title: Engineering of p450pyr hydroxylase for the highly regio- and enantioselective subterminal hydroxylation of alkanes.
abstract: Terminal-selective cytochrome P450pyr has been successfully engineered through directed evolution for the subterminal hydroxylation of alkanes with excellent regio- and enantioselectivity. A sensitive colorimetric high-throughput screening (HTS) assay was developed for the measurement of both the regioselectivity and the enantioselectivity of a hydroxylation reaction. By using the HTS assay and iterative saturation mutagenesis, sextuple-mutant P450pyrSM1 was created for the hydroxylation of n-octane (1) to give (S)-2-octanol (2) with 98 % ee and >99 % subterminal selectivity. The engineered P450 is the first enzyme for this type of highly selective alkane hydroxylation, being useful for the C-H activation and functionalization of alkanes and the preparation of enantiopure alcohols. Molecular modeling provided structure-based understanding of the fully altered regioselectivity and the excellent enantioselectivity. Another sextuple-mutant P450pyrSM2 catalyzed the hydroxylation of propylbenzene (3) to afford (S)-1-phenyl-2-propanol (4) with 95 % ee and 98 % subterminal selectivity.
authors.affiliation: Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117585 (Singapore) http://www.chbe.nus.edu.sg/people/faculty_liz.html.
authors.forename: Yi
authors.initials: Y
authors.lastname: Yang
authors.forename: Ji
authors.initials: J
authors.lastname: Liu
authors.forename: Zhi
authors.initials: Z
authors.lastname: Li
mesh_terms: D000473:Alkanes / Q000737:chemistry*; D057166:High-Throughput Screening Assays; D006900:Hydroxylation; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D015394:Molecular Structure; D009154:Mutation; D013237:Stereoisomerism
chemical_list: D000473:Alkanes; D006899:Mixed Function Oxygenases
keywords: CH activation; P450 enzymes; biohydroxylation; directed evolution; high-throughput screening