Modulating D-amino acid oxidase substrate specificity: production of an enzyme for analytical determination of all D-amino acids by directed evolution.
Recent research on the flavoenzyme D-amino acid oxidase from Rhodotorula gracilis (RgDAAO) has revealed new, intriguing properties of this catalyst and offers novel biotechnological applications. Among them, the reaction of RgDAAO has been exploited in the analytical determination of the D-amino acid content in biological samples. However, because the enzyme does not oxidize acidic D-amino acids, it cannot be used to detect the total amount of D-amino acids. We now present the results obtained using a random mutagenesis approach to produce RgDAAO mutants with a broader substrate specificity. The libraries of RgDAAO mutants were generated by error-prone PCR, expressed in BL21(DE3)pLysS Escherichia coli cells and screened for their ability to oxidize different substrates by means of an activity assay. Five random mutants that have a 'modified' substrate specificity, more useful for the analytical determination of the entire content of D-amino acids than wild-type RgDAAO, have been isolated. With the only exception of Y223 and G199, none of the effective amino acid substitutions lie in segments predicted to interact directly with the bound substrate. The substitutions appear to cluster on the protein surface: it would not have been possible to predict that these substitutions would enhance DAAO activity. We can only conclude that these substitutions synergistically generate small structural changes that affect the dynamics and/or stability of the protein in a way that enhances substrate binding or subsequently catalytic turnover.
Sacchi
Silvia
S
Department of Biotechnology and Molecular Sciences, University of Insubria, via J.H. Dunant 3, 21100 Varese, Italy.
Rosini
Elena
E
Molla
Gianluca
G
Pilone
Mirella S
MS
Pollegioni
Loredano
L
D000409:Alanine / Q000235:genetics / Q000378:metabolism; D019943:Amino Acid Substitution; D000596:Amino Acids / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D001120:Arginine / Q000235:genetics / Q000378:metabolism; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism; D001565:Benzoates / Q000378:metabolism; D002384:Catalysis; D003605:D-Amino-Acid Oxidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019020:Directed Molecular Evolution; D004791:Enzyme Inhibitors / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics / Q000502:physiology; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012248:Rhodotorula / Q000201:enzymology* / Q000235:genetics; D013237:Stereoisomerism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D062367:ortho-Aminobenzoates / Q000378:metabolism
D000596:Amino Acids; D001565:Benzoates; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D062367:ortho-Aminobenzoates; C031385:anthranilic acid; D001224:Aspartic Acid; D001120:Arginine; D003605:D-Amino-Acid Oxidase; D000409:Alanine