Controlling the Regioselectivity of Fatty Acid Hydroxylation (C₁₀) at α‐ and β‐Position by CYP152A1 (P450Bsβ) Variants

Abstract

Regioselective hydroxylation on inactivated C−H bonds is among the dream reactions of organic chemists. Cytochrome P450 enzymes (CYPs) perform this reaction in general with high regio‐ and stereoselectivity (e. g. for steroids as substrates). Furthermore, enzyme engineering may allow to tune the properties of the enzyme. Regioselective hydroxylation of shorter or linear molecules (fatty acids), however, remains challenging even with this enzyme class, due to the high similarity of the substrate's backbone carbons and their conformational flexibility. CYPs hydroxylating fatty acids selectively in the chemically more distinct α‐ or ω‐ position are well described. In contrast, selective in‐chain hydroxylation of fatty acids lacks precedence. The peroxygenase CYP152A1 (P450Bsβ) is a family member that displays fatty acid hydroxylation at both, the α‐ and β‐position, with preference for the α‐position. Herein we report the influence of hydrophobic active site residues on the hydroxylation pattern of this enzyme. By site directed mutagenesis and combination of the libraries, double and triple mutation variants were identified, which hydroxylated decanoic acid (C10) with improved regio‐selectivity in the β‐position. Variants were identified with a 10‐fold increase of the β‐regioselectivity (expressed as α/β‐ratio) compared to the wild type. In total 103 variants of CYP152A1 (P450Bsβ) were investigated.

Enzymatic Regio- and Enantioselective C–H Oxyfunctionalization of Fatty Acids

“…The G290I mutant plasmid was then used as the template for the next round screening at residues L78 and V170, as both residues contained the second-best hits in the initial screening. The P450 BSβ -L78A mutation was reported to aid the β-selectivity in hydroxylation of decanoic acid (67:33 Cβ/Cα) . We discovered that the double variant, P450 BSβ -L78A/G290I, more effectively boosted the β-regioselectivity (84:7 Cβ/Cα) with 67% conversion (Figure b).…”

“…This highly reactive species then abstracts Cα/Cβ hydrogen atoms of FAs, followed by either an ·OH rebound step to give the respective α/β-OH FAs or a C–C bond scission to yield 1-alkene . Previous search for improved regioselective P450 BSβ variants with hydrophobic amino acid substitutions (Ala, Phe, Val, Leu, Ile, and Trp) and combination of beneficial mutations neither realized a universally satisfying regioselectivity (β-OH/(α-OH+β-OH)<90%) that is sufficient for preparing chiral β-hydroxy FAs nor rationalized the stereochemical outcome of the transformation from a mechanistic perspective . We envisioned that directed evolution of P450 BSβ could possibly remodel its catalytic pathway to favor a predominant Cβ-H abstraction and concurrently shut down the C–C bond scission route, thereby achieving the desired highly regio- and enantioselective β-hydroxylation of fatty acids.…”

Light-driven biocatalytic oxidation

“…15,58,59 In contrast, CYP152A1 shows a preference for the b-position of fatty acids. 15,59,60 On the other hand, CYP152L isozymes mainly perform oxidative fatty acid decarboxylation, producing terminal alkenes, along with aand b-hydroxylation reactions. 61,62 CYP152N1 catalyzes oxidative decarboxylation reaction of fatty acids to produce one-carbon shorter fatty acids.…”

Functional characterization of a novel CYP119 variant to explore its biocatalytic potential

“…As an alternative to NAD(P)H and molecular oxygen, P450s can also utilize hydrogen peroxide (H 2 O 2 ), through the H 2 O 2 ‐shunt pathway. However, this pathway is very inefficient for most P450s, except for the P450s in the CYP152 subfamily 12,13 . In addition, recent studies have shown that the P450 domain of some self‐sufficient monooxygenases can also utilize peroxides for generation of drug metabolites 14,15 .…”

“…However, this pathway is very inefficient for most P450s, except for the P450s in the CYP152 subfamily. 12,13 In addition, recent studies have shown that the P450 domain of some self-sufficient monooxygenases can also utilize peroxides for generation of drug metabolites. 14,15 Obtaining soluble and stable P450s that can utilize H 2 O 2 has been an appealing target for engineering these enzymes for industrial utilization.…”