N-acetylmannosamine-6-phosphate 2-epimerase uses a novel substrate-assisted mechanism to catalyze amino sugar epimerization
| Autor: | Michael J. Currie, Phillip M. Rendle, Lavanyaa Manjunath, Ramaswamy Subramanian, Christopher R Horne, Antony J. Fairbanks, Andrew C. Muscroft-Taylor, Renwick C. J. Dobson, Rachel A. North, Rosmarie Friemann |
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| Rok vydání: | 2021 |
| Předmět: |
Staphylococcus aureus
crystal structure Amino sugar Stereochemistry SaNanE NanE from Staphylococcus aureus Lysine Mutation Missense ManNAc-6P N-acetylmannosamine-6-phosphate methicillin-resistant Staphylococcus aureus CpNanE NanE enzyme from Clostridium perfringens G6PD glucose-6-phosphate dehydrogenase GlcNAc-6P N-acetylglucosamine-6-phosphate Biochemistry Catalysis Triosephosphate isomerase Stereocenter Bacterial Proteins Protein Domains PDB Protein Data Bank TIM triosephosphate isomerase energy metabolism G6P glucose-6-phosphate enzyme mechanism PGI phosphoglucoisomerase GlcNAc-6P Molecular Biology chemistry.chemical_classification epimerase SAXS small-angle X-ray scattering NagB glucosamine-6-phosphate deaminase Substrate (chemistry) Hexosamines Cell Biology Protein engineering ManNAc-6P 6PG 6-phosphogluconate NAL N-acetylneuraminate lyase NanE N-acetylmannosamine-6-phosphate 2-epimerase Enzyme Amino Acid Substitution chemistry sialic acid GlcN-6P glucosamine-6-phosphate Protein Conformation beta-Strand Sugar Phosphates N-acetylneuraminate lyase Carbohydrate Epimerases NagA GlcNAc-6P deacetylase Research Article |
| Zdroj: | The Journal of Biological Chemistry |
| ISSN: | 0021-9258 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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