Cloning, expression, and site-directed mutagenesis of the propene monooxygenase genes from Mycobacterium sp. strain M156.
Propene monooxygenase has been cloned from Mycobacterium sp. strain M156, based on hybridization with the amoABCD genes of Rhodococcus corallinus B276. Sequencing indicated that the mycobacterial enzyme is a member of the binuclear nonheme iron monooxygenase family and, in gene order and sequence, is most similar to that from R. corallinus B-276. Attempts were made to express the pmoABCD operon in Escherichia coli and Mycobacterium smegmatis mc(2)155. In the former, there appeared to be a problem resolving overlapping reading frames between pmoA and -B and between pmoC and -D, while in the latter, problems were encountered with plasmid instability when the pmoABCD genes were placed under the control of the hsp60 heat shock promoter in the pNBV1 vector. Fortuitously, constructs with the opposite orientation were constitutively expressed at a level sufficient to allow preliminary mutational analysis. Two PMO active-site residues (A94 and V188) were targeted by site-directed mutagenesis to alter their stereoselectivity. The results suggest that changing the volume occupied by the side chain at V188 leads to a systematic alteration in the stereoselectivity of styrene oxidation, presumably by producing different orientations for substrate binding during catalysis. Changing the volume occupied by the side chain at A94 produced a nonsystematic change in stereoselectivity, which may be attributable to the role of this residue in expansion of the binding site during substrate binding. Neither set of mutations changed the enzyme's specificity for epoxidation.
Chan Kwo Chion
Chan K
CK
Department of Biological Sciences, Imperial College London, London, United Kingdom.
Askew
Sarah E
SE
Leak
David J
DJ
D000475:Alkenes / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D008969:Molecular Sequence Data; D005810:Multigene Family; D016297:Mutagenesis, Site-Directed; D009161:Mycobacterium / Q000201:enzymology* / Q000235:genetics; D010105:Oxygenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D013237:Stereoisomerism
D000475:Alkenes; C013658:propylene; D010105:Oxygenases