title: Catalytic mechanism and allosteric regulation of an oligomeric (p)ppGpp synthetase by an alarmone.
abstract: Nucleotide-based second messengers serve in the response of living organisms to environmental changes. In bacteria and plant chloroplasts, guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) [collectively named "(p)ppGpp"] act as alarmones that globally reprogram cellular physiology during various stress conditions. Enzymes of the RelA/SpoT homology (RSH) family synthesize (p)ppGpp by transferring pyrophosphate from ATP to GDP or GTP. Little is known about the catalytic mechanism and regulation of alarmone synthesis. It also is unclear whether ppGpp and pppGpp execute different functions. Here, we unravel the mechanism and allosteric regulation of the highly cooperative alarmone synthetase small alarmone synthetase 1 (SAS1) from Bacillus subtilis. We determine that the catalytic pathway of (p)ppGpp synthesis involves a sequentially ordered substrate binding, activation of ATP in a strained conformation, and transfer of pyrophosphate through a nucleophilic substitution (SN2) reaction. We show that pppGpp-but not ppGpp-positively regulates SAS1 at an allosteric site. Although the physiological significance remains to be elucidated, we establish the structural and mechanistic basis for a biological activity in which ppGpp and pppGpp execute different functional roles.
authors.affiliation: LOEWE Center for Synthetic Microbiology, Philipps University Marburg, 35043 Marburg, Germany; Department of Chemistry, Philipps University Marburg, 35043 Marburg, Germany.
authors.forename: Wieland
authors.initials: W
authors.lastname: Steinchen
authors.affiliation: LOEWE Center for Synthetic Microbiology, Philipps University Marburg, 35043 Marburg, Germany; Department of Chemistry, Philipps University Marburg, 35043 Marburg, Germany.
authors.forename: Jan S
authors.initials: JS
authors.lastname: Schuhmacher
authors.affiliation: LOEWE Center for Synthetic Microbiology, Philipps University Marburg, 35043 Marburg, Germany; Department of Chemistry, Philipps University Marburg, 35043 Marburg, Germany.
authors.forename: Florian
authors.initials: F
authors.lastname: Altegoer
authors.affiliation: LOEWE Center for Synthetic Microbiology, Philipps University Marburg, 35043 Marburg, Germany; Department of Chemistry, Philipps University Marburg, 35043 Marburg, Germany.
authors.forename: Christopher D
authors.initials: CD
authors.lastname: Fage
authors.affiliation: LOEWE Center for Synthetic Microbiology, Philipps University Marburg, 35043 Marburg, Germany; Department of Chemistry, Philipps University Marburg, 35043 Marburg, Germany.
authors.forename: Vasundara
authors.initials: V
authors.lastname: Srinivasan
authors.affiliation: LOEWE Center for Synthetic Microbiology, Philipps University Marburg, 35043 Marburg, Germany; Department of Chemistry, Philipps University Marburg, 35043 Marburg, Germany.
authors.forename: Uwe
authors.initials: U
authors.lastname: Linne
authors.affiliation: LOEWE Center for Synthetic Microbiology, Philipps University Marburg, 35043 Marburg, Germany; Department of Chemistry, Philipps University Marburg, 35043 Marburg, Germany.
authors.forename: Mohamed A
authors.initials: MA
authors.lastname: Marahiel
authors.affiliation: LOEWE Center for Synthetic Microbiology, Philipps University Marburg, 35043 Marburg, Germany; Department of Chemistry, Philipps University Marburg, 35043 Marburg, Germany gert.bange@synmikro.uni-marburg.de.
authors.forename: Gert
authors.initials: G
authors.lastname: Bange
mesh_terms: D000494:Allosteric Regulation / Q000502:physiology*; D001412:Bacillus subtilis / Q000235:genetics*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D003460:Crystallization; D004926:Escherichia coli; D006158:Guanosine Pentaphosphate / Q000096:biosynthesis*; D006159:Guanosine Tetraphosphate / Q000096:biosynthesis*; D008025:Ligases / Q000378:metabolism / Q000502:physiology*; D013058:Mass Spectrometry; D016296:Mutagenesis
chemical_list: D001426:Bacterial Proteins; D006159:Guanosine Tetraphosphate; D006158:Guanosine Pentaphosphate; D008025:Ligases; C028812:guanosine 3',5'-polyphosphate synthetases
keywords: (p)ppGpp; alarmone; crystallography; hydrogen–deuterium exchange mass spectrometry; stringent response