Zähringer, Franziska. Structural and biochemical investigations into c-di-GMP signaling in "E.coli" : Zinc dependent regulation of the diguanylate cyclase YdeH and characterization of PgaA and PgaB, involved in c-di-GMP controlled exopolysaccharide synthesis. 2013, PhD Thesis, University of Basel, Faculty of Science.
Official URL: http://edoc.unibas.ch/diss/DissB_10661
In the structures of YdeH, substrate and product binding to the active site could be shown, however the dimeric arrangement of the two DGC domains, each harboring only one half of the active site, are not in a competent constellation. With the help of the determined structures of YdeH a model of a competent dimer was generated, which provides insights into the regulation of YdeH. Product binding to the inhibitory site of YdeH was shown in the crystal structures and inhibition by c-di-GMP was demonstrated in enzymatic experiments. YdeH represents the first example of a biological zinc-sensor that exerts its downstream effects post-transcriptionally and the first example of a metal sensory c-di-GMP signaling protein.
A protocol for the enzymatic large-scale synthesis of c-di-GMP by using the DGC YdeH from E. coli was developed and optimized. In contrast to the chemical synthesis of c-di- GMP, enzymatic c-di-GMP production is a one-step reaction that can easily be performed with the equipment of a standard biochemical lab. The protocol allows the production of milligram amounts of c-di-GMP within one day and paves the way for extensive biochemical and biophysical studies on c-di-GMP-mediated processes.
In biofilms cells are entrapped within a extracellular polymeric matrix. One component of this matrix is the poly-β-1,6-N-Acetyl-glucosamine (poly-1,6-GlcNAc), which is synthesized and exported by the four proteins of the pgaABCD operon. PgaC and PgaD are responsible for the synthesis of poly-1,6-GlcNAc and are allosterically regulated by c-di-GMP. The deacetylase PgaB and the outer membrane protein PgaA are involved in the modification and export of the poly-1,6-GlcNAc chain.
For PgaA and PgaB an expression and purification protocol was established and resulted in stable and homogenous proteins. The predicted deacetylase activity of PgaB was demonstrated in vitro with an activity assay, which is suitable for rapid screening of different reaction conditions and for the search of inhibitors for PgaB and PgaC, which are of specific pharmaceutical interest.
|Committee Members:||Jenal, Urs|
|Faculties and Departments:||05 Faculty of Science > Departement Biozentrum > Structural Biology & Biophysics > Structural Biology (Schirmer)|
|Bibsysno:||Link to catalogue|
|Number of Pages:||169 S.|
|Last Modified:||30 Jun 2016 10:55|
|Deposited On:||04 Apr 2014 13:24|
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