Pseudomonas Cytochromes P-450

Unger, B.; Sligar, Stephen G.; Gunsalus, I. C.

doi:10.1016/b978-0-12-307210-8.50021-x

Cited by 48 publications

(56 citation statements)

References 86 publications

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“…These two constitutive FR activities are then potentially supplemented by two additional camphor-induced quanta -Fred, a homodimeric 37.0 kDa FR recently identified in camphor-grown NCIMB 10007 (Iwaki et al, 2013), and putidaredoxin reductase (Pdr), a protein first reported in camphor-grown P. putida nearly 50 years ago . Whereas camphor-induced Fred has been recognized previously to serve such a DKCMOsupporting role (Iwaki et al, 2013), the widely accepted role of Pdr in camphor-grown P. putida is as the supplier of FMNH 2 to the haem moiety of the cytochrome P450cam biooxygenating subunit of the multimeric camphor-5-monooxygenase complex (Unger et al, 1986), the camphor-induced hydroxylase that initiates the catabolic pathway for the assimilation of both (+)-and (2)-camphor (Gunsalus et al, 1967;Sokatch, 1986;Fig. S1).…”

Section: Discussionmentioning

confidence: 99%

Multiple native flavin reductases in camphor-metabolizing Pseudomonas putida NCIMB 10007: functional interaction with two-component diketocamphane monooxygenase isoenzymes

Willetts

Kelly

2014

Microbiology

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Although they have been studied for nearly 50 years, the source of the FMNH 2 needed for effective biooxidation by the 2,5-and 3,6-diketocamphane monooxygenase (DKCMO) isoenzymes induced by the growth of Pseudomonas putida NCIMB 10007 (ATCC 17453) on camphor remains incompletely characterized. Prior studies have focussed exclusively on enzymes present in cells harvested during late-exponential-phase growth despite considerable circumstantial evidence that the flavin reductase (FR) component of these multicomponent monooxygenases is subject to growth-phase-dependent variation. In this study, a number of alternative FMNH 2 -generating activities, including both conventional FRs and enzymes also able to serve as ferric reductases, were isolated from camphor-grown cells, and the relative level, and hence potential contribution, of these various proteins shown to vary considerably depending on the point of harvest of NCIMB 10007 within exponential-phase growth. While two constitutive monomeric ferric reductases (molecular masses 27.0 and 28.5 kDa) were found to be the major relevant sources of FMNH 2 during the initial stages of growth on camphor-based media, a significant subsequent contribution throughout the mid-to late-exponential phases of growth was also made by the camphor-induced homodimeric 37.0 kDa FR Fred, recently reported to serve such a role exclusively. The possible involvement of camphor-induced putidaredoxin reductase (51.0 kDa) as a contributory activity was also investigated and considered. Studies with highly purified preparations of the isofunctional DKCMOs confirmed the potential of the various reductases to function effectively as sources of the requisite FMNH 2 to both monooxygenases at different times throughout growth on camphor.

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Section: Discussionmentioning

confidence: 99%

Multiple native flavin reductases in camphor-metabolizing Pseudomonas putida NCIMB 10007: functional interaction with two-component diketocamphane monooxygenase isoenzymes

Willetts

Kelly

2014

Microbiology

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“…Plasmids-pUS200 plasmid carrying the wild type P450cam gene from P. putida was used as described (16). The plasmid carrying the Y96F mutant gene was described previously (17).…”

Section: Methodsmentioning

confidence: 99%

Tyrosine Radical Formation in the Reaction of Wild Type and Mutant Cytochrome P450cam with Peroxy Acids

Schünemann

Lendzian²,

Jung

et al. 2004

Journal of Biological Chemistry

108

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“…Yeast (1), COS 1 (2), and eukaryotic cells infected with a viral vector (3,4) have been used as hosts for the heterologous expression of cytochrome P450 molecules; however, each has limitations to their usefulness as systems for structure-function analysis. Although the bacterium Escherichia coli has demonstrated great usefulness in the expression of many prokaryotic and eukaryotic proteins, E. coli as an expression system for cytochrome P450 has been limited primarily to the soluble prokaryotic forms of this gene superfamily (5). We have used the cDNA encoding bovine 17a-hydroxylase cytochrome P450 (P45017a) to examine the utility of E. coli as an expression system for eukaryotic cytochromes P450 in the hopes that such a system might prove suitable for both enzymatic and structural studies.…”

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confidence: 99%

Expression and enzymatic activity of recombinant cytochrome P450 17 alpha-hydroxylase in Escherichia coli.

