Gas-Selective Catalytic Regulation by a Newly Identified Globin-Coupled Sensor Phosphodiesterase Containing an HD-GYP Domain from the Human Pathogen <i>Vibrio fluvialis</i>

Globin-coupled sensors constitute an important family of heme-based gas sensors, an emerging class of heme proteins. In this study, we have identified and characterized a globin-coupled sensor phosphodiesterase containing an HD-GYP domain (GCS-HD-GYP) from the human pathogen Vibrio fluvialis, which is an emerging foodborne pathogen of increasing public health concern. The amino acid sequence encoded by the AL536_01530 gene from V. fluvialis indicated the presence of an N-terminal globin domain and a C-terminal HD-GYP domain, with HD-GYP domains shown previously to display phosphodiesterase activity toward bis(3′,5′)-cyclic dimeric guanosine monophosphate (c-di-GMP), a bacterial second messenger that regulates numerous important physiological functions in bacteria, including in bacterial pathogens. Optical absorption spectral properties of GCS-HD-GYP were found to be similar to those of myoglobin and hemoglobin and of other bacterial globin-coupled sensors. The binding of O2 to the Fe(II) heme iron complex of GCS-HD-GYP promoted the catalysis of the hydrolysis of c-di-GMP to its linearized product, 5′-phosphoguanylyl-(3′,5′)-guanosine (pGpG), whereas CO and NO binding did not enhance the catalysis, indicating a strict discrimination of these gaseous ligands. These results shed new light on the molecular mechanism of gas-selective catalytic regulation by globin-coupled sensors, with these advances apt to lead to a better understanding of the family of globin-coupled sensors, a still growing family of heme-based gas sensors. In addition, given the importance of c-di-GMP in infection and virulence, our results suggested that GCS-HD-GYP could play an important role in the ability of V. fluvialis to sense O2 and NO in the context of host–pathogen interactions

Kinetic Analysis of a Globin-Coupled Histidine Kinase, AfGcHK: Effects of the Heme Iron Complex, Response Regulator, and Metal Cations on Autophosphorylation Activity

International audienceThe globin-coupled histidine kinase, AfGcHK, is a part of the two-component signal transduction system from the soil bacterium Anaeromyxobacter sp. Fw109-5. Activation of its sensor domain significantly increases its autophosphorylation activity, which targets the His183 residue of its functional domain. The phosphate group of phosphorylated AfGcHK is then transferred to the cognate response regulator. We investigated the effects of selected variables on the autophosphorylation reaction’s kinetics. The kcat values of the heme Fe(III)-OH–, Fe(III)-cyanide, Fe(III)-imidazole, and Fe(II)-O2 bound active AfGcHK forms were 1.1–1.2 min–1, and their KmATP values were 18.9–35.4 μM. However, the active form bearing a CO-bound Fe(II) heme had a kcat of 1.0 min–1 but a very high KmATP value of 357 μM, suggesting that its active site structure differs strongly from the other active forms. The Fe(II) heme-bound inactive form had kcat and KmATP values of 0.4 min–1 and 78 μM, respectively, suggesting that its low activity reflects a low affinity for ATP relative to that of the Fe(III) form. The heme-free form exhibited low activity, with kcat and KmATP values of 0.3 min–1 and 33.6 μM, respectively, suggesting that the heme iron complex is essential for high catalytic activity. Overall, our results indicate that the coordination and oxidation state of the sensor domain heme iron profoundly affect the enzyme’s catalytic activity because they modulate its ATP binding affinity and thus change its kcat/KmATP value. The effects of the response regulator and different divalent metal cations on the autophosphorylation reaction are also discusse

Pressure effects reveal that changes in the redox states of the heme iron complexes in the sensor domains of two heme-based oxygen sensor proteins, EcDOS and YddV, have profound effects on their flexibility

