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Substrate Recognition Properties of Oligopeptidase B from Salmonella enterica Serovar Typhimurium. Rory E. Morty, 2002.Oligopeptidase B (OpdB) is a serine peptidase broadly distributed among unicellular eukaryotes, gram-negative bacteria, and spirochetes which has emerged as an important virulence factor and potential therapeutic target in infectious diseases . We report here the cloning and expression of the opdB homologue from Salmonella enterica serovar Typhimurium and demonstrate that it exhibits amidolytic activity exclusively against substrates with basic residues in P1 . While similar to its eukaryotic homologues in terms of substrate specificity, Salmonella OpdB differs significantly in catalytic power and inhibition and activation properties . In addition to oligopeptide substrates, restricted proteolysis of histone proteins was observed, although no cleavage was seen at or near residues that had been posttranslationally modified or at defined secondary structures . This supports the idea that the catalytic site of OpdB may be accessible only to unstructured oligopeptides, similar to the closely related prolyl oligopeptidase (POP) . Salmonella OpdB was employed as a model enzyme to define determinants of substrate specificity that distinguish OpdB from POP, which hydrolyzes substrates exclusively at proline residues . Using site-directed mutagenesis, nine acidic residues that are conserved in OpdBs but absent from POPs were converted to their corresponding residues in POP . In this manner, we identified a pair of glutamic acid residues, Glu576 and Glu578, that define P1 specificity and direct OpdB cleavage C terminal to basic residues . We have also identified a second pair of residues, Asp460 and Asp462, that may be involved in defining P2 specificity and thus direct preferential cleavage by OpdB after pairs of basic residues . The PrpC Serine-Threonine Phosphatase and PrkC Kinase Have Opposing Physiological Roles in Stationary-Phase Bacillus subtilis Cells. Tatiana A. Gaidenko, 2002.Loss of the PrpC serine-threonine phosphatase and the associated PrkC kinase of Bacillus subtilis were shown to have opposite effects on stationary-phase physiology by differentially affecting cell density, cell viability, and accumulation of ß-galactosidase from a general stress reporter fusion . These pleiotropic effects suggest that PrpC and PrkC have important regulatory roles in stationary-phase cells . Elongation factor G (EF-G) was identified as one possible target of the PrpC and PrkC pair in vivo, and purified PrpC and PrkC manifested the predicted phosphatase and kinase activities against EF-G in vitro . Identification and Characterization of a pSLA2 Plasmid Locus Required for Linear DNA Replication and Circular Plasmid Stable Inheritance in Streptomyces lividans. Zhongjun Qin, 2003.Streptomyces linear plasmids and linear chromosomes can replicate also in a circular form when their telomeres are deleted . The 17-kb linear plasmid pSLA2 has been a useful model in studies of such replicons . Here we report that the minimal origin initiating replication of pSLA2-derived plasmids as circular molecules cannot propagate these plasmids in a linear mode unless they also contain a novel plasmid-encoded locus, here named rlrA (required for linear replication) . In contrast with the need for rlrA to accomplish replication of telomere-containing linear plasmids, expression of rlrA, which encodes two LuxR family regulatory domains, interferes with the establishment of pSLA2 in circular form in Streptomyces lividans transformants . The additional presence of an adjacent divergently transcribed locus, rorA (rlrA override), which strongly resembles the kor (kil override) transcription control genes identified previously on Streptomyces plasmids, reversed the detrimental effects of rlrA on plasmid establishment and additionally stabilized circular plasmid inheritance by spores during the S . lividans life cycle . While the effects of the rlrA/rorA locus of pSLA2 were seen also on linear plasmids derived from the unrelated SLP2 replicon, they did not extend to plasmids whose replication was initiated at a cloned chromosomal origin . Our results establish the existence of, and provide the initial description of, a novel plasmid-borne regulatory system that differentially affects the propagation of linear and circular plasmids in Streptomyces . A Novel NAD-Dependent Dehydrogenase, Highly Specific for 1,5-Anhydro-D-Glucitol, from Trichoderma longibrachiatum Strain 11-3. Nobuyuki Yoshida, 2003.A novel NAD-dependent dehydrogenase highly specific for 1,5-anhydro-D-glucitol (1,5-AG) was found in the cell extract of an imperfect fungus, Trichoderma longibrachiatum strain 11-3 . This fungus used 1,5-AG as a sole carbon source for growth and transformed 1,5-AG into glucose . 1,5-AG dehydrogenase (AGH) was purified to homogeneity, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) . The molecular mass of the purified enzyme was estimated to be 36 and 141 kDa by SDS-PAGE and by gel filtration, respectively, suggesting that the enzyme was homotetrameric . The enzyme was highly specific for 1,5-AG and did not exhibit activity with any sugar or sugar alcohol tested in this study other than 1,5-AG . A linear relationship between the initial rate of the enzyme reaction and the concentration of 1,5-AG at the physiological level was observed . The presence of glucose in abundance did not interfere with the relationship . The optimum temperature for the enzyme reaction was 50°C, and the enzyme was stable at temperatures up to 70°C . These results suggested that AGH is a novel enzyme and is useful for specifically diagnosing diabetes mellitus .
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