Biochemical studies of the enzymes involved in deoxysugar D-forosamine biosynthesis
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Deoxysugars are indispensable structural components of many biologically active natural products and are essential for many significant cellular processes. In this work, the biosynthetic pathway of TDP-forosamine, which is a 2,3,4,6-tetradeoxy sugar component of the potent, and environmentally benign insecticide spinosyn, has been established without ambiguity. Five genes, spnO, spnN, spnQ, spnR, and spnS have been cloned from Saccharopolyspora spinosa chromosomal DNA and expressed in E. coli. The encoded proteins have been purified and characterized by in vitro assays. The products of four reactions have been isolated and characterized with MS and/or NMR. SpnO was demonstrated to be a 2,3-dehydratase, while SpnN was shown to be a 3- ketoreductase, catalyzing equatorial hydroxyl formation at C-3. Studies of the combined action of SpnO and SpnN reinforce the previous finding that C-2 deoxygenation is accomplished through a -elimination/reduction type mechanism. SpnQ is a homologue of the well-characterized CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase (E1) which in combination with CDP-6-deoxy-4-keto-3,4-glucoseen reductase (E3) catalyzes the C-O bond cleavage at C-3 in the biosynthesis of CDP-ascarylose in Yersinia pseudotuberculosis. In the biosynthesis of TDP-forosamine, SpnQ was shown to catalyze an analogous C-3 deoxygenation but instead using ferredoxin/ferredoxin reductase pair as the electron transfer media. Additionally, in the absence of ferredoxin/ferredoxin reductase to allow C-3 deoxygenation, and in the presence of Lglutamate as an amino donor, SpnQ was shown to act as a 4-aminotransferase, converting TDP-2,6-dideoxy-4-keto-D-glucose to TDP-4-amino-2,4,6-trideoxy-D-glucose. Due to the instability of 2-deoxysugars, an analog of TDP-4-amino-2,3,4,6-tetradeoxy-glucose was used to characterize the late steps in the pathway. SpnR was shown to be a 4- aminotransferase which, interestingly, can recognize both the SpnQ product and the SpnN product as substrates. SpnS was shown to be a 4-amino-N,N-dimethyltransferase. Mechanistic investigation revealed that both N-methyl groups of TDP-forosamine are installed by SpnS in a step-wise manner via a monomethylated intermediate. Together, studies described in this thesis have extensively enriched our knowledge of the enzymes involved in deoxysugar biosynthesis. The newly discovered substrate flexibility of TDPforosamine pathway enzymes will also be very useful in generating engineered sugar biosynthetic pathway for use in generating novel natural products.