Decrypting the biochemical function of an essential gene from Streptococcus pneumoniae using ThermoFluor technology.
J Biol Chem
; 280(12): 11704-12, 2005 Mar 25.
Article
en En
| MEDLINE
| ID: mdl-15634672
ABSTRACT
The protein product of an essential gene of 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 phosphate and pyridoxamine phosphate as equilibrium binding ligands (K(d) approximately 50 pM, K(d) approximately 2.5 microM, respectively), consistent with an enzymatic cofactor function. Several nucleotides and nucleotide sugars were 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 phosphate binding were strictly conserved. Screening a collection of generic drugs and 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 functions of a protein, an approach that is both independent of and complementary to existing genomic and proteomic technologies.
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Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Streptococcus pneumoniae
/
Proteínas Bacterianas
/
Genes Esenciales
/
Transaminasas
/
Azúcares de Nucleósido Difosfato
Idioma:
En
Revista:
J Biol Chem
Año:
2005
Tipo del documento:
Article
País de afiliación:
Estados Unidos