Structural basis for the dynamics of human methionyl-tRNA synthetase in multi-tRNA synthetase complexes.

In mammals, eight aminoacyl-tRNA synthetases (AARSs) and three AARS-interacting multifunctional proteins (AIMPs) form a multi-tRNA synthetase complex (MSC). MSC components possess extension peptides for MSC assembly and specific functions. Human cytosolic methionyl-tRNA synthetase (MRS) has appended peptides at both termini of the catalytic main body. The N-terminal extension includes a glutathione transferase (GST) domain responsible for interacting with AIMP3, and a long linker peptide between the GST and catalytic domains. Herein, we determined crystal structures of the human MRS catalytic main body, and the complex of the GST domain and AIMP3. The structures reveal human-specific structural details of the MRS, and provide a dynamic model for MRS at the level of domain orientation. A movement of zinc knuckles inserted in the catalytic domain is required for MRS catalytic activity. Depending on the position of the GST domain relative to the catalytic main body, MRS can either block or present its tRNA binding site. Since MRS is part of a huge MSC, we propose a dynamic switching between two possible MRS conformations; a closed conformation in which the catalytic domain is compactly attached to the MSC, and an open conformation with a free catalytic domain dissociated from other MSC components.

Structure and Dynamics of the Human Multi-tRNA Synthetase Complex.

Aminoacyl-tRNA synthetases (ARSs) are essential enzymes that ligate amino acids to their cognate tRNAs during protein synthesis. A growing body of scientific evidence acknowledges that ubiquitously expressed ARSs act as crossover mediators of biological processes, such as immunity and metabolism, beyond translation. In particular, a cytoplasmic multi-tRNA synthetase complex (MSC), which consists of eight ARSs and three ARS-interacting multifunctional proteins in humans, is recognized to be a central player that controls the complexity of biological systems. Although the role of the MSC in biological processes including protein synthesis is still unclear, maintaining the structural integrity of MSC is essential for life. This chapter deals with current knowledge on the structural aspects of the human MSC and its protein components. The main focus is on the regulatory functions of MSC beyond its catalytic activity.

New staining method using methionyl-tRNA synthetase 1 antibody for brushing cytology of bile duct cancer.

BACKGROUND AND AIMS: Identifying malignant biliary strictures using endobiliary brushing cytology specimens is important for treatment decision-making and prognosis prediction. The sensitivity of brushing cytology specimens based on Papanicolaou (Pap) staining is low, which hampers accurate diagnosis of indeterminate strictures. Here, we assessed the diagnostic value of immunohistochemical (IHC) and immunofluorescence (IF) staining for methionyl-tRNA synthetase 1 (MARS1). METHODS: Endobiliary brushing cytology specimens were obtained during ERCP from 80 patients with an extrahepatic biliary stricture. Pap and MARS1 IF staining were performed on liquid-based cytology slides derived from these specimens. Sections of bile duct adenocarcinoma and normal bile duct tissue were obtained from 45 patients who underwent surgery for malignant biliary stricture, and MARS1 levels were evaluated by IHC staining. RESULTS: MARS1 IF staining was applied to brushing cytology specimens, and the results showed strong signals in malignant biliary structures but not in the negative for malignancy specimens. Sensitivity, specificity, positive predictive value, negative predictive value, and accuracy were 70.4%, 96.2%, 97.4%, 56.8%, and 78.8%, respectively, for conventional Pap staining and 98.1%, 96.1%, 98.1%, 96.2%, and 97.5%, respectively, for MARS1 IF (P < .0001). When IHC staining was used, MARS1 was detected in 45 bile duct adenocarcinoma sections but not in 15 normal bile duct sections. Moreover, MARS1 mRNA and protein levels were significantly higher in bile duct adenocarcinoma sections according to polymerase chain reaction and Western blot, respectively. CONCLUSIONS: The high sensitivity and accuracy of MARS1 IF staining enabled detection of malignancy in patients with indeterminate biliary stricture. Further prospective studies are needed to validate our findings. (Clinical trial registration number: KCT 0003285.).

NADP+-dependent cytosolic isocitrate dehydrogenase provides NADPH in the presence of cadmium due to the moderate chelating effect of glutathione.

