title: On the effect of alkaline pH and cofactor availability in the conformational and oligomeric state of Escherichia coli glutamate decarboxylase.
abstract: Escherichia coli glutamate decarboxylase (EcGad) is a homohexameric pyridoxal 5'-phosphate (PLP)-dependent enzyme. It is the structural component of the major acid resistance system that protects E. coli from strong acid stress (pH < 3), typically encountered in the mammalian gastrointestinal tract. In fact EcGad consumes one proton/catalytic cycle while yielding γ-aminobutyrate and carbon dioxide from the decarboxylation of l-glutamate. Two isoforms of Gad occur in E. coli (GadA and GadB) that are 99% identical in sequence. GadB is the most intensively investigated. Prompted by the observation that some transcriptomic and proteomic studies show EcGad to be expressed in conditions far from acidic, we investigated the structural organization of EcGadB in solution in the pH range 7.5-8.6. Small angle X-ray scattering, combined with size exclusion chromatography, and analytical ultracentrifugation analysis show that the compact and entangled EcGadB hexameric structure undergoes dissociation into dimers as pH alkalinizes. When PLP is not present, the dimeric species is the most abundant in solution, though evidence for the occurrence of a likely tetrameric species was also obtained. Trp fluorescence emission spectra as well as limited proteolysis studies suggest that PLP plays a key role in the acquisition of a folding necessary for the canonical catalytic activity.
authors.affiliation: Istituto Pasteur Italia - Fondazione Cenci Bolognetti, Dipartimento di Scienze e Biotecnologie Medico-Chirurgiche, Sapienza Università di Roma, I-04100 Latina, Italy.
authors.forename: F
authors.initials: F
authors.lastname: Giovannercole
authors.affiliation: Institut de Biologie Intégrative de la Cellule, UMR 9198, Université Paris-Sud, F-91405 ORSAY Cedex, France.
authors.forename: C
authors.initials: C
authors.lastname: Mérigoux
authors.affiliation: Dipartimento di Scienze Biochimiche 'A. Rossi Fanelli', Sapienza Università di Roma, I-00185 Roma, Italy.
authors.forename: C
authors.initials: C
authors.lastname: Zamparelli
authors.affiliation: CNR Istituto di Biologia e Patologia Molecolari, Dipartimento di Scienze Biochimiche 'A. Rossi Fanelli', Sapienza Università di Roma, I-00185 Roma, Italy.
authors.forename: D
authors.initials: D
authors.lastname: Verzili
authors.affiliation: Istituto Pasteur Italia - Fondazione Cenci Bolognetti, Dipartimento di Scienze e Biotecnologie Medico-Chirurgiche, Sapienza Università di Roma, I-04100 Latina, Italy.|Present address at: Laboratoire de Chimie Bactérien (ACB), Aix-Marseille University.
authors.forename: G
authors.initials: G
authors.lastname: Grassini
authors.affiliation: LBPA, ENS Cachan, CNRS, Université Paris-Saclay, F-94235, Cachan, France.
authors.forename: M
authors.initials: M
authors.lastname: Buckle
authors.affiliation: Institut de Biologie Intégrative de la Cellule, UMR 9198, Université Paris-Sud, F-91405 ORSAY Cedex, France.
authors.forename: P
authors.initials: P
authors.lastname: Vachette
authors.affiliation: Istituto Pasteur Italia - Fondazione Cenci Bolognetti, Dipartimento di Scienze e Biotecnologie Medico-Chirurgiche, Sapienza Università di Roma, I-04100 Latina, Italy.
authors.forename: D
authors.initials: D
authors.lastname: De Biase
mesh_terms: D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D005968:Glutamate Decarboxylase / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008565:Membrane Proteins / Q000737:chemistry*; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D014961:X-Ray Diffraction
chemical_list: D029968:Escherichia coli Proteins; D008565:Membrane Proteins; C121044:gadA protein, E coli; C584507:gadB protein, E coli; D005968:Glutamate Decarboxylase
keywords: analytic ultracentrifugation; glutamate decarboxylase; limited proteolysis; pyridoxal 5′-phosphate (PLP); small angle X-ray scattering (SAXS)