Evolving P450pyr Monooxygenase for Regio- and Stereoselective Hydroxylations

Abstract

P450pyr monooxygenase from Sphingomonas sp. HXN-200 catalysed the regio- and stereoselective hydroxylation at a non-activated carbon atom, a useful but challenging reaction in classic chemistry, with unique substrate specificity for a number of alicyclic compounds. New P450pyr mutants were developed by directed evolution with improved catalytic performance, thus significantly extending the application of the P450pyr monooxygenase family in biohydroxylation to prepare useful and valuable chiral alcohols. Directed evolution of P450pyr created new enzymes with improved S-enantioselectivity or R-enantioselectivity for the hydroxylation of N-benzyl pyrrolidine, enhanced regioselectivity for the hydroxylation of N-benzyl pyrrolidinone, and increased enantioselectivity for the hydroxylation of N-benzyl piperidinone, respectively. Directed evolution of P450pyr generated also mutants with fully altered regioselectivity (from terminal to subterminal) and newly created excellent S-enantioselectivity for the biohydroxylation of n-octane and propylbenzene, respectively, providing new opportunities for the regio- and enantioselective alkane functionalization. New P450pyr mutants were engineered as the first catalyst for highly selective terminal hydroxylation of n-butanol to 1,4-butanediol. Several novel, accurate, sensitive, simple, and HTS assays based on colorimetric or MS detection for measuring the enantio- and/or regioselectivity of hydroxylation were developed and proven to be practical in directed evolution. The P450pyr X-ray structure was obtained and used to guide the evolution. In silico modelling and substrate docking provided some insight into the influence of several important amino acid mutations of the engineered P450pyr mutants on the altered or enhanced regio- and enantioselectivity as well as new substrate acceptance. The obtained information and knowledge is useful for further engineering of P450pyr for other hydroxylations and oxidations.

Highly Enantioselective Hydroxylation of 3-Arylpropanenitriles to Access Chiral β-Hydroxy Nitriles by Engineering of P450pyr Monooxygenase

“…In our previous study, we expressed several P450 monooxygenases in Escherichia coli BL21­(DE3) and investigated their catalytic activity and enantioselectivity in the benzylic hydroxylation of halohydrocarbons and aromatic hydrocarbons. , P450pyr monooxygenase is a unique hydroxylase that catalyzes the asymmetric hydroxylation of N-heterocycles at nonactivated C–H bonds as well as alkanes at terminal or subterminal C–H bonds. − Meanwhile, we found that some E. coli BL21­(DE3) strains harboring P450pyr variants , exhibited good enantioselectivity in the benzylic hydroxylation of halohydrocarbons.…”

Deconstruction of the CYP153A6 Alkane Hydroxylase System: Limitations and Optimization of In Vitro Alkane Hydroxylation

“…Members of the CYP153 class of enzymes have long been recognized for their ability to catalyze the selective terminal oxidation of alkanes and fatty acids. This selectivity and specificity have been expanded on in recent years [28][29][30][31][32][33]. Despite their impressive regioselectivity, their application in industrial processes have been hindered by various limitations, including among others, requirement for redox partner proteins, cofactor regeneration, stability, and low activity.…”

Crystal Structure of a Putative Cytochrome P450 Alkane Hydroxylase (CYP153D17) from Sphingomonas sp. PAMC 26605 and Its Conformational Substrate Binding

“…Previous studies also demonstrated that Sphingomonas sp. HXN-200 bacteria contain hydrocarbon-degrading enzymes [17,18]. The results of these studies suggest that Sphingomonas sp.…”