The Critical Role of the Conserved Thr 247 Residue in the Functioning of the Osmosensor EnvZ, a Histidine Kinase/Phosphatase, in Escherichia coli *

The histidine kinase/phosphatase EnvZ helps Escherichia coli adapt to osmotic shock by controlling the phosphorylation state of the transcription factor OmpR, which regulates the levels of the outer membrane porin proteins OmpF and OmpC. We examined the effects of mutating the highly conserved Thr 247 residue in EnvZ. Using purified C-terminal domains of wild-type and mutant EnvZ proteins, we demonstrate that Thr 247 plays a vital role in EnvZ function, variously affecting its autokinase and phosphotransferase activities, but mostly its function as a phosphatase. The cytoplasmic domain of EnvZ (EnvZc) is composed of three segments: the linker domain (residues 180–222), domain A (residues 223–289), and domain B (residues 290–450). It has been shown that the isolated domain A itself can dephosphorylate phosphorylated OmpR. Here we show that mutating Thr 247 to Arg in domain A abolishes its phosphatase activity. Furthermore, using an in vivo b -galactosidase activity assay of Taz1-1 (hybrid of the aspartate receptor Tar and EnvZ) constructs of the Thr 247 mutants in RU1012 cells expressing ompC-lacZ , we demonstrate that the external signal primarily down-regulates the phosphatase activity of EnvZ. Of the nine EnvZc(T247 X ) mutants ( X 5 Ser, Ala, Cys, Lys, Asn,

A sensor transducer histidine kinase and a response regulator protein constitute the two basic components of the His-Asp phosphorelay system, a ubiquitous signal transduction system in bacteria (1)(2)(3)(4)(5). Typically, the dimeric histidine kinase undergoes ATP-dependent trans-autophosphorylation on a specific histidine residue and subsequently transfers the phosphoryl group to an aspartate residue on its cognate response regulator. Phosphorylation of the response regulator alters its transcriptional, enzymatic, or mechanistic properties, bringing about a change in cellular behavior. Currently, Ͼ300 such signaling systems are known in prokaryotes (6). His-Asp phosphorelay systems also find highly specialized applications in lower eukaryotes (7).
One of the most extensively studied His-Asp phosphorelay signal transduction systems is the EnvZ/OmpR-mediated osmosensory pathway in Escherichia coli (8 -10). EnvZ, located in the inner cytoplasmic membrane, undergoes ATP-dependent trans-autophosphorylation (11,12) on the conserved His 243 residue (13). It subsequently transfers this phosphoryl group to the conserved Asp 55 residue on OmpR (14 -17). Phosphorylated OmpR (OmpR-P) 1 functions as a transcription factor for ompF and ompC genes, encoding the outer membrane porin proteins OmpF and OmpC, respectively (18,19). The level of OmpR-P in the cell reciprocally regulates the production of these porins, with OmpF and OmpC being predominantly produced under low and high osmolar conditions, respectively (18, 20 -22).
Representing a large class of bifunctional histidine kinase/ phosphatases, EnvZ exhibits dual opposing functions, both phosphorylation and dephosphorylation of OmpR (1). It has been proposed that the osmotic signal regulates the OmpR-P level in the cell by modulating the ratio of the kinase to phosphatase activity of EnvZ (11,12,23), altering primarily the phosphatase activity (24).
Structurally, EnvZ consists of an N-terminal short cytosolic segment, the first membrane-spanning segment (TM1), a periplasmic domain, the second transmembrane segment (TM2), and a C-terminal cytosolic domain. The cytosolic domain has the highly conserved regions for histidine kinases: the H, N, F, G 1 , and G 2 boxes (3) and the recently recognized G 3 box (25). Domain dissection studies revealed that the enzymatically active kinase/phosphatase segment (residues 223-450) of EnvZ consists of two complementary functional domains (26): the 67-residue dimerization and histidine phosphotransfer domain A (the DHp domain, residues 223-289) and the 161residue catalytic and ATP-binding domain B (the CA domain, residues 289 -450) (27). The linker domain (residues 180 -222) connects the kinase/phosphatase segment to TM2 and is responsible for transducing the signal (see Fig. 1A) (24,28). Recently, the NMR solution structures of domain B (29) and domain A (30) of EnvZ have been solved.