The catalytic activity of a heme-based oxygen sensor phosphodiesterase from Escherichia coli (EcDOS) towards cyclic diGMP is regulated by the redox state of the heme iron complex in the enzyme's sensing domain and the association of external ligands with the iron center. Specifically, the Fe (II) complex is more active towards cyclic diGMP than the Fe(III) complex, and its activity is further enhanced by O-2 or CO binding. In order to determine how the redox state and coordination of the heme iron atom regulate the catalytic activity of EcDOS, we investigated the flexibility of its isolated N-terminal heme-binding domain (EcDOS-heme) by monitoring its spectral properties at various hydrostatic pressures. The most active form of the heme-containing domain, i.e. the Fe(II)-CO complex, was found to be the least flexible. Conversely, the oxidized Fe(III) forms of EcDOS-heme and its mutants had relatively high flexibilities, which appeared to be linked to the low catalytic activity of the corresponding intact enzymes. These findings corroborate the suggestion, made on the basis of crystallographic data, that there is an inverse relationship between the flexibility of the heme-containing domain of EcDOS and its catalytic activity. The Fe(II)-CO form of the heme domain of a second heme-based oxygen sensor, diguanylate cyclase (YddV), was also found to be quite rigid. Interestingly, the incorporation of a water molecule into the heme complex of YddV caused by mutation of the Leu65 residue reduced the flexibility of this heme domain. Conversely, mutation of the Tyr43 residue increased its flexibility

Structures of human peptidylarginine deiminase type III provide insights into substrate recognition and inhibitor design

Peptidylarginine deiminase type III (PAD3) is an isozyme belonging to the PAD enzyme family that converts arginine to citrulline residue(s) within proteins. PAD3 is expressed in most differentiated keratinocytes of the epidermis and hair follicles, while S100A3, trichohyalin, and filaggrin are its principal substrates. In this study, the X-ray crystal structures of PAD3 in six states, including its complex with the PAD inhibitor Cl-amidine, were determined. This structural analysis identified a large space around Gly374 in the PAD3-Ca(2+)-Cl-amidine complex, which may be used to develop novel PAD3-selective inhibitors. In addition, similarities between PAD3 and PAD4 were found based on the investigation of PAD4 reactivity with S100A3 in vitro. A comparison of the structures of PAD1, PAD2, PAD3, and PAD4 implied that the flexibility of the structures around the active site may lead to different substrate selectivity among these PAD isozymes

Kinetic Analysis of a Globin-Coupled Histidine Kinase, <i>Af</i>GcHK: Effects of the Heme Iron Complex, Response Regulator, and Metal Cations on Autophosphorylation Activity

The globin-coupled histidine kinase, <i>Af</i>GcHK, is a part of the two-component signal transduction system from the soil bacterium <i>Anaeromyxobacter</i> sp. Fw109-5. Activation of its sensor domain significantly increases its autophosphorylation activity, which targets the His183 residue of its functional domain. The phosphate group of phosphorylated <i>Af</i>GcHK is then transferred to the cognate response regulator. We investigated the effects of selected variables on the autophosphorylation reaction’s kinetics. The <i>k</i>_cat values of the heme Fe­(III)-OH^–, Fe­(III)-cyanide, Fe­(III)-imidazole, and Fe­(II)-O₂ bound active <i>Af</i>GcHK forms were 1.1–1.2 min^–1, and their <i>K</i>_m^ATP values were 18.9–35.4 μM. However, the active form bearing a CO-bound Fe­(II) heme had a <i>k</i>_cat of 1.0 min^–1 but a very high <i>K</i>_m^ATP value of 357 μM, suggesting that its active site structure differs strongly from the other active forms. The Fe­(II) heme-bound inactive form had <i>k</i>_cat and <i>K</i>_m^ATP values of 0.4 min^–1 and 78 μM, respectively, suggesting that its low activity reflects a low affinity for ATP relative to that of the Fe­(III) form. The heme-free form exhibited low activity, with <i>k</i>_cat and <i>K</i>_m^ATP values of 0.3 min^–1 and 33.6 μM, respectively, suggesting that the heme iron complex is essential for high catalytic activity. Overall, our results indicate that the coordination and oxidation state of the sensor domain heme iron profoundly affect the enzyme’s catalytic activity because they modulate its ATP binding affinity and thus change its <i>k</i>_cat/<i>K</i>_m^ATP value. The effects of the response regulator and different divalent metal cations on the autophosphorylation reaction are also discussed