Cloning, expression, and site-directed mutagenesis of the propene monooxygenase genes from Mycobacterium sp. 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.

FIG. 1.

Genetic map and features of the alkene monooxygenase genes of Mycobacterium sp. strain M156. (A) pPMOA903, pPMOAC85, and pPMOK201 are constructs created by cloning of the respective SmaI, EcoRI, and PstI fragments, as indicated by the horizontal bars. The E. coli-mycobacterium shuttle constructs for homologous expression are also shown as pNBVpmo11 and pNBVpmo15, with the location of the hsp60 promoter (gray triangles) indicated. Propene oxide formation activities obtained from whole-cell assays are indicated on the right. (B) DNA region showing the end of pmoA overlapping with the start of pmoB. The stop codon and start codon are underlined. (C) Region at the end of pmoC displaying the slippage codons (in italics), the overlapping start and stop codons (underlined), and the plausible stem-loop (inverted arrows).

FIG. 2.

Active-site residues of Mycobacterium sp. strain M156 β subunit (PmoC) aligned with homologs. The sequences aligned with PmoC are as follows: MEMA_METTR from Methylosinus trichosporium OB3b (GenBank accession no. P27353); MEMA_METCA from Methylococcus capsulatus Bath (GenBank accession no. P22869); AmoC from Rhodococcus corallinus B-276 (GenBank accession no. BAA07114); ETNC from M. rhodesiae JS60 (GenBank accession no. AAO48576); the rsph3375 protein, a Rhodococcus sphaeroides hypothetical oxygenase (GenBank accession no. ZP_00007442); the blr3677 protein, a Bradyrhizobium japonicum hypothetical oxygenase (GenBank accession no. BAC48942); PRMA from Gordonia sp. strain TY-5 (GenBank accession no. BAD03956); THMA from Pseudonocardia K1 (GenBank accession no. CAC10506); and TMOA_PSEME from Pseudomonas mendocina (GenBank accession no. Q00456). The alignment shows only some of the amino acids within the active site (Leu110 to Ile239), as previously defined (5, 21, 22). Ligands of the binuclear iron center are joined with black lines. The residues forming the active site are highlighted with white diamonds (⋄).The positions of Ala 94 and Val 188 in PmoC, which were subjected to mutagenesis in this study, are indicated with black triangles. Residues that are known or predicted to form helices (H) are indicated. Secondary structure information was taken from M. capsulatus Bath (pdb code 1mty chain D) and Methylosinus trichosporium (pdb code 1mhy chain D). PmoCpred is the predicted secondary structure of PmoC, as described in Materials and Methods.

FIG. 3.

Stereoselectivities of styrene epoxidation catalyzed by the natural isoform (WT) and mutant isoforms of PMO. The results are from capillary GC-FID analysis of the reaction products obtained from resting cell biotransformation assays. Each experiment was performed in triplicate, and the mean data are presented.