The NMR solution structure of domain A reveals that the dimerization core of EnvZ is formed by a parallel association of helical hairpins (helixes I and II), resulting in a four-helix bundle (30). The invariant His 243 residue is located in the middle of helix I of each subunit, completely exposed to the solvent. His 243 is involved in both the kinase and phosphatase activities of EnvZ (31)(32)(33). No other amino acid residues have been implicated in the enzymatic functions of EnvZ or any other histidine kinase/phosphatase.
In this study, we report the identification of Thr 247 in the H box on helix I of domain A as a critical active-site residue in EnvZ. Using EnvZc(T247X) mutant proteins, generated by sitedirected mutagenesis, we provide clear evidence that Thr 247 plays an important role, affecting all EnvZ catalytic activities, most importantly, the phosphatase function.
Purification of EnvZc and EnvZc(T247X) Mutant Proteins-E. coli BL21(DE3) was used for the expression and purification of the cytoplasmic domain of EnvZ (EnvZc, residues 180 -450) and the EnvZc(T247X) mutant proteins. Cells harboring different plasmids were cultured to mid-log phase when 1 mM isopropyl-␤-D-thiogalactopyranoside was added. After another 3-h incubation, the cells were harvested and broken with a French press. The EnvZc and EnvZc(T247X) mutant proteins were purified by 40% ammonium sulfate fractionation and subsequent Sephacryl S-100HR gel-filtration column chromatography (15). Proteins were quantified using the Bio-Rad protein assay reagent.
Phosphotransfer Assays-Each protein (50 g) was first autophos-phorylated using autokinase reaction buffer in a 200-l final reaction volume for 15 min at room temperature. The reaction contents were exchanged into 50 mM Tris-HCl (pH 8.0) containing 50 mM KCl, 1 mM EDTA, 5 mM ␤-mercaptoethanol, and 15% glycerol, and free ATP was removed using Ultrafree centrifugal filter tubes (UFV5BGC00, Millipore Corp.). The reaction mixture was concentrated to 50 l. Phosphorylated EnvZc protein (wild-type/mutant; 2 M) thus prepared was incubated with 4 M OmpR in phosphatase buffer (50 mM Tris-HCl (pH 8.0) containing 50 mM KCl, 10 mM MgCl 2 , 5 mM ␤-mercaptoethanol, and 5% glycerol) at room temperature. Aliquots were removed at 0, 20, 40, 120, and 300 s. Another set of experiments was conducted on ice, and aliquots were removed at 15 and 30 s. Reactions were stopped with 5ϫ SDS loading buffer. The products were separated by 17.5% SDS-PAGE. The gel was dried and exposed for autoradiography.
Phosphatase Assays-Phosphorylated OmpR was prepared as previously reported (26). To remove free ATP from OmpR-P, it was washed several times with 50 mM Tris-HCl (pH 8.0) containing 50 mM KCl, 1 mM EDTA, 5 mM ␤-mercaptoethanol, and 15% glycerol using Ultrafree centrifugal filter tubes. Phosphatase activities were determined essentially as described previously (11) in the presence of 10 mM MgCl 2 .
Kinase/Phosphatase (ATPase) Assays-EnvZc or EnvZc(T247X) mutant proteins (4 M) and OmpR (8 M) were each incubated with 50 M ATP and 0.25 of l [␥-32 P]ATP (3000 Ci/mmol, 10 mCi/ml) in autophosphorylation buffer with 5 mM MgCl 2 . The mixture was incubated at room temperature. Aliquots were removed at 30 and 60 min. The reaction was stopped with 5ϫ SDS loading buffer, and the products were separated by 17.5% SDS-PAGE. The gel was dried, analyzed with a PhosphorImager, and also exposed for autoradiography.

RESULTS
Significance of Thr 247 in EnvZ-Threonine is the most preferred amino acid residue at the Hϩ4 position in the conserved H box of histidine kinases (6). The consensus sequence of the H box is as follows: h-HahbTPL. The highly conserved Thr 247 residue is strategically positioned just one turn below the phospho-accepting His 243 residue on helix I (Fig. 1B). Historically, a mutant of Thr 247 , EnvZ(T247R) (EnvZ11), was first reported as a TP-1-resistant mutant (tpo-11) that pleiotropically repressed synthesis of OmpF, alkaline phosphatase, and several proteins of the maltose regulon, but increased OmpC production (18,37). In vitro biochemical studies established that the EnvZ11 protein is a "superkinase" that is deficient in dephosphorylating OmpR-P (17). ompR77 (OmpR(L16Q) mutant) was later isolated as a suppressor for the EnvZ11 phenotype (38), and the phosphotransfer reaction between the two proteins was found to be markedly low (17).
