In vivo and in vitro hydroxylation of cineole and camphor by cytochromes P450CYP101A1, CYP101B1 and N242A CYP176A1

Stok, Jeanette E.; Hall, Emma A.; Stone, Isobella S. J.; Noble, Margaret C.; Wong, Siew Hoon; Bell, Stephen; Voss, James J. De

doi:10.1016/j.molcatb.2016.03.004

Cited by 10 publications

(29 citation statements)

References 73 publications

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“…This observation, combined with the minimal shifts triggered by the camphor enantiomers, indicates that 1,8-cineole is the physiological substrate for the newly discovered P450s. Furthermore, despite being shown to oxidize 1,8-cineole, CYP101A has a K d value that is higher and a spin-state shift that is smaller (14) than those of the P450s from S. yanoikuyae. These functional variations due to sequence divergence and presumably structural differences warrant further investigations and comparison.…”

Section: Discussionmentioning

confidence: 90%

“…CYP101A1 was char-acterized based on its activity with camphor (18). More recently, both CYP101B1 and CYP101A1 also have been shown to hydroxylate 1,8-cineole, producing a range of hydroxy-1,8-cineole isomers (14). The P450 from C. braakii is unique, as no other bacterial enzymes have been found that belong to the CYP176 family or use similar electron transfer partners.…”

Section: Discussionmentioning

confidence: 99%

“…Interestingly, even though the 1,8-cineole-hydroxylating proteins from the two different microorganisms share limited sequence similarity and hydroxylate in different orientations, both are able to use electron transfer chains present in E. coli (11). Compared to the N242A P450 cin mutant CYP101A1 and CYP101B1, which each produce a series of hydroxylated 1,8-cineoles, the CYP101J enzymes from S. yanoikuyae B2 appear to hydroxylate 1,8-cineole more specifically (14).…”

Section: Discussionmentioning

confidence: 99%

“…The initial oxidation of 1,8-cineole by this Gram-negative bacterium is catalyzed by a P450 designated P450 cin (CYP176A1), and with the aid of suitable electron transport partners it yielded (1R)-6␤-hydroxy-1,8-cineole (7,11). More recently, other P450s, including CYP101A1, CYP101B1, and the N252A P450 cin mutant, were shown to hydroxylate 1,8-cineole and yield a range of hydroxy-1,8-cineole derivatives (14,15).…”

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confidence: 99%

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CYP101J2, CYP101J3, and CYP101J4, 1,8-Cineole-Hydroxylating Cytochrome P450 Monooxygenases from Sphingobium yanoikuyae Strain B2

Unterweger

Bulach

Scoble

et al. 2016

Appl Environ Microbiol

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We report the isolation and characterization of three new cytochrome P450 monooxygenases: CYP101J2, CYP101J3, and CYP101J4. These P450s were derived from Sphingobium yanoikuyae B2, a strain that was isolated from activated sludge based on its ability to fully mineralize 1, 8 T he bicyclic monoterpenoid 1,8-cineole (1,3,3-trimethyl-2-oxabicyclo[2,2,2]octane) is a chemically stable saturated ditertiary ether (1) that is an abundant natural resource. It is a major component of many essential oils, in particular Eucalyptus oil (2). While 1,8-cineole has its main applications in pharmaceutical, fragrance, food, and cleaning industries, its oxidation provides a potential route to value-added chemicals. Hydroxylated 1,8-cineole derivatives are chiral compounds that can serve as building blocks and precursors with uses in organic chemistry (3). Hydroxylation of 1,8-cineole can be achieved chemically; however, these reactions often use a range of environmentally hazardous reagents and/or yield product mixtures (4-6). On the other hand, biooxygenation of 1,8-cineole using enzymes or whole cells can be performed under mild reaction conditions with the potential to yield products with high enantiopurity (6, 7).A potential route to bio-derived 1,8-cineole derivatives is the utilization of enzymes involved in bacterial 1,8-cineole metabolism. Bacterial 1,8-cineole metabolism was first examined in the late 1970s, when the isolation of a Pseudomonas flava strain capable of growing on 1,8-cineole as a sole source of carbon was reported (8). Subsequently, 1,8-cineole biohydroxylation has also been observed in several other bacterial species, including Rhodococcus sp. strain C1 (9), Bacillus cereus (10), Citrobacter braakii (11), Novosphingobium subterranea (12), and a Sphingomonas species (13). The first bacterial 1,8-cineole-hydroxylating enzyme which was isolated and characterized was isolated from C. braakii (11). The initial oxidation of 1,8-cineole by this Gram-negative bacterium is catalyzed by a P450 designated P450 cin (CYP176A1), and with the aid of suitable electron transport partners it yielded (1R)-6␤-hydroxy-1,8-cineole (7, 11). More recently, other P450s, including CYP101A1, CYP101B1, and the N252A P450 cin mutant,

