PURIFICATION AND CHARACTERIZATION OF NADH OXIDASE FROM SERPULINA-(TREPONEMA)-HYODYSENTERIAE

NADH oxidase (EC 1.6.99.3) was purified from cell lysates of Serpulina (Treponema) hyodysenteriae B204 by differential ultracentrifugation, ammonium sulfate precipitation, and chromatography on anion-exchange, dye-ligand-affinity, and size-exclusion columns. Purified NADH oxidase had a specific activity 119-fold higher than that of cell lysates and migrated as a single band during denaturing gel electrophoresis (sodi um dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE]). The enzyme was a monomeric protein with an estimated molecular mass of 47 to 48 kDa, as determined by SDS-PAGE and size-exclusion chromatograph y. Optimum enzyme activity occurred in buffers with a pH between 5.5 a nd 7.0. In the presence of oxygen, beta-NADH but not alpha-NADH, alpha -NADPH, or beta-NADPH was rapidly oxidized by the enzyme (K(m) = 10 mu M beta-NADH; V(max) = 110 mumol beta-NADH min-1 mg of protein-1). Oxyg en was the only identified electron acceptor for the enzyme. On isoele ctric focusing gels, the enzyme separated into three subforms, with is oelectric pH values of 5.25, 5.35, and 5.45. Purified NADH oxidase had a typical flavoprotein absorption spectrum, with peak absorbances at wavelengths of 274, 376, and 448 nm. Flavin adenine dinucleotide was i dentified as a cofactor and was noncovalently associated with the enzy me at a molar ratio of 1:1. Assays of the enzyme after various chemica l treatments indicated that a flavin cofactor and a sulfhydryl group(s ), but not a metal cofactor, were essential for activity. Hydrogen per oxide and superoxide were not yielded in significant amounts by the S. hyodysenteriae NADH oxidase, indirect evidence that the enzyme produc es water from reduction of oxygen with NADH. The N-terminal amino acid sequence of the NADH oxidase was determined to be MKVIVIGCNHAGTWAAK. In its biochemical properties, the NADH oxidase of S. hyodysenteriae r esembles the NADH oxidase of another intestinal bacterium, Enterococcu s faecalis.