In the NMR solution structure of domain A of EnvZ, the segment (residues 242-248) containing the invariant His 243 residue and the conserved residues Thr 247 and Pro 248 is poorly defined (root mean square deviation of 1.14 Å for backbone atoms and 1.99 Å for heavy atoms when calculated with only these residues; compare with the average pairwise root mean square deviation of 1.10 Å for backbone atoms and 1.90 Å for heavy atoms for the rest of the NMR-derived structure of domain A of EnvZ). Moreover, the backbone NH groups in this region exhibit a fast hydrogen-deuterium rate, again indicating that this region is structurally dynamic, probably undergoing a conformational equilibrium between helical and unfolded states. It has been proposed that the structural dynamism observed in this segment in helix I might play a role in the catalytic function of EnvZ (30). Taken together, the previous genetic and biochemical analyses and the recent structural studies strongly implicate the conserved Thr 247 residue in EnvZ function. To further investigate the role of this residue, we have characterized EnvZc(T247X) mutants generated by site-directed mutagenesis.
Construction and Purification of EnvZc(T247X) Mutant Proteins-We mutated plasmid pET11a-EnvZc, which expresses the cytoplasmic domain of EnvZ (residues 180 -450), and replaced Thr 247 with Ala, Glu, Lys, Arg, Cys, Ser, or Tyr by site-directed mutagenesis. The various plasmids were transformed into E. coli BL21(DE3) cells. The proteins were overexpressed and subsequently purified by a two-step procedure involving 40% ammonium sulfate precipitation and Sephacryl S-100 gel-filtration chromatography. All the mutant proteins were soluble and stable upon storage at Ϫ20°C.

EnvZc(T247X) Mutant Proteins Have Trypsin Digestion Profiles Similar to That of EnvZc-
To check whether the mutation of Thr 247 in EnvZ resulted in any major alteration of conformation in the mutant proteins, they were subjected to trypsin digestion. 30 g of each protein was digested with 0.3 g of trypsin at room temperature. Aliquots were removed at 30 and 60 min and analyzed by Tricine/SDS-PAGE. No remarkable change was observed in the digestion profiles of the EnvZc and EnvZc(T247X) mutant proteins (Fig. 2). Whereas the EnvZc-(T247E), EnvZc(T247K), and EnvZc(T247A) mutant proteins were more susceptible to trypsin digestion (in decreasing order) than EnvZc, the EnvZc(T247R) protein was more resistant. These results indicate that the substitutions we made (Ala, Glu, Lys, Cys, Ser, Tyr, and Arg) at Thr 247 in EnvZ did not significantly affect the conformation of the protein.
Mutation of Thr 247 in EnvZ Variously Affects Its Autokinase Activity-The autophosphorylation activities of the EnvZc-(T247X) mutant proteins were compared with that of EnvZc in an in vitro biochemical assay using [␥-32 P]ATP as described under "Experimental Procedures." The time course of incorporation of the phosphoryl group into the proteins was followed and is shown in Fig. 3 (A and B). Interestingly, the incorporation of the phosphoryl group into the mutant protein was variable depending on the specific substitution (Tyr/Arg Ͼ Ͼ Thr Ͼ Ser Ͼ Cys Ͼ Ͼ Ala Ͼ Lys Ͼ Glu). Densitometric analysis revealed that the maximum level of incorporation of the phosphoryl group into EnvZc(T247Y) and EnvZc(T247R) was ϳ1.6 times higher than that into EnvZc (Fig. 3B), whereas the incorporation into the EnvZc(T247E), EnvZc(T247K), and EnvZc-(T247A) mutant proteins was significantly lower that that into wild-type EnvZc. Of the mutant proteins, EnvZc(T247S) followed by EnvZc(T247C) exhibited levels of autokinase activity that were close to those of wild-type EnvZc (Fig. 3B). Since the autokinase levels varied significantly with the nature of the substituting amino acid, it is unlikely that Thr 247 plays a catalytic role in the autokinase activity of EnvZ. It is clear though that Thr 247 significantly influences the autophosphorylation reaction.

