Cooperative Substrate Binding by a Diguanylate Cyclase

• Published in: Journal of molecular biology Vol. 427; no. 2; pp. 415 - 432
• Main Authors: Oliveira, Maycon C., Teixeira, Raphael D., Andrade, Maxuel O., Pinheiro, Glaucia M.S., Ramos, Carlos H.I., Farah, Chuck S.
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 30.01.2015
• Subjects: Amino Acid Sequence; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Base Sequence; c-di-GMP; Circular Dichroism; cooperativity; Cyclic GMP - analogs & derivatives; Cyclic GMP - genetics; Cyclic GMP - metabolism; dissociation; enzyme kinetics; enzymes; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; Gene Expression Regulation, Bacterial; GGDEF; guanosine triphosphate; hydrogen peroxide; Hydrogen Peroxide - metabolism; Hydrogen-Ion Concentration; lysine; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Phosphorus-Oxygen Lyases - genetics; Phosphorus-Oxygen Lyases - metabolism; Plasmids - genetics; Protein Binding; Sequence Alignment; site-directed mutagenesis; Substrate Specificity; Xanthomonas - chemistry; Xanthomonas - genetics; Xanthomonas citri; Xanthomonas citri subsp. citri; PDE; SEC; DGC; c-di-GMP; Xanthomonas citri; CFU; cooperativity; SEC-MALS; GGDEF; enzyme kinetics; CR; SB
• ISSN: 0022-2836; 1089-8638; 1089-8638
• DOI: 10.1016/j.jmb.2014.11.012

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. [Display omitted] •DGCs catalyze the formation of c-di-GMP from two GTP molecules.•XAC0610 is an active DGC that binds its two GTP substrates with different affinities and displays positive cooperativity.•Cooperative substrate binding could have an important role for the in vivo regulation of DGC activity.•We present a scheme appropriate for use when analyzing DGC enzyme kinetics data.