Physiology of sialic acid capsular polysaccharide synthesis in serogroup B Neisseria meningitidis.
The pathway for biosynthesis of sialic acid capsular polysaccharide was examined in Neisseria meningitidis serogroup B strain M986 and in strain PRM102, an isogenic mutant defective in polysaccharide production. Strain PRM102 was found to possess only 25% of the level of sialyltransferase activity that was found in strain M986, but it had wild-type levels of both the N-acetylneuraminic acid (NANA) condensing enzyme and the CMP-NANA synthetase. A new meningococcal enzyme, a CMP-NANA hydrolase, was found in both meningococcal strains. This enzyme generated CMP and NANA from CMP-NANA, had a Km of 0.88 microM, had a Vmax of 10.75 nmol of NANA produced per h per mg of protein, and was completely inhibited by 45.3 microM CMP. The sialyltransferase, which also had CMP-NANA as substrate, was insensitive to CMP addition. Subcellular fractionation and purification of cytoplasmic and outer membranes on sucrose density gradients revealed that both the sialyltransferase and the CMP-NANA hydrolase were cytoplasmic membrane associated. The NANA condensing enzyme and the CMP-NANA synthetase were found to be cytosolic. A working hypothesis for the regulation of sialic acid polysaccharide synthesis was developed. The CMP-NANA hydrolase with its high affinity for CMP-NANA regulates polysaccharide formation by the sialyltransferase, whereas CMP, a product of both the sialyltransferase and the CMP-NANA hydrolase, modulates the activity of the hydrolase on the cytoplasmic membrane.
D002462:Cell Membrane / Q000201:enzymology; D003568:Cytidine Monophosphate / Q000378:metabolism; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology; D007700:Kinetics; D019158:N-Acetylneuraminic Acid; D000216:N-Acylneuraminate Cytidylyltransferase / Q000378:metabolism; D009345:Neisseria meningitidis / Q000145:classification / Q000378:metabolism*; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D011135:Polysaccharides, Bacterial / Q000096:biosynthesis*; D012794:Sialic Acids / Q000378:metabolism; D012799:Sialyltransferases / Q000378:metabolism*; D014166:Transferases / Q000378:metabolism*; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase
D011135:Polysaccharides, Bacterial; D012794:Sialic Acids; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid; D014166:Transferases; D012799:Sialyltransferases; D000216:N-Acylneuraminate Cytidylyltransferase; D010727:Phosphoric Diester Hydrolases; C010265:CMP-N-acylneuraminate phosphodiesterase; D003568:Cytidine Monophosphate; D019158:N-Acetylneuraminic Acid; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase