Cloning, expression, and characterization of sialic acid synthases.
The most commonly occurring sialic acid, N-acetylneuraminic acid, is the repeating unit in polysialic acid chain of human neuronal cell adhesion molecule as well as in capsular polysialic acid of neuroinvasive bacteria, Escherichia coli K1 and Neisseria meningitidis. Sialic acid synthesis and polymerization occur in slightly different pathways in animals and bacteria. N-Acetylneuraminic acid (NeuNAc) is synthesized by the condensation of phosphoenolpyruvate and N-acetylmannosamine by NeuNAc synthase in bacteria. The mammalian homologue N-acetylneuraminic acid-9-phosphate (NeuNAc-9-P) synthase uses N-acetylmannosamine-6-phosphate in the condensation reaction to produce NeuNAc-9-P. Both subfamilies of sialic acid synthases possess N-terminal triosephosphate isomerase barrel domain and C-terminal antifreeze protein domain. We report cloning of the genes, expression, purification, and characterization of human NeuNAc-9-P synthase and N. meningitidis NeuNAc synthase. Stability of the purified enzymes and effects of pH and temperature on their activities were evaluated. Enzyme kinetics and preliminary mutagenesis experiments reveal the importance of C-terminal antifreeze protein domain and a conserved cysteine residue for the enzyme activities.
Hao
Jijun
J
Division of Nephrology, Department of Medicine, Center for Matrix Biology, Nashville, TN 37232-2372, USA.
Balagurumoorthy
Pichumani
P
Sarilla
Suryakala
S
Sundaramoorthy
Munirathinam
M
D003001:Cloning, Molecular; D004795:Enzyme Stability; D015870:Gene Expression / Q000235:genetics*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D019216:Metals, Heavy / Q000494:pharmacology; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009345:Neisseria meningitidis / Q000201:enzymology / Q000235:genetics; D007652:Oxo-Acid-Lyases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D013696:Temperature
D019216:Metals, Heavy; C018730:N-acetylneuraminate synthase; D007652:Oxo-Acid-Lyases