Purification and properties of N-acyl-D-mannosamine dehydrogenase from Flavobacterium sp. 141-8.
A new enzyme, N-acyl-D-mannosamine dehydrogenase, was purified to apparent homogeneity from a cell-free extract of Flavobacterium sp. 141-8 and some of its properties were investigated. The enzyme showed optimum activity at pH 8.0-9.5. N-Acetyl- and N-glycolyl-D-mannosamine were oxidized but other commonly existing sugars, such as N-acetylglucosamine, N-acetylgalactosamine, amino sugars, neutral hexoses, and pentoses, were not oxidized. NAD+ was specifically utilized as an effective hydrogen acceptor. The apparent Km values for N-acetyl- and N-glycolyl-D-mannosamine, and NAD+ were 1.0, 13.3, and 0.41 mM, respectively. The stoichiometry data showed that 1 mol each of N-acetyl-D-mannosamine and NAD+ were converted to 1 mol each of N-acetyl-D-mannosaminic acid and NADH, respectively. Although the formation of lactone was detected in the enzyme reaction mixture, the reverse reaction of the enzyme, the reduction of N-acetyl-D-mannosamino-lactone, was not observed. The enzyme activity was strongly inhibited by Hg2+ and SDS, but metal-chelating reagents and sulfhydryl-group-blocking reagents had almost no effect. The molecular weight of the enzyme was estimated to be 120,000 on gel filtration and 29,000 on SDS-polyacrylamide gel electrophoresis. Its isoelectric point was at pH 4.8. On trial application of the enzyme, it was indicated that N-acetylneuraminic acid can be determined quantitatively with the combined enzyme system involving the new enzyme and N-acetylneuraminic acid aldolase.
Horiuchi
T
T
Noda Institute for Scientific Research, Chiba.
Kurokawa
T
T
D001426:Bacterial Proteins / Q000032:analysis; D002413:Cations, Divalent; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005417:Flavobacterium / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D007525:Isoelectric Focusing; D007783:Lactones / Q000032:analysis; D008970:Molecular Weight; D013401:Sugar Alcohol Dehydrogenases / Q000032:analysis / Q000302:isolation & purification*
D001426:Bacterial Proteins; D002413:Cations, Divalent; D007202:Indicators and Reagents; D007783:Lactones; D013401:Sugar Alcohol Dehydrogenases; C059050:N-acyl-D-mannosamine dehydrogenase