EnvZc(T247R) Mutant Protein (EnvZ11) Has a Lower Requirement for Divalent Metal Ions than EnvZc for Its
Autokinase Activity-The autophosphorylation reaction of EnvZ is metal ion-dependent (39). To further investigate the higher rate of autophosphorylation observed in EnvZc(T247R) and EnvZc(T247Y), we compared the divalent metal ion dependences of the autokinase activities of these two mutant proteins with those of EnvZc and EnvZc(T247S). Each protein was autophosphorylated in autokinase reaction buffer containing either 10 mM EDTA or 10 mM MgCl 2 . Aliquots were removed at 1 and 3 min, and the course of the reaction was followed as shown in Fig. 4. EnvZc(T247R) was unique in being able to autophosphorylate well not only in the presence of Mg 2ϩ ions (Fig. 4, lanes 9 -12), but also in the absence of divalent metal ions (in 10 mM EDTA). Interestingly, EnvZc(T247Y), which also has a higher rate of autophosphorylation compared with wildtype EnvZc, did not show a similar divalent metal ion independence (Fig. 4, lanes 13-16). This aspect is being separately investigated.
EnvZc(T247X) Mutant Proteins Are Impaired in Their Phosphotransferase Activity-Phosphorylated EnvZ transfers its phosphoryl group from His 243 to Asp 55 on OmpR (39). We compared the phosphotransferase activities of the EnvZc-(T247X) mutant proteins with that of wild-type EnvZc. Individual proteins were autophosphorylated and purified of free ATP contamination as described under "Experimental Procedures." Each phosphoprotein was then incubated with OmpR at room temperature. Aliquots were removed at 0, 20, 40, 120, and 300 s, and the course of the reaction was followed as shown in Fig. 5A. In all the mutant proteins with the exception of EnvZc(T247S) and EnvZc(T247A), the transfer of the phosphoryl group from the phosphorylated EnvZ donor protein was distinctly impaired. The EnvZc(T247K) mutant protein displayed the least phosphotransferase activity, with very little OmpR-P detectable even 5 min after the reaction (Fig. 5A). These differences became much more pronounced when the phosphotransferase experiment was conducted on ice (Fig. 5B). Following the decrease in phosphorylated EnvZ with time, it was evident that at the end of 30 s, hardly any loss of the phosphoryl group from the EnvZc(T247K/Y/C/R/E) mutant proteins had occurred. Only the conservative mutations EnvZc-(T247S) and EnvZc(T247A) exhibited phosphotransferase activities that were close to wild-type levels (Fig. 5, A and B). These results clearly indicate that residue 247 in EnvZ strongly influences the phosphotransfer reaction between EnvZ and OmpR. Only EnvZc(T247S) Exhibits Phosphatase Activity Similar to That of Wild-type EnvZc-Not only does EnvZ phosphorylate OmpR, it also dephosphorylates OmpR-P (16, 41). A compari-son of the in vitro phosphatase activities of the mutant proteins was performed. OmpR-P purified of free ATP was incubated with each EnvZc(T247X) protein at room temperature in phosphatase buffer. Aliquots were removed at 1, 2.5, 5, and 10 min, and the time course of the reaction was followed as shown in Fig. 6 (A and B). Most significantly, of all the mutant proteins, only EnvZc(T247S) displayed phosphatase activity comparable to that of EnvZc. The phosphatase activities of EnvZc(T247A) and EnvZc(T247C) were negligible compared with that of wildtype EnvZc. Furthermore, no dephosphorylation of OmpR was detected with the EnvZc(T247Y) mutant protein. Interestingly, EnvZc(T247K), EnvZc(T247E), and EnvZc(T247R) displayed a reverse phosphotransfer reaction, where part of the phosphoryl group was transferred from OmpR-P back to the respective EnvZ mutant (Fig. 6A).
Comparison of the Enzymatic Activities of EnvZc(T247N) and EnvZc(T247Q) Mutant Proteins with That of EnvZc-The biochemical characterization of the EnvZc(T247A/E/K/C/S/Y/R) mutant proteins strongly suggested that the conserved Thr 247 residue is critical for the phosphatase activity of EnvZ. Interestingly, the Hϩ4 residue in the H box of the nitrogen sensor NtrB is replaced with asparagine. NtrB is the only bifunctional histidine kinase/phosphatase other than EnvZ whose phosphatase activity has been localized to an isolated domain equivalent to domain A of EnvZ (42,43). Therefore, we also investigated two other substitution mutations of Thr 247 , T247N and T247Q.
