Ahangar, Mohd Syed, Furze, Christopher M., Guy, Collette S., Cooper, Charlotte, Maskew, Kathryn S, Graham, Ben, Cameron, Alexander and Fullam, Elizabeth (2018) Structural and functional determination of homologs of the Mycobacterium tuberculosis N-acetylglucosamine-6-phosphate deacetylase (NagA). Journal of Biological Chemistry, 293 (25). pp. 9770-9783. doi:10.1074/jbc.RA118.002597 ISSN 0021-9258.

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Abstract

The (Mtb) pathogen encodes an -acetylglucosamine-6-phosphate deacetylase enzyme, NagA (Rv3332), that belongs to the amidohydrolase superfamily. NagA enzymes catalyze the deacetylation of -acetylglucosamine-6-phosphate (GlcNAc6P) to glucosamine-6-phosphate (GlcN6P). NagA is a potential anti-tubercular drug target because it represents the key enzymatic step in the generation of essential amino-sugar precursors required for cell wall biosynthesis and also influences recycling of cell wall peptidoglycan fragments. Here, we report the structural and functional characterization of NagA from (MSNagA) and (MMNagA), close relatives of Using a combination of X-ray crystallography, site-directed mutagenesis, and biochemical and biophysical assays, we show that these mycobacterial NagA enzymes are selective for GlcNAc6P. Site-directed mutagenesis studies revealed crucial roles of conserved residues in the active site that underpin stereo-selective recognition, binding, and catalysis of substrates. Moreover, we report the crystal structure of MSNagA in both ligand-free form and in complex with the GlcNAc6P substrate at 2.6 Å and 2.0 Å resolutions, respectively. The GlcNAc6P-complex structure disclosed the precise mode of GlcNAc6P binding and the structural framework of the active site, including two divalent metals located in the α/β binuclear site. Furthermore, we observed a cysteine residue located on a flexible loop region that occludes the active site. This cysteine is unique to mycobacteria and may represent a unique subsite for targeting mycobacterial NagA enzymes. Our results provide critical insights into the structural and mechanistic properties of mycobacterial NagA enzymes having an essential role in amino-sugar and nucleotide metabolism in mycobacteria.

Item Type:	Journal Article
Subjects:	Q Science > QR Microbiology
Divisions:	Faculty of Science, Engineering and Medicine > Science > Life Sciences (2010- )
SWORD Depositor:	Library Publications Router
Library of Congress Subject Headings (LCSH):	Mycobacterium tuberculosis, Tuberculosis, Enzyme kinetics
Journal or Publication Title:	Journal of Biological Chemistry
Publisher:	American Society for Biochemistry and Molecular Biology
ISSN:	0021-9258
Official Date:	22 June 2018
Dates:	Date Event 22 June 2018 Published 4 May 2018 Available 4 May 2018 Accepted
Volume:	293
Number:	25
Page Range:	pp. 9770-9783
DOI:	10.1074/jbc.RA118.002597
Status:	Peer Reviewed
Publication Status:	Published
Reuse Statement (publisher, data, author rights):
Access rights to Published version:	Open Access (Creative Commons)
Date of first compliant deposit:	27 June 2018
Date of first compliant Open Access:	27 June 2018
RIOXX Funder/Project Grant:	Project/Grant ID RIOXX Funder Name Funder ID 201442/Z/16/Z Wellcome Trust http://dx.doi.org/10.13039/100010269 104193/Z/14/Z [RS] Royal Society http://dx.doi.org/10.13039/501100000288 RG120405 [RS] Royal Society http://dx.doi.org/10.13039/501100000288 BB/M017982/1 [BBSRC] Biotechnology and Biological Sciences Research Council http://dx.doi.org/10.13039/501100000268 105627/Z/14/Z Wellcome Trust Institutional Strategic Support Fund (ISSF), UNSPECIFIED BB/M01116X/1 [BBSRC] Biotechnology and Biological Sciences Research Council http://dx.doi.org/10.13039/501100000268

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