Turning pyridoxal-5'-phosphate-dependent enzymes into thermostable binding proteins: D-Serine dehydratase from baker's yeast as a case study

Abstract

D-serine dehydratase from Saccharomyces cerevisae is a recently discovered dimeric enzyme catalyzing the β-elimination of D-serine to pyruvate and ammonia. The reaction is highly enantioselective and depends on cofactor pyridoxal-5'-phosphate (PLP) and Zn(2+). In our work, the aldimine linkage tethering PLP to recombinant, tagged D-serine dehydratase (Dsd) has been reduced by treatment with NaBH(4) so as to yield an inactive form of the holoenzyme (DsdR), which was further treated with a protease in order to remove the amino-terminal purification tag. Fourier Transform infrared (FT-IR) spectroscopic analysis revealed that both the reduced form (DsdR) and the reduced/detagged form (DsdRD) maintain the overall secondary structure of Dsd, but featured a significant increased thermal stability. The observed T(m) values for DsdR and for DsdRD shifted to 71.5 °C and 73.3 °C, respectively, resulting in nearly 11 °C and 13 °C higher than the one measured for Dsd. Furthermore, the analysis of the FT-IR spectra acquired in the presence of D-serine and L-serine indicates that, though catalytically inert, DsdRD retains the ability to enantioselectively bind its natural substrate. Sequence analysis of D-serine dehydratase and other PLP-dependent enzymes also highlighted critical residues involved in PLP binding. In virtue of its intrinsic properties, DsdRD represents an ideal candidate for the design of novel platforms based on stable, non-consuming binding proteins aimed at measuring d-serine levels in biological fluids.