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Engineering of P450pyr Hydroxylase for the Highly Regio‐ and Enantioselective Subterminal Hydroxylation of Alkanes
Author(s) -
Yang Yi,
Liu Ji,
Li Zhi
Publication year - 2014
Publication title -
angewandte chemie international edition
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.831
H-Index - 550
eISSN - 1521-3773
pISSN - 1433-7851
DOI - 10.1002/anie.201311091
Subject(s) - hydroxylation , regioselectivity , enantiopure drug , chemistry , enantioselective synthesis , selectivity , saturated mutagenesis , cytochrome p450 , stereochemistry , octane , combinatorial chemistry , enzyme , mutant , organic chemistry , biochemistry , catalysis , gene
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 CH 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.