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Identification and characterization of important residues in the catalytic mechanism of CMP-Neu5Ac synthetase from Neisseria meningitidis.

Horsfall LE, Nelson A, Berry A - FEBS J. (2010)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, UK.

ABSTRACT
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.

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Cross-eye stereo view of the active site of CNS from Neisseria meningitidis (blue) with CDP (yellow) in the active site (PDB 1EYR) showing the residues highlighted previously in the literature and those mutated in this study.
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fig02: Cross-eye stereo view of the active site of CNS from Neisseria meningitidis (blue) with CDP (yellow) in the active site (PDB 1EYR) showing the residues highlighted previously in the literature and those mutated in this study.

Mentions: Mutation of Arg199, Arg202 or Gln203 to alanine in murine CNS was shown to abolish activity [17]. Similarly, mutation of the equivalent N. meningitidis residues (R165A and Q166A; Fig. 1) resulted in enzymes with either no, or strongly reduced, activity [17]. In silico docking simulations revealed that Arg165 forms a salt bridge with the carboxylate of the second substrate, Neu5Ac, whereas Gln166 does not interact with either substrate but is required in the quaternary organization of the enzyme [12,17]. This docking also highlighted several other residues (Ser82, Gln104, Thr106, Lys142, Arg165, Tyr179, Phe192 and Phe193) as important in binding the sugar, Neu5Ac [12]. Some of these residues are conserved in the structures of murine CNS and the related CKS enzymes (see Figs 1 and 2) [11,18,19].


Identification and characterization of important residues in the catalytic mechanism of CMP-Neu5Ac synthetase from Neisseria meningitidis.

Horsfall LE, Nelson A, Berry A - FEBS J. (2010)

Cross-eye stereo view of the active site of CNS from Neisseria meningitidis (blue) with CDP (yellow) in the active site (PDB 1EYR) showing the residues highlighted previously in the literature and those mutated in this study.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2901514&req=5

fig02: Cross-eye stereo view of the active site of CNS from Neisseria meningitidis (blue) with CDP (yellow) in the active site (PDB 1EYR) showing the residues highlighted previously in the literature and those mutated in this study.
Mentions: Mutation of Arg199, Arg202 or Gln203 to alanine in murine CNS was shown to abolish activity [17]. Similarly, mutation of the equivalent N. meningitidis residues (R165A and Q166A; Fig. 1) resulted in enzymes with either no, or strongly reduced, activity [17]. In silico docking simulations revealed that Arg165 forms a salt bridge with the carboxylate of the second substrate, Neu5Ac, whereas Gln166 does not interact with either substrate but is required in the quaternary organization of the enzyme [12,17]. This docking also highlighted several other residues (Ser82, Gln104, Thr106, Lys142, Arg165, Tyr179, Phe192 and Phe193) as important in binding the sugar, Neu5Ac [12]. Some of these residues are conserved in the structures of murine CNS and the related CKS enzymes (see Figs 1 and 2) [11,18,19].

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, UK.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus