Genetic and biochemical characterization of a novel monoterpene epsilon-lactone hydrolase from Rhodococcus erythropolis DCL14

A monoterpene E-lactone hydrolase (MLH) from Rhodococcus erythropolis DCL14 , catalyzing the ring opening of lactones which are formed during degradati on of several monocyclic monoterpenes, including carvone and menthol, was p urified to apparent homogeneity, It is a monomeric enzyme of 31 kDa that is active with (4R)-4-isopropenyl-7-methyl-2-oxo-oxepanone and (6R)-6-isoprop enyl-3-methyl-2-oxo-oxepanone, lactones derived from (4R)-dihydrocarvone, a nd 7-isopropyl-4-methyl-2-oxo-oxepanone, the lactone derived from menthone, Both enantiomers of 4-, 5-, 6-, and 7-methyl-2-oxo-oxepanone were converte d at equal rates, suggesting that the enzyme is not stereoselective. Maxima l enzyme activity was measured at pH 9.5 and 30 degreesC. Determination of the N-terminal amino acid sequence of purified MLH enabled cloning of the c orresponding gene by a combination of PCR and colony screening. The gene, d esignated mlhB (monoterpene lactone hydrolysis), showed up to 43% similarit y to members of the GDXG family of lipolytic enzymes. Sequencing of the adj acent regions revealed two other open reading frames, one encoding a protei n with similarity to the short-chain dehydrogenase reductase family and the second encoding a protein with similarity to acyl coenzyme A dehydrogenase s, Both enzymes are possibly also involved in the monoterpene degradation p athways of this microorganism.