Biotransformations catalyzed by cloned p-cymene monooxygenase from Pseudomonas putida F1.
p-Cymene monooxygenase (CMO) from Pseudomonas putida F1 consists of a hydroxylase (CymA1) and a reductase component (CymA2) which initiate pcymene (p-isopropyltoluene) catabolism by oxidation of the methyl group to p-isopropylbenzyl alcohol (p-cumic alcohol). To study the possible diverse range of substrates catalyzed by CMO, the cymA1A2 genes were cloned in an Escherichia coli pT7-5 expression system and the cells were used in transformation experiments. The tested substrates include different substituents on the aromatic ring at the 2 (ortho), 3 (meta) or 4 (para) position relative to the methyl moiety. As a result, a distinct preference was observed for substrates containing at least an alkyl or heteroatom substituent at the para-position of toluene. The conversion rate of 4-chlorotoluene or 4-methylthiotoluene to the corresponding benzyl alcohol was found to be as good as the canonical substrate, p-cymene. But 3-chlorotoluene, 4-fluorotoluene and 4-nitrotoluene were relatively poor substrates. CMO is also capable of producing styrene oxide from styrene. However, the oxidation of 4-chlorostyrene to 4-chlorostyrene oxide was by far the fastest among the substrates used in this study. The various biotransformation products were identified by a combined solid phase microextraction/gas chromatographic-mass spectrometric analytical technique.
Nishio
T
T
Biotechnology Research Institute, National Research Council Canada, Montreal, Quebec.
Patel
A
A
Wang
Y
Y
Lau
P C
PC
D001426:Bacterial Proteins; D019905:Benzyl Alcohol / Q000032:analysis; D001711:Biotransformation; D003001:Cloning, Molecular; D004852:Epoxy Compounds / Q000032:analysis; D004926:Escherichia coli / Q000235:genetics; D008401:Gas Chromatography-Mass Spectrometry; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D016958:Pseudomonas putida / Q000201:enzymology*; D020058:Styrene / Q000494:pharmacology; D013379:Substrate Specificity; D014050:Toluene / Q000031:analogs & derivatives* / Q000494:pharmacology
D001426:Bacterial Proteins; D004852:Epoxy Compounds; D014050:Toluene; D020058:Styrene; C013690:styrene oxide; C105942:CymA protein, Pseudomonas aureofaciens; D010088:Oxidoreductases; D019905:Benzyl Alcohol; C029049:4-chlorotoluene