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Section: Discussionmentioning

confidence: 90%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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CYP101J2, CYP101J3, and CYP101J4, 1,8-Cineole-Hydroxylating Cytochrome P450 Monooxygenases from Sphingobium yanoikuyae Strain B2

Unterweger

Bulach

Scoble

et al. 2016

Appl Environ Microbiol

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“…Recently, it has been shown that P450 enzymes can hydroxylate 1,8‐cineole . 1,8‐Cineole is a renewable compound found in eucalyptus oil that belongs to the diverse class of monoterpenoids .…”

Section: Introductionmentioning

confidence: 99%

X‐ray crystal structure of cytochrome P450 monooxygenase CYP101J2 fromSphingobium yanoikuyaestrain B2

et al. 2017

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The cytochrome P450 monooxygenases (P450s) catalyze a vast array of oxygenation reactions that can be useful in biocatalytic applications. CYP101J2 from Sphingobium yanoikuyae is a P450 that catalyzes the hydroxylation of 1,8-cineole. Here we report the crystallization and X-ray structure elucidation of recombinant CYP101J2 to 1.8 Å resolution. The CYP101J2 structure shows the canonical P450-fold and has an open conformation in the absence of substrate. Analysis of the structure revealed that CYP101J2, in the absence of substrate, forms a well-ordered substrate-binding channel that suggests a unique form of substrate guidance in comparison to other bacterial 1,8-cineole-hydroxylating P450 enzymes. Proteins 2017; 85:945-950. © 2016 Wiley Periodicals, Inc.

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The Use of Directing Groups Enables the Selective and Efficient Biocatalytic Oxidation of Unactivated Adamantyl C‐H Bonds

et al. 2016

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Adamantane, 1‐ and 2‐adamantanol and 2‐adamantanone, were poor substrates for the cytochrome P450 enzyme CYP101B1. The CYP101B1 catalysed oxidation of 1‐adamantyl methyl ketone, and methyl 2‐(1‐adamantyl acetate), were more active generating a majority of the 4‐hydroxy metabolite. Substrate engineering using acetate and isobutyrate ester directing groups significantly increased the affinity, activity and coupling efficiency of CYP101B1 for the esters compared to the parent adamantanols, resulting in enhanced product formation rates (720 to 1350 nmol.(nmol‐P450)−1.min−1). The majority of the turnovers were selective for C−H bond hydroxylation with 4‐hydroxy‐1‐adamantyl isobutyrate and the 5‐hydroxy‐2‐adamantyl esters being generated as the sole majority product, 97 %, with high total turnover numbers, ranging from 4130 to 16500. In addition N‐(1‐adamantyl)acetamide, was oxidised by CYP101B1 whereas 1‐adamantylamine, was not. Whole‐cell biocatalytic reactions were used to generate the products in good yield. Overall the use of ester protecting groups and the modification of the amine to an amide enabled the more efficient and selective biocatalytic oxidation of adamantane frameworks.

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In vivo and in vitro hydroxylation of cineole and camphor by cytochromes P450CYP101A1, CYP101B1 and N242A CYP176A1

Abstract: In vivo and In vitro hydroxylation of Cineole and Camphor by Cytochromes P450CYP101A1, CYP101B1 and N242A CYP176A1

Cited by 10 publications

References 73 publications

CYP101J2, CYP101J3, and CYP101J4, 1,8-Cineole-Hydroxylating Cytochrome P450 Monooxygenases from Sphingobium yanoikuyae Strain B2

CYP101J2, CYP101J3, and CYP101J4, 1,8-Cineole-Hydroxylating Cytochrome P450 Monooxygenases from Sphingobium yanoikuyae Strain B2

X‐ray crystal structure of cytochrome P450 monooxygenase CYP101J2 fromSphingobium yanoikuyaestrain B2

The Use of Directing Groups Enables the Selective and Efficient Biocatalytic Oxidation of Unactivated Adamantyl C‐H Bonds

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