Nucleoside Oxidase, a Hydrogen Peroxide-Forming Oxidase, from Flavobacterium meningosepticum

Abstract

A novel enzyme which catalyzes the oxidation of nucleosides to nucleoside-5-carboxylic acids, forming hydrogen peroxide, was purified to homogeneity from Flavobacterium meningosepticum T-2799. The enzyme has a molecular weight of about 500,000, and four nonidentical subunits (molecular weights of 81,000, 69,000, 33,000, and 16,000) were detected on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On the basis of visible absorption spectra of the purified enzyme, the enzyme is concluded to be a hemoprotein. It also contains covalently bound flavin adenine dinucleotide. The various nucleosides, such as adenosine (K m ‫؍‬ 48 M), inosine (K m ‫؍‬ 66 M), guanosine (K m ‫؍‬ 21 M), thymidine (K m ‫؍‬ 50 M), uridine (K m ‫؍‬ 80 M), and cytidine (K m ‫؍‬ 50 M), were oxidized by the enzyme, but nucleotides, bases, and ribose were not.

Coordinate induction of peroxidase and H2O2-forming nucleoside oxidase in Flavobacterium meningosepticum

“…H P O P appeared not to be involved in the induction, because some substrates of nucleoside oxidase which are oxidized with the formation of H P O P , such as adenosine, guanosine and inosine [5], enhanced neither the peroxidase production nor the nucleoside oxidase production. The time course of peroxidase production coincided with that of nucleoside oxidase production.…”

“…The activity of nucleoside oxidase was assayed by measuring the formation of H P O P with horseradish peroxidase (Boehringer, Mannheim Germany) in the presence of inosine as a substrate, as described previously [5]. One unit (U) of the nucleoside oxidase was de¢ned as the amount catalyzing the formation of 1 Wmol H P O P per min under the assay conditions.…”

“…The Pseudomonas maltophilia enzyme [1^4] was shown to catalyze the oxidation of nucleosides to the corresponding nucleoside-5P-carboxylic acids via nucleoside-5P-aldehydes using molecular O P as a primary electron acceptor without the formation of H P O P . Recently, we found another type of nucleoside oxidase in Flavobacterium meningosepticum which produces H P O P in addition to the reaction products, nucleoside-5P-aldehyde and nucleoside-5P-carboxylic acid [5].…”

Coordinate induction of peroxidase and H2O2-forming nucleoside oxidase inFlavobacterium meningosepticum

“…The activity of nucleoside oxidase was assayed by measuring the formation of H 2 O 2 with horseradish peroxidase (Boehringer, Mannheim Germany) in the presence of inosine as a substrate, as described previously [5]. One unit (U) of the nucleoside oxidase was defined as the amount catalyzing the formation of 1 μmol H 2 O 2 per min under the assay conditions.…”

“…The Pseudomonas maltophilia enzyme [1–4] was shown to catalyze the oxidation of nucleosides to the corresponding nucleoside‐5′‐carboxylic acids via nucleoside‐5′‐aldehydes using molecular O 2 as a primary electron acceptor without the formation of H 2 O 2 . Recently, we found another type of nucleoside oxidase in Flavobacterium meningosepticum which produces H 2 O 2 in addition to the reaction products, nucleoside‐5′‐aldehyde and nucleoside‐5′‐carboxylic acid [5].…”