| Spectroscopic and biochemical studies on protein variants of quinaldine 4-oxidase: Role of E736 in catalysis and effects of serine ligands on the FeSI and FeSII clusters. | |
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MedLine Citation:
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PMID: 17144679 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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Quinaldine 4-oxidase (Qox), which catalyzes the hydroxylation of quinaldine to 1H-4-oxoquinaldine, is a heterotrimeric (LMS)2 molybdo-iron/sulfur flavoprotein belonging to the xanthine oxidase family. Variants of Qox were generated by site-directed mutagenesis. Replacement in the large subunit at E736, which is presumed to be located close to the molybdenum, by aspartate (QoxLE736D) resulted in a marked decrease in kcat app for quinaldine, while Km app was largely unaffected. Although a minor reduction of the glutamine substituted variant QoxLE736Q by quinaldine occurred, its activity was below detection, indicating that the carboxylate group of E736 is crucial for catalysis. Replacement of cysteine ligands C40, C45, or C60 (FeSII) and of the C120 or C154 ligands to FeSI in the small subunit of Qox by serine led to decreased iron contents of the protein preparations. Substitutions C40S and C45S (Fe1 of FeSII) suppressed the characteristic FeSII EPR signals and significantly reduced catalytic activity. In QoxSC154S (Fe1 of FeSI), the g-factor components of FeSI were drastically changed. In contrast, Qox proteins with substitutions of C48 and C60 (Fe2 of FeSII), and of the C120 ligand at Fe2 of FeSI, retained considerable activity and showed less pronounced changes in their EPR parameters. Taken together, the properties of the Qox variants suggest that Fe1 of both FeSI and FeSII are the reducible iron sites, whereas the Fe2 ions remain in the ferric state. The location of the reducible iron sites of FeSI and FeSII appears to be conserved in enzymes of the xanthine oxidase family. |
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Authors:
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Reinhard Kappl; Sonja Sielker; Kalina Ranguelova; Jeannine Wegner; Katja Parschat; Jürgen Hüttermann; Susanne Fetzner |
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Publication Detail:
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Type: Journal Article; Research Support, Non-U.S. Gov't |
Journal Detail:
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Title: Biochemistry Volume: 45 ISSN: 0006-2960 ISO Abbreviation: Biochemistry Publication Date: 2006 Dec |
Date Detail:
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Created Date: 2006-12-05 Completed Date: 2007-01-30 Revised Date: 2008-11-21 |
Medline Journal Info:
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Nlm Unique ID: 0370623 Medline TA: Biochemistry Country: United States |
Other Details:
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Languages: eng Pagination: 14853-68 Citation Subset: IM |
Affiliation:
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Institut für Biophysik, Universität des Saarlandes, D-66421 Homburg, Germany. |
Export Citation:
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APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
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Amino Acid Substitution Bacterial Proteins / chemistry, metabolism Base Sequence DNA Primers Electron Spin Resonance Spectroscopy Flavin-Adenine Dinucleotide / chemistry, metabolism Genetic Variation Iron / metabolism Ligands Metalloproteins / chemistry, genetics*, metabolism* Molecular Conformation Mutagenesis, Site-Directed Oxidoreductases / chemistry, genetics*, metabolism* Plasmids Protein Conformation Protein Subunits / chemistry, metabolism Pseudomonas putida / enzymology Recombinant Proteins / chemistry, metabolism Spectrophotometry |
| Chemical | |
Reg. No./Substance:
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0/Bacterial Proteins; 0/DNA Primers; 0/Ligands; 0/Metalloproteins; 0/Protein Subunits; 0/Recombinant Proteins; 146-14-5/Flavin-Adenine Dinucleotide; 7439-89-6/Iron; EC 1.-/Oxidoreductases; EC 1.5.99.-/quinaldine oxidoreductase |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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