The EnvZc(T247N) and EnvZc(T247Q) mutant proteins were overexpressed and purified following the same protocol used for the other EnvZc(T247X) proteins. The purified proteins were both stable upon storage at Ϫ20°C. Limited trypsin digestion of the two proteins showed no significant difference in the digestion patterns (Fig. 7A). EnvZc(T247Q) appeared to be slightly more resistant to digestion by trypsin than wild-type EnvZc (Fig. 7A). The two proteins were then analyzed for their autokinase, phosphotransferase, and phosphatase activities using the same methods and protein concentrations as were used for the other EnvZc(T247X) mutants. Comparison of these activities with those of wild-type EnvZc is shown in Fig. 7 (B-E). Fig. 7B shows the comparison of the autophosphorylation activities: both EnvZc(T247N) and EnvZc(T247Q) exhibited very similar abilities to utilize ATP. The maximum level of incorporation of the phosphoryl group into EnvZc(T247N) and EnvZc(T247Q) was, however, 60% of that of wild-type EnvZc. Fig. 7C represents the phosphotransferase activities: whereas replacement of threonine with glutamine did not affect the ability of EnvZc to transfer its phosphoryl group to OmpR, the transfer was slow in the EnvZc(T247N) mutant. Importantly, the results of the phosphatase assays of the EnvZc(T247N) and Aliquots were removed at 1 and 3 min, and the reaction was stopped by adding 5ϫ SDS loading buffer. The products were separated by 17.5% SDS-PAGE. The dried gel was exposed for autoradiography. Lanes 1-4, wild-type EnvZc (WT); lanes 5-8, EnvZc(T247S); lanes 9 -12, EnvZc(T247R); lanes 13-16, EnvZc(T247Y). EnvZc(T247Q) proteins revealed that both mutants exhibited low but detectable phosphatase activities (Fig. 7, D and E). Whereas the half-life of OmpR-P was ϳ30 s in the presence of EnvZc and 50 s in the presence of EnvZc(T247S), it was prolonged to 2 min with EnvZc(T247N) and still further to ϳ4 min with EnvZc(T247Q). Thus, EnvZc(T247N) exhibited 16.7% and EnvZc(T247Q) only 8.3% of the wild-type EnvZc phosphatase activity (Fig. 7E). Interestingly, the phosphatase activity of NtrB, in which the Hϩ4 residue is asparagine, is negligible in the absence of the accessory protein PII (41). A(T247R))-We have recently demonstrated that the isolated domain A of EnvZ (residues 223-289) itself exhibits OmpR-P phosphatase activity (33). Domain B of EnvZ modulates this activity in the presence of cofactors like ADP and AMP-PNP. Since the EnvZc(T247X) mutants appear to be severely impaired in this function, we decided to test whether replacing threonine at position 247 in domain A would also cause a similar reduction in phosphatase function. As shown in Fig. 8, indeed, the domain A(T247R) mutant was also impaired in its ability to dephosphorylate OmpR-P.

Phosphatase Activity of the T247R Mutant in Domain A of EnvZ (Domain
Comparison of Kinase/Phosphatase (ATPase) Activities of EnvZc and EnvZc(T247X) Mutants-The net result of the autophosphorylation, phosphotransferase, and phosphatase activities of EnvZ can also be termed as EnvZ/OmpR-dependent ATPase activity, the final products of which are inorganic phosphate and ADP. We followed the course of the EnvZ/OmpRdependent ATPase reaction for the wild-type EnvZc and EnvZc-(T247S/A/N/Q/R) mutant proteins. The EnvZc and EnvZc-(T247X) mutant proteins were added together with OmpR in the ATPase reaction buffer, and the mixture was incubated at room temperature. Aliquots were removed at 30 and 60 min; reactions were stopped; and products were analyzed as described under "Experimental Procedures." As shown in Fig. 9A, OmpR-P was barely detectable at the end of 1 h of incubation for EnvZc and EnvZc(T247S) protein reactions. This indicates that under the conditions used, the rate of dephosphorylation of OmpR-P was equal to or higher than the rate of OmpR phosphorylation in these reactions. In contrast, high levels of OmpR-P accumulated in the reaction mixtures of the EnvZc-(T247A/N/Q/R) mutants. The highest accumulation of OmpR-P was observed for the EnvZc(T247R) mutant protein, which has enhanced autokinase activity, but negligible phosphatase activity. Note that the EnvZc(T247N) protein, which has 60% autokinase activity and only 16.7% of the wild-type phosphatase activity, accumulated appreciable amounts of OmpR-P. Although both EnvZc(T247S) and EnvZc(T247N) reaction mixtures showed low levels of OmpR-P accumulation at 30 min, very little OmpR-P was detectable at 60 min in the case of EnvZc(T247S), whereas in the EnvZc(T247N) reaction, higher OmpR-P levels were maintained. The relative level of OmpR-P accumulation was estimated by PhosphorImager analysis by taking the OmpR-P value of the EnvZc(T247Q) reaction at 30 min as 100 (Fig. 9B). When the amount of OmpR-P produced in the wild-type EnvZc reaction was taken as 1, the relative OmpR-P levels were calculated to be 1,4,8,6,159, and 365 at 30 min and 1,2,8,7,116, and 393 at 60 min for EnvZc, EnvZc(T247S), EnvZc(T247A), EnvZc(T247N), EnvZc(T247Q), and EnvZc(T247R), respectively.
