| dc.contributor.author |
Andreadeli, A |
en |
| dc.contributor.author |
Platis, D |
en |
| dc.contributor.author |
Tishkov, V |
en |
| dc.contributor.author |
Popov, V |
en |
| dc.contributor.author |
Labrou, NE |
en |
| dc.date.accessioned |
2014-06-06T06:48:41Z |
|
| dc.date.available |
2014-06-06T06:48:41Z |
|
| dc.date.issued |
2008 |
en |
| dc.identifier.issn |
1742464X |
en |
| dc.identifier.uri |
http://dx.doi.org/10.1111/j.1742-4658.2008.06533.x |
en |
| dc.identifier.uri |
http://62.217.125.90/xmlui/handle/123456789/4236 |
|
| dc.subject |
Coenzyme |
en |
| dc.subject |
Coenzyme specificity |
en |
| dc.subject |
Enzyme redesign |
en |
| dc.subject |
Formate dehydrogenase |
en |
| dc.subject |
Saturation mutagenesis |
en |
| dc.subject.other |
cytochrome P450 |
en |
| dc.subject.other |
formate dehydrogenase |
en |
| dc.subject.other |
formic acid |
en |
| dc.subject.other |
nicotinamide adenine dinucleotide |
en |
| dc.subject.other |
nicotinamide adenine dinucleotide phosphate |
en |
| dc.subject.other |
unspecific monooxygenase |
en |
| dc.subject.other |
article |
en |
| dc.subject.other |
Bacillus subtilis |
en |
| dc.subject.other |
Candida boidinii |
en |
| dc.subject.other |
coenzyme |
en |
| dc.subject.other |
controlled study |
en |
| dc.subject.other |
enzyme specificity |
en |
| dc.subject.other |
enzyme structure |
en |
| dc.subject.other |
nonhuman |
en |
| dc.subject.other |
nucleotide sequence |
en |
| dc.subject.other |
priority journal |
en |
| dc.subject.other |
protein interaction |
en |
| dc.subject.other |
sequence alignment |
en |
| dc.subject.other |
site directed mutagenesis |
en |
| dc.subject.other |
Amino Acid Sequence |
en |
| dc.subject.other |
Base Sequence |
en |
| dc.subject.other |
Candida |
en |
| dc.subject.other |
Chromatography, Affinity |
en |
| dc.subject.other |
Coenzymes |
en |
| dc.subject.other |
DNA Primers |
en |
| dc.subject.other |
Formate Dehydrogenases |
en |
| dc.subject.other |
Kinetics |
en |
| dc.subject.other |
Models, Molecular |
en |
| dc.subject.other |
Molecular Sequence Data |
en |
| dc.subject.other |
Mutagenesis |
en |
| dc.subject.other |
NADP |
en |
| dc.subject.other |
Protein Conformation |
en |
| dc.subject.other |
Sequence Homology, Amino Acid |
en |
| dc.subject.other |
Substrate Specificity |
en |
| dc.subject.other |
Bacillus subtilis |
en |
| dc.subject.other |
Candida boidinii |
en |
| dc.subject.other |
Kobus |
en |
| dc.title |
Structure-guided alteration of coenzyme specificity of formate dehydrogenase by saturation mutagenesis to enable efficient utilization of NADP+ |
en |
| heal.type |
journalArticle |
en |
| heal.identifier.primary |
10.1111/j.1742-4658.2008.06533.x |
en |
| heal.publicationDate |
2008 |
en |
| heal.abstract |
Formate dehydrogenase from Candida boidinii (CboFDH) catalyses the oxidation of formate anion to carbon dioxide with concomitant reduction of NAD+ to NADH. CboFDH is highly specific to NAD+ and virtually fails to catalyze the reaction with NADP+. Based on structural information for CboFDH, the loop region between β-sheet 7 and α-helix 10 in the dinucleotide-binding fold was predicted as a principal determinant of coenzyme specificity. Sequence alignment with other formate dehydrogenases revealed two residues (Asp195 and Tyr196) that could account for the observed coenzyme specificity. Positions 195 and 196 were subjected to two rounds of site-saturation mutagenesis and screening and enabled the identification of a double mutant Asp195Gln/Tyr196His, which showed a more than 2 × 107-fold improvement in overall catalytic efficiency with NADP+ and a more than 900-fold decrease in the efficiency with NAD+ as cofactors. The results demonstrate that the combined polar interactions and steric factors comprise the main structural determinants responsible for coenzyme specificity. The double mutant Asp195Gln/Tyr196His was tested for practical applicability in a cofactor recycling system composed of cytochrome P450 monooxygenase from Bacillus subtilis, (CYP102A2), NADP +, formic acid and ω-(p-nitrophenyl)dodecanoic acid (12-pNCA). Using a 1250-fold excess of 12-pNCA over NADP+ the first order rate constant was determined to be equal to kobs = 0.059 ± 0.004 min-1. © 2008 The Authors. |
en |
| heal.journalName |
FEBS Journal |
en |
| dc.identifier.issue |
15 |
en |
| dc.identifier.volume |
275 |
en |
| dc.identifier.doi |
10.1111/j.1742-4658.2008.06533.x |
en |
| dc.identifier.spage |
3859 |
en |
| dc.identifier.epage |
3869 |
en |