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A spectrophotometric method to measure hydrolysis of the bacterial second messenger cyclic dimeric guanosine monophosphate is described for characterization of enzymes under aerobic and anaerobic conditions. The method allows for obtaining all necessary data to calculate KM and kcat from reactions within a single 96-well plate that be can measured using a standard plate reader. The spectrophotometric assay has been used to measure the rates and obtain Michaelis-Menten for the c-di-GMP phosphodiesterase DcpG with the sensor domain in various ligation states. 

Cyclic dimeric guanosine monophosphate (c-di-GMP) serves as a second messenger that modulates bacterial cellular processes, including biofilm formation. While proteins containing both c-di-GMP synthesizing (GGDEF) and c-di-GMP hydrolyzing (EAL) domains are widely predicted in bacterial genomes, it is poorly understood how domains with opposing enzymatic activity are regulated within a single polypeptide. Herein, we report the characterization of a globin-coupled sensor protein (GCS) fromPaenibacillus dendritiformis(DcpG) with bifunctional c-di-GMP enzymatic activity. DcpG contains a regulatory sensor globin domain linked to diguanylate cyclase (GGDEF) and phosphodiesterase (EAL) domains that are differentially regulated by gas binding to the heme; GGDEF domain activity is activated by the Fe(II)-NO state of the globin domain, while EAL domain activity is activated by the Fe(II)-O₂state. The in vitro activity of DcpG is mimicked in vivo by the biofilm formation ofP. dendritiformisin response to gaseous environment, with nitric oxide conditions leading to the greatest amount of biofilm formation. The ability of DcpG to differentially control GGDEF and EAL domain activity in response to ligand binding is likely due to the unusual properties of the globin domain, including rapid ligand dissociation rates and high midpoint potentials. Using structural information from small-angle X-ray scattering and negative stain electron microscopy studies, we developed a structural model of DcpG, providing information about the regulatory mechanism. These studies provide information about full-length GCS protein architecture and insight into the mechanism by which a single regulatory domain can selectively control output domains with opposing enzymatic activities.