Functional characterization and substrate specificity of spinosyn rhamnosyltransferase by in vitro reconstitution of spinosyn biosynthetic enzymes.
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 structure-activity 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.
Chen
Yi-Lin
YL
Department of Biological Science and Technology, 75 Po-Ai St., National Chiao Tung University, Hsinchu 300, Taiwan.
Chen
Yi-Hsine
YH
Lin
Yu-Chin
YC
Tsai
Kuo-Chung
KC
Chiu
Hsien-Tai
HT
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D006602:Hexosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007306:Insecticides / Q000737:chemistry* / Q000378:metabolism; D018942:Macrolides / Q000737:chemistry* / Q000378:metabolism; D009702:Nucleoside Diphosphate Sugars / Q000096:biosynthesis / Q000737:chemistry; D017434:Protein Structure, Tertiary / Q000502:physiology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016930:Saccharopolyspora / Q000201:enzymology* / Q000235:genetics; D013329:Structure-Activity Relationship; D013379:Substrate Specificity / Q000502:physiology; D013942:Thymine Nucleotides / Q000096:biosynthesis / Q000737:chemistry
D001426:Bacterial Proteins; D007306:Insecticides; D018942:Macrolides; D009702:Nucleoside Diphosphate Sugars; D011994:Recombinant Proteins; D013942:Thymine Nucleotides; C073293:thymidine diphosphate rhamnose; D006602:Hexosyltransferases