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Some GPCRs transmit signals through adenylyl cyclase (AC), a transmembrane enzyme. AC helps synthesize second messenger cyclic adenosine monophosphate (cAMP). AC catalyzes cyclization reaction and converts ATP to cAMP by releasing a pyrophosphate. The pyrophosphate is further hydrolyzed to phosphate by the enzyme pyrophosphatase, which drives cAMP synthesis to completion. However, cAMP is rapidly degraded to 5′ AMP by the enzymes phosphodiesterase (PDE), preventing overstimulation of...
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Guanine nucleotide-binding proteins (G-proteins), also known as GTPases, are a superfamily of proteins that regulate many cellular processes, such as cell signaling, vesicular transport, and the regulation of cell shape and motility. Mutation or dysfunction of these proteins can lead to disease. There are around 40,000 known G-proteins that can broadly be classified into two groups ‒  small G-proteins consisting of a single domain and large multi-domain G-proteins.
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Heterotrimeric G proteins are guanine nucleotide-binding proteins. As the name suggests, heterotrimeric G proteins are composed of three subunits: alpha, beta, and gamma. They remain GDP-bound or GTP-bound inside the cells and switch between inactive/active states. The Gα subunit possesses the nucleotide-binding pocket that binds guanine nucleotides and switches between GDP or GTP-bound states. In contrast, the Gꞵ and Gγ subunits are always bound together with high...
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Vesicles incorporate different coat protein subunits in different cell locations, which changes the properties of the coat, such as the shape and geometry of the transport vesicles. Thus, vesicle coat proteins also play a significant role in cargo selection.
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Comparing the Affinity of GTPase-binding Proteins using Competition Assays
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Cooperative substrate binding by a diguanylate cyclase.

Maycon C Oliveira1, Raphael D Teixeira1, Maxuel O Andrade1

  • 1Department of Biochemistry, Institute of Chemistry, University of São Paulo, São Paulo, SP 05508-070, Brazil.

Journal of Molecular Biology
|December 3, 2014
PubMed
Summary
This summary is machine-generated.

XAC0610, a Xanthomonas protein, exhibits diguanylate cyclase (DGC) activity crucial for bacterial signaling. Its GTP substrate binding shows positive cooperativity, suggesting a novel regulatory mechanism for cyclic di-GMP production.

Keywords:
GGDEFXanthomonas citric-di-GMPcooperativityenzyme kinetics

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Area of Science:

  • Bacterial molecular biology
  • Enzymology
  • Signal transduction

Background:

  • XAC0610 is a multi-domain protein from Xanthomonas citri subsp. citri.
  • It possesses diguanylate cyclase (DGC) activity, producing the signaling molecule cyclic di-GMP (c-di-GMP).
  • DGCs regulate various bacterial processes, but their regulatory mechanisms are not fully understood.

Purpose of the Study:

  • To characterize the DGC activity of XAC0610.
  • To investigate the regulatory mechanisms governing XAC0610's DGC activity.
  • To explore the role of XAC0610 in Xanthomonas citri subsp. citri.

Main Methods:

  • Site-directed mutagenesis of conserved residues.
  • In vitro and in vivo assays for DGC activity.
  • Kinetic analysis of substrate binding and product inhibition.
  • Bioinformatic analysis.

Main Results:

  • XAC0610 knockout affects bacterial motility and H2O2 resistance.
  • Mutagenesis of Lys759 significantly reduced DGC activity.
  • XAC0610 is not subject to allosteric product inhibition.
  • GTP exhibits positive cooperative binding kinetics (K1 > K2).

Conclusions:

  • XAC0610 plays a role in regulating bacterial motility and stress resistance.
  • Cooperative GTP binding is a potential regulatory mechanism for DGCs.
  • A new kinetic model for DGCs is proposed.
  • This finding may offer insights into bacterial signaling regulation.