Cloning and characterization of a Nocardia corallina B-276 gene cluster encoding alkene monooxygenase

Abstract Alkene monooxygenase (AMO) of Nocardia corallina B-276, which consists of three components, expoxidase, reductase and coupling protein, catalyses the oxidation of alkenes to the corresponding epoxides. The NH 2 terminal amino acid sequences of the large and small subunits of the epoxidase were used for the preparation of synthetic oligonucleotides as hybridization probes. A 6.4-kb Bam HI fragment, which contained DNA sequences hybridizing to the probes, was cloned in Escherichia coli . DNA sequencing and comparison of the determined NH 2 terminal amino acid sequences identified a four-gene cluster, amoABCD , within this region. The subunits of epoxidase (small and large), reductase and coupling protein were encoded by amoA, amoC, amoD and amoB , respectively. When the cloned amoABCD gene cluster was placed under the control of a lac promoter on a recombinant pUC18 plasmid in E. coli and induced with isopropyl β- d -thiogalactopyranoside, epoxidation activities were expressed. The amoABCD genes show a homology to the reported nucleotide sequence for methane monooxygenase from methanotrophic bacteria. The existence of a conserved pair of the amino acid sequence Glu-X-X-His in the large subunit of epoxidase is consistent with the assignment of the epoxidase to the class of O 3 -activating proteins containing diiron-oxo clusters.