Efficient enzymatic production of the bacterial second messenger c-di-GMP by the diguanylate cyclase YdeH from E. coli.
Cyclic di-GMP (c-di-GMP) is an almost universal bacterial second messenger involved in the regulation of cell surface-associated traits and the persistence of infections. GGDEF and EAL domain-containing proteins catalyse c-di-GMP synthesis and degradation, respectively. We report the enzymatic large-scale synthesis of c-di-GMP, making use of the GGDEF domain-containing protein YdeH from Escherichia coli. Overexpression and purification of YdeH have been established, and the conditions for c-di-GMP synthesis were optimised. 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 1 day and paves the way for extensive biochemical and biophysical studies on c-di-GMP-mediated processes.
Zähringer
Franziska
F
Core Program Structural Biology and Biophysics, Biozentrum, University of Basel, Klingelbergstrasse 70, Basel, Switzerland.
Massa
Claudia
C
Schirmer
Tilman
T
D006152:Cyclic GMP / Q000096:biosynthesis*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D019761:Phosphorus-Oxygen Lyases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D015290:Second Messenger Systems
D029968:Escherichia coli Proteins; D019761:Phosphorus-Oxygen Lyases; C060720:diguanylate cyclase; D006152:Cyclic GMP