title: Biochemical studies on Francisella tularensis RelA in (p)ppGpp biosynthesis.
abstract: The bacterial stringent response is induced by nutrient deprivation and is mediated by enzymes of the RSH (RelA/SpoT homologue; RelA, (p)ppGpp synthetase I; SpoT, (p)ppGpp synthetase II) superfamily that control concentrations of the 'alarmones' (p)ppGpp (guanosine penta- or tetra-phosphate). This regulatory pathway is present in the vast majority of pathogens and has been proposed as a potential anti-bacterial target. Current understanding of RelA-mediated responses is based on biochemical studies using Escherichia coli as a model. In comparison, the Francisella tularensis RelA sequence contains a truncated regulatory C-terminal region and an unusual synthetase motif (EXSD). Biochemical analysis of F. tularensis RelA showed the similarities and differences of this enzyme compared with the model RelA from Escherichia coli. Purification of the enzyme yielded a stable dimer capable of reaching concentrations of 10 mg/ml. In contrast with other enzymes from the RelA/SpoT homologue superfamily, activity assays with F. tularensis RelA demonstrate a high degree of specificity for GTP as a pyrophosphate acceptor, with no measurable turnover for GDP. Steady state kinetic analysis of F. tularensis RelA gave saturation activity curves that best fitted a sigmoidal function. This kinetic profile can result from allosteric regulation and further measurements with potential allosteric regulators demonstrated activation by ppGpp (5',3'-dibisphosphate guanosine) with an EC50 of 60±1.9 μM. Activation of F. tularensis RelA by stalled ribosomal complexes formed with ribosomes purified from E. coli MRE600 was observed, but interestingly, significantly weaker activation with ribosomes isolated from Francisella philomiragia.
authors.affiliation: Department of Chemistry, University of Southampton, Southampton, SO17 1BJ, U.K.
authors.forename: Rachael C
authors.initials: RC
authors.lastname: Wilkinson
authors.affiliation: Department of Chemistry, University of Southampton, Southampton, SO17 1BJ, U.K.
authors.forename: Laura E
authors.initials: LE
authors.lastname: Batten
authors.affiliation: Department of Chemistry, University of Southampton, Southampton, SO17 1BJ, U.K.
authors.forename: Neil J
authors.initials: NJ
authors.lastname: Wells
authors.affiliation: Institute for Life Sciences, University of Southampton, Southampton, SO17 1BJ, U.K. DSTL, Porton Down, Salisbury, SP4 OJQ, U.K.
authors.forename: Petra C F
authors.initials: PC
authors.lastname: Oyston
authors.affiliation: Department of Chemistry, University of Southampton, Southampton, SO17 1BJ, U.K. Institute for Life Sciences, University of Southampton, Southampton, SO17 1BJ, U.K. plr2@soton.ac.uk.
authors.forename: Peter L
authors.initials: PL
authors.lastname: Roach
mesh_terms: D000494:Allosteric Regulation / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology; D005604:Francisella tularensis / Q000201:enzymology*; D007700:Kinetics; D008025:Ligases / Q000235:genetics / Q000378:metabolism*; D012270:Ribosomes / Q000201:enzymology*
chemical_list: D008025:Ligases; C028812:guanosine 3',5'-polyphosphate synthetases
keywords: 3′-dibisphosphate guanosine (ppGpp); 5′; Francisella; RelA (p)ppGpp synthetase I; kinetics; stringent response; synthetase