O-GlcNAc is an abundant post-translational modification of serine and threonine residues of nucleocytoplasmic proteins. This modification, found only within higher eukaryotes, is a dynamic modification that is often reciprocal to phosphorylation. In a manner analogous to phosphatases, a glycoside hydrolase termed O-GlcNAcase cleaves O-GlcNAc from modified proteins. Enzymes with high sequence similarity to human O-GlcNAcase are also found in human pathogens and symbionts. We report the three-dimensional structure of O-GlcNAcase from the human gut symbiont Bacteroides thetaiotaomicron both in its native form and in complex with a mimic of the reaction intermediate. Mutagenesis and kinetics studies show that the bacterial enzyme, very similarly to its human counterpart, operates via an unusual 'substrate-assisted' catalytic mechanism, which will inform the rational design of enzyme inhibitors.
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R.J.D. thanks the Biotechnology and Biological Sciences Research Council (BBSRC) for a PhD fellowship. This work was supported by grants from the BBSRC to G.J.D. and from the Natural Sciences and Engineering Research Council of Canada (NSERC) and the Protein Engineering Network of Centres of Excellence to D.J.V. D.J.V. is supported as a Tier II Canada Research Chair in Chemical Glycobiology. M.S.M. thanks NSERC and the Michael Smith Foundation for Health Research for fellowships. We also thank A.J. Bennet for access to equipment. This work was funded by the BBSRC, the Royal Society of the UK and the NSERC.
The authors declare no competing financial interests.
Inhibitors used in the study of BtGH84 and key interactions within the enzyme active site (PDF 197 kb)
Kinetic analyses of site-directed mutants of BtGH84 (PDF 124 kb)
Electrostatic potential surface figure of the active center environment of BtGH84 (PDF 123 kb)
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Dennis, R., Taylor, E., Macauley, M. et al. Structure and mechanism of a bacterial β-glucosaminidase having O-GlcNAcase activity. Nat Struct Mol Biol 13, 365–371 (2006). https://doi.org/10.1038/nsmb1079
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