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Protein glycosylation plays acrucial role in various biological processes in all domain of life.In human,aberrant O-GlcNAcylations are associated with many challenging diseases,including diabetes,cardiovascular disease,neurodegenerative diseases and cancer.Whereas,the O-GlcNAcylation of glycoprotein in bacteria is closely related with the physiology and pathogenesis,especially for the pathogenic bacterial strains Prominently,serine-rich repeat glycoproteins(SRRPs)exclusively exist in Gram positive pathogens are a large family of surface adhesins,which are important for bacterial adhesion,immune evasion,colonization,biofilm formation and virulence.PsrP is highly glycosylated by a series of glycosyltransferases.Beyond the well-studied first fourth steps of the glycosylation of PsrP,the subsequent glycosylation steps remains unknown. GlyB and GlyG form S.pneumoniae TIGR4are45.4KDa and35.1KDa are involoved in Psrp glycosylation,through the present study we crystallized Apo-form of GlyB and GlyG along with a complex form with UDP,UDP-Glc/UDP-Gal,UDP and Glc/GlcNAc.Unfortunately we failed to determine the structure of the two protein due to poor X-ray diffraction that were5.0-6.0(A)for GlyB and3.5(A)was obtained by crystal of GlyG,therefore we were not able to unravel the catalytic subunit of GlyB and GlyG structurally.However the structure based homologus revealed that the N-terminal core domain of GlyB and GlyG are typical metal-dependent GT-A fold while the C-terminus of GlyB adopt GT-B type fold. Moreover,enzymatic assays indicate that GlyB is a glucosyltransferase transferring glucose/galactose moiety by hydrolyzed UDP-glucose and UDP-galactose suggesting that may be divaricated enzyme,while GlyG contains a single GT2domain could only hydrolyzed UDP-Glc and transfer glucose.Study of the structure and function of these glycosyltransferases will aid to understand the glycosylation mechanism and biogenesis of PsrP,which might be the starting point to design a novel antibiotics drugs.