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Structural engineering of the active site of diguanylate cyclase (GGDEF) domains to produce new second messengers

Grant number: 15/13318-4
Support type:Scholarships in Brazil - Doctorate (Direct)
Effective date (Start): October 01, 2015
Effective date (End): November 30, 2020
Field of knowledge:Biological Sciences - Biochemistry - Chemistry of Macromolecules
Principal researcher:Shaker Chuck Farah
Grantee:Santiago Justo Arevalo
Home Institution: Instituto de Química (IQ). Universidade de São Paulo (USP). São Paulo , SP, Brazil
Associated research grant:11/07777-5 - Cyclic di-GMP signaling and the Type IV macromolecule secretion system in Xanthomonas citri, AP.TEM

Abstract

The cyclic nucleotides are important second messengers involved in several pathways including bacterial virulence, biofilm formation and immune evasion. They can be divided into two main groups: the cyclic nucleotides (cAMP and cGMP) and the cyclic dinucleotides: c-di-GMP, c-di-AMP and cGAMP. The control of cellular levels is mediated through protein domains that synthesize and others that degrade these molecules and their downstream effects are mediated by several receptors in the cell. GGDEF domains are responsible for the condensation of two GTP molecules to produce c-di-GMP. This domain is ubiquitous in the prokaryote kingdom and, although it has only one described enzymatic function, the diversity of its sequences raise the possibility that other enzymatic functions may yet be discovered. The high resolution structure of the canonical GGDEF protein PleD from Caulobacter crescentus and the available database of protein sequences with GGDEF domains, allows us to test the hypothesis that certain structural changes introduced by changing specific aminoacids would produce GGDEF domains that recognize other nucleotide substrates or can retain nucleotide binding with loss of enzymatic activity. The main goal of this work is to test these hypotheses, first by introducing specific point mutations in PleD, thereby creating enzymes that synthesize new dinucleotide molecules, and then searching if these or similar enzyme variants occur naturally in the large database of over 42 thousand GGDEF domains annotated so far. Finally we will use the phytopathogen Xanthomonas citri to test the activities of these new molecules in vivo. (AU)

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