| dc.contributor.advisor |
Λάμπρου, Νικόλαος Ε. |
el |
| dc.contributor.advisor |
Labrou, Nikolaos E. |
en |
| dc.contributor.author |
Rigden, Daniel J. |
en |
| dc.date.accessioned |
2014-06-06T06:44:34Z |
|
| dc.date.available |
2014-06-06T06:44:34Z |
|
| dc.date.issued |
2001 |
en |
| dc.identifier.issn |
02646021 |
en |
| dc.identifier.uri |
http://dx.doi.org/10.1042/0264-6021:3540455 |
en |
| dc.identifier.uri |
http://62.217.125.90/xmlui/handle/123456789/1953 |
|
| dc.title |
Active-site characterization of Candida boidinii formate dehydrogenase |
en |
| heal.type |
journalArticle |
en |
| heal.keyword |
Kinetic mechanism |
en |
| heal.keyword |
Oxidoreductase |
en |
| heal.keyword |
Candida boidinii |
en |
| heal.keyword |
Enzyme activity |
en |
| heal.keyword |
Catalysis |
en |
| heal.keyword |
Escherichia coli |
en |
| heal.keyword |
Dehydrogenases |
en |
| heal.keyword |
Kinetics |
en |
| heal.keyword |
NAD |
en |
| heal.keyword |
Pseudomonas |
en |
| heal.keyword |
Hydrogen-Ion Concentration |
en |
| heal.keyword |
Protein engineering |
en |
| heal.keyword |
Site-directed mutagenesis |
en |
| heal.identifier.primary |
10.1042/0264-6021:3540455 |
en |
| heal.recordProvider |
Γεωπονικό Πανεπιστήμιο Αθηνών/Τμήμα Γεωπονικής Βιοτεχνολογίας |
el |
| heal.recordProvider |
Universida de Catolica de Brasılia/Bioinformatics Laboratory |
en |
| heal.publicationDate |
2001 |
en |
| heal.bibliographicCitation |
Labrou, Nikolaos E. Active-site characterization of Candida boidinii formate dehydrogenase, Biochemical Journal vol. 354 (2) pp. 456-463, Portland Press 2001 |
en |
| heal.abstract |
NAD+-dependent formate dehydrogenase (FDH) from Candida boidinii was cloned and expressed to a high level in Escherichia coli (20% of soluble E. coli protein). Molecular modelling studies were used to create a three-dimensional model of C. boidinii FDH, based on a known structure of the Pseudomonas sp. 101 enzyme. This model was used for investigating the catalytic mechanism by site-directed mutagenesis. Eleven forms of C. boidinii FDH were characterized by steady-state kinetic analysis: the wild type as well as 10 mutants involving single (Phe-69-Ala, Asn-119-His, Ile-175-Ala, Gln-197-Leu, Arg-258-Ala, Gln-287-Glu and His-311-Gln) and double amino acid substitutions (Asn-119-His/His-311-Gln, Gln-287-Glu/His-311-Gln and Gln-287-Glu/Pro-288-Thr). The kinetic results of the mutant enzymes provide the first experimental support that hydrophobic patches, formed by Phe-69 and Ile-175, destabilize substrates and stabilize products. Also, the key role of Arg-258 in stabilization of the negative charge on the migrating hydride was established. Asn-119, besides being an anchor group for formate, also may comprise one of the hinge regions around which the two domains shift on binding of NAD+. The more unexpected results, obtained for the His-311-Gln and Gln-287-Glu/His-311-Gln mutants, combined with molecular modelling, suggest that steric as well as electrostatic properties of His-311 are important for enzyme function. An important structural role has also been attributed to cis-Pro-288. This residue may provide the key residues Gln-287 and His-311 with the proper orientation for productive binding of formate. |
en |
| heal.publisher |
Portland Press |
en |
| heal.journalName |
Biochemical Journal |
en |
| dc.identifier.doi |
10.1042/0264-6021:3540455 |
en |