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Mutant uridine phosphorylase genes from Shewanella oneidensis MR-1 (S. oneidensis) were constructed by site-directed mutagenesis and strains-producers of the corresponding recombinant (F5I and F5G) proteins were obtained on the basis of Escherichia coli cells. The mutant proteins were purified and their physicochemical and enzymatic properties were studied. It was shown that the N-terminal fragment of uridine phosphorylase plays an important role in the thermal stabilization of the enzyme as a whole. The role of the aminoacid (a.a.) residue phenylalanine (F5) in the formation of thermotolerance of uridine phosphorylases from gamma-proteobacteria was revealed. [ABSTRACT FROM AUTHOR]

Crystals of mutants of uridine phosphorylase from Shewanella oneidensis MR-1 at the active-site threonine residue were obtained, and the three-dimensional structures of the mutants were determined. It was shown that the loop 161–179 responsible for the nucleoside recognition is involved in stabilization of the hexameric structure of the protein and its disorder significantly facilitates the access of nucleoside to the enzyme active site. The role of conformational changes in the enzyme function is discussed. [ABSTRACT FROM AUTHOR]

A comparison of the primary structures of the protein translocation Sec-system proteins in the Shewanella oneidensis MR-1 and Escherichia coli bacteria was carried out. The process of translocation of recombinant pro-enterotoxins ( SEB and SEH) from Staphylococcus aureus and pro-streptavidin ( SAV) from Streptomyces avidinii in the S. oneidensis MR-1 and E. coli cell periplasm was studied. It was demonstrated that these marker proteins are transferred into the periplasmic space of the S. oneidensis MR-1 transformant strain cells. The identity of N-terminal amino acid sequences of mature recombinant SEB, SEH, and SAV proteins (generated during post-translation proteolysis of leader peptide by the Sec-system both in E. coli and S. oneidensis MR-1) was established. [ABSTRACT FROM AUTHOR]

The article presents a study on the physicochemical investigation of recombinant uridine phosphorylases (UP) from the free living bacterium Shewanella oneidensis MR-1 (S. oneidensis). It offers a comparison of the primary sequence and thermal stability of S. oneidensis MR-1 uridine phosphorylase (SUP) and Escherichia coli uridine phosphorylase (EUP). The authors also successfully collected X-ray diffraction data sets at high resolution using synchrotron radiation due to a high crystal quality.