Linked Life Data

8774727

Source:http://linkedlifedata.com/resource/pubmed/id/8774727

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
3
pubmed:dateCreated
1996-10-24
pubmed:abstractText
Xanthine dehydrogenase, a molybdenum, iron-sulfur flavoenzyme encoded in the fruit fly Drosophila melanogaster by the rosy gene, has been characterised both from the wild-type and mutant files. Enzyme assays, using a variety of different oxidising and reducing substrates were supplemented by limited molecular characterisation. Four rosy strains showed no detectable activity in any enzyme assay tried, whereas from four wild-type and three rosy mutant strains, those for the [E89K], [L127F] and [L157P]xanthine dehydrogenases (in all of which the mutation is in the iron-sulfur domain), the enzyme molecules, although present at different levels, had extremely similar or identical properties. This was confirmed by purification of one wild-type and one mutant enzyme. [E89K]xanthine dehydrogenase. These both had ultraviolet-visible absorption spectra similar to milk xanthine oxidase. Both were found to be quite stable molecules, showing very high catalytic-centre activities and with little tendency to become degraded by proteolysis or modified by conversion to oxidase or desulfo forms. In three further rosy strains, giving [G353D]xanthine dehydrogenase and [S357F]xanthine dehydrogenase mutated in the flavin domain, and [G1011E]xanthine dehydrogenase mutated in the molybdenum domain, enzyme activities were selectively diminished in certain assays. For the G353D and S357F mutant enzymes activities to NAD+ as oxidising substrate were diminished, to zero for the latter. In addition for [G353D]xanthine dehydrogenase, there was an increase in apparent Km values both for NAD+ and NADH. These findings indicate involvement of this part of the sequence in the NAD(+)-binding site. The G1011E mutation has a profound effect on the enzyme. As isolated and as present in crude extracts of the files, this xanthine dehydrogenase variant lacks activity to xanthine or pterin as reducing substrate, indicating an impairment of the functioning of its molybdenum centre. However, it retains full activity to NADH with dyes as oxidising substrate. Mild oxidation of the enzyme converts it, apparently irreversibly, to a form showing full activity to xanthine and pterin. The nature of the group that is oxidised is discussed in the light of redox potential data. It is proposed that the process involves oxidation of the pterin of the molybdenum cofactor from the tetrahydro to a dihydro oxidation state. This conclusion is fully consistent with recent information [Romäo, M. J., Archer, M., Moura, I., Moura. J.J.G., LeGall, J., Engh, R., Schneider, M., Hof, P. & Huber, R. (1995) Science 270. 1170-1176) from X-ray crystallography on the structure of a closely related enzyme from Desulfovibrio gigas. It is proposed, that apparent irreversibility of the oxidative activating process for [G1011E]xanthine dehydrogenase, is due to conversion of its pterin to the tricyclic derivative detected by these workers. The data thus provide the strongest evidence available, that the oxidation state of the pterin can have a controlling influence on the activity of a molybdenum cofactor enzyme. Implications regarding pterin incorporation into xanthine dehydrogenase and in relation to other molybdenum enzymes are discussed.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Aug
pubmed:issn
0014-2956
pubmed:author
pubmed:issnType
Print
pubmed:day
1
pubmed:volume
239
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
782-95
pubmed:dateRevised
2008-11-21
pubmed:meshHeading
pubmed-meshheading:8774727-Amino Acid Sequence, pubmed-meshheading:8774727-Animals, pubmed-meshheading:8774727-Binding Sites, pubmed-meshheading:8774727-Coenzymes, pubmed-meshheading:8774727-Conserved Sequence, pubmed-meshheading:8774727-Cross Reactions, pubmed-meshheading:8774727-Drosophila melanogaster, pubmed-meshheading:8774727-Enzyme Activation, pubmed-meshheading:8774727-Genetic Variation, pubmed-meshheading:8774727-Kinetics, pubmed-meshheading:8774727-Metalloproteins, pubmed-meshheading:8774727-Molecular Sequence Data, pubmed-meshheading:8774727-Mutagenesis, Site-Directed, pubmed-meshheading:8774727-Mutation, pubmed-meshheading:8774727-NAD, pubmed-meshheading:8774727-Oxidation-Reduction, pubmed-meshheading:8774727-Pteridines, pubmed-meshheading:8774727-Sequence Homology, Amino Acid, pubmed-meshheading:8774727-Structure-Activity Relationship, pubmed-meshheading:8774727-Xanthine, pubmed-meshheading:8774727-Xanthine Dehydrogenase, pubmed-meshheading:8774727-Xanthines
pubmed:year
1996
pubmed:articleTitle
Properties of xanthine dehydrogenase variants from rosy mutant strains of Drosophila melanogaster and their relevance to the enzyme's structure and mechanism.
pubmed:affiliation
School of Biological Sciences, University of Sussex, Brighton, UK.
pubmed:publicationType
Journal Article, Comparative Study, Research Support, Non-U.S. Gov't