New access to complex glycoconjugates through engineering and evolution of glycosyltransferases and glycosynthases
报 告 人：Stephen G. Withers
Extreme Gene Machines: how hyperthermophiles maintain genome integrity
报 告 人：马尔科姆•怀特（Malcolm F White）
英国University of St Andrews教授
联 系 人：于晴
Stephen G. Withers教授是加拿大不列颠哥伦比亚大学Khorana讲席教授、英国皇家学会院士、加拿大皇家学会院士、加拿大化学会院士，现任加拿大化学生物首席研究科学家(Canada Research Chair)，UBC高通量生物学中心主任。他主要从事糖类代谢酶的作用机制、糖类合成酶分子改造、糖代谢酶小分子药物设计研究。他在糖化学生物领域有着卓越成就和重大影响。1992年获加拿大皇家学会卢瑟福奖，2002年获国际碳水化合物组织惠斯勒奖，2012年获英国皇家学会百年奖。
Malcolm F White是英国University of St Andrews教授、英国爱丁堡皇家科学院院士、欧洲分子生物学组织 (EMBO)成员。现任圣安德鲁斯大学生物学院科研院长，英国生物技术与生物科学研究理事会(BBSRC)委员，兼任生化领域著名国际期刊Biochemical Journal副主编。他综合运用生物化学、分子生物学、蛋白质组学、生物信息学等方法，研究核酸相关代谢机制，在古菌DNA损伤修复和基于CRISPR的抗病毒机制方面取得了令人瞩目的研究成就，是活跃在微生物遗传学研究前沿领域的著名学者，在古菌遗传学和结构生物学研究领域具有很大影响力。
Glycans on the surfaces of cells play key roles in the interaction of that cell with its environment, primarily through interaction with specific protein-based receptors. Study of these interactions requires access to these complex glycans, while interference with these interactions, most likely through the use of competing glycans, is a possible therapeutic approach. Such studies require synthesis of glycans, and on a large scale in the case of therapeutics. Traditional routes to the enzymatic synthesis of oligosaccharides have either involved the use of Nature’s own biosynthetic enzymes, the glycosyl transferases, or glycosidases run in transglycosylation mode. Each approach has its drawbacks. Glycosynthases are mutant glycosidases in which the catalytic nucleophile has been removed. When used in conjunction with glycosyl fluorides of the opposite anomeric configuration to that of the substrate, these enzymes function as highly efficient transferases, frequently giving stoicheometric yields. Thioglycoligases are a new class of mutant glycosidases in which the acid/base catalyst has been mutated. These enzymes synthesise sulfur-linked oligosaccharides when an activated donor is used in conjunction with a thiosugar acceptor. Recent results in the engineering of these two classes of mutant enzymes, as well as of “classical” glycosyl transferases, will be discussed. Particular attention will be paid to their application to oligosaccharides and glycolipids. Emphasis will be placed upon the directed evolution of these enzymes using a variety of screening methodologies including robot-assisted ELISA assays and FACS cell sorting.
Archaea frequently inhabit extreme environments and thus face challenges in maintaining functional cellular structures and macromolecules, in particular DNA which must be repaired efficiently. The fundamental relationship between the archaea and eukarya means that studies of the former can shed light on processes and proteins essential for human health. This talk will focus on recent research in our laboratory on the Nucleotide Excision Repair (NER) pathway in the model crenarchaeon Sulfolobus solfataricus, which grows in volcanic pools at 80°C. NER removes bulky lesions such as photoproducts from DNA. Lesions are detected, DNA unwound locally and nicks introduced on either side of the damage to release an oligonucleotide “patch” containing the damage. The gapped product is then repaired by DNA synthesis. The enzymatic components of eukaryal NER are the XPB and XPD helicases (components of transcription factor TFIIH) and the XPF and XPG nucleases. By studying these enzymes in archaea we can gain important insights into human health and disease. A second challenge facing most living things is the threat posed by viruses and other mobile genetic elements. The recently discovered CRISPR system is a prokaryotic adaptive immune system that provides RNA-mediated defence against invading nucleic acid entities. Recent work in our laboratory on the Type III Interference machines of the CRISPR system will be described.