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Structural elucidation of a hy-droxy-cineole product obtained from cytochrome P450 monooxygenase CYP101J2 catalysed transformation of 1,8-cineole.

ABSTRACT: 1,8-Cineole is an abundant natural product that has the potential to be transformed into other building blocks that could be suitable alternatives to petroleum-based chemicals. Mono-hydroxy-lation of 1,8-cineole can potentially occur at eight different carbon sites around the bicyclic ring system. Using cytochrome P450 monooxygenase CYP101J2 from Sphingobium yanoikuyae B2, the hy-droxy-lation can be regioselectively directed at the C atom adjacent to the methyl-substituted quaternary bridgehead atom of 1,8-cineole. The unambiguous location of the hydroxyl functionality and the stereochemistry at this position was determined by X-ray crystal analysis. The mono-hydroxy-lated compound derived from this microorganism was determined to be (1S)-2a-hy-droxy-1,8-cineole (trivial name) or (1S,4R,6S)-1,3,3-trimethyl-2-oxabi-cyclo-[2.2.2]octan-6-ol (V) (systematic), C10H18O2. In the solid state this compound exhibits an inter-esting O-H?O hydrogen-bonding motif.

SUBMITTER: Collis GE

PROVIDER: S-EPMC5598857 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature

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Structural elucidation of a hy-droxy-cineole product obtained from cytochrome P450 monooxygenase CYP101J2 catalysed transformation of 1,8-cineole.

Collis Gavin E GE Unterweger Birgit B Dumsday Geoff J GJ Forsyth Craig M CM

Acta crystallographica. Section E, Crystallographic communications 20170721 Pt 8

1,8-Cineole is an abundant natural product that has the potential to be transformed into other building blocks that could be suitable alternatives to petroleum-based chemicals. Mono-hydroxy-lation of 1,8-cineole can potentially occur at eight different carbon sites around the bicyclic ring system. Using cytochrome P450 monooxygenase CYP101J2 from <i>Sphingobium yanoikuyae</i> B2, the hy-droxy-lation can be regioselectively directed at the C atom adjacent to the methyl-substituted quaternary brid ...[more]

PMID: 28932445

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Project description: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-cineole. Genome sequencing of this strain in combination with purification of native 1,8-cineole-binding proteins enabled identification of 1,8-cineole-binding P450s. The P450 enzymes were cloned, heterologously expressed (N-terminally His6 tagged) in Escherichia coli BL21(DE3), purified, and spectroscopically characterized. Recombinant whole-cell biotransformation in E. coli demonstrated that all three P450s hydroxylate 1,8-cineole using electron transport partners from E. coli to yield a product putatively identified as (1S)-2α-hydroxy-1,8-cineole or (1R)-6α-hydroxy-1,8-cineole. The new P450s belong to the CYP101 family and share 47% and 44% identity with other 1,8-cineole-hydroxylating members found in Novosphingobium aromaticivorans and Pseudomonas putida Compared to P450cin (CYP176A1), a 1,8-cineole-hydroxylating P450 from Citrobacter braakii, these enzymes share less than 30% amino acid sequence identity and hydroxylate 1,8-cineole in a different orientation. Expansion of the enzyme toolbox for modification of 1,8-cineole creates a starting point for use of hydroxylated derivatives in a range of industrial applications.ImportanceCYP101J2, CYP101J3, and CYP101J4 are cytochrome P450 monooxygenases from S. yanoikuyae B2 that hydroxylate the monoterpenoid 1,8-cineole. These enzymes not only play an important role in microbial degradation of this plant-based chemical but also provide an interesting route to synthesize oxygenated 1,8-cineole derivatives for applications as natural flavor and fragrance precursors or incorporation into polymers. The P450 cytochromes also provide an interesting basis from which to compare other enzymes with a similar function and expand the CYP101 family. This could eventually provide enough bacterial parental enzymes with similar amino acid sequences to enable in vitro evolution via DNA shuffling.

Dataset Information

Structural elucidation of a hy-droxy-cineole product obtained from cytochrome P450 monooxygenase CYP101J2 catalysed transformation of 1,8-cineole.

Publications

Structural elucidation of a hy-droxy-cineole product obtained from cytochrome P450 monooxygenase CYP101J2 catalysed transformation of 1,8-cineole.

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