Substrate specificity of two cytochrome P450 monooxygenases involved in lankamycin biosynthesis.

Abstract

To elucidate the gross lankamycin biosynthetic pathway including two cytochrome P450 monooxygenases, LkmK and LkmF, we constructed two double mutants of P450 genes in combination with glycosyltransferase genes, lkmL and lkmI. An aglycon 8,15-dideoxylankanolide, a possible substrate for LkmK, was prepared from an lkmK-lkmL double mutant, while a monoglycoside 3-O-l-arcanosyl-8-deoxylankanolide, a substrate for LkmF, was from an lkmF-lkmI double mutant. Bioconversion of lankamycin derivatives was performed in the Escherichia coli recombinant for LkmK and the Streptomyces lividans recombinant for LkmF, respectively. LkmK catalyzes the C-15 hydroxylation on all 15-deoxy derivatives, including 8,15-dideoxylankanolide (a possible substrate), 8,15-dideoxylankamycin, and 15-deoxylankamycin, suggesting the relaxed substrate specificity of LkmK. On the other hand, LkmF hydroxylates the C-8 methine of 3-O-l-anosyl-8-deoxylankanolide. Other 8-deoxy lankamycin/lankanolide derivatives were not oxidized, suggesting the importance of a C-3 l-arcanosyl moiety for substrate recognition by LkmF in lankamycin biosynthesis. Thus, LkmF has a strict substrate specificity in lankamycin biosynthesis.