Characterization of FdmV as an amide synthetase for fredericamycin A biosynthesis in Streptomyces griseus ATCC 43944 |
| |
Authors: | Chen Yihua Wendt-Pienkowski Evelyn Ju Jianhua Lin Shuangjun Rajski Scott R Shen Ben |
| |
Institution: | Division of Pharmaceutical Sciences, University of Wisconsin, Madison, Wisconsin 53705-2222, USA. |
| |
Abstract: | Fredericamycin (FDM) A is a pentadecaketide natural product that features an amide linkage. Analysis of the fdm cluster from Streptomyces griseus ATCC 43944, however, failed to reveal genes encoding the types of amide synthetases commonly seen in natural product biosynthesis. Here, we report in vivo and in vitro characterizations of FdmV, an asparagine synthetase (AS) B-like protein, as an amide synthetase that catalyzes the amide bond formation in FDM A biosynthesis. This is supported by the findings that (i) inactivation of fdmV in vivo afforded the ΔfdmV mutant strain SB4027 that abolished FDM A and FDM E production but accumulated FDM C, a biosynthetic intermediate devoid of the characteristic amide linkage; (ii) FdmV in vitro catalyzes conversion of FDM C to FDM B, a known intermediate for FDM A biosynthesis (apparent K(m) = 162 ± 67 μM and k(cat) = 0.11 ± 0.02 min(-1)); and (iii) FdmV also catalyzes the amidation of FDM M-3, a structural analog of FDM C, to afford amide FDM M-6 in vitro, albeit at significantly reduced efficiency. Preliminary enzymatic studies revealed that, in addition to the common nitrogen sources (L-Gln and free amine) of class II glutamine amidotransferases (to which AS B belongs), FdmV can also utilize L-Asn as a nitrogen donor. The amide bond formation in FDM A biosynthesis is proposed to occur after C-8 hydroxylation but before the carbaspirocycle formation. |
| |
Keywords: | Antibiotics Bacterial Genetics Bacterial Metabolism Enzyme Catalysis Enzyme Mechanisms Enzymes Asparagine Synthetase Natural Product Biosynthesis |
本文献已被 PubMed 等数据库收录! |
|