Structural and kinetic analyses of the H121A mutant of cholesterol oxidase

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LIM, Louis, Gianluca MOLLA, Nicole GUINN, Sandro GHISLA, Loredano POLLEGIONI, Alice VRIELINK, 2006. Structural and kinetic analyses of the H121A mutant of cholesterol oxidase. In: Biochemical Journal. 400(1), pp. 13-22. ISSN 0264-6021. eISSN 1470-8728. Available under: doi: 10.1042/BJ20060664

@article{Lim2006Struc-7776, title={Structural and kinetic analyses of the H121A mutant of cholesterol oxidase}, year={2006}, doi={10.1042/BJ20060664}, number={1}, volume={400}, issn={0264-6021}, journal={Biochemical Journal}, pages={13--22}, author={Lim, Louis and Molla, Gianluca and Guinn, Nicole and Ghisla, Sandro and Pollegioni, Loredano and Vrielink, Alice} }

<rdf:RDF xmlns:dcterms="http://purl.org/dc/terms/" xmlns:dc="http://purl.org/dc/elements/1.1/" xmlns:rdf="http://www.w3.org/1999/02/22-rdf-syntax-ns#" xmlns:bibo="http://purl.org/ontology/bibo/" xmlns:dspace="http://digital-repositories.org/ontologies/dspace/0.1.0#" xmlns:foaf="http://xmlns.com/foaf/0.1/" xmlns:void="http://rdfs.org/ns/void#" xmlns:xsd="http://www.w3.org/2001/XMLSchema#" > <rdf:Description rdf:about="https://kops.uni-konstanz.de/rdf/resource/123456789/7776"> <dcterms:rights rdf:resource="https://creativecommons.org/licenses/by-nc-nd/2.0/legalcode"/> <dc:format>application/pdf</dc:format> <bibo:uri rdf:resource="http://kops.uni-konstanz.de/handle/123456789/7776"/> <dcterms:issued>2006</dcterms:issued> <dcterms:available rdf:datatype="http://www.w3.org/2001/XMLSchema#dateTime">2011-03-24T17:37:27Z</dcterms:available> <foaf:homepage rdf:resource="http://localhost:8080/jspui"/> <dcterms:bibliographicCitation>First publ. in: Biochemical Journal 400 (2006), pp. 13-22</dcterms:bibliographicCitation> <dc:creator>Vrielink, Alice</dc:creator> <void:sparqlEndpoint rdf:resource="http://localhost/fuseki/dspace/sparql"/> <dcterms:abstract xml:lang="eng">Cholesterol oxidase is a monomeric flavoenzyme that catalyses the oxidation of cholesterol to cholest-5-en-3-one followed by isomerization to cholest-4-en-3-one. The enzyme from Brevibacterium sterolicum contains the FAD cofactor covalently bound to His121. It was previously demonstrated that the H121A substitution results in a ≈100 mV decrease in the midpoint redox potential and a ≈40-fold decrease in turnover number compared to wild-type enzyme [Motteran, Pilone, Molla, Ghisla and Pollegioni (2001) Journal of Biological Chemistry 276, 18024 18030]. A detailed kinetic analysis of the H121A mutant enzyme shows that the decrease in turnover number is largely due to a corresponding decrease in the rate constant of flavin reduction, whilst the re-oxidation reaction is only marginally altered and the isomerization reaction is not affected by the substitution and precedes product dissociation. The X-ray structure of the mutant protein, determined to 1.7 Å resolution (1 Å≡0.1 nm), reveals only minor changes in the overall fold of the protein, namely: two loops have slight movements and a tryptophan residue changes conformation by a rotation of 180° about χ1 compared to the native enzyme. Comparison of the isoalloxazine ring moiety of the FAD cofactor between the structures of the native and mutant proteins shows a change from a non-planar to a planar geometry (resulting in a more tetrahedral-like geometry for N5). This change is proposed to be a major factor contributing to the observed alteration in redox potential. Since a similar distortion of the flavin has not been observed in other covalent flavoproteins, it is proposed to represent a specific mode to facilitate flavin reduction in covalent cholesterol oxidase.</dcterms:abstract> <dspace:isPartOfCollection rdf:resource="https://kops.uni-konstanz.de/rdf/resource/123456789/28"/> <dc:contributor>Guinn, Nicole</dc:contributor> <dc:creator>Molla, Gianluca</dc:creator> <dc:contributor>Vrielink, Alice</dc:contributor> <dcterms:isPartOf rdf:resource="https://kops.uni-konstanz.de/rdf/resource/123456789/28"/> <dc:date rdf:datatype="http://www.w3.org/2001/XMLSchema#dateTime">2011-03-24T17:37:27Z</dc:date> <dc:creator>Lim, Louis</dc:creator> <dc:contributor>Pollegioni, Loredano</dc:contributor> <dc:creator>Pollegioni, Loredano</dc:creator> <dc:language>eng</dc:language> <dcterms:title>Structural and kinetic analyses of the H121A mutant of cholesterol oxidase</dcterms:title> <dc:creator>Guinn, Nicole</dc:creator> <dc:rights>deposit-license</dc:rights> <dcterms:hasPart rdf:resource="https://kops.uni-konstanz.de/bitstream/123456789/7776/1/Structural_and_kinetic_analyses_of_the_H121A_mutant_of_cholesterol_oxidase.pdf"/> <dc:contributor>Molla, Gianluca</dc:contributor> <dc:contributor>Ghisla, Sandro</dc:contributor> <dc:creator>Ghisla, Sandro</dc:creator> <dspace:hasBitstream rdf:resource="https://kops.uni-konstanz.de/bitstream/123456789/7776/1/Structural_and_kinetic_analyses_of_the_H121A_mutant_of_cholesterol_oxidase.pdf"/> <dc:contributor>Lim, Louis</dc:contributor> </rdf:Description> </rdf:RDF>

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