Experimental and Computational Studies on the Biotransformation of Pseudopyronines with Human Cytochrome P450 CYP4F2

Abstract

The secondary metabolite pseudopyronine B, isolated from Pseudomonas mosselii P33, was biotransformed by human P450 enzymes, heterologously expressed in the fission yeast Schizosaccharomyces pombe. Small-scale studies confirmed that both CYP4F2 and CYP4F3A were capable of oxidizing the substrate, with the former achieving a higher yield. In larger-scale studies using CYP4F2, three new oxidation products were obtained, the structures of which were elucidated by UV–vis, 1D and 2D NMR, and HR-MS spectroscopy. These corresponded to hydroxylated, carboxylated, and ester derivatives (1–3) of pseudopyronine B, all of which had been oxidized exclusively at the ω-position of the C-6 alkyl chain. In silico homology modeling experiments highlighted key interactions between oxygen atoms of the pyrone ring and two serine residues and a histidine residue of CYP4F2, which hold the substrate in a suitable orientation for oxidation at the terminus of the C-6 alkyl chain. Additional modeling studies with all three pseudopyronines revealed that the seven-carbon alkyl chain of pseudopyronine B was the perfect length for oxidation, with the terminal carbon lying close to the heme iron. The antibacterial activity of the substrates and three oxidation products was also assessed, revealing that oxidation at the ω-position removes all antimicrobial activity. This study both increases the range of known substrates for human CYF4F2 and CYP4F3A enzymes and demonstrates their utility in producing additional natural product derivatives.

Expected and Unexpected Products from the Biochemical Oxidation of Bacterial Alkylquinolones with CYP4F11

“…Meanwhile, the methyl ester 1d could be formed from compound 1b via action of an endogenous carboxyl methyltransferase in the yeast strain . Similar acetate and methyl ester derivatives were isolated in previous biotransformation studies. , …”

“…46 Similar acetate and methyl ester derivatives were isolated in previous biotransformation studies. 26,27 However, the other ester conjugates are reported for the first time here. First, there was a leucic acid unit attached to the ωoxidation products in 1e, 3e, and 4e.…”

“…The final class of esters were formed by conjugation of the quinolones with 5-hydroxy-4-ketohexanoic acid, another common product in yeast metabolism . While minor products with masses consistent with similar esters were observed in the previous biotransformation study of pseudopyronine B, yields were too low for isolation (not shown). These small acids are likely produced by the heterologous host S. pombe and could be attached to the oxidized quinolones via the action of an endogenous alcohol acyltransferase.…”

“…We recently analyzed the binding behavior of pseudopyronines, dialkylresorcinols, and, here, 2-alkylquinolone derivatives to the CYP4 family member enzymes CYP4F2, 4F3A and 4F11. From the CYP4F subfamily, these three enzymes all physiologically ω-hydroxylate fatty acids .…”

“…They are a superfamily of heme-thiolate proteins and catalyze the biotransformation of a diverse range of molecules . There are 57 functional CYP genes in humans, which are crucial in drug metabolism and biosynthetic processes in vivo. − The metabolism of 4-quinolones with CYP enzymes has been studied previously, though this has largely focused on drug metabolism in the liver . The work of Yasutake et al demonstrated that the cytochrome P450 RauA from Rhodococcus erythropolis JCM 6824 catalyzed the hydroxylation of a nitrogen atom in the quinolone ring of the 4-quinolone aurachin .…”