Cadmium (Cd2+) is toxic to living organisms because it causes the malfunction of essential proteins and induces oxidative stress. NADP+-dependent cytosolic isocitrate dehydrogenase (IDH) provides reducing energy to counteract oxidative stress via oxidative decarboxylation of isocitrate. Intriguingly, the effects of Cd2+ on the activity of IDH are both positive and negative, and to understand the molecular basis, we determined the crystal structure of NADP+-dependent cytosolic IDH in the presence of Cd2+. The structure includes two Cd2+ ions, one coordinated by active site residues and another near a cysteine residue. Cd2+ presumably inactivates IDH due to its high affinity for thiols, leading to a covalent enzyme modification. However, Cd2+ also activates IDH by providing a divalent cation required for catalytic activity. Inactivation of IDH by Cd2+ is less effective when the enzyme is activated with Cd2+ than Mg2+. Although reducing agents cannot restore activity following inactivation by Cd2+, they can maintain IDH activity by chelating Cd2+. Glutathione, a cellular sulphydryl reductant, has a moderate affinity for Cd2+, allowing IDH to be activated with residual Cd2+, unlike dithiothreitol, which has a much higher affinity. In the presence of Cd2+-consuming cellular antioxidants, cells must continually supply reductants to protect against oxidative stress. The ability of IDH to utilise Cd2+ to generate NADPH could allow cells to protect themselves against Cd2+.

Peroxiredoxin I maintains luteal function by regulating unfolded protein response.

BACKGROUND: Mounting evidence shows that ROS regulation by various antioxidants is essential for the expression of enzymes involved in steroidogenesis and maintenance of progesterone production by the corpus luteum (CL). However, the underlying mechanisms of peroxiredoxin 1 (PRDX1), an antioxidant enzyme, in luteal function for progesterone production in mice have not been reported. The aim of this study was to evaluate the functional link between PRDX1 and progesterone production in the CL of Prdx1 knockout (K/O) mice in the functional stage of CL. METHODS: The expression pattern of the unfolded protein response (UPR) signaling pathways, endoplasmic reticulum (ER) stress-induced apoptosis related genes and peroxiredoxins 1 (PRDX1) were investigated by western blotting analysis in CL tissue of 10 weeks mice during functional stage of CL. The protein levels of these genes after ER-stress inducer tunicamycin (Tm), ER-stress inhibitor tauroursodeoxycholic acid (TUDCA) and ROS scavenger, N-acetylcysteine (NAC) stimulation by intraperitoneal (i.p) injection were also investigated in CL tissue of wild type (WT) mice. Finally, we examined progesterone production and UPR signaling related gene expression in CL tissue of Prdx1 K/O mice. RESULTS: We demonstrated that PRDX1 deficiency in the functional stage activates the UPR signaling pathways in response to ER stress-induced apoptosis. Interestingly, CL number, serum progesterone levels, and steroidogenic enzyme expression in Prdx1 K/O mice decreased significantly, compared to those in wild type mice. Levels of UPR signaling pathway markers (GRP78/BIP, P50ATF6, and phosphorylated (p)-eIF2) and ER-stress associated apoptotic factors (CHOP, p-JNK, and cleaved caspase-3) were dramatically increased in the CL tissue of Prdx1 K/O mice. In addition, administration of the NAC, reduced progesterone production and activated ER-stress-induced UPR signaling in the CL tissue obtained from the ovary of Prdx1 K/O mice. Taken together, these results indicated that reduction in serum progesterone levels and activation of ER-stress-induced UPR signaling are restored by NAC injection in the CL of Prdx1 K/O mice. CONCLUSION: These observations provide the first evidence regarding the basic mechanisms connecting PRDX1 and progesterone production in the functional stage of CL.

Loss of Function in Zeaxanthin Epoxidase of Dunaliella tertiolecta Caused by a Single Amino Acid Mutation within the Substrate-Binding Site.

The zea1 mutant of marine microalga Dunaliella tertiolecta accumulates zeaxanthin under normal growth conditions, and its phenotype has been speculated to be related to zeaxanthin epoxidase (ZEP). In this study, we isolated the ZEP gene from both wild-type D. tertiolecta and the mutant. We found that the zea1 mutant has a point mutation of the 1337th nucleotide of the ZEP sequence (a change from guanine to adenine), resulting in a change of glycine to aspartate in a highly conserved region in the catalytic domain. Similar expression levels of ZEP mRNA and protein in both wild-type and zea1 were confirmed by using qRT-PCR and western blot analysis, respectively. Additionally, the enzyme activity analysis of ZEPs in the presence of cofactors showed that the inactivation of ZEP in zea1 was not caused by deficiency in the levels of cofactors. From the predicted three-dimensional ZEP structure of zea1, we observed a conformational change on the substrate-binding site in the ZEP. A comparative analysis of the ZEP structures suggested that the conformational change induced by a single amino acid mutation might impact the interaction between the substrate and substrate-binding site, resulting in loss of zeaxanthin epoxidase function.

