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DOI	10.1073/pnas.1703397114
	Analysis of a dual domain phosphoglycosyl transferase reveals a ping-pong mechanism with a covalent enzyme intermediate
	Das D.; Kuzmic P.; Imperiali B.
发表日期	2017
ISSN	0027-8424
起始页码	7019
结束页码	7024
卷号	114 期号:27
英文摘要	Phosphoglycosyl transferases (PGTs) are integralmembrane proteins with diverse architectures that catalyze the formation of polyprenol diphosphate-linked glycans via phosphosugar transfer from a nucleotide diphosphate-sugar to a polyprenol phosphate. There are two PGT superfamilies that differ significantly in overall structure and topology. The polytopic PGT superfamily, represented by MraY and WecA, has been the subject of many studies because of its roles in peptidoglycan and O-antigen biosynthesis. In contrast, less is known about a second, extensive superfamily of PGTs that reveals a core structure with dual domain architecture featuring a C-terminal soluble globular domain and a predicted N-terminal membraneassociated domain. Representative members of this superfamily are the Campylobacter PglCs, which initiate N-linked glycoprotein biosynthesis and are implicated in virulence and pathogenicity. Despite the prevalence of dual domain PGTs, their mechanism of action is unknown. Here, we present the mechanistic analysis of PglC, a prototypic dual domain PGT from Campylobacter concisus. Using a luminescence-based assay, together with substrate labeling and kinetics-based approaches, complementary experiments were carried out that support a ping-pong mechanism involving a covalent phosphosugar intermediate for PglC. Significantly, mass spectrometrybased approaches identified Asp93, which is part of a highly conserved AspGlu dyad found in all dual domain PGTs, as the active-site nucleophile of the enzyme involved in the formation of the covalent adduct. The existence of a covalent phosphosugar intermediate provides strong support for a ping-pong mechanism of PglC, differing fundamentally from the ternary complex mechanisms of representative polytopic PGTs.
英文关键词	Covalent intermediate; Dual domain PGT; Glycoconjugate biosynthesis; Membrane protein; Phosphoglycosyl transferase
语种	英语
scopus关键词	bacterial enzyme; dual domain phosphoglycosyl transferase pglc; glycoconjugate; sugar phosphate; transferase; unclassified drug; uridine phosphate; antiinfective agent; aspartic acid; bacterial protein; carbohydrate; glutamic acid; peptidoglycan; transferase; amino terminal sequence; Article; bacterial survival; bacterial virulence; Campylobacter concisus; carbohydrate synthesis; carboxy terminal sequence; controlled study; covalent bond; enzyme active site; enzyme analysis; enzyme kinetics; enzyme substrate; nonhuman; priority journal; protein glycosylation; Campylobacter; chemical model; chemistry; enzyme specificity; enzymology; kinetics; luminescence; metabolism; Anti-Bacterial Agents; Aspartic Acid; Bacterial Proteins; Campylobacter; Catalytic Domain; Glutamic Acid; Kinetics; Luminescence; Models, Chemical; Peptidoglycan; Substrate Specificity; Sugars; Transferases
来源期刊	Proceedings of the National Academy of Sciences of the United States of America
文献类型	期刊论文
条目标识符	http://gcip.llas.ac.cn/handle/2XKMVOVA/160618
作者单位	Das, D., Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139s, United States, Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, United States; Kuzmic, P., BioKin Ltd., Watertown, MA 02472, United States; Imperiali, B., Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139s, United States, Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, United States
推荐引用方式 GB/T 7714	Das D.,Kuzmic P.,Imperiali B.. Analysis of a dual domain phosphoglycosyl transferase reveals a ping-pong mechanism with a covalent enzyme intermediate[J],2017,114(27).
APA	Das D.,Kuzmic P.,&Imperiali B..(2017).Analysis of a dual domain phosphoglycosyl transferase reveals a ping-pong mechanism with a covalent enzyme intermediate.Proceedings of the National Academy of Sciences of the United States of America,114(27).
MLA	Das D.,et al."Analysis of a dual domain phosphoglycosyl transferase reveals a ping-pong mechanism with a covalent enzyme intermediate".Proceedings of the National Academy of Sciences of the United States of America 114.27(2017).