title: Cooperative substrate binding by a diguanylate cyclase.
abstract: XAC0610, from Xanthomonas citri subsp. citri, is a large multi-domain protein containing one GAF (cGMP-specific phosphodiesterases, adenylyl cyclases and FhlA) domain, four PAS (Per-Arnt-Sim) domains and one GGDEF domain. This protein has a demonstrable in vivo and in vitro diguanylate cyclase (DGC) activity that leads to the production of cyclic di-GMP (c-di-GMP), a ubiquitous bacterial signaling molecule. Analysis of a XacΔ0610 knockout strain revealed that XAC0610 plays a role in the regulation of Xac motility and resistance to H2O2. Site-directed mutagenesis of a conserved DGC lysine residue (Lys759 in XAC0610) resulted in a severe reduction in XAC0610 DGC activity. Furthermore, experimental and in silico analyses suggest that XAC0610 is not subject to allosteric product inhibition, a common regulatory mechanism for DGC activity control. Instead, steady-state kinetics of XAC0610 DGC activity revealed a positive cooperative effect of the GTP substrate with a dissociation constant for the binding of the first GTP molecule (K1) approximately 5× greater than the dissociation constant for the binding of the second GTP molecule (K2). We present a general kinetics scheme that should be used when analyzing DGC kinetics data and propose that cooperative GTP binding could be a common, though up to now overlooked, feature of these enzymes that may in some cases offer a physiologically relevant mechanism for regulation of DGC activity in vivo.
authors.affiliation: Department of Biochemistry, Institute of Chemistry, University of São Paulo, São Paulo, SP 05508-070, Brazil.
authors.forename: Maycon C
authors.initials: MC
authors.lastname: Oliveira
authors.affiliation: Department of Biochemistry, Institute of Chemistry, University of São Paulo, São Paulo, SP 05508-070, Brazil.
authors.forename: Raphael D
authors.initials: RD
authors.lastname: Teixeira
authors.affiliation: Department of Biochemistry, Institute of Chemistry, University of São Paulo, São Paulo, SP 05508-070, Brazil.
authors.forename: Maxuel O
authors.initials: MO
authors.lastname: Andrade
authors.affiliation: Institute of Chemistry, State University of Campinas, Campinas, SP 13083-970, Brazil.
authors.forename: Glaucia M S
authors.initials: GM
authors.lastname: Pinheiro
authors.affiliation: Institute of Chemistry, State University of Campinas, Campinas, SP 13083-970, Brazil.
authors.forename: Carlos H I
authors.initials: CH
authors.lastname: Ramos
authors.affiliation: Department of Biochemistry, Institute of Chemistry, University of São Paulo, São Paulo, SP 05508-070, Brazil. Electronic address: chsfarah@iq.usp.br.
authors.forename: Chuck S
authors.initials: CS
authors.lastname: Farah
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002942:Circular Dichroism; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D006861:Hydrogen Peroxide / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019761:Phosphorus-Oxygen Lyases / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D011485:Protein Binding; D016415:Sequence Alignment; D013379:Substrate Specificity; D014974:Xanthomonas / Q000737:chemistry / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C062025:bis(3',5')-cyclic diguanylic acid; D006861:Hydrogen Peroxide; D019761:Phosphorus-Oxygen Lyases; C060720:diguanylate cyclase; D006152:Cyclic GMP
keywords: GGDEF; Xanthomonas citri; c-di-GMP; cooperativity; enzyme kinetics