Structural insights into oxidation of medium-chain fatty acids and flavanone by myxobacterial cytochrome P450 CYP267B1

Abstract

Oxidative biocatalytic reactions performed by cytochrome P450 enzymes (P450s) are of high interest for the chemical and pharmaceutical industries. CYP267B1 is a P450 enzyme from myxobacterium So ce56 displaying a broad substrate scope. In this work, a search for new substrates was performed, combined with product characterization and a structural analysis of substrate-bound complexes using X-ray crystallography and computational docking. The results demonstrate the ability of CYP267B1 to perform in-chain hydroxylations of medium-chain saturated fatty acids (decanoic acid, dodecanoic acid and tetradecanoic acid) and a regioselective hydroxylation of flavanone. The fatty acids are mono-hydroxylated at different in-chain positions, with decanoic acid displaying the highest regioselectivity towardsω-3 hydroxylation. Flavanone is preferably oxidized to 3-hydroxyflavanone. High-resolution crystal structures of CYP267B1 revealed a very spacious active site pocket, similarly to other P450s capable of converting macrocyclic compounds. The pocket becomes more constricted near to the heme and is closed off from solvent by residues of the F and G helices and the B-C loop. The crystal structure of the tetradecanoic acid-bound complex displays the fatty acid bound near to the heme, but in a nonproductive conformation. Molecular docking allowed modeling of the productive binding modes for the four investigated fatty acids and flavanone, as well as of two substrates identified in a previous study (diclofenac and ibuprofen), explaining the observed product profiles. The obtained structures of CYP267B1 thus serve as a valuable prediction tool for substrate hydroxylations by this highly versatile enzyme and will encourage future selectivity changes by rational protein engineering.

Novel insights into oxidation of fatty acids and fatty alcohols by cytochrome P450 monooxygenase CYP4B1

“…Similar observations of shifted regioselectivities have been made previously: Jóźwig et al [46] reported that for CYP267B1 from Sorangium cellulosum the regioselectivity of hydroxylation was shifted from positions located closer to the methyl terminal carbon (ω−1, ω−2, ω−3) towards the in-chain positions (ω−3 and ω−4) with increasing chain length of the fatty acid. The authors also found evidence for C14 6 to be bound U-shaped and in two different orientations [46].…”

“…Similar observations of shifted regioselectivities have been made previously: Jóźwig et al [46] reported that for CYP267B1 from Sorangium cellulosum the regioselectivity of hydroxylation was shifted from positions located closer to the methyl terminal carbon (ω−1, ω−2, ω−3) towards the in-chain positions (ω−3 and ω−4) with increasing chain length of the fatty acid. The authors also found evidence for C14 6 to be bound U-shaped and in two different orientations [46]. Khatri et al [19] investigated fatty acid hydroxylation by CYP267A1 from the same organism and noticed that the wild-type enzyme contains a natural heme-signature variant (a conserved phenylalanine at position 366 exchanged by a leucine residue) that led to a shift in regioselectivity from subterminal (ω−1 to ω−3) positions to in-chain (ω−4 to ω−9) positions.…”