Rational Design of a Minimal and Highly Enriched CYP102A1 Mutant Library with Improved Regio‐, Stereo‐ and Chemoselectivity

Abstract

A minimal CYP102A1 mutant library of only 24 variants plus wild type was constructed by combining five hydrophobic amino acids (alanine, valine, phenylalanine, leucine and isoleucine) in two positions. Both positions are located close to the centre of the haem group. The first, position 87, has been shown to mediate substrate specificity and regioselectivity in CYP102A1. The second hotspot, position 328, was predicted to interact with all substrates during oxidation and has previously been identified by systematic analysis of 31 crystal structures and 6300 sequences of cytochrome P450 monooxygenases. By systematically altering the size of the side chains, a broad range of binding site shapes was generated. All variants were functionally expressed in E. coli. The library was screened with four terpene substrates geranylacetone, nerylacetone, (4R)-limonene and (+)-valencene. Only three variants showed no activity towards all four terpenes, while eleven variants demonstrated either a strong shift or improved regio- or stereoselectivity during oxidation of at least one substrate as compared to CYP102A1 wild type.

Dearomatisation of o‐Xylene by P450BM3 (CYP102A1)

“…[7] Variants with enhanced activity towards non-natural substrates have been created. [8][9][10][11][12] Selectivity control has also been addressed, [13][14][15][16] leading to the development of variants with the ability to promote phenol formation, [17,18] terminal hydroxylation [19] and epoxidation. [20] Wild-type P450 BM3 (WT) [21] and a number of evolved variants [22][23][24] have been shown to generate the same metabolites as mammalian P450s from certain pharmaceuticals.…”

A Single Active Site Mutation Inverts Stereoselectivity of 16‐Hydroxylation of Testosterone Catalyzed by Engineered Cytochrome P450 BM3

“…Synergistic effects of P450 BM3 double mutants have previously been shown to affect regio-and stereoselectivity. For example, hydrophobic substitutions at positions 87 and 328 improved selectivity of terpene hydroxylation [18] and combined substitutions at residues 82 and 438 have been shown to control stereoselectivity of styrene epoxidation. [19] To evaluate if introduction of S72I in other 16 b-OH-TES-selective mutants could also produce similar changes in stereoselectivity, we introduced the S72I mutation in BM3 variant M11 V87I, which has been shown previously to hydroxylate testosterone selectively at the 16 bposition.…”

Systematic Analysis of Large Enzyme Families: Identification of Specificity‐ and Selectivity‐Determining Hotspots

“…[19,20] Thus, a focused CYP102A1 variant library was constructed that consisted of 25 combinations of five hydrophobic residues in the two hotspot positions, F87 and A328 (Figure 2). [22] This focused library was screened with four terpenes [geranylacetone, nerylacetone, (4R)-limonene, and (+)-valencene]. Although the wildtype enzyme converted all four terpenes with poor regioselectivity, 11 variants demonstrated either a strong shift or an improved regio-or stereoselectivity during oxidation of at least one substrate.…”