title: FK506 maturation involves a cytochrome p450 protein-catalyzed four-electron C-9 oxidation in parallel with a C-31 O-methylation.
abstract: FK506, structurally similar to FK520 and rapamycin, is an α-keto amide bonding-containing, macrolide natural product that exhibits potent immunosuppressive activity and moderate antifungal activity. FK506 biosynthesis requires a hybrid polyketide synthase (PKS)-nonribosomal peptide synthetase (NRPS) system to construct the skeleton of the macrolide. The mechanism for tailoring this macrolide to furnish FK506 remains poorly understood. In this study, we report a maturation paradigm common for FK506, FK520, and rapamycin, by characterizing two conserved regiospecific, post-PKS-NRPS modifications in an FK506-producing Streptomyces tsukubaensis strain. A cytochrome P450 protein, FkbD, catalyzes a less common, four-electron oxidation at C-9 to give a rarely found α-keto amide group, whereas a methyltransferase, FkbM, is responsible for O-methylation at C-31 to afford a methoxy group. Both FkbD and FkbM are highly tolerant in their substrate choice; therefore, the order of FkbD- and FkbM-catalyzed reactions is interchangeable in the FK506 biosynthetic pathway. Inactivation of fkbD produced a new intermediate, 9-deoxo-FK506, which displayed antifungal activity lower than that of FK506. Taking previously reported bioassay results regarding the intermediates 9-deoxo-31-O-demethyl-FK506 and 31-O-demethyl-FK506 into account, it is clear that the modifications catalyzed by FkbD and FkbM are of importance to reach the full biological activity of FK506 by forming a key structure motif that is necessary for interaction of the molecule with the receptor and, subsequently, the downstream intracellular responses.
authors: State Key Laboratory of Bioorganic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai, China.
authors: Dandan
authors: D
authors: Chen
authors: Lihan
authors: L
authors: Zhang
authors: Bo
authors: B
authors: Pang
authors: Jing
authors: J
authors: Chen
authors: Zhinan
authors: Z
authors: Xu
authors: Ikuro
authors: I
authors: Abe
authors: Wen
authors: W
authors: Liu
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D004583:Electrons; D008745:Methylation; D015394:Molecular Structure; D010084:Oxidation-Reduction; D020123:Sirolimus / Q000737:chemistry / Q000378:metabolism; D013302:Streptomyces / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D016559:Tacrolimus / Q000737:chemistry / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D003577:Cytochrome P-450 Enzyme System; D020123:Sirolimus; D016559:Tacrolimus