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SummaryProtein glycosylation is a widespread post-translational modification. The first committed step to the lipid-linked glycan used for this process is catalysed by dolichyl-phosphate N-acetylglucosamine-phosphotransferase DPAGT1 (GPT/E.C. 2.7.8.15). Missense DPAGT1 variants cause congenital myasthenic syndrome and congenital disorders of glycosylation. In addition, naturally-occurring bactericidal nucleoside analogues such as tunicamycin are toxic to eukaryotes due to DPAGT1 inhibition, preventing their clinical use as antibiotics. However, little is known about the mechanism or the effects of disease-associated mutations in this essential enzyme. Our structures of DPAGT1 with the substrate UDP-GlcNAc and tunicamycin reveal substrate binding modes, suggest a mechanism of catalysis, provide an understanding of how mutations modulate activity (and thus cause disease) and allow design of non-toxic ‘lipid-altered’ tunicamycins. The structure-tuned activity of these analogues against several bacterial targets allowed design of potent antibiotics forMycobacterium tuberculosis, enabling treatmentin vitro,in celluloandin vivothereby providing a promising new class of antimicrobial drug.HighlightsStructures of DPAGT1 with UDP-GlcNAc and tunicamycin reveal mechanisms of catalysisDPAGT1 mutants in patients with glycosylation disorders modulate DPAGT1 activityStructures, kinetics and biosynthesis reveal role of lipid in tunicamycinLipid-altered, tunicamycin analogues give non-toxic antibiotics against TB

Protein glycosylation is a widespread post-translational modification. The first committed step to the lipid-linked glycan used for this process is catalysed by dolichyl-phosphate N-acetylglucosamine-phosphotransferase DPAGT1 (GPT/E.C. 2.7.8.15). Missense DPAGT1 variants cause congenital myasthenic syndrome and congenital disorders of glycosylation. In addition, naturally-occurring bactericidal nucleoside analogues such as tunicamycin are toxic to eukaryotes due to DPAGT1 inhibition, preventing their clinical use as antibiotics. However, little is known about the mechanism or the effects of disease-associated mutations in this essential enzyme. Our structures of DPAGT1 with the substrate UDP-GlcNAc and tunicamycin reveal substrate binding modes, suggest a mechanism of catalysis, provide an understanding of how mutations modulate activity (and thus cause disease) and allow design of non-toxic ‘lipid-altered’ tunicamycins. The structure-tuned activity of these analogues against several bacterial targets allowed design of potent antibiotics for Mycobacterium tuberculosis, enabling treatment in vitro, in cellulo and in vivo thereby providing a promising new class of antimicrobial drug.

The tunicamycins are archetypal nucleoside antibiotics targeting bacterial peptidoglycan biosynthesis and eukaryotic protein N-glycosylation. Understanding the biosynthesis of their unusual carbon framework may lead to variants with improved selectivity. Here, we demonstrate in vitro recapitulation of key sugar-manipulating enzymes from this pathway. TunA is found to exhibit unusual regioselectivity in the reduction of a key α,β-unsaturated ketone. The product of this reaction is shown to be the preferred substrate for TunF--an epimerase that converts the glucose derivative to a galactose. In Streptomyces strains in which another gene (tunB) is deleted, the biosynthesis is shown to stall at this exo-glycal product. These investigations confirm the combined TunA/F activity and delineate the ordering of events in the metabolic pathway. This is the first time these surprising exo-glycal intermediates have been seen in biology. They suggest that construction of the aminodialdose core of tunicamycin exploits their enol ether motif in a mode of C-C bond formation not previously observed in nature, to create an 11-carbon chain.

A rare example of beta- over gamma-C-H selectivity during Rh-catalysed sulfamate ester cyclisation is presented; from derivatives of 1,6-anhydro-beta-d-mannopyranose, five-membered sulfamidates were formed in preference to the typical six-membered oxathiazinane intramolecular insertion products. A 3D structure of sulfamate 1 helps to rationalise this unusual selectivity and analyses suggest that n-->sigma*(CH) interactions may be a key controlling factor.

Tunicamycin nucleoside antibiotics were the first known to target the formation of peptidoglycan precursor lipid I in bacterial cell wall biosynthesis. They have also been used extensively as inhibitors of protein N-glycosylation in eukaryotes, blocking the biogenesis of early intermediate dolichyl-pyrophosphoryl-N-acetylglucosamine. Despite their unusual structures and useful activities, little is known about their biosynthesis. Here we report identification of the tunicamycin biosynthetic genes in Streptomyces chartreusis following genome sequencing and a chemically-guided strategy for in silico genome mining that allowed rapid identification and unification of an operon fractured across contigs. Heterologous expression established a likely minimal gene set necessary for antibiotic production, from which a detailed metabolic pathway for tunicamycin biosynthesis is proposed. These studies unlock a comprehensive and unusual toolbox of biosynthetic machinery with which to create variants of this important natural product, allowing possible improved understanding of the mode of action and facilitating future redesign. We anticipate that these results will enable the generation of altered specific inhibitors of diverse carbohydrate-processing enzymes, including improved targeting of lipid I biosynthesis. © 2010 The Royal Society of Chemistry.