Differential ligand-selective control of opposing enzymatic activities within a bifunctional c-di-GMP enzyme

Abstract

Significance Bifunctional enzymes are widely distributed throughout bacteria and are involved in modulating bacterial phenotypes; however, regulatory mechanisms that control the activities of the opposing output domains have remained elusive. Studies on DcpG demonstrate that binding of ligands to the sensor globin domain differentially affect GGDEF and EAL domain activities and highlight a role for protein conformational changes in modulating enzymatic activity. Unusual sensor globin domain characteristics, including heme midpoint potentials, are likely important for the unique regulatory properties of DcpG. As Paenibacillus dendritiformis responds to changes in the gaseous environment by modulating biofilm formation, DcpG is likely important in modulating physiological responses to changes in O2 and NO levels, identifying a role for heme sensor signaling in the bacterium. 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) from Paenibacillus 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)-O2 state. The in vitro activity of DcpG is mimicked in vivo by the biofilm formation of P. dendritiformis in 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.