Document Detail


Investigating the role of active site residues of Rhodotorula gracilis D-amino acid oxidase on its substrate specificity.
MedLine Citation:
PMID:  17145127     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
D-amino acid oxidase (DAAO) is a flavoprotein that catalyzes stereospecifically the oxidative deamination of D-amino acids. The wild-type DAAO is mainly active on neutral D-amino acids, while basic D-amino acids are poor substrates and the acidic ones are virtually not oxidized. To present a comprehensive picture of how the active site residues can modulate the substrate specificity a number of mutants at position M213, Y223, Y238, R285, S335, and Q339 were prepared in the enzyme from the yeast Rhodotorula gracilis. All DAAO mutants have spectral properties similar to those of the wild-type enzyme and are catalytically active, thus excluding an essential role in catalysis; a lower activity on neutral and basic amino acids was observed. Interestingly, an increase in activity and (k(cat)/K(m))(app) ratio on D-aspartate was observed for all the mutants containing an additional charged residue in the active site. The active site of yeast DAAO appears to be a highly evolved scaffold built up through evolution to optimize the oxidative deamination of neutral D-amino acids without limiting its substrate specificity. It is noteworthy, that introduction of a sole, additional, positively charged residue in the active site is sufficient to optimize the reactivity on acidic D-amino acids, giving rise to kinetic properties similar to those of D-aspartate oxidase.
Authors:
Angelo Boselli; Luciano Piubelli; Gianluca Molla; Mirella S Pilone; Loredano Pollegioni; Silvia Sacchi
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2006-11-27
Journal Detail:
Title:  Biochimie     Volume:  89     ISSN:  0300-9084     ISO Abbreviation:  Biochimie     Publication Date:  2007 Mar 
Date Detail:
Created Date:  2007-03-26     Completed Date:  2007-08-13     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  1264604     Medline TA:  Biochimie     Country:  France    
Other Details:
Languages:  eng     Pagination:  360-8     Citation Subset:  IM    
Affiliation:
Department of Biotechnology and Molecular Sciences, University of Insubria, via J.H. Dunant 3, 21100 Varese, Italy.
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MeSH Terms
Descriptor/Qualifier:
Amino Acid Substitution
Binding Sites / genetics
D-Amino-Acid Oxidase / chemistry,  genetics,  metabolism*
Kinetics
Models, Molecular
Mutagenesis, Site-Directed
Mutant Proteins / chemistry,  metabolism
Oxidation-Reduction
Protein Binding
Rhodotorula / enzymology*,  genetics
Spectrophotometry / methods
Structure-Activity Relationship
Substrate Specificity
Thermodynamics
Chemical
Reg. No./Substance:
0/Mutant Proteins; EC 1.4.3.3/D-Amino-Acid Oxidase

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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