Functional characterization and substrate specificity of spinosyn rhamnosyltransferase by in Vitro reconstitution of spinosyn biosynthetic enzymes

Yi Lin Chen, Yi Hsine Chen, Yu Chin Lin, Kuo Chung Tsai, Hsien Tai Chiu

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27 Citations (Scopus)

Abstract

Spinosyn, a potent insecticide, is a novel tetracyclic polyketide decorated with D-forosamine and tri-O-methyl-L-rhamnose. Spinosyn rhamnosyltransferase (SpnG) is a key biocatalyst with unique sequence identity and controls the biosynthetic maturation of spinosyn. The rhamnose is critical for the spinosyn insecticidal activity and cell wall biosynthesis of the spinosyn producer, Saccharopolyspora spinosa. In this study, we have functionally expressed and characterized SpnG and the three enzymes, Gdh, Epi, and Kre, responsible for dTDP-L-rhamnose biosynthesis in S. spinosa by purified enzymes from Escherichia coli. Most notably, the substrate specificity of SpnG was thoroughly characterized by kinetic and inhibition experiments using various NDP sugar analogs made by an in situ combination of NDP-sugar-modifying enzymes. SpnG was found to exhibit striking substrate promiscuity, yielding corresponding glycosylated variants. Moreover, the critical residues presumably involved in catalytic mechanism of Gdh and SpnG were functionally evaluated by site-directed mutagenesis. The information gained from this study has provided important insight into molecular recognition and mechanism of the enzymes, especially SpnG. The results have made possible the structureactivity characterization of SpnG, as well as the use of SpnG or its engineered form to serve as a combinatorial tool to make spinosyn analogs with altered biological activities and potency.

Original languageEnglish
Pages (from-to)7352-7363
Number of pages12
JournalJournal of Biological Chemistry
Volume284
Issue number11
DOIs
Publication statusPublished - 2009 Mar 13

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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