【摘要】 大肠杆菌O152抗原的寡糖重复单位中含葡萄糖-β-1,3-N-乙酰葡萄糖胺(Glc-β-1,3-GlсNAc)连接键。本研究采用电喷雾离子化多级串联质谱技术对以人工合成的天然受体底物的结构类似物苯氧基十一烷二磷酸-N-乙酰葡萄糖胺(GlcNAc-β-PO3-PO3-(CH2)11-O-phenyl(GlcNAc-PP-PhU))为受体底物,尿苷二磷酸葡萄糖(UDP-Glc)为给予体底物的酶促反应产物进行了详细的结构表征。电喷雾离子化多级串联质谱图中观察到的主要碎片源于磷酸二酯键部分和糖苷键的裂解。此外,由观察到的二糖产物非还原端碎片获得了序列信息;跨环断裂碎片及源于吡喃环取代基消除的‘内在’碎裂离子可提供组成产物的单糖残基连接方式信息。广泛的碎裂信息表明wfgD基因编码UDP-Glc:GlcNAc-pyrophosphate-lipid中的β-1-3葡萄糖基转移酶。更多还原

【Abstract】 The O152 antigens of Escherichia coli contains a Glc-β-1,3-GlcNAc linkage within the repeating unit.The wfgD gene in E.coli O152 O antigen gene cluster had been demonstrated utilizing NMR technique to encode a glucosyltransferase which is responsible for the synthesis of Glc-β-1,3-GlcNAc linkage.In this study,a synthetic substrate analog of the natural acceptor substrate undecaprenol-pyrophosphate-lipid [GlcNAc-α-PO₃-PO₃-（CH₂）₁1-O-phenyl] was used as an acceptor and UDP-Glc as a donor substrate,and electrospray ionization tandem mass spectrometry（ESI-MS-MS） was used for the detailed structural characte-rization of the enzyme product.A systematic study was conducted on product to allow rationalization of the fragmentation processes.The major fragments observed in the ESI-MS-MS spectra result from cleavage of glycosidic bond and diphosphate moiety.The fragment originating from the nonreducing end of the product yields information on sequence.Cross-ring cleavages,which are very informative of the linkages of the monosaccharide residues constituting the product,and "internal" cleavage ions which are derived from elimination of substituents from around the pyranose ring,were also observed.This extensive fragmentation was shown that the expected Glc-β-1,3-GlcNAc linkage in the product,confirming that wfgD is in the form of UDP-Glc: GlcNAc-pyrophosphate-lipid β-1,3-glucosyltransferase.更多还原