title: CYP101J2, CYP101J3, and CYP101J4, 1,8-Cineole-Hydroxylating Cytochrome P450 Monooxygenases from Sphingobium yanoikuyae Strain B2.
abstract: 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 His6tagged) 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.
  
  
  
  CYP101J2, 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.
authors: Department of Microbiology, Monash University, Clayton, VIC, Australia Infection and Immunity Program, Monash Biomedicine Discovery Institute, Monash University, Clayton, VIC, Australia CSIRO Manufacturing, Clayton, VIC, Australia.
authors: Birgit
authors: B
authors: Unterweger
authors: Department of Microbiology, Monash University, Clayton, VIC, Australia Victorian Life Sciences Computation Initiative, The University of Melbourne, Carlton, VIC, Australia.
authors: Dieter M
authors: DM
authors: Bulach
authors: CSIRO Manufacturing, Parkville, VIC, Australia.
authors: Judith
authors: J
authors: Scoble
authors: CSIRO Energy, North Ryde, NSW, Australia.
authors: David J
authors: DJ
authors: Midgley
authors: CSIRO Oceans & Atmosphere, North Ryde, NSW, Australia.
authors: Paul
authors: P
authors: Greenfield
authors: Department of Microbiology, Monash University, Clayton, VIC, Australia Infection and Immunity Program, Monash Biomedicine Discovery Institute, Monash University, Clayton, VIC, Australia.
authors: Dena
authors: D
authors: Lyras
authors: Department of Microbiology, Monash University, Clayton, VIC, Australia Infection and Immunity Program, Monash Biomedicine Discovery Institute, Monash University, Clayton, VIC, Australia.
authors: Priscilla
authors: P
authors: Johanesen
authors: CSIRO Manufacturing, Clayton, VIC, Australia geoff.dumsday@csiro.au.
authors: Geoffrey J
authors: 0000-0002-5080-8791
authors: GJ
authors: Dumsday
mesh_terms: D001711:Biotransformation; D019475:Camphor 5-Monooxygenase / Q000145:classification / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D002954:Citrobacter / Q000201:enzymology / Q000235:genetics; D003511:Cyclohexanols / Q000378:metabolism*; D004579:Electron Transport; D004926:Escherichia coli / Q000235:genetics; D000077591:Eucalyptol; D016680:Genome, Bacterial; D006900:Hydroxylation; D007218:Industrial Microbiology; D039821:Monoterpenes / Q000378:metabolism*; D011485:Protein Binding; D016958:Pseudomonas putida / Q000201:enzymology / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D012722:Sewage / Q000382:microbiology*; D042301:Sphingomonadaceae / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D003511:Cyclohexanols; D039821:Monoterpenes; D011994:Recombinant Proteins; D012722:Sewage; D019475:Camphor 5-Monooxygenase; D000077591:Eucalyptol