Journal
ENZYME AND MICROBIAL TECHNOLOGY
Volume 111, Issue -, Pages 29-37Publisher
ELSEVIER SCIENCE INC
DOI: 10.1016/j.enzmictec.2018.01.002
Keywords
Cytochrome P450; Isophorone; Hydroxylation; Stereoselective; Regioselective
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Funding
- Australian Research Council (ARC) [FT140100355]
- Australian Government Research Training Program Scholarship
- University of Adelaide International Postgraduate Award
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The stereoselective oxidation of hydrocarbons is an area of research where enzyme biocatalysis can make a substantial impact. The cyclic ketone isophorone was stereoselectively hydroxylated (>= 95%) by wild-type CYP102A1 to form (R)-4-hydroxyisophorone, an important chiral synthon and flavour and fragrance compound. CYP102A1 variants were also selective for 4-hydroxyisophorone formation and the product formation rate increased over the wild-type enzyme by up to 285-fold, with the best mutants being R47L/Y51F/1401P and A74G/F87V/L188Q. The latter variant, which contained mutations in the distal substrate binding pocket, was marginally less selective. Combining perfluorodecanoic acid decoy molecules with the rate accelerating variant R47L/Y51F/1401P engendered further improvement with the purified enzymes. However when the decoy molecules were used with A74G/F87V/L188Q the amount of product generated by the enzyme was reduced. Addition of decoy molecules to whole-cell turnovers did not improve the productivity of these CYP102A1 systems. WT CYP101A1 formed significant levels of 7-hydroxyisophorone as a minor product alongside 4-hydroxyisophorone. However the F87W/Y96F/L244A/V247L CYP101A1 mutant was >= 98% selective for (R)-4-hydroxyisophorone. A comparison of the two enzyme systems using whole-cell oxidation reactions showed that the best CYP101A1 variant was able to generate more product. We also characterised that the further oxidation metabolite 4-ketoisophorone was produced and then subsequently reduced to levodione by an endogenous Escherichia colt ene reductase.
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