title: P450 fingerprinting method for rapid discovery of terpene hydroxylating P450 catalysts with diversified regioselectivity.
abstract: Engineered P450 enzymes constitute attractive catalysts for the selective oxidation of unactivated C-H bonds in complex molecules. A current bottleneck in the use of P450 catalysis for chemical synthesis is the time and effort required to identify the P450 variant(s) with the desired level of activity and selectivity. In this report, we describe a method to map the active site configuration of engineered P450 variants in high throughput using a set of semisynthetic chromogenic probes. Through analysis of the resulting 'fingerprints', reliable predictions can be made regarding the reactivity of these enzymes toward complex substrates structurally related to the fingerprint probes. In addition, fingerprint analysis offers a convenient and time-effective means to assess the regioselectivity properties of the fingerprinted P450s. The described approach can represent a valuable tool to expedite the discovery of P450 oxidation catalysts for the functionalization of relevant natural products such as members of the terpene family.
authors.affiliation: Department of Chemistry, University of Rochester, Rochester, New York 14627, United States.
authors.forename: Kaidong
authors.initials: K
authors.lastname: Zhang
authors.forename: Shady
authors.initials: S
authors.lastname: El Damaty
authors.forename: Rudi
authors.initials: R
authors.lastname: Fasan
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D002384:Catalysis; D020134:Catalytic Domain; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics; D057166:High-Throughput Screening Assays; D015335:Molecular Probes / Q000737:chemistry; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry* / Q000235:genetics; D010084:Oxidation-Reduction; D015202:Protein Engineering; D013379:Substrate Specificity; D013729:Terpenes / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; D015335:Molecular Probes; D013729:Terpenes; D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases