A dedicated phosphopantetheinyl transferase for the fredericamycin polyketide synthase from Streptomyces griseus.
Polyketide synthases cannot be functional unless their apo-acyl carrier proteins (apo-ACPs) are post-translationally modified by covalent attachment of the 4'-phosphopantetheine group to the highly conserved serine residue, and this reaction is catalyzed by phosphopantetheinyl transferases (PPTases). Cloning and sequence analysis of the 33-kb fredericamycin (FDM) biosynthetic gene cluster from Streptomyces griseus revealed fdmW, whose deduced gene product showed significant sequence homology to known PPTases. Biochemical characterization of FdmW in vitro confirmed that it is a PPTase. Inactivation of fdmW resulted in approximately 93% reduction of FDM production, and complementation of the fdmW::aac (3)IV mutant by expressing fdmW in trans restored FDM production to a level comparable with that of the wild-type strain. Although FdmW can phosphopantetheinylate various ACPs, it prefers its cognate substrate, the FdmH ACP, with a K(m) of 5.8 microM and a k(cat)/K(m) of 8.1 microM(-1) x min(-1), to heterologous ACPs, such as the TcmM ACP with a K(m) of 1.0 x 10(2) microM and a k(cat) /K(m) of 0.6 microM(-1) x min(-1). These findings suggest that FdmW is specific for FDM biosynthesis. FdmW therefore represents the first holo-ACP synthase-type PPTase identified from an aromatic polyketide biosynthetic gene cluster.
Huang
Yong
Y
Division of Pharmaceutical Sciences, University of Wisconsin, Madison, Wisconsin 53705, USA.
Wendt-Pienkowski
Evelyn
E
Shen
Ben
B
D000475:Alkenes / Q000138:chemical synthesis / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D007546:Isoquinolines / Q000138:chemical synthesis / Q000378:metabolism*; D008969:Molecular Sequence Data; D005810:Multigene Family; D048630:Polyketide Synthases / Q000737:chemistry / Q000502:physiology*; D013305:Streptomyces griseus / Q000201:enzymology* / Q000235:genetics; D017855:Transferases (Other Substituted Phosphate Groups) / Q000737:chemistry / Q000235:genetics / Q000502:physiology*
D000475:Alkenes; D001426:Bacterial Proteins; D007546:Isoquinolines; C082662:phosphopantetheinyl transferase; D048630:Polyketide Synthases; D017855:Transferases (Other Substituted Phosphate Groups)