Barnes

Arlotto

Waterman

1991

Proc. Natl. Acad. Sci. U.S.A.

520

355

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When the cDNA encoding bovine microsomal 17a-hydroxylase cytochrome P450 (P45017a) containing modifications within the first seven codons which favor expression in Escherichia coli is placed in a highly regulated tac promoter expression plasmid, as much as 16 mg of spectrally detectable P45017a per liter of culture can be synthesized and integrated into E. coli membranes. The known enzymatic activities of bovine P45017a can be reconstituted by addition of purified rat liver NADPH-cytochrome P450 reductase to isolated E. coli membrane fractions containing the recombinant P45017a enzyme. Surprisingly, it is found that E. coli contain an electrontransport system that can substitute for the mammalian microsomal NADPH-cytochrome P450 reductase in supporting both the 17a-hydroxylase and 17,20-lyase activities of P45017a. Thus, not only can E. coli express this eukaryotic membrane protein at relatively high levels, but as evidenced by metabolism of steroids added directly to the cells, the enzyme is catalytically active in vivo. These studies establish E. coli as an efficacious heterologous expression system for structurefunction analysis of the cytochrome P450 system. Microsomal cytochromes P450 are integral membrane hemoproteins that catalyze the oxidative metabolism of a wide variety of endogenous and exogenous compounds. Deriving reducing equivalents from NADPH via a membrane-bound flavoprotein oxidoreductase (NADPH-cytochrome P450 reductase), these mixed-function oxidases activate molecular oxygen so as to insert one atom into a lipophilic substrate and the other atom into water. Recent study of the molecular aspects underlying eukaryotic cytochrome P450 structure and function has relied on the techniques of molecular biology to synthesize specific individual forms ofcytochrome P450 in heterologous expression systems. Yeast (1), COS 1 (2), and eukaryotic cells infected with a viral vector (3, 4) have been used as hosts for the heterologous expression of cytochrome P450 molecules; however, each has limitations to their usefulness as systems for structure-function analysis. Although the bacterium Escherichia coli has demonstrated great usefulness in the expression of many prokaryotic and eukaryotic proteins, E. coli as an expression system for cytochrome P450 has been limited primarily to the soluble prokaryotic forms of this gene superfamily (5). We have used the cDNA encoding bovine 17a-hydroxylase cytochrome P450 (P45017a) to examine the utility of E. coli as an expression system for eukaryotic cytochromes P450 in the hopes that such a system might prove suitable for both enzymatic and structural studies. This microsomal cytochrome P450 catalyzes the regiospecific and stereospecific 17a-hydroxylation of the C21 steroids pregnenolone and progesterone in the pathway leading to the production of cortisol and the 17,20-lyase conversion of 17a-hydroxypregnenolone to the C19 adrenal androgen dehydroepiandrosterone (DHEA) in the adrenal cortex of most mammalian species. P45017a also converts these 17a-hydroxylat...

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Pseudomonas Cytochromes P-450

Cited by 48 publications

References 86 publications

Multiple native flavin reductases in camphor-metabolizing Pseudomonas putida NCIMB 10007: functional interaction with two-component diketocamphane monooxygenase isoenzymes

Multiple native flavin reductases in camphor-metabolizing Pseudomonas putida NCIMB 10007: functional interaction with two-component diketocamphane monooxygenase isoenzymes

Tyrosine Radical Formation in the Reaction of Wild Type and Mutant Cytochrome P450cam with Peroxy Acids

Expression and enzymatic activity of recombinant cytochrome P450 17 alpha-hydroxylase in Escherichia coli.

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