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.Several bacteria are capable of aerobic growth on shortchain alkene substrates (C 2 to C 4 ) as sole carbon and energy sources. These include strains of Rhodococcus spp., Mycobacterium spp., Nocardia spp., and Xanthobacter autotrophicus (10). Mycobacterium sp. strain M156 was isolated on propene as the sole carbon source and was shown to initiate alkene oxidation by an O 2 -and NAD(P)H-dependent monooxygenase reaction (32). In propene-utilizing organisms that have been characterized thus far, the further metabolism of propene involves carboxylation to acetoacetate (6), and the absolute requirement of carbon dioxide for the growth of M156 on propene in sparged bioreactors (32) suggests that this is also the case for this strain.Alkene monooxygenases have interesting prospects as biocatalysts for asymmetric synthesis (15). Although they belong to the same family of binuclear nonheme iron monooxygenases as soluble methane monooxygenase (sMMO) (14), they are able to discriminate between nonactivated C-H bonds and double bonds, specifically epoxidizing the latter, often with high enantiomeric excess (20). This discrimination appears to be a fundamental part of the catalytic mechanism, but stereoselectivity is probably a reflection of substrate binding. Therefore, it should be possible to modify stereoselectivity through protein engineering without a loss of reaction specificity, which is not likely to be the case with alkane or methane monooxygenases, for which epoxidation...

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