Identification of two distinct NADH oxidases corresponding to H2O2-forming oxidase and H2O-forming oxidase induced in Streptococcus mutans.
Two distinct NADH oxidases, corresponding to H2O2-forming and H2O-forming enzymes were purified to homogeneity from Streptococcus mutans and their basic properties determined. The H2O2-forming enzyme was a tetramer with a subunit molecular mass of about 56 kDa and required flavin adenine dinucleotide (FAD) for full activity. The enzyme had an isoelectric point of 6.6 and exhibited optimal activity at pH 6.0. The H2O-forming enzyme was a monomer with a molecular mass of 50 kDa and activity independent of exogenously added flavin. The enzyme had an isoelectric point of 4.8 and exhibited optimal activity between pH 7.0 and 7.5. Both enzymes oxidized NADH (Km 0.05 and 0.025 mM for the H2O2- and H2O-forming enzyme, respectively) but not NADPH and contained 1 mol of FAD per monomer. Spectra of the oxidized enzymes exhibited maxima at 271, 383 and 449 nm for the H2O2-forming enzyme and 271, 375 and 447 nm for the H2O-forming enzyme. Antibodies raised against the H2O2-forming enzyme or the H2O-forming enzyme reacted with their corresponding antigen, but did not cross-react. The amino-terminal regions of the two enzymes had completely different amino acid sequences.
Higuchi
M
M
Research Centre, Nippon Paint Co., Ltd, Osaka, Japan.
Shimada
M
M
Yamamoto
Y
Y
Hayashi
T
T
Koga
T
T
Kamio
Y
Y
D000595:Amino Acid Sequence; D003429:Cross Reactions; D004789:Enzyme Activation / Q000187:drug effects; D004790:Enzyme Induction; D004795:Enzyme Stability; D005182:Flavin-Adenine Dinucleotide / Q000032:analysis; D015964:Gene Expression Regulation, Bacterial; D006358:Hot Temperature; D006861:Hydrogen Peroxide / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000276:immunology / Q000302:isolation & purification*; D009247:NADH, NADPH Oxidoreductases / Q000037:antagonists & inhibitors / Q000276:immunology / Q000302:isolation & purification*; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D013056:Spectrophotometry, Ultraviolet; D013295:Streptococcus mutans / Q000201:enzymology*; D013379:Substrate Specificity; D014867:Water / Q000378:metabolism*
D009097:Multienzyme Complexes; D014867:Water; D005182:Flavin-Adenine Dinucleotide; D006861:Hydrogen Peroxide; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases