RESUMO
Protein N-glycosylation is a widespread post-translational modification. The first committed step in 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 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. 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 (thus causing disease) and allow design of non-toxic "lipid-altered" tunicamycins. The structure-tuned activity of these analogues against several bacterial targets allowed the design of potent antibiotics for Mycobacterium tuberculosis, enabling treatment in vitro, in cellulo and in vivo, providing a promising new class of antimicrobial drug.
Assuntos
Antibióticos Antituberculose/farmacologia , Defeitos Congênitos da Glicosilação/metabolismo , Inibidores Enzimáticos/farmacologia , N-Acetilglucosaminiltransferases/química , Animais , Antibióticos Antituberculose/química , Sítios de Ligação , Defeitos Congênitos da Glicosilação/genética , Inibidores Enzimáticos/química , Feminino , Células HEK293 , Células Hep G2 , Humanos , Metabolismo dos Lipídeos , Camundongos , Simulação de Acoplamento Molecular , Mutação , N-Acetilglucosaminiltransferases/antagonistas & inibidores , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , Ligação Proteica , Células Sf9 , Spodoptera , Tunicamicina/química , Tunicamicina/farmacologia , Uridina Difosfato Ácido Glucurônico/química , Uridina Difosfato Ácido Glucurônico/metabolismoRESUMO
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,ß-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.
Assuntos
Antibacterianos/biossíntese , Tunicamicina/biossíntese , Antibacterianos/química , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Biocatálise , Carboidratos Epimerases/química , Carboidratos Epimerases/metabolismo , Biologia Computacional , Hidroliases/química , Hidroliases/metabolismo , Cetonas/química , Cetonas/metabolismo , Família Multigênica , Estrutura Terciária de Proteína , Pseudomonas aeruginosa/enzimologia , Estereoisomerismo , Especificidade por Substrato , Tunicamicina/químicaRESUMO
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.