ompC-lacZ Expression of Taz1-1(T247X) Mutants-Taz1-1 is a derivative of the hybrid receptor Taz1, in which the receptor domain of Tar, an aspartate chemoreceptor, is fused with the cytoplasmic signaling domain of EnvZ (24,35). It has been previously demonstrated that Taz1-1 responds to aspartate in the medium by inducing the expression of ompC-lacZ in E. coli RU1012 cells (24). In the absence of a known ligand for osmolarity, the Taz constructs have been successfully employed to study the regulation of EnvZ function in vivo by monitoring the production of ␤-galactosidase.
We took advantage of this system to investigate the phenotypic effect of introducing the T247X mutations in Taz1-1. The plasmids carrying mutant taz1-1 genes were transformed into E. coli RU1012 cells. The transformed cells were incubated in the presence of 0, 1, 2, 5, and 7 mM aspartate in M9 medium until they reached mid-log phase and then were assayed for ␤-galactosidase activity. As shown in Fig. 10, Taz1-1(T247S) was the only aspartate-regulatable mutant. All other Taz1-1(T247X) mutants resulted in an OmpC ϩ (LacZ ϩ ) constitutive phenotype. It is important to note that both Taz1-1(T247N) and Taz1-1(T247Q) also exhibited OmpC ϩ constitutive phenotypes. DISCUSSION EnvZ, a histidine kinase/phosphatase, displays three enzymatic activities: autokinase, phosphotransferase (OmpR kinase), and OmpR-P phosphatase. The results presented in this study indicate that a balance of these activities is critical in modulating the cellular level of OmpR-P, which in turn reciprocally regulates the ompF and ompC genes encoding outer membrane porins (Figs. 9 and 10). Mutations of the conserved Thr 247 residue in EnvZ tested in this study variously affect the three enzymatic activities of EnvZ, resulting in changes in the OmpR-P level (Fig. 9).
Substituting Thr 247 alters the autokinase activity, from negligible (e.g. EnvZc(T247E)) to 1.6-fold higher than that of wildtype EnvZc (EnvZc(T247R) and EnvZc(T247Y)) (Fig. 3). EnvZc(T247R) is unique in that its autokinase is highly active even in the absence of Mg 2ϩ , whereas wild-type EnvZc and all the other EnvZc(T247X) mutant proteins, including EnvZc- The reaction was stopped with 5ϫ SDS loading buffer. The products were separated by 17.5% SDS-PAGE. The dried gel was exposed for autoradiography. B, aliquots were removed at 15 and 30 s, and the reaction was stopped and analyzed as described for A. EnvZ-P, phosphorylated EnvZ.
(T247Y), require Mg 2ϩ ion for optimal autokinase activity (Fig.  4). It seems plausible that the guanidino group of arginine replaces the Mg 2ϩ ion of the MgATP complex that binds wildtype EnvZc. Such a replacement has been proposed for histidyl-tRNA synthetases, which harbor an arginine at a position where the other aminoacyl-tRNA synthetases bind a catalytic Mg 2ϩ ion (44). This interesting possibility for the EnvZc-(T247R) mutant is currently under investigation.
