Abstract
Globin-coupled diguanylate cyclases contain globin, middle, and diguanylate cyclase domains that sense O2 to synthesize c-di-GMP and regulate bacterial motility, biofilm formation, and virulence. However, relatively few studies have extensively examined the roles of individual residues and domains of globin-coupled diguanylate cyclases, which can shed light on their signaling mechanisms and provide drug targets. Here, we report the critical residues of two globin-coupled diguanylate cyclases, EcGReg from Escherichia coli and BpeGReg from Bordetella pertussis, and show that their diguanylate cyclase activity requires an intact globin domain. In the distal heme pocket of the globin domain, residues Phe42, Tyr43, Ala68 (EcGReg)/Ser68 (BpeGReg), and Met69 are required to maintain full diguanylate cyclase activity. The highly conserved amino acids His223/His225 and Lys224/Lys226 in the middle domain of EcGReg/BpeGReg are essential to diguanylate cyclase activity. We also identified sixteen important residues (Leu300, Arg306, Asp333, Phe337, Lys338, Asn341, Asp342, Asp350, Leu353, Asp368, Arg372, Gly374, Gly375, Asp376, Glu377, and Phe378) in the active site and inhibitory site of the diguanylate cyclase domain of EcGReg. Moreover, BpeGReg266 (residues 1–266) and BpeGReg296 (residues 1–296), which only contain the globin and middle domains, can inhibit bacterial motility. Our findings suggest that the distal residues of the globin domain affect diguanylate cyclase activity and that BpeGReg may interact with other c-di-GMP-metabolizing proteins to form mixed signaling teams.
Highlights
Globin-coupled diguanylate cyclases (GCDCs) form a subfamily of globin-coupled sensors (GCS) that are heme-binding sensors linked to variable signaling domains [1,2,3,4,5]
C-di-GMP is synthesized by diguanylate cyclase (DGC) containing a GGDEF domain and degraded by phosphodiesterases (PDEs) with either an EAL or HD-GYP domain [11]
To address related questions on how globins regulate DGC activity, here we report the critical residues identified in the three domains of GCDCs and that GCDCs require an intact globin domain for their enzyme activities
Summary
Globin-coupled diguanylate cyclases (GCDCs) form a subfamily of globin-coupled sensors (GCS) that are heme-binding sensors linked to variable signaling domains [1,2,3,4,5]. Critical residues of globin-coupled diguanylate cyclases terminal diguanylate cyclase (DGC) domains of GCDCs, the highly conserved GGD/EEF (Gly-Gly-Asp/Glu-Glu-Phe) motif serves as the active site to synthesize the second messenger bis-(3’-5’)-cyclic diguanosine monophosphate (c-di-GMP) [6, 8]. C-di-GMP is synthesized by DGCs (including GCDCs) containing a GGDEF domain and degraded by phosphodiesterases (PDEs) with either an EAL or HD-GYP domain [11]. Multiple DGCs and PDEs are found in most bacteria and are often associated with sensory or regulatory domains that allow them to modulate their activities in response to internal and environmental stimuli [11]. Studies on how DGCs and PDEs sense environmental signals to regulate c-di-GMP levels will shed light on the mechanisms of bacterial behavior and provide potential drug targets to attenuate the virulence of pathogens
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