Limits...
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.

Show MeSH

Related in: MedlinePlus

Partial amino acid sequence alignment of CNS enzymes from Neisseria meningitidis [23], Escherichia coli [29], Haemophilus ducreyi [20], Haemophilus influenza [30], mouse [31] and rainbow trout [24], and the two types of CKS enzymes from E. coli [32–34]. Sequences were aligned using the ClustalW program [35]. Residues highlighted in black have been identified as important in the literature and have roles assigned [12–18,20]: *, CTP-binding residue of the P-loop; (*), CTP-binding residue; ^, Neu5Ac-binding residue; #, Neu5Ac-binding residue forming part of the hydrophobic pocket; °, residue required in the quaternary organization of the molecule; -, residue lining the active site; +, Mg2+-binding residue. Residues highlighted in grey share identity with those highlighted in black.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2901514&req=5

fig01: Partial amino acid sequence alignment of CNS enzymes from Neisseria meningitidis [23], Escherichia coli [29], Haemophilus ducreyi [20], Haemophilus influenza [30], mouse [31] and rainbow trout [24], and the two types of CKS enzymes from E. coli [32–34]. Sequences were aligned using the ClustalW program [35]. Residues highlighted in black have been identified as important in the literature and have roles assigned [12–18,20]: *, CTP-binding residue of the P-loop; (*), CTP-binding residue; ^, Neu5Ac-binding residue; #, Neu5Ac-binding residue forming part of the hydrophobic pocket; °, residue required in the quaternary organization of the molecule; -, residue lining the active site; +, Mg2+-binding residue. Residues highlighted in grey share identity with those highlighted in black.

Mentions: Previous studies have identified Lys19 in the Haemophilus ducreyi CNS, and Lys21 and Arg12 in CNS from Escherichia coli (Fig. 1) as important catalytic residues, indicating a role in binding the nucleotide into the active site [15,16]. The crystal structure of CNS from Neisseria meningitidis crystallized in the presence of the substrate analogue CDP confirmed the interaction between these residues and the first substrate [12].


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)

Partial amino acid sequence alignment of CNS enzymes from Neisseria meningitidis [23], Escherichia coli [29], Haemophilus ducreyi [20], Haemophilus influenza [30], mouse [31] and rainbow trout [24], and the two types of CKS enzymes from E. coli [32–34]. Sequences were aligned using the ClustalW program [35]. Residues highlighted in black have been identified as important in the literature and have roles assigned [12–18,20]: *, CTP-binding residue of the P-loop; (*), CTP-binding residue; ^, Neu5Ac-binding residue; #, Neu5Ac-binding residue forming part of the hydrophobic pocket; °, residue required in the quaternary organization of the molecule; -, residue lining the active site; +, Mg2+-binding residue. Residues highlighted in grey share identity with those highlighted in black.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig01: Partial amino acid sequence alignment of CNS enzymes from Neisseria meningitidis [23], Escherichia coli [29], Haemophilus ducreyi [20], Haemophilus influenza [30], mouse [31] and rainbow trout [24], and the two types of CKS enzymes from E. coli [32–34]. Sequences were aligned using the ClustalW program [35]. Residues highlighted in black have been identified as important in the literature and have roles assigned [12–18,20]: *, CTP-binding residue of the P-loop; (*), CTP-binding residue; ^, Neu5Ac-binding residue; #, Neu5Ac-binding residue forming part of the hydrophobic pocket; °, residue required in the quaternary organization of the molecule; -, residue lining the active site; +, Mg2+-binding residue. Residues highlighted in grey share identity with those highlighted in black.
Mentions: Previous studies have identified Lys19 in the Haemophilus ducreyi CNS, and Lys21 and Arg12 in CNS from Escherichia coli (Fig. 1) as important catalytic residues, indicating a role in binding the nucleotide into the active site [15,16]. The crystal structure of CNS from Neisseria meningitidis crystallized in the presence of the substrate analogue CDP confirmed the interaction between these residues and the first substrate [12].

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