Xanthine dehydrogenase from Pseudomonas putida 86: specificity, oxidation-reduction potentials of its redox-active centers, and first EPR characterization.
Xanthine dehydrogenase (XDH) from Pseudomonas putida 86, which was induced 65-fold by growth on hypoxanthine, was purified to homogeneity. It catalyzes the oxidation of hypoxanthine, xanthine, purine, and some aromatic aldehydes, using NAD+ as the preferred electron acceptor. In the hypoxanthine:NAD+ assay, the specific activity of purified XDH was 26.7 U (mg protein)(-1). Its activity with ferricyanide and dioxygen was 58% and 4%, respectively, relative to the activity observed with NAD+. XDH from P. putida 86 consists of 91.0 kDa and 46.2 kDa subunits presumably forming an alpha4beta4 structure and contains the same set of redox-active centers as eukaryotic XDHs. After reduction of the enzyme with xanthine, electron paramagnetic resonance (EPR) signals of the neutral FAD semiquinone radical and the Mo(V) rapid signal were observed at 77 K. Resonances from FeSI and FeSII were detected at 15 K. Whereas the observable g factors for FeSII resemble those of other molybdenum hydroxylases, the FeSI center in contrast to most other known FeSI centers has nearly axial symmetry. The EPR features of the redox-active centers of P. putida XDH are very similar to those of eukaryotic XDHs/xanthine oxidases, suggesting that the environment of each center and their functionality are analogous in these enzymes. The midpoint potentials determined for the molybdenum, FeSI and FAD redox couples are close to each other and resemble those of the corresponding centers in eukaryotic XDHs.
Parschat
K
K
AG Mikrobiologie, Ossietzky Universität Oldenburg, Germany.
Canne
C
C
Hüttermann
J
J
Kappl
R
R
Fetzner
S
S
D000447:Aldehydes / Q000378:metabolism; D001665:Binding Sites; D004578:Electron Spin Resonance Spectroscopy; D004790:Enzyme Induction; D010084:Oxidation-Reduction; D016958:Pseudomonas putida / Q000201:enzymology*; D013379:Substrate Specificity; D014968:Xanthine Dehydrogenase / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*
D000447:Aldehydes; D014968:Xanthine Dehydrogenase