In Vivo Investigation of the Roles of FdmM and FdmM1 in Fredericamycin Biosynthesis Unveiling a New Family of Oxygenases

Abstract

Fredericamycin (FDM) A, a highly oxidized aromatic pentadecaketide natural product, exhibits potent cytotoxicity and has been studied as a new anticancer drug lead. The FDM biosynthetic gene cluster has been previously cloned from Streptomyces griseus ATCC 49344 and successfully expressed in the heterologous host Streptomyces albus J1074. The fdmM and fdmM1 genes code for two proteins with high sequence homology to each other but unknown function. In-frame deletion of each of the genes from the fdm cluster was accomplished in the S. albus host. Each mutant failed to produce FDM A and the key biosynthetic intermediate FDM E but produced various new metabolites, the titers of which were dramatically increased via overexpression of an fdm pathway-specific activator fdmR1. The ⌬fdmM mutant strain accumulated three new compounds FDM M-1, FDM M-2, and FDM M-3, whereas the ⌬fdmM1 mutant strain produced one new compound FDM M1-1. Isolation and structural characterization of these compounds enable us to propose that FdmM and FdmM1 catalyze the C-6 and C-8 hydroxylations for FDM biosynthesis, respectively. Homologs of FdmM and FdmM1 can be found in biosynthetic gene clusters of many other aromatic polyketides, ranging from dodecaketides to pentadecaketides, but to date all of them were annotated as proteins of unknown function. Based on the findings reported here for FdmM and FdmM1, we now propose similar functions for those proteins, and FdmM and FdmM1 therefore represent an emerging family of novel oxygenases responsible for hydroxylation of aromatic polyketide natural products.Studies on natural product biosynthetic pathways have uncovered a number of intriguing oxygenases. For instance, the first reported metal-and cofactor-free monooxygenase TcmH is responsible for quinone moiety formation in tetracenomycin D3 and represents a rapidly growing family of antibiotic biosynthetic monooxygenases (1, 2). Another example is DpgC, which is involved in the biosynthesis of vancomycin building block 3, 5-dihydroxyphenylglycine. The crystal structure of DpgC has been used to understand the reaction mechanism of metal-and cofactor-free dioxygenases (3). MomA, involved in mompain biosynthesis (4) is a metal ion-dependent monooxygenase belonging to the cupin superfamily of oxidases bearing no prosthetic group. The discovery of enzymes such as TcmH, DpgC, and MomA continually encourage the community to search for and characterize new oxygenases from proteins of unknown functions involved in natural products biosynthesis.Whereas octaketides (C-16), nonaketides (C-18), and decaketides (C-20) are the most common aromatic polyketide natural products whose biosyntheses have been extensively investigated, aromatic polyketides ranging from dodecaketides (C-24) to pentadecaketides (C-30) are also known including biologically active compounds such as fredericamycin (FDM) 2 A (1) (5, 6), pradimicin A (2) (7), lysolipin X (3) (8), griseorhodin A (4) (9), benastatin A (5) (10), and ␥-rubromycin (6) ( Fig. 1 and Ref. 11). The highly oxidized nat...

Cloning of the biosynthetic gene cluster for naphthoxanthene antibiotic FD-594 from Streptomyces sp. TA-0256

“…Recently, Shen and co-workers reported that PnxE1 and PnxE2 homologs FdmM and FdmM1 were responsible for the oxygenation at C6 and C8 of the polyketide intermediate by gene inactivation studies. 33 However, this oxygenation activity is the same as that proposed for PnxH and PnxI described above. Therefore, at the moment, we propose that three of PnxH, PnxI, PnxE1 and PnxE2 might be responsible for the oxygenation at C6, C8 and C10.…”

Functional Characterization of ttnI Completing the Tailoring Steps for Tautomycetin Biosynthesis in Streptomyces griseochromogenes

“…Both the TtnD decarboxylase and TtnI P-450 oxygenase also shown some degree of substrate promiscuity, as exemplified by the accumulation of TTN D-4 in the ΔttnD mutant strain SB13013 and subsequent conversion of TTN D-4 to TTN in the ΔttnD complementation strain SB13013. 6b Substrate promiscuity is common for enzymes from secondary metabolite biosynthetic machinery, 9 a property that has been extensively exploited for natural product structural diversity by combinatorial biosynthesis methods. In the absence of the TtnD carboxylase activity, TTN D-4 apparently could undergo C-5 reduction to afford TTN D-2 and TTN D-3, shunt metabolites whose formation most likely resulted from adventitious activities in the ΔttnD mutant SB13013 fermentation.…”

Characterization of FdmV as an Amide Synthetase for Fredericamycin A Biosynthesis in Streptomyces griseus ATCC 43944

“…These facts support the proposal that the amide nitrogen of FDM A is incorporated following installation of the polycyclic ring system. The isolation of four new FDM analogs, FDM M-1, FDM M-2, FDM M-3, and FDM M1-1, all sharing the same FDM C scaffold, from the ⌬fdmM or ⌬fdmM1 mutant strains further supports the idea of amide formation as a postpolyketide synthase tailoring step (20) (Fig. 2).…”

“…1) (20). Among these aromatic polyketides, lysolipin X (LlpA) (13) possesses a lactam ring like FDM A. Pradimicin A (PdmN) (28) contains an amide bond formed putatively using D-Ala as the amino donor.…”

“…2) (20) and selected monocyclic benzoic acid derivatives (Fig. S4) were next tested as potential substrates of FdmV.…”