A novel secondary alcohol dehydrogenase from Micrococcus luteus WIUJH20: purification, cloning, and properties

Abstract

A novel NAD+‐dependent secondary alcohol dehydrogenase (2o‐ADH) has been purified to homogeneity from Micrococcus luteus WIUJH20 (M. luteus WIUJH20). It has a molecular weight of ~ 140 kDa as determined by gel filtration chromatography, and a molecular weight of ~ 34 kDa as determined by SDS‐PAGE. Thus, the enzyme is a homotetramer. The gene encodes for this enzyme has been amplified by PCR from the genome of M. luteus WIUJH20, cloned into pGEX2T expression vector, and recombinant protein overexpressed as a GST fusion protein (rGST‐2o‐ADH). Based on sequence homology, the enzyme is a NAD+‐dependent L‐3‐hydroxyacyl‐CoA dehydrogenase. But based on its catalytic activity, the enzyme belongs to secondary alcohol dehydrogenase. The rGST‐2o‐ADH fusion protein, the recombinant 2o‐ADH (r2o‐ADH) with GST‐tag removed, and the 2o‐ADH isolated from M. Luteus WIUJH20 had broad substrate specificity. Enzyme kinetics analysis showed that the r2o‐ADH possesses a Km and Vmax of 3.25 x 10−6 M and 150.3 μmol/min/mg respectively, when 12‐hydroxyoctadecanoic acid was the substrate. This r2o‐ADH prefers to use linear secondary alcohols as substrates over the cyclic secondary alcohols. The enzyme prefers long‐chain alcohol over short‐chain alcohol as shown by the decreasing in Km from 4‐heptanol (7 mM) to 6‐dodecanol (8 μM). Primary alcohols (particularly ethanol) are inhibitors to the 2o‐ADH and the inhibition constant Ki increases as the hydrocarbon chain length decreases.The research was supported in part by grants from USDA from USDA CSREES 04‐35504‐14712 and 02‐015470, Western Illinois University Foundation, College of Arts and Sciences and the Honors College at Western Illinois University.