Synthesis of Heptaprenyl-Lipid Ⅳ to Analyze Peptidoglycan Glycosyltransferases

摘要

Peptidoglycan (PG) is a crosslinked carbohydrate polymer that surrounds and protects bacterial cell membranes, enabling bacteria to withstand large fluctuations in internal osmotic pressure. Because peptidoglycan is essential for bacterial cell survival, the enzymes involved in the biosynthesis of PG are targets for antibiotics. Over the past fifteen years, great progress has been made toward understanding the different steps of PG synthesis, but there is still one family of enzymes that remains poorly characterized: the peptidoglycan glycosyltransferases (PGTs) that catalyze formation of the carbohydrate chains of peptidoglycan from a disaccharide precursor called Lipid Ⅱ (1, Figure 1). Bacteria typically contain several different PGTs whose biological roles are poorly understood. Biochemical studies of PGTs have been hampered by difficulties in obtaining substrates to dissect the polymerization mechanism. The first coupling catalyzed by PGTs involves the condensation of two Lipid Ⅱ substrates to form a tetrasaccharide, Lipid Ⅳ (2, Figure 1). Subsequent coupling cycles involve the elongation of the growing polymer by addition of Lipid Ⅱ subunits. Therefore, after the first coupling cycle, the substrates used by PGTs are different. To probe the mechanism of glycosyltransfer and to characterize enzyme inhibitors, it is essential to have Lipid Ⅱ substrates (1) as well as a longer substrate representing the growing polymer (such as Lipid Ⅳ, 2). We and others have previously developed approaches to obtain Lipid Ⅱ, but longer substrates have not been reported. Here we describe the total synthesis of heptaprenyl-Lipid Ⅳ (2b) and we show that both major E. coli PGTs, PBP1a and PBP1b, couple this substrate to heptaprenyl-Lipid Ⅱ (1b). Unexpectedly, PBPla also couples Lipid Ⅳ subunits to one another, suggesting that some PGTs may be able to ligate longer glycan polymers in addition to building glycan chains from Lipid Ⅱ.