Structural and functional characterisation of cytidine monophosphate-N-acetylneuraminic acid hydroxylase - BfH, IBB, ENWW

胞苷一磷酸-N-乙酰神经氨酸羟基酶(CMAH)在动物细胞中催化唾液酸N-乙酰神经氨酸(Neu5Ac)转化为N-羟基神经氨酸(Neu5Gc)。唾液酸构成细胞表面糖蛋白和糖脂的最外层成分，参与细胞-细胞识别和细胞-病原体相互作用。CMAH是金属内酰胺酶(MBL)折叠超家族的一员，最早发现于降解内酰胺类抗生素的细菌酶中，在耐药中起重要作用。MBL折叠超家族成员广泛存在于动物、植物和真菌中，具有包括水解和氧化还原反应在内的多种功能。CMAH是一种独特的氧化还原酶，含有一个Rieske[2Fe2S]中心和一个单核铁原子，预计需要分子氧才能进行反应。人类CMAH是由于N末端缺失事件而催化失活的，该事件发生在大约300万年前，在人类和类人猿的最后一个共同祖先之后，但在现代人出现之前。CMAH功能的丧失可能在涉及唾液酸的各种过程中产生影响，例如细胞-细胞和细胞-病原体的相互作用导致对某些识别病原体(包括疟疾寄生虫和流感病毒)的唾液酸敏感性的变化以及糖的代谢。尽管没有催化活性，但人类CMAH仍然被表达，这表明它可能有另一种功能。关于活性动物CMAH的作用机制和催化失活的人类酶的潜在替代功能知之甚少，到目前为止还没有可用的CMAH同源物的三维结构。在这个项目中，我将使用X射线结晶学、动力学和生物物理特性以及基于细胞的分析来研究截短的人CMAH及其催化活性的小鼠同源物的结构和功能。了解CMAH在人类中的功能和失活缺失的影响将为病原体的易感性、糖尿病和人类进化提供新的线索，而活性CMAH也是工业生物技术的研究热点，因为它催化N-乙酰基团的困难化学过程-氧化(即羟化)。BBSRC优先领域抗菌素耐药性、营养和健康，工业生物技术的新战略方法

Cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) catalyses the conversion of sialic acid N-acetylneuraminic acid (Neu5Ac) to N-glycolylneuraminic acid (Neu5Gc) in animal cells. Sialic acids constitute the outermost components of cell surface glycoproteins and glycolipids and are involved in cell-cell recognition and cell-pathogen interactions. CMAH is a member of metallo-lactamase (MBL) fold superfamily that was first discovered in bacterial enzymes hydrolysing -lactam antibiotics and which is important in drug resistance. Members of MBL fold superfamily are found in animals, plants and fungi and have diverse functions including hydrolysis and redox reactions. CMAH is a unique redox enzyme containing a Rieske [2Fe2S] centre and a mononuclear iron atom and is predicted to require molecular oxygen for the reaction to proceed. Human CMAH is catalytically inactive due to an N-terminal deletion event which occurred around 3 million years ago after the last common ancestor of humans and great apes but before emergence of modern humans. Loss of CMAH function likely had implications in various processes involving sialic acids, such as cell-cell and cell-pathogen interactions leading to changes in susceptibility to some sialic acid recognising pathogens (including malaria parasites and influenza viruses) as well as sugar metabolism. Despite being catalytically inactive, human CMAH is still expressed suggesting it may have an alternative function. Little is known about the mechanism of action of the active animal CMAH and the potential alternative function of the catalytically inactive human enzyme and to date there is no three-dimensional structure of any CMAH homologue available. During this project I am going to investigate structure and function of both the truncated human CMAH and its catalytically active mouse homologue using X-ray crystallography, kinetic and biophysical characterisation as well as cell-based assays. Understanding CMAH function and the effect of the inactivating deletion in humans will shed new light on susceptibility to pathogens, diabetes and human evolution while active CMAH is also interesting from and industrial biotechnology perspective because it catalyses difficult chemical process - oxidation (i.e. hydroxylation) of an N-acetyl group.BBSRC priority areasCombating antimicrobial resistance, Nutrition and health, New strategic approaches to industrial biotechnology