title: Stereoselective carveol dehydrogenase from Rhodococcus erythropolis DCL14. A novel nicotinoprotein belonging to the short chain dehydrogenase/reductase superfamily.
abstract: A novel nicotinoprotein, catalyzing the dichlorophenolindophenol-dependent oxidation of carveol to carvone, was purified to homogeneity from Rhodococcus erythropolis DCL14. The enzyme is specifically induced after growth on limonene and carveol. Dichlorophenolindophenol-dependent carveol dehydrogenase (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 degrees C and displays a broad substrate specificity with a preference for substituted 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 showed that this is due to large differences in V(max)/K(m) values and simultaneous product inhibition by (R)- or (S)-carvone. The R. erythropolis CDH gene (limC) was identified in an operon encoding the enzymes involved in limonene 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 members of the short chain dehydrogenase/reductase superfamily. Structure homology modeling with trihydroxynaphthalene reductase from Magnaporthe grisea 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 flexible C-terminal part involved in monoterpene binding.
authors.affiliation: Division of Industrial Microbiology, Department of Food Technology and Nutritional Sciences, Wageningen University, Bomenweg 2, 6703 HD Wageningen, The Netherlands. mariet.vanderwerf@imb.ftns.wau.nl
authors.forename: M J
authors.initials: MJ
authors.lastname: van der Werf
authors.forename: C
authors.initials: C
authors.lastname: van der Ven
authors.forename: F
authors.initials: F
authors.lastname: Barbirato
authors.forename: M H
authors.initials: MH
authors.lastname: Eppink
authors.forename: J A
authors.initials: JA
authors.lastname: de Bont
authors.forename: W J
authors.initials: WJ
authors.lastname: van Berkel
mesh_terms: D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004790:Enzyme Induction; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D012240:Rhodococcus / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity
chemical_list: D004269:DNA, Bacterial; D009243:NAD; D000429:Alcohol Oxidoreductases; C121536:carveol dehydrogenase