The efficient and selective biocatalytic oxidation of norisoprenoid and aromatic substrates by CYP101B1 from Novosphingobium aromaticivorans DSM12444

Hall, Emma A.; Bell, Stephen

doi:10.1039/c4ra14010a

Cited by 22 publications

(52 citation statements)

References 52 publications

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“…The CYP101 family of P450 enzymes are able to bind norisoprenoids, camphor and certain terpenoids with high affinity . A class I electron transfer system from N. aromaticivorans , consisting of a flavin‐dependent ferredoxin reductase, ArR, and a [2Fe‐2S] ferredoxin, Arx, has been used to support the activity the CYP101B1, C1, D1 and D2 enzymes –. Whole‐cell systems containing these CYP enzymes coupled to, ArR and Arx, are capable of product formation on the gram‐per‐litre scale in shake flasks .…”

Section: Introductionmentioning

confidence: 99%

“…Whole‐cell systems containing these CYP enzymes coupled to, ArR and Arx, are capable of product formation on the gram‐per‐litre scale in shake flasks . CYP101B1 is a highly efficient biocatalyst for the C−H bond oxidation of norisoprenoids and monoterpenoid acetates . The acetate side chain of the esters resembles the butenone side chain of norisoprenoids allowing substrate binding with high affinity which holds the substrate in place for efficient and selective oxidation.…”

Section: Introductionmentioning

confidence: 99%

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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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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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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“…cellulosum with titres in the lower mg l −1 range (Khatri et al ., ; Litzenburger and Bernhardt, ). Compared to the most active β‐ionone‐biocatalyst known today, CYP101B1 (0.83 g l −1 ), this is only a slightly lower titre (Hall and Bell, ). The ratio of the products was equivalent to that of the in vitro biotransformations.…”

Section: Discussionmentioning

confidence: 99%

Characterization and structure‐guided engineering of the novel versatile terpene monooxygenase CYP109Q5 from Chondromyces apiculatus DSM436

Klenk

Dubiel

Sharma

et al. 2018

Microbial Biotechnology

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Summary One of the major challenges in chemical synthesis is the selective oxyfunctionalization of non‐activated C‐H bonds, which can be enabled by biocatalysis using cytochrome P450 monooxygenases. In this study, we report on the characterization of the versatile CYP 109Q5 from Chondromyces apiculatus DSM 436, which is able to functionalize a wide range of substrates (terpenes, steroids and drugs), including the ring of β‐ionone in non‐allylic positions. The crystal structure of CYP 109Q5 revealed flexibility within the active site pocket that permitted the accommodation of bulky substrates, and enabled a structure‐guided approach to engineering the enzyme. Some variants of CYP 109Q5 displayed a switch in selectivity towards the non‐allylic positions of β‐ionone, allowing the simultaneous production of 2‐ and 3‐hydroxy‐β‐ionone, which are chemically challenging to synthesize and are important precursors for carotenoid synthesis. An efficient whole‐cell system finally enabled the production of up to 0.5 g l −1 hydroxylated products of β‐ionone; this system can be applied to product identification in further biotransformations. Overall, CYP 109Q5 proved to be highly evolvable and active. The studies in this work demonstrate that, using rational mutagenesis, the highly versatile CYP 109Q5 generalist can be progressively evolved to be an industrially valuable specialist for the synthesis of specific products.

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“…Compared to previously characterized members of the CYP101 family, all three newly discovered P450s are most closely related to CYP101B1 and CYP101A1, sharing 45 to 47% and 44% sequence identity, respectively. CYP101B1 has been shown to oxidize a broad range of substrates, including norisoprenoids (e.g., ␤-ionone), various aromatic compounds, and camphor (39,43). CYP101A1 was char-acterized based on its activity with camphor (18).…”

Section: Discussionmentioning

confidence: 99%

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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The efficient and selective biocatalytic oxidation of norisoprenoid and aromatic substrates by CYP101B1 from Novosphingobium aromaticivorans DSM12444

Abstract: CYP101B1 from Novosphingobium aromaticivorans oxidises ionone derivatives and phenylcyclohexane with high activity and regioselectivity.

Cited by 22 publications

References 52 publications

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

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

Characterization and structure‐guided engineering of the novel versatile terpene monooxygenase CYP109Q5 from Chondromyces apiculatus DSM436

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

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