Molecular characterization of cytochrome P450 genes in the polycyclic aromatic hydrocarbon degrading Mycobacterium vanbaalenii PYR-1.
Mycobacterium vanbaalenii PYR-1 has the ability to degrade low- and high-molecular-weight polycyclic aromatic hydrocarbons (PAHs). In addition to dioxygenases, cytochrome P450 monooxygenases have been implicated in PAH degradation. Three cytochrome P450 genes, cyp151 (pipA), cyp150, and cyp51, were detected and amplified by polymerase chain reaction from M. vanbaalenii PYR-1. The complete sequence of these genes was determined. The translated putative proteins were > or = 80% identical to other GenBank-listed mycobacterial CYP151, CYP150, and CYP51. Genes pipA and cyp150 were cloned, and the proteins partially expressed in Escherichia coli as soluble heme-containing cytochrome P450s that exhibited a characteristic peak at 450 nm in reduced carbon monoxide difference spectra. Monooxygenation metabolites of pyrene, dibenzothiophene, and 7-methylbenz[alpha]anthracene were detected in whole cell biotransformations, with E. coli expressing pipA or cyp150 when analyzed by gas chromatography/mass spectrometry. The cytochrome P450 inhibitor metyrapone strongly inhibited the S-oxidation of dibenzothiophene. Thirteen other Mycobacterium strains were screened for the presence of pipA, cyp150, and cyp51 genes, as well as the initial PAH dioxygenase (nidA and nidB). The results indicated that many of the Mycobacterium spp. surveyed contain both monooxygenases and dioxygenases to degrade PAHs. Our results provide further evidence for the diverse enzymatic capability of Mycobacterium spp. to metabolize polycyclic aromatic hydrocarbons.
Brezna
Barbara
B
Division of Microbiology, National Center for Toxicological Research, US Food and Drug Administration, 3900 NCTR Road, Jefferson, AR 72079, USA.
Kweon
Ohgew
O
Stingley
Robin L
RL
Freeman
James P
JP
Khan
Ashraf A
AA
Polek
Bystrik
B
Jones
Richard C
RC
Cerniglia
Carl E
CE
D000595:Amino Acid Sequence; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics; D008401:Gas Chromatography-Mass Spectrometry; D008969:Molecular Sequence Data; D009161:Mycobacterium / Q000201:enzymology* / Q000235:genetics*; D010802:Phylogeny; D011084:Polycyclic Aromatic Hydrocarbons / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013056:Spectrophotometry, Ultraviolet
D004269:DNA, Bacterial; D011084:Polycyclic Aromatic Hydrocarbons; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System