Genetic and biochemical characterization of a novel monoterpene epsilon-lactone hydrolase from Rhodococcus erythropolis DCL14.
A monoterpene epsilon-lactone hydrolase (MLH) from Rhodococcus erythropolis DCL14, catalyzing the ring opening of lactones which are formed during degradation of several monocyclic monoterpenes, including carvone and menthol, was purified 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-isopropenyl-3-methyl-2-oxo-oxepanone, lactones derived from (4R)-dihydrocarvone, and 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 converted at equal rates, suggesting that the enzyme is not stereoselective. Maximal enzyme activity was measured at pH 9.5 and 30 degrees C. Determination of the N-terminal amino acid sequence of purified MLH enabled cloning of the corresponding gene by a combination of PCR and colony screening. The gene, designated mlhB (monoterpene lactone hydrolysis), showed up to 43% similarity to members of the GDXG family of lipolytic enzymes. Sequencing of the adjacent regions revealed two other open reading frames, one encoding a protein with similarity to the short-chain dehydrogenase reductase family and the second encoding a protein with similarity to acyl coenzyme A dehydrogenases. Both enzymes are possibly also involved in the monoterpene degradation pathways of this microorganism.
van der Vlugt-Bergmans
C J
CJ
Division of Industrial Microbiology, Department of Food Technology and Nutritional Sciences, Wageningen University, Wageningen, The Netherlands.
van der Werf
M J
MJ
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D005798:Genes, Bacterial; D006867:Hydrolases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D007783:Lactones / Q000378:metabolism*; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D012240:Rhodococcus / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D017422:Sequence Analysis, DNA; D013729:Terpenes / Q000378:metabolism
D001426:Bacterial Proteins; D007783:Lactones; D013729:Terpenes; C429780:MlhA protein, Rhodococcus erythropolis; D010088:Oxidoreductases; D006867:Hydrolases; D002265:Carboxylic Ester Hydrolases; C429781:MlhB protein, Rhodococcus erythropolis