Cloning, Expression, and Site-Directed Mutagenesis of the Propene Monooxygenase Genes fromMycobacteriumsp. Strain M156

Abstract

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...

Reconstitution of Active Mycobacterial Binuclear Iron Monooxygenase Complex in Escherichia coli

“…PmoABCD from Mycobacterium sp. strain M156 also shows epoxidation activity toward alkenes (10). In addition, propane monooxygenase (PrmABCD) (11,12) and tetrahydrofuran monooxygenase (ThmABCD) (13) from actinomycetous strains have high catalytic potential for applications in biocatalysis and biodegradation.…”

“…The gene clusters encoding the actinomycetous monooxygenases described above have been successfully identified and cloned, while attempts to express these gene clusters in heterologous hosts have encountered difficulties (10,14). In particular, expression of these gene clusters in Escherichia coli has been unsuccessful, although this extensively characterized and developed model microorganism is an ideal host for biochemical characterization and biotechnological applications of enzymes.…”

“…Similarly, E. coli cells transformed with pmoABCD from Mycobacterium sp. strain M156 were not able to acquire oxidation activity (10). Chan Kwo Chion et al suggested that the unsuccessful expression could be attributed to overlapping reading frames between pmoA and pmoB and between pmoC and pmoD (10).…”

Protocol for Mutagenesis of Alkene Monooxygenase and Screening for Modified Enantiocomposition of the Epoxypropane Product

“…during styrene epoxidation. 7 In another study, mutagenesis at position 103 in toluene 4-monooxygenase, a proposed gating residue controlling access to the active site, increased yield but not stereoselectivity during epoxygenation of butadiene. 9 The substrate specificity of toluene 4-monooxygenase has been manipulated by directed evolution 11 and that of sMMO by site-directed mutagenesis.…”

“…4,5 The AMO oxygenase has the same number of subunits as the propene monooxygenase (PMO) of Mycobacterium sp. strain M156, 7 which we propose should become the 2nd member of the AMO subclass of BMMs. X-ray structures for the terminal oxygenases of 2 sMMOs and 1 TMO have been solved, indicating a common global fold for the diiron-containing subunits.…”