Identification and characterization of important residues in the catalytic mechanism of CMP-Neu5Ac synthetase from Neisseria meningitidis.
Sialylated oligosaccharides, present on mammalian outer-cell surfaces, play vital roles in cellular interactions and some bacteria are able to mimic these structures to evade their host's immune system. It would be of great benefit to the study of infectious and autoimmune diseases and cancers, to understand the pathway of sialylation in detail to enable the design and production of inhibitors and mimetics. Sialylation occurs in two stages, the first to activate sialic acid and the second to transfer it to the target molecule. The activation step is catalysed by the enzyme CMP-Neu5Ac synthetase (CNS). Here we used crystal structures of CNS and similar enzymes to predict residues of importance in the CNS from Neisseria meningitidis. Nine residues were mutated to alanine, and the steady-state enzyme kinetic parameters were measured using a continuous assay to detect one of the products of the reaction, pyrophosphate. Mutations that caused the greatest loss in activity included K142A, D211A, D209A and a series of mutations at residue Q104, highlighted from sequence-alignment studies of related enzymes, demonstrating significant roles for these residues in the catalytic mechanism of CNS. The mutations of D211A and D209A provide strong evidence for a previously proposed metal-binding site in the enzyme, and the results of our mutations at residue Q104 lead us to include this residue in the metal-binding site of an intermediate complex. This suggests that, like the sugar-activating lipopolysaccharide-synthesizing CMP-2-keto-3-deoxy-manno-octonic acid synthetase enzyme KdsB, CNS recruits two Mg(2+) ions during the catalytic cycle.
Horsfall
Louise E
LE
Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, UK.
Nelson
Adam
A
Berry
Alan
A
D055162:Biocatalysis; D020134:Catalytic Domain; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid / Q000138:chemical synthesis / Q000737:chemistry; D007700:Kinetics; D000216:N-Acylneuraminate Cytidylyltransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009345:Neisseria meningitidis / Q000201:enzymology*; D017354:Point Mutation
D003569:Cytidine Monophosphate N-Acetylneuraminic Acid; D000216:N-Acylneuraminate Cytidylyltransferase