Biosynthesis of N-glycolylneuraminic acid-containing glycoconjugates. Purification and characterization of the key enzyme of the cytidine monophospho-N-acetylneuraminic acid hydroxylation system.
We have proposed that cytidine monophospho-N-acetylneuraminic acid (CMP-NeuAc) hydroxylation is carried out by a multienzyme system involving CMP-NeuAc hydroxylase (the terminal enzyme of the system), cytochrome b5, and an NADH-dependent cytochrome b5-reducing factor (Kozutsumi, Y., Kawano, T., Yamakawa, T., and Suzuki, A. (1990) J. Biochem. 108, 704-706). The CMP-NeuAc hydroxylase was purified to homogeneity from the cytosolic fraction of mouse liver, using ion exchange columns, a Red-Sepharose column, and a soluble cytochrome b5-immobilized Sepharose column. The purified enzyme exhibited a single band (64 kDa) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a single peak (58 kDa) on gel permeation chromatography, indicating that it is composed of a single polypeptide chain. The absorption spectrum did not indicate the presence of a heme prosthetic group in the enzyme. Atomic absorption spectrometry and an inhibition test using an iron chelator indicate that the enzyme contains non-heme iron as an electron acceptor. A reconstitution experiment with the purified CMP-NeuAc hydroxylase, soluble cytochrome b5, and recombinant NADH-cytochrome b5 reductase revealed that these three factors are essential for the reaction. The hydroxylase exhibited high affinity to CMP-NeuAc (Km = 5 microM) and was greatly stabilized by CMP-NeuAc. The molecular activity of the enzyme (approximately 500/min) is much lower than that reported for NADH-cytochrome b5 reductase, suggesting that the activity or amount of hydroxylase is rate-limiting in CMP-N-glycolylneuraminic acid (NeuGc) biosynthesis. These results, together with the previous observation that the level of CMP-NeuAc hydroxylase activity was associated with the expression of NeuGc in various tissues, support the notion that the enzyme is the key for regulation of the overall velocity of CMP-NeuAc hydroxylation and consequently for the expression of NeuGc-containing glycoconjugates.
Kawano
T
T
Department of Membrane Biochemistry, Tokyo Metropolitan Institute of Medical Science, Japan.
Kozutsumi
Y
Y
Kawasaki
T
T
Suzuki
A
A
D000818:Animals; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid / Q000378:metabolism; D006001:Glycoconjugates / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D051379:Mice; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D009097:Multienzyme Complexes; D013057:Spectrum Analysis
D006001:Glycoconjugates; D009097:Multienzyme Complexes; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid; D006899:Mixed Function Oxygenases; C074383:CMPacetylneuraminate monooxygenase