Silibinin Ameliorates O-GlcNAcylation and Inflammation in a Mouse Model of Nonalcoholic Steatohepatitis.

The mechanisms underlying the progression to non-alcoholic steatohepatitis (NASH) remain to be elucidated. In the present study, we aimed to identify the proteins involved in the pathogenesis of liver tissue inflammation and to investigate the effects of silibinin, a natural polyphenolic flavonoid, on steatohepatitis. We performed comparative proteomic analysis using methionine and choline-deficient (MCD) diet-induced NASH model mice. Eighteen proteins were identified from the two-dimensional proteomic analysis, which are not only differentially expressed, but also significantly improved, by silibinin treatment. Interestingly, seven of these proteins, including keratin cytoskeletal 8 and 18, peroxiredoxin-4, and protein disulfide isomerase, are known to undergo GlcNAcylation modification, most of which are related to structural and stress-related proteins in NASH model animals. Thus, we primarily focused on how the GlcNAc modification of these proteins is involved in the progression to NASH. Remarkably, silibinin treatment alleviates the severity of hepatic inflammation along with O-GlcNAcylation in steatohepatitis. In particular, the reduction of inflammation by silibinin is due to the inhibition of the O-GlcNAcylation-dependent NF-κB-signaling pathway. Therefore, silibinin is a promising therapeutic agent for hyper-O-GlcNAcylation as well as NASH.

An Algorithm for Computing Side Chain Conformational Variations of a Protein Tunnel/Channel.

In this paper, a novel method to compute side chain conformational variations for a protein molecule tunnel (or channel) is proposed. From the conformational variations, we compute the flexibly deformed shapes of the initial tunnel, and present a way to compute the maximum size of the ligand that can pass through the deformed tunnel. By using the two types of graphs corresponding to amino acids and their side chain rotamers, the suggested algorithm classifies amino acids and rotamers which possibly have collisions. Based on the divide and conquer technique, local side chain conformations are computed first, and then a global conformation is generated by combining them. With the exception of certain cases, experimental results show that the algorithm finds up to 327,680 valid side chain conformations from 128~1233 conformation candidates within three seconds.

New Potential Biomarker Proteins for Alcoholic Liver Disease Identified by a Comparative Proteomics Approach.

Chronic alcohol consumption causes hepatic steatosis, which is characterized by a considerable increase in free fatty acid (FFA) and triglyceride levels. To identify the possible proteins involved in the progression to alcoholic hepatosteatosis, we performed proteomic analysis on livers of mice exposed to alcohol. 2D-based proteomic analysis revealed that EtOH exposure in mice changed the expression of 43 proteins compared with that in mice fed a normal diet (ND). The most notable protein changes were proteins involved in Met metabolism and oxidative stress, most of which were significantly downregulated in alcohol-exposed animals. Although non-alcoholic fatty liver disease (NAFLD) and alcoholic liver disease (ALD) seem to share the same molecular processes, the difference between these conditions is still unclear. To address this question, we explored the features of alcoholic hepatosteatosis that were different compared with those of methionine and choline deficient (MCD) diet-induced mice with nonalcoholic liver damage. Although most of the differentially expressed proteins associated with ALD did not significantly differ from those of NAFLD, nine proteins showed considerably different patterns. Of these, ornithine aminotransferase, vitamin D binding protein, and phosphatidylethanolamine-binding protein were considerably upregulated in ALD mice, compared to that in NAFLD and ND mice. However, other proteins including inorganic pyrophosphatase were differentially regulated in MCD mice; however, they did not differ significantly between the alcoholic model and ND control mice. These results suggested that the identified proteins might be useful candidate markers to differentiate ALD from NAFLD. J. Cell. Biochem. 118: 1189-1200, 2017. © 2016 Wiley Periodicals, Inc.

