Publications

• Biotransformations catalyzed by cloned p-cymene monooxygenase from Pseudomonas putida F1.

  Nishio T., Patel A., Wang Y., Lau P.C.

  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 ... >> More

  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. << Less

  Appl Microbiol Biotechnol 55:321-325(2001) [PubMed] [EuropePMC]

• Reductase gene sequences and protein structures: p-cymene methyl hydroxylase.

  Dutta T.K., Gunsalus I.C.

  Oxygenases are critical to cycling carbon in the biosphere and dependent on reductase action, principally from flavoprotein enzymes. Oxygenase diversity among organisms and strains carries a common theme of protein sequence and folding. p-Cymene (para-isopropyl toluene) was chosen as a point of co ... >> More

  Oxygenases are critical to cycling carbon in the biosphere and dependent on reductase action, principally from flavoprotein enzymes. Oxygenase diversity among organisms and strains carries a common theme of protein sequence and folding. p-Cymene (para-isopropyl toluene) was chosen as a point of convergence in terpene-aromatic mineralization to characterize a methyl hydroxylase electron transport system with the aerobe Pseudomonas aureofaciens. The cymA hydroxylase reductase gene was isolated and sequenced and the protein primary structure deduced. Optimized amino acid sequence alignments of flavoprotein reductases revealed major similarities over protein length, in the binding domains for NAD(P)H, and the flavine centers of pro- and eukaryote systems. << Less

  Biochem Biophys Res Commun 233:502-506(1997) [PubMed] [EuropePMC]

• p-Cymene catabolic pathway in Pseudomonas putida F1: cloning and characterization of DNA encoding conversion of p-cymene to p-cumate.

  Eaton R.W.

  Pseudomonas putida F1 utilizes p-cymene (p-isopropyltoluene) by an 11-step pathway through p-cumate (p-isopropylbenzoate) to isobutyrate, pyruvate, and acetyl coenzyme A. The cym operon, encoding the conversion of p-cymene to p-cumate, is located just upstream of the cmt operon, which encodes the ... >> More

  Pseudomonas putida F1 utilizes p-cymene (p-isopropyltoluene) by an 11-step pathway through p-cumate (p-isopropylbenzoate) to isobutyrate, pyruvate, and acetyl coenzyme A. The cym operon, encoding the conversion of p-cymene to p-cumate, is located just upstream of the cmt operon, which encodes the further catabolism of p-cumate and is located, in turn, upstream of the tod (toluene catabolism) operon in P. putida F1. The sequences of an 11,236-bp DNA segment carrying the cym operon and a 915-bp DNA segment completing the sequence of the 2,673-bp DNA segment separating the cmt and tod operons have been determined and are discussed here. The cym operon contains six genes in the order cymBCAaAbDE. The gene products have been identified both by functional assays and by comparing deduced amino acid sequences to published sequences. Thus, cymAa and cymAb encode the two components of p-cymene monooxygenase, a hydroxylase and a reductase, respectively; cymB encodes p-cumic alcohol dehydrogenase; cymC encodes p-cumic aldehyde dehydrogenase; cymD encodes a putative outer membrane protein related to gene products of other aromatic hydrocarbon catabolic operons, but having an unknown function in p-cymene catabolism; and cymE encodes an acetyl coenzyme A synthetase whose role in this pathway is also unknown. Upstream of the cym operon is a regulatory gene, cymR. By using recombinant bacteria carrying either the operator-promoter region of the cym operon or the cmt operon upstream of genes encoding readily assayed enzymes, in the presence or absence of cymR, it was demonstrated that cymR encodes a repressor which controls expression of both the cym and cmt operons and is inducible by p-cumate but not p-cymene. Short (less than 350 bp) homologous DNA segments that are located upstream of cymR and between the cmt and tod operons may have been involved in recombination events that led to the current arrangement of cym, cmt, and tod genes in P. putida F1. << Less

  J. Bacteriol. 179:3171-3180(1997) [PubMed] [EuropePMC]

• Cloning and characterization of a p-cymene monooxygenase from Pseudomonas chlororaphis subsp. aureofaciens.

  Dutta T.K., Chakraborty J., Roy M., Ghosal D., Khara P., Gunsalus I.C.

  p-Cymene monooxygenase is the enzyme system that catalyzes the hydroxylation of p-cymene to 4-isopropylbenzyl alcohol (p-cumic alcohol), the initial step in the assimilation of p-cymene by Pseudomonas chlororaphis subsp. aureofaciens. Cloning and sequencing of single NADH-dependent cytochrome c re ... >> More

  p-Cymene monooxygenase is the enzyme system that catalyzes the hydroxylation of p-cymene to 4-isopropylbenzyl alcohol (p-cumic alcohol), the initial step in the assimilation of p-cymene by Pseudomonas chlororaphis subsp. aureofaciens. Cloning and sequencing of single NADH-dependent cytochrome c reductase gene (cymA) present in P. chlororaphis subsp. aureofaciens was described earlier. In this study, analysis of the upstream sequence of cymA revealed two open reading frames, designated as cymB (495 bp) and cymM (1128 bp). Database searches with the cymM gene product showed similarity to integral-membrane di-iron enzymes, while that with cymB showed no significant similarity to other known proteins with the exception of epoxystyrene isomerases. Expression of all three components (cymMBA) in Escherichia coli confirmed its ability for p-cymene methyl group hydroxylation, while expression of cymM and cymA along with the partially truncated cymB gene showed an 85% decrease in the hydroxylation capacity. Our results suggest for the first time that the small protein, CymB, having no conserved domains in protein databases, is involved as enhancer/activator in p-cymene hydroxylation. << Less

  Res Microbiol 161:876-882(2010) [PubMed] [EuropePMC]