The consequence of mutations of Thr 247 was most remarkable for the phosphatase activity. Of the nine mutant proteins, only EnvZc(T247S) exhibited phosphatase activity comparable to that of wild-type EnvZc (Figs. 6 and 7D). The hierarchy of the activity was as follows: EnvZc(T247S), EnvZc(T247N), and EnvZc(T247Q), 40, 16.7, and 8.3% of the wild-type EnvZc activity, respectively. All others displayed negligible phosphatase activity (Figs. 6 and 7, D and E). Interestingly, EnvZc(T247K), EnvZc(T247E), and EnvZc(T247R) showed some reverse phosphotransfer of the phosphoryl group from OmpR-P (Fig. 6). Such reverse phosphotransfer activity from OmpR-P to EnvZ has been previously reported for the EnvZc(N347D) mutant protein (32). In each of these cases, a charged residue substitutes for a polar residue, suggesting that the charge environment at the active-site histidine plays a role in shifting the reaction equilibrium in favor of the back-reaction.
The isolated domain A of EnvZ carrying the T247R mutation (domain A(T247R) protein) exhibits autokinase and phosphotransferase activities similar to those observed for the C-terminal domain of EnvZ carrying the same T247R mutation (EnvZc(T247R) protein) (data not shown). Replacing Thr with Arg in domain A of EnvZ also abolished its intrinsic phosphatase function (Fig. 8), strongly supporting the notion that Thr 247 plays a critical role in EnvZ function. The role of domain A in EnvZ function has been analyzed recently (33). The results obtained with the domain A(T247R) protein strengthen the view that domain A of EnvZ is not only the dimerization and histidine phosphotransfer domain, but also the phosphatase domain. Although domain B was considered to be the catalytic and ATP-binding domain (27), the present evidence substantiates the notion that domain A is the true catalytic unit responsible for all three activities of EnvZ, whereas domain B is the ATP-binding and regulatory unit.
The present results demonstrating that Thr 247 can be replaced only with Ser to retain comparable levels of all three EnvZ activities clearly indicate that Thr 247 is a critical residue at the active center of EnvZ. However, the extent of involvement of Thr 247 in the three activities is likely to be different. It appears that Thr 247 might be directly involved in catalyzing the phosphatase reaction while facilitating the autokinase and phosphotransferase reactions. Whereas the exact mechanism of the phosphatase reaction is unknown, we speculate that the imidazole of the proximal His 243 residue could be functioning as an acid-base catalyst. It could enhance the nucleophilicity of the hydroxyl group of Thr 247 , thereby enabling it to directly attack the phosphorus of the phosphoryl moiety on Asp 55 of OmpR-P, forming a highly reactive ester acyl-enzyme, which is rapidly hydrolyzed. Alternatively, the hydroxyl group of Thr 247 could potentiate the oxygen of a bound water molecule to make a nucleophilic attack on the phosphorus atom on Asp 55 . The structure of the N-terminal domain of OmpR has not been solved. However, five water molecules are found in the active site of the Mg 2ϩ -bound structure of the homologous response regulator CheY (45). On the other hand, the use of the threonine hydroxyl rather than the direct attack of a water molecule on the substrate is considered to be more favorable as alcohols are often better nucleophiles than water molecules (46). However, it has been clearly demonstrated that His 243 plays an important role in the phosphatase function of EnvZ (31, 33, 47). Therefore, a model that involves both the invariant His 243 residue and the highly conserved Thr 247 residue in the phosphatase function of EnvZ seems to be more likely. Note that the structural dynamism of the H box segment (residues 242-248), which includes Thr 247 , might also play an indirect role in this process.
The role of the histidine kinase in the phosphotransfer reactions between the kinase and the response regulator in the His-Asp signaling systems has been debated. Response regulators can use small molecule phosphodonors such as phosphoramidate and acetyl phosphate directly (48,49). The half-lives of phosphoaspartate groups under denaturing conditions are generally much longer (49,51) than those observed with the native response regulator proteins when such studies have been conducted (40,50,52). Therefore, the role of histidine kinases in the dephosphorylation of their phosphorylated response regulators has often been considered to be just the modulation of the autophosphatase activity of the response regulator. The results presented in this study, however, clearly indicate that EnvZ might not be a passive partner in the dephosphorylation of OmpR-P, as Thr 247 in domain A of EnvZ might be actively engaged in the hydrolysis of OmpR-P.
One intriguing aspect of the results of the in vivo ␤-galactosidase assays is that, although the soluble C-terminal EnvZc-(T247N) mutant protein exhibited reasonable levels of both kinase and phosphatase activities, the membrane-bound Taz1-1(T247N) mutant protein expressed in RU1012 cