydecanoic acid of known stereochemistry, OleD was shown to have a marked stereochemical preference for the (2R,3S)-isomer. Finally, experiments involving OleA and OleD demonstrate the first 3 steps and stereochemical course in olefin formation from acyl thioesters; condensation to form a 2-alkyl-3-ketoacyl thioester, subsequent thioester hydrolysis, and ketone reduction.