The Rhodococcus erythropolis DCL14 limonene-1,2-epoxide hydrolase gene encodes an enzyme belonging to a novel class of epoxide hydrolases.
Recently, we reported the purification of the novel enzyme limonene-1,2-epoxide hydrolase involved in limonene degradation by Rhodococcus erythropolis DCL14. The N-terminal amino acid sequence of the purified enzyme was used to design two degenerate primers at the beginning and the end of the 50 amino acids long stretch. Subsequently, the complete limonene-1,2-epoxide hydrolase gene (limA) was isolated from a genomic library of R. erythropolis DCL14 using a combination of PCR and colony hybridization. The limA gene encoded a 149-residue polypeptide with a deduced molecular mass of 16.5 kDa. It was functionally expressed in Escherichia coli. The amino acid sequence of limA contains neither any of the conserved regions of the alpha,beta-hydrolase fold enzymes, to which most of the previously reported epoxide hydrolases belong, nor any of the conserved motifs present in leukotriene A4 hydrolase. The structural data presented in this paper confirm previous physical and biochemical findings [van der Werf et al. (1998) J. Bacteriol. 180, 5052-5057] that limonene-1,2-epoxide hydrolase is the first member of a new class of epoxide hydrolases.
Barbirato
F
F
Department of Food Technology and Nutritional Sciences, Wageningen University and Research Centre, The Netherlands.
Verdoes
J C
JC
de Bont
J A
JA
van der Werf
M J
MJ
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004851:Epoxide Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli; D005798:Genes, Bacterial; D015698:Genomic Library; D007700:Kinetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D012240:Rhodococcus / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development
D001426:Bacterial Proteins; D017931:DNA Primers; D011994:Recombinant Proteins; D004851:Epoxide Hydrolases; C115926:limonene-1,2-epoxide hydrolase