Abstract
There are five known general catalytic mechanisms used by enzymes to catalyze carbohydrate epimerization. The amino sugar epimerase N-acetylmannosamine-6-phosphate 2-epimerase (NanE) has been proposed to use a deprotonation–reprotonation mechanism, with an essential catalytic lysine required for both steps. However, the structural determinants of this mechanism are not clearly established. We characterized NanE from Staphylococcus aureus using a new coupled assay to monitor NanE catalysis in real time and found that it has kinetic constants comparable with other species. The crystal structure of NanE from Staphylococcus aureus, which comprises a triosephosphate isomerase barrel fold with an unusual dimeric architecture, was solved with both natural and modified substrates. Using these substrate-bound structures, we identified the following active-site residues lining the cleft at the C-terminal end of the β-strands: Gln11, Arg40, Lys63, Asp124, Glu180, and Arg208, which were individually substituted and assessed in relation to the mechanism. From this, we re-evaluated the central role of Glu180 in this mechanism alongside the catalytic lysine. We observed that the substrate is bound in a conformation that ideally positions the C5 hydroxyl group to be activated by Glu180 and donate a proton to the C2 carbon. Taken together, we propose that NanE uses a novel substrate-assisted proton displacement mechanism to invert the C2 stereocenter of N-acetylmannosamine-6-phosphate. Our data and mechanistic interpretation may be useful in the development of inhibitors of this enzyme or in enzyme engineering to produce biocatalysts capable of changing the stereochemistry of molecules that are not amenable to synthetic methods.
Highlights
There are five known general catalytic mechanisms used by enzymes to catalyze carbohydrate epimerization
The third step in the sialic acid catabolic pathway is mediated by the enzyme N-acetylmannosamine-6-phosphate 2-epimerase (NanE) [8], which catalyzes an inversion of stereochemistry at the C2 position of N-acetylmannosamine-6-phosphate (ManNAc-6P), yielding N-acetylglucosamine-6-phosphate (GlcNAc6P) (Fig. 1A)
The data were processed in the space group P21212 and solved by molecular replacement (Protein Data Bank [PDB] ID: 6VVA)
Summary
Rendle , Ramaswamy Subramanian, Rosmarie Friemann, Antony J. J. Dobson1,8,* From the 1Biomolecular Interaction Centre and School of Biological Sciences, University of Canterbury, Christchurch, New Zealand; 2Institute of Stem Cell Science and Regenerative Medicine, NCBS, Bangalore, Karnataka, India; 3Ferrier Research Institute, Victoria University of Wellington, Lower Hutt, New Zealand; 4Fujirebio Diagnostics, Gothenburg, Sweden; 5Centre for Antibiotic Resistance Research (CARe), University of Gothenburg, Gothenburg, Sweden; 6Biomolecular Interaction Centre and School of Physical and Chemical Sciences, University of Canterbury, Christchurch, New Zealand; 7Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden; 8Department of Biochemistry and Molecular Biology, Bio Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria, Australia
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.