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

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.