Stereoselective carveol dehydrogenase from Rhodococcus erythropolis DCL14 - A novel nicotinoprotein belonging to the short chain dehydrogenase/reductase superfamily

A novel nicotinoprotein, catalyzing the dichlorophenolindophenol-dependent oxidation of carveol to carvone, was purified to homogeneity from Rhodococc us erythropolis DCL14. The enzyme is specifically induced after growth on l imonene and carveol Dichlorophenolindophenol-dependent carveol dehydrogenas e (CDH) is a homotetramer of 120 kDa with each subunit containing a tightly bound NAD(H) molecule. The enzyme is optimally active at pH 5.5 and 50 deg rees C and displays a broad substrate specificity with a preference for sub stituted cyclohexanols. When incubated with a diastereomeric mixture of (4R )- or (4S)-carveol, CDH stereoselectively catalyzes the conversion of the ( 6S)-carveol stereoisomers only, Kinetic studies with pure stereoisomers sho wed that this is due to large differences in V-max/K-m values and simultane ous product inhibition by CR)- or (S)-carvone. The R. erythropolis CDH gene (limC) was identified in an operon encoding the enzymes involved in Iimone ne degradation. The CDH nucleotide sequence revealed an open reading frame of 831 base pairs encoding a 277-amino acid protein with a deduced mass of 29,531 Da. The CDH primary structure shares 10-30% sequence identity with m embers of the short chain dehydrogenase/reductase superfamily. Structure ho mology modeling with trihydroxynaphthalene reductase fi om Magnaporthe gris ea suggests-that CDH from R. erythropolis DCL14 is an alpha/beta one-domain protein with an extra loop insertion involved in NAD binding and a flexibl e C-terminal part involved in monoterpene binding.