Cloning and Characterizaton of a Nocardia corallina B-276 Gene Cluster Encoding Alken Monooxygenase

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Alkene monooxygenase (AMO) of Nocardia corallina B-276,which consists of three componensts, epoxidase, reductase and coupling protein, catalyses the oxidation of alkanes to the corresponding epocides. 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 BamHI 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 indentified 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 culster was placed under the control of a lac promoter on a recombinant pUC18 plasmid in E. coli and induced with isopropyl β-D-thiogalactopyranotide, 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 epoxidae to the class of O_2-activating proteins containing diiron-oxo clusters.
社団法人日本生物工学会の論文
1994-12-25