Decrypting the Biochemical Function of an Essential Gene from Streptococcus pneumoniae Using ThermoFluor® Technology

Abstract The protein product of an essential geneof unknown function from Streptococcus pneumoniae was expressed and purified for screening in the ThermoFluor® affinity screening assay. This assay can detect ligand binding to proteins of unknown function. The recombinant protein was found to be in a dimeric, native-like folded state and to unfold cooperatively. ThermoFluor was used to screen the protein against a library of 3000 compounds that were specifically selected to provide information about possible biological functions. The results of this screen identified pyridoxal phosphateand pyridoxamine phosphateas equilibrium binding ligands (Kd ∼ 50 pm, Kd ∼ 2.5 μm, respectively), consistent with an enzymatic cofactor function. Several nucleotides and nucleotide sugarswere also identified as ligands of this protein. Sequence comparison with two enzymes of known structure but relatively low overall sequence homology established that several key residues directly involved in pyridoxal phosphatebinding were strictly conserved. Screening a collection of generic drugsand natural products identified the antifungal compound canescin A as an irreversible covalent modifier of the enzyme. Our investigation of this protein indicates that its probable biological role is that of a nucleoside diphospho-keto-sugar aminotransferase, although the preferred keto-sugar substrate remains unknown. These experiments demonstrate the utility of a generic affinity-based ligand binding technology in decrypting possible biological functionsof a protein, an approach that is both independent of and complementary to existing genomic and proteomic technologies.