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

胞苷单磷酸- n -乙酰神经氨酸羟化酶（CMAH）在动物细胞中催化唾液酸n -乙酰神经氨酸（Neu5Ac）转化为n -糖基神经氨酸（Neu5Gc）。唾液酸构成细胞表面糖蛋白和糖脂的最外层成分，并参与细胞-细胞识别和细胞-病原体相互作用。CMAH是最早在细菌酶水解-内酰胺类抗生素中发现的金属内酰胺酶（MBL）折叠超家族成员，在耐药过程中起重要作用。MBL折叠超家族成员存在于动物、植物和真菌中，具有水解和氧化还原反应等多种功能。CMAH是一种独特的氧化还原酶，含有Rieske [2Fe2S]中心和单核铁原子，预计需要分子氧才能进行反应。人类CMAH的催化活性是由于大约300万年前发生的n端缺失事件，该事件发生在人类和类人猿最后一个共同祖先之后，但在现代人类出现之前。CMAH功能的丧失可能影响与唾液酸有关的各种过程，例如细胞-细胞和细胞-病原体相互作用，导致对某些唾液酸识别病原体（包括疟疾寄生虫和流感病毒）的易感性发生变化，以及糖代谢。尽管没有催化活性，但人类CMAH仍然表达，这表明它可能具有替代功能。目前对活性动物CMAH的作用机制和催化失活的人酶的潜在替代功能知之甚少，迄今为止还没有任何CMAH同源物的三维结构。在这个项目中，我将使用x射线晶体学、动力学和生物物理表征以及基于细胞的分析来研究截断的人类CMAH及其催化活性小鼠同源物的结构和功能。了解人类CMAH的功能和失活缺失的影响将有助于了解对病原体、糖尿病和人类进化的易感性，而从工业生物技术的角度来看，活性CMAH也很有趣，因为它能催化n -乙酰基的氧化（即羟基化）这一困难的化学过程。重点领域：抗微生物药物耐药性、营养与健康、工业生物技术的新战略途径

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