Investigating the role of active site residues of Rhodotorula gracilis D-amino acid oxidase on its substrate specificity.
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.
Boselli
Angelo
A
Department of Biotechnology and Molecular Sciences, University of Insubria, via J.H. Dunant 3, 21100 Varese, Italy.
Piubelli
Luciano
L
Molla
Gianluca
G
Pilone
Mirella S
MS
Pollegioni
Loredano
L
Sacchi
Silvia
S
D019943:Amino Acid Substitution; D001665:Binding Sites / Q000235:genetics; D003605:D-Amino-Acid Oxidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D011485:Protein Binding; D012248:Rhodotorula / Q000201:enzymology* / Q000235:genetics; D013053:Spectrophotometry / Q000379:methods; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013816:Thermodynamics
D050505:Mutant Proteins; D003605:D-Amino-Acid Oxidase