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

Lead Research Organisation: University of Oxford
Department Name: Interdisciplinary Bioscience DTP


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 areas
Combating antimicrobial resistance, Nutrition and health, New strategic approaches to industrial biotechnology


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Studentship Projects

Project Reference Relationship Related To Start End Student Name
BB/M011224/1 30/09/2015 31/03/2024
1810160 Studentship BB/M011224/1 30/09/2015 29/09/2019
Description I have developed a mammalian cell expression and purification procedure for mouse CMAH protein production, as well as a new mass spectrometry-based activity assay.
For my side project focusing on Escherichia coli glyoxalase II (GloB), I have determined a previously unsolved crystal structure which is the second bacterial glyoxalase II structure reported. Metal content analysis showed binding of a mixture of transition metals (as oppossed to the predicted Zn) in the active site which may potentially alter activity and inhibition profiles. I have also performed inihibitor screens and identifies several mid-range inhibitors.
Exploitation Route Better understanding of CMAH could lead to potential used for biocatalysis for otherwise difficult hydroxylation reaction which could be useful for biotechnology applications.
GloB could be explored as an additional antimicrobial target in combination with out proteins in pharmaceutical sector.
Additionally, both of the proteins are members of the metallo-beta-lactamase (MBL) superfamily and share structural similarity to enzymes involved in antimicrobial resistance. Better understanding of more MBL proteins could lead to less off-target inhibition and hence less side effects in therapeutics targeting resistant bacteria.
Sectors Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology