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Cofactor determination and spectroscopic characterization of the selenium-dependent purine hydroxylase from Clostridium purinolyticum.
Biochemistry. 2003 Sep 30; 42(38):11382-90.B

Abstract

Purine hydroxylase (PH) from Clostridium purinolyticum contains a labile selenium cofactor and belongs to a class of enzymes known as the selenium-dependent molybdenum hydroxylases. The presence of approximately 1.1 mol of molybdenum, 0.87 mol of selenium, and 3.3 mol of iron per mol of PH was determined by atomic absorption spectroscopy. Enzyme preparations with lower than stoichiometric amounts of selenium exhibited correspondingly lower hydroxylase activities. Bound FAD, 1 mol per mol enzyme, was confirmed by UV-vis and fluorescence spectroscopy. CMP, released by acid hydrolysis, indicated the presence of a molybdopterin cytosine dinucleotide cofactor. The fully active PH utilized NADP(+) as an electron acceptor, and kinetic analysis revealed an optimal k(cat) of 412 s(-1) using hypoxanthine as the hydroxylase substrate. Xanthine, NAD(+), and NADPH had no significant effect on this reaction rate. A selenium-independent NADPH oxidase activity was exhibited by native PH. Electron paramagnetic resonance spectroscopy revealed the presence of a Mo(V) desulfo signal, FAD radical, and 2Fe-2S centers in hypoxanthine-reduced PH. No hyperfine coupling of selenium, using (77)Se isotope-enriched PH, was observed in any of the EPR active signals studied. The appearance of the desulfo signal suggests that the ligands of Mo in selenium-dependent molybdenum hydroxylases are different from the well-studied mammalian xanthine oxidoreductases (XOR) and aldehyde oxidoreductases (AOR) and suggests a unique role for Se in catalysis.

Authors+Show Affiliations

Laboratory of Biochemistry, National Heart, Lung and Blood Institute, National Institutes of Health, 50 South Drive MSC 8012, Bethesda, Maryland 20892-8012, USA.No affiliation info availableNo affiliation info available

Pub Type(s)

Comparative Study
Journal Article

Language

eng

PubMed ID

14503889

Citation

Self, William T., et al. "Cofactor Determination and Spectroscopic Characterization of the Selenium-dependent Purine Hydroxylase From Clostridium Purinolyticum." Biochemistry, vol. 42, no. 38, 2003, pp. 11382-90.
Self WT, Wolfe MD, Stadtman TC. Cofactor determination and spectroscopic characterization of the selenium-dependent purine hydroxylase from Clostridium purinolyticum. Biochemistry. 2003;42(38):11382-90.
Self, W. T., Wolfe, M. D., & Stadtman, T. C. (2003). Cofactor determination and spectroscopic characterization of the selenium-dependent purine hydroxylase from Clostridium purinolyticum. Biochemistry, 42(38), 11382-90.
Self WT, Wolfe MD, Stadtman TC. Cofactor Determination and Spectroscopic Characterization of the Selenium-dependent Purine Hydroxylase From Clostridium Purinolyticum. Biochemistry. 2003 Sep 30;42(38):11382-90. PubMed PMID: 14503889.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Cofactor determination and spectroscopic characterization of the selenium-dependent purine hydroxylase from Clostridium purinolyticum. AU - Self,William T, AU - Wolfe,Matt D, AU - Stadtman,Thressa C, PY - 2003/9/25/pubmed PY - 2003/10/30/medline PY - 2003/9/25/entrez SP - 11382 EP - 90 JF - Biochemistry JO - Biochemistry VL - 42 IS - 38 N2 - Purine hydroxylase (PH) from Clostridium purinolyticum contains a labile selenium cofactor and belongs to a class of enzymes known as the selenium-dependent molybdenum hydroxylases. The presence of approximately 1.1 mol of molybdenum, 0.87 mol of selenium, and 3.3 mol of iron per mol of PH was determined by atomic absorption spectroscopy. Enzyme preparations with lower than stoichiometric amounts of selenium exhibited correspondingly lower hydroxylase activities. Bound FAD, 1 mol per mol enzyme, was confirmed by UV-vis and fluorescence spectroscopy. CMP, released by acid hydrolysis, indicated the presence of a molybdopterin cytosine dinucleotide cofactor. The fully active PH utilized NADP(+) as an electron acceptor, and kinetic analysis revealed an optimal k(cat) of 412 s(-1) using hypoxanthine as the hydroxylase substrate. Xanthine, NAD(+), and NADPH had no significant effect on this reaction rate. A selenium-independent NADPH oxidase activity was exhibited by native PH. Electron paramagnetic resonance spectroscopy revealed the presence of a Mo(V) desulfo signal, FAD radical, and 2Fe-2S centers in hypoxanthine-reduced PH. No hyperfine coupling of selenium, using (77)Se isotope-enriched PH, was observed in any of the EPR active signals studied. The appearance of the desulfo signal suggests that the ligands of Mo in selenium-dependent molybdenum hydroxylases are different from the well-studied mammalian xanthine oxidoreductases (XOR) and aldehyde oxidoreductases (AOR) and suggests a unique role for Se in catalysis. SN - 0006-2960 UR - https://www.unboundmedicine.com/medline/citation/14503889/Cofactor_determination_and_spectroscopic_characterization_of_the_selenium_dependent_purine_hydroxylase_from_Clostridium_purinolyticum_ L2 - https://doi.org/10.1021/bi030136k DB - PRIME DP - Unbound Medicine ER -