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PEDS Advance Access published online on August 13, 2004
Protein Engineering Design and Selection, doi:10.1093/protein/gzh064
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Received June 1, 2004
Revised July 21, 2004
Accepted August 4, 2004

Article

Modulating D-amino acid oxidase substrate specificity: production of an enzyme for analytical determination of all D-amino acids by directed evolution

Silvia Sacchi , Elena Rosini , Gianluca Molla , Mirella S. Pilone , Loredano Pollegioni 1

1 Department of Structural and Functional Biology, University of Insubria, via J.H. Dunant 3, 21100 Varese (Italy)


  Abstract

Recent research on the flavoenzyme D-amino acid oxidase from R. 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 E. 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.

Keywords: directed evolution; flavoprotein; function-structure relationships; substrate recognition.
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