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A new approach to understanding structure-function relationships in cytochromes P450 by targeting terpene metabolism in the wild.

ABSTRACT: A strategy for elucidating sequence determinants of function in the class of cytochrome P450 (CYP) enzymes that catalyze the first steps of terpene metabolism in wild microbiomes is described. Wild organisms that can use camphor, terpineol, pinene and limonene were isolated from soils rich in coniferous waste. Cell free extracts and growth beers were analyzed by gas chromatography/mass spectrometry to identify primary oxidative metabolites. For one organism, Pseudomonas nitroreducens TPJM, a cytochrome P450 (CYP108B1) isolated from cell free extracts was demonstrated to catalyze the oxidation of ?-terpineol in assays combining the native ferredoxin and putidaredoxin reductase, and the resulting oxidation products identified by gas chromatography/mass spectrometry. Shotgun sequencing of PnTPJM identified four candidate P450 genes, including an apparently fragmentary gene with a high degree of homology with the known enzyme CYP108 (P450terp).

SUBMITTER: Wong NR

PROVIDER: S-EPMC6258033 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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A new approach to understanding structure-function relationships in cytochromes P450 by targeting terpene metabolism in the wild.

Wong Nathan R NR Liu Xinyue X Lloyd Hannah H Colthart Allison M AM Ferrazzoli Alexander E AE Cooper Deani L DL Zhuang Yihao Y Esquea Phillix P Futcher Jeffrey J Pochapsky Theodore M TM Matthews Jeffrey M JM Pochapsky Thomas C TC

Journal of inorganic biochemistry 20180806

A strategy for elucidating sequence determinants of function in the class of cytochrome P450 (CYP) enzymes that catalyze the first steps of terpene metabolism in wild microbiomes is described. Wild organisms that can use camphor, terpineol, pinene and limonene were isolated from soils rich in coniferous waste. Cell free extracts and growth beers were analyzed by gas chromatography/mass spectrometry to identify primary oxidative metabolites. For one organism, Pseudomonas nitroreducens TPJM, a cyt ...[more]

PMID: 30170307

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Project description:Benzalkonium chlorides (BACs) are widely used as disinfectants in cleaning products, medical products, and the food processing industry. Despite a wide range of reported toxicities, limited studies have been conducted on the metabolism of these compounds in animal models and none in human-derived cells or tissues. In this work, we report on the metabolism of BACs in human liver microsomes (HLM) and by recombinant human hepatic cytochrome P450 (CYP) enzymes. BAC metabolism in HLM was NADPH-dependent and displayed apparent half-lives that increased with BAC alkyl chain length (C10 < C12 < C14 < C16), suggesting enhanced metabolic stability of the more lipophilic, longer chain BACs. Metabolites of d7-benzyl labeled BAC substrates retained all deuteriums and there was no evidence of N-dealkylation. Tandem mass spectrometry fragmentation of BAC metabolites confirmed that oxidation occurs on the alkyl chain region. Major metabolites of C10-BAC were identified as ?-hydroxy-, (?-1)-hydroxy-, (?, ?-1)-diol-, (?-1)-ketone-, and ?-carboxylic acid-C10-BAC by liquid chromatography-mass spectrometry comparison with synthetic standards. In a screen of hepatic CYP isoforms, recombinant CYP2D6, CYP4F2, and CYP4F12 consumed substantial quantities of BAC substrates and produced the major microsomal metabolites. The use of potent pan-CYP4 inhibitor HET0016, the specific CYP2D6 inhibitor quinidine, or both confirmed major contributions of CYP4- and CYP2D6-mediated metabolism in the microsomal disappearance of BACs. Kinetic characterization of C10-BAC metabolite formation in HLM demonstrated robust Michaelis-Menten kinetic parameters for ?-hydroxylation (Vmax = 380 pmol/min/mg, Km = 0.69 ?M) and (?-1)-hydroxylation (Vmax = 126 pmol/min/mg, Km = 0.13 ?M) reactions. This work illustrates important roles for CYP4-mediated ?-hydroxylation and CYP2D6/CYP4-mediated (?-1)-hydroxylation during the hepatic elimination of BACs, an environmental contaminant of emerging concern. Furthermore, we demonstrate that CYP-mediated oxidation of C10-BAC mitigates the potent inhibition of cholesterol biosynthesis exhibited by this short-chain BAC.

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Dataset Information

A new approach to understanding structure-function relationships in cytochromes P450 by targeting terpene metabolism in the wild.

Publications

A new approach to understanding structure-function relationships in cytochromes P450 by targeting terpene metabolism in the wild.

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