Binding Direction-Based Two-Dimensional Flattened Contact Area Computing Algorithm for Protein-Protein Interactions.

Interactions between protein molecules are essential for the assembly, function, and regulation of proteins. The contact region between two protein molecules in a protein complex is usually complementary in shape for both molecules and the area of the contact region can be used to estimate the binding strength between two molecules. Although the area is a value calculated from the three-dimensional surface, it cannot represent the three-dimensional shape of the surface. Therefore, we propose an original concept of two-dimensional contact area which provides further information such as the ruggedness of the contact region. We present a novel algorithm for calculating the binding direction between two molecules in a protein complex, and then suggest a method to compute the two-dimensional flattened area of the contact region between two molecules based on the binding direction.

Assembly of Multi-tRNA Synthetase Complex via Heterotetrameric Glutathione Transferase-homology Domains.

Many multicomponent protein complexes mediating diverse cellular processes are assembled through scaffolds with specialized protein interaction modules. The multi-tRNA synthetase complex (MSC), consisting of nine different aminoacyl-tRNA synthetases and three non-enzymatic factors (AIMP1-3), serves as a hub for many signaling pathways in addition to its role in protein synthesis. However, the assembly process and structural arrangement of the MSC components are not well understood. Here we show the heterotetrameric complex structure of the glutathione transferase (GST) domains shared among the four MSC components, methionyl-tRNA synthetase (MRS), glutaminyl-prolyl-tRNA synthetase (EPRS), AIMP2 and AIMP3. The MRS-AIMP3 and EPRS-AIMP2 using interface 1 are bridged via interface 2 of AIMP3 and EPRS to generate a unique linear complex of MRS-AIMP3:EPRS-AIMP2 at the molar ratio of (1:1):(1:1). Interestingly, the affinity at interface 2 of AIMP3:EPRS can be varied depending on the occupancy of interface 1, suggesting the dynamic nature of the linear GST tetramer. The four components are optimally arranged for maximal accommodation of additional domains and proteins. These characteristics suggest the GST tetramer as a unique and dynamic structural platform from which the MSC components are assembled. Considering prevalence of the GST-like domains, this tetramer can also provide a tool for the communication of the MSC with other GST-containing cellular factors.

Promiscuous methionyl-tRNA synthetase mediates adaptive mistranslation to protect cells against oxidative stress.

Aminoacyl-tRNA synthetases (ARSs) acylate transfer (t)RNAs with amino acids. Charging tRNAs with the right amino acids is the first step in translation; therefore, the accurate and error-free functioning of ARSs is an essential prerequisite for translational fidelity. A recent study found that methionine (Met) can be incorporated into non-Met residues of proteins through methionylation of non-cognate tRNAs under conditions of oxidative stress. However, it was not understood how this mis-methionylation is achieved. Here, we report that methionyl-tRNA synthetase (MRS) is phosphorylated at Ser209 and Ser825 by extracellular signal-related kinase (ERK1/2) under conditions of stress caused by reactive oxygen species (ROS), and that this phosphorylated MRS shows increased affinity for non-cognate tRNAs with lower affinity for tRNA(Met), leading to an increase in Met residues in cellular proteins. The expression of a mutant MRS containing the substitutions S209D and S825D, mimicking dual phosphorylation, reduced ROS levels and cell death. This controlled inaccuracy of MRS seems to serve as a defense mechanism against ROS-mediated damage at the cost of translational fidelity.

The splicing factor SRSF1 modulates pattern formation by inhibiting transcription of tissue specific genes during embryogenesis.

Alternative splicing is a major mechanism regulating pattern of gene expression through the production of multiple mRNAs from a single gene transcript. Any misregulation can cause various human diseases and also have severe effects on embryogenesis. SRSF1 is one of the critical factors regulating alternative splicing at many stages of vertebrate development and any disturbance in SRSF1 leads to serious consequences. In current study, we investigated the effects of loss of the SRSF1 gene using antisense morpholino oligonucleotides (MO) in Xenopus embryogenesis. It is evident from the results of RT-PCR and whole-mount in situ hybridization that SRSF1 is a maternal gene having strong expression in head, eyes and central nervous system. Moreover, SRSF1 morphants exhibited malformed phenotypes, including miscoiled guts, heart and cartilage formation, edema in the head and heart, and small eyes. Especially, in SRSF1 morphants, bone cartilage formation was reduced in the brain and Nkx-2.5 expression was dramatically reduced in the heart of SRSF1 morphants. In addition, a dramatic reduction in functional chordin RNA in SRSF1 morphants was observed suggesting that chordin is one of the targets of SRSF1. Thus, we concluded that SRSF1 is an essential factor for pattern formation including heart, cartilage and germ layers through the regulation of specific genes.

IFT46 plays crucial roles in craniofacial and cilia development.

The intraflagellar transport (IFT) system is essential for bidirectional movement of ciliary components from the basal body to the tip beneath the ciliary sheath and is conserved for cilia and flagella formation in most vertebrates. IFT complex A is involved in anterograde trafficking, whereas complex B is involved in retrograde trafficking. IFT46 is well known as a crucial component of IFT complex B, however, its developmental functions are poorly understood. In this study, we investigated the novel functions of IFT46 during vertebrate development, especially, ciliogenesis and neurogenesis, because IFT46 is strongly expressed in both multiciliated cells of epithelial and neural tissues. Knockdown of IFT46 using morpholino microinjections caused shortening of the body axis as well as the formation of fewer and shorter cilia. Furthermore, loss of IFT46 down-regulated the expression of the neural plate and neural tube markers, thus may influence Wnt/planar cell polarity and the sonic hedgehog signaling pathway during neurogenesis. In addition, loss of IFT46 caused craniofacial defects by interfering with cartilage formation. In conclusion, our results depict that IFT46 plays important roles in cilia as well as in neural and craniofacial development.

Enhanced catalytic site thermal stability of cold-adapted esterase EstK by a W208Y mutation.

Hydrophobic interactions are known to play an important role for cold-adaptation of proteins; however, the role of amino acid residue, Trp, has not been systematically investigated. The extracellular esterase, EstK, which was isolated from the cold-adapted bacterium Pseudomonas mandelii, has 5 Trp residues. In this study, the effects of Trp mutation on thermal stability, catalytic activity, and conformational change of EstK were investigated. Among the 5 Trp residues, W(208) was the most crucial in maintaining structural conformation and thermal stability of the enzyme. Surprisingly, mutation of W(208) to Tyr (W(208)Y) showed an increased catalytic site thermal stability at ambient temperatures with a 13-fold increase in the activity at 40°C compared to wild-type EstK. The structure model of W(208)Y suggested that Y(208) could form a hydrogen bond with D(308), which is located next to catalytic residue H(307), stabilizing the catalytic domain. Interestingly, Tyr was conserved in the corresponding position of hyper-thermophilic esterases EstE1 and AFEST, which are active at high temperatures. Our study provides a novel insight into the engineering of the catalytic site of cold-adapted enzymes with increased thermal stability and catalytic activity at ambient temperatures.

Construction of target-specific virus-like particles for the delivery of algicidal compounds to harmful algae.

Harmful algal blooms (HABs) can lead to substantial socio-economic losses and extensive damage to aquatic ecosystems, drinking water sources and human health. Common algicidal techniques, including ozonation, ultrasonic treatment and dispersion of algae-killing chemicals, are unsatisfactory both economically and ecologically. This study therefore presents a novel alternative strategy for the efficient control of deleterious algae via the use of host-specific virus-like particles (VLPs) combined with chemically synthesized algicidal compounds. The capsid protein of HcRNAV34, a single-stranded RNA virus that infects the toxic dinoflagellate, Heterocapsa circularisquama, was expressed in and purified from Escherichia coli and then self-assembled into VLPs in vitro. Next, the algicidal compound, thiazolidinedione 49 (TD49), was encapsidated into HcRNAV34 VLPs for specific delivery to H. circularisquama. Consequently, HcRNAV34 VLPs demonstrated the same host selectivity as naturally occurring HcRNAV34 virions, while TD49-encapsidated VLPs showed a more potent target-specific algicidal effect than TD49 alone. These results indicate that target-specific VLPs for the delivery of cytotoxic compounds to nuisance algae might provide a safe, environmentally friendly approach for the management of HABs in aquatic ecosystems.

RGS19 converts iron deprivation stress into a growth-inhibitory signal.

Iron chelation is a promising therapeutic strategy for cancer that works, in part, by inducing overexpression of N-myc downstream-regulated gene 1 protein (NDRG1), a known growth inhibitor and metastasis suppressor. However, details of the signaling cascades that convert physical stress into a biological response remain elusive. We investigated the role of RGS19, a regulator of G-protein signaling, in iron chelator-induced NDRG1 overexpression in HeLa cells. Knockdown of RGS19 diminished the expression of genes involved in desferrioxamine (DFO)-induced growth inhibition. Conversely, overexpression of RGS19 enhanced the expression of these genes. Moreover, overexpression of RGS19 reduced cell viability. Overexpression of G-protein alpha subunit i3 (Gαi3) repressed the induction of NDRG1 expression. Selective inhibition of downstream targets of Gαi3 abrogated DFO-induced overexpression of NDRG1. DFO protected RGS19 from proteolysis induced by GAIP interacting protein N terminus (GIPN); moreover, an iron-deficient RGS19 mutant was stable in the presence of GIPN and retained GTPase-activating protein activity. RGS19 was co-purified with iron and showed unique UV-absorption characteristics frequently observed in iron-binding proteins. This study demonstrates that RGS19 senses cellular iron availability and is stabilized under iron-depleted conditions, resulting in the induction of a growth-inhibitory signal.

Functional Identification of OphR, an IclR Family Transcriptional Regulator Involved in the Regulation of the Phthalate Catabolic Operon in Rhodococcus sp. Strain DK17.

A putative gene for a transcriptional regulator (ophR) was detected near each copy of the duplicated phthalate-degrading operon of Rhodococcus sp. DK17. Sequence analysis and molecular modeling indicate that OphR belongs to the IclR family of transcriptional regulators and possesses the N-terminal DNA-binding and C-terminal effector-binding domains. DNA-binding assays demonstrate that OphR regulates the phthalate operon by binding to the ophA1-ophR intergenic region.

Activation of ATP binding for the autophosphorylation of DosS, a Mycobacterium tuberculosis histidine kinase lacking an ATP lid motif.

The sensor histidine kinases of Mycobacterium tuberculosis, DosS and DosT, are responsible for sensing hypoxic conditions and consist of sensor and kinase cores responsible for accepting signals and phosphorylation activity, respectively. The kinase core contains a dimerization and histidine phosphate-accepting (DHp) domain and an ATP binding domain (ABD). The 13 histidine kinase genes of M. tuberculosis can be grouped based on the presence or absence of the ATP lid motif and F box (elements known to play roles in ATP binding) in their ABDs; DosS and DosT have ABDs lacking both these elements, and the crystal structures of their ABDs indicated that they were unsuitable for ATP binding, as a short loop covers the putative ATP binding site. Although the ABD alone cannot bind ATP, the kinase core is functional in autophosphorylation. Appropriate spatial arrangement of the ABD and DHp domain within the kinase core is required for both autophosphorylation and ATP binding. An ionic interaction between Arg(440) in the DHp domain and Glu(537) in the short loop of the ABD is available and may open the ATP binding site, by repositioning the short loop away from the site. Mutations at Arg(440) and Glu(537) reduce autophosphorylation activity. Unlike other histidine kinases containing an ATP lid, which protects bound ATP, DosS is unable to accept ATP until the ABD is properly positioned relative to the histidine; this may prevent unexpected ATP reactions. ATP binding can, therefore, function as a control mechanism for histidine kinase activity.

Serine 83 in DosR, a response regulator from Mycobacterium tuberculosis, promotes its transition from an activated, phosphorylated state to an inactive, unphosphorylated state.

A sensor kinase, DosS, and its corresponding response regulator, DosR, constitute a two component system for regulating gene expression under hypoxic conditions in Mycobacterium tuberculosis. Among response regulators in M. tuberculosis, NarL has high sequence similarity to DosR, and autophosphorylated DosS transfers its phosphate group not only to DosR but also to NarL. Phosphorylated DosR is more rapidly dephosphorylated than phosphorylated NarL. DosR and NarL differ with respect to the amino acids at positions T+1 and T+2 around the phosphorylation sites in the N-terminal phosphoacceptor domain; NarL has S83 and Y84, whereas DosR has A90 and H91. A DosR S83A mutant shows prolonged phosphorylation. Structural comparison with a histidinol phosphate phosphatase suggests that the hydroxyl group of DosR S83 could play a role in activating the water molecule involved in the triggering of autodephosphorylation.