Cooperative Substrate Binding by a Diguanylate Cyclase

Autor(es)

Oliveira, Maycon C. ^[1] ; Teixeira, Raphael D. ^[1] ; Andrade, Maxuel O. ^[1] ; Pinheiro, Glaucia M. S. ^[2] ; Ramos, Carlos H. I. ^[2] ; Farah, Chuck S. ^[1]

Afiliação do(s) autor(es)

^[1] Univ Sao Paulo, Inst Chem, Dept Biochem, BR-05508070 Sao Paulo - Brazil

^[2] Univ Estadual Campinas, Inst Chem, BR-13083970 Campinas, SP - Brazil

• Tipo de documento: Artigo Científico
• Fonte: Journal of Molecular Biology; v. 427, n. 2, p. 415-432, JAN 30 2015.
• Citações Web of Science: 10

Resumo

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 Xac Delta 0610 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 (K-1) approximately 5x greater than the dissociation constant for the binding of the second GTP molecule (K-2). 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. (C) 2014 Elsevier Ltd. All rights reserved. (AU)

• Processo FAPESP: 11/07777-5 - Sinalização por c-di-GMP e o sistema de secreção de macromoléculas do tipo IV em Xanthomonas citri
• Beneficiário: Shaker Chuck Farah
• Modalidade de apoio: Auxílio à Pesquisa - Temático