Structural investigation of the thymidine phosphorylase from Salmonella typhimurium in the unliganded state and its complexes with thymidine and uridine.

Highly specific thymidine phosphorylases catalyze the phosphorolytic cleavage of thymidine, with the help of a phosphate ion, resulting in thymine and 2-deoxy-α-D-ribose 1-phosphate. Thymidine phosphorylases do not catalyze the phosphorolysis of uridine, in contrast to nonspecific pyrimidine nucleoside phosphorylases and uridine phosphorylases. Understanding the mechanism of substrate specificity on the basis of the nucleoside is essential to support rational drug-discovery investigations of new antitumour and anti-infective drugs which are metabolized by thymidine phosphorylases. For this reason, X-ray structures of the thymidine phosphorylase from Salmonella typhimurium were solved and refined: the unliganded structure at 2.05 Å resolution (PDB entry 4xr5), the structure of the complex with thymidine at 2.55 Å resolution (PDB entry 4yek) and that of the complex with uridine at 2.43 Å resolution (PDB entry 4yyy). The various structural features of the enzyme which might be responsible for the specificity for thymidine and not for uridine were identified. The presence of the 2'-hydroxyl group in uridine results in a different position of the uridine furanose moiety compared with that of thymidine. This feature may be the key element of the substrate specificity. The specificity might also be associated with the opening/closure mechanism of the two-domain subunit structure of the enzyme.

Preliminary investigation of the three-dimensional structure of Salmonella typhimurium uridine phosphorylase in the crystalline state.

Uridine phosphorylase (UPh) catalyzes the phosphorolytic cleavage of the C-N glycosidic bond of uridine to ribose 1-phosphate and uracil in the pyrimidine-salvage pathway. The crystal structure of the Salmonella typhimurium uridine phosphorylase (StUPh) has been determined at 2.5 A resolution and refined to an R factor of 22.1% and an Rfree of 27.9%. The hexameric StUPh displays 32 point-group symmetry and utilizes both twofold and threefold non-crystallographic axes. A phosphate is bound at the active site and forms hydrogen bonds to Arg91, Arg30, Thr94 and Gly26 of one monomer and Arg48 of an adjacent monomer. The hexameric StUPh model reveals a close structural relationship to Escherichia coli uridine phosphorylase (EcUPh).

NMR structure of the ribosomal protein L23 from Thermus thermophilus.

The ribosomal protein L23 is a component of the large ribosomal subunit in which it is located close to the peptide exit tunnel. In this position L23 plays a central role both for protein secretion and folding. We have determined the solution structure of L23 from Thermus thermophilus. Uncomplexed L23 consists of a well-ordered part, with four anti-parallel beta-strands and three alpha-helices connected as beta-alpha-beta-alpha-beta-beta-alpha, and a large and flexible loop inserted between the third and fourth beta-strand. The observed topology is distantly related to previously known structures, primarily within the area of RNA biochemistry. A comparison with RNA-complexed crystal structures of L23 from T. thermophilus, Deinococcus radiodurans and Haloarcula marismourtui, shows that the conformation of the well-ordered part is very similar in the uncomplexed and complexed states. However, the flexible loop found in the uncomplexed solution structure forms a rigid extended structure in the complexed crystal structures as it interacts with rRNA and becomes part of the exit tunnel wall. Structural characteristics of importance for the interaction with rRNA and with the ribosomal protein L29, as well as the functional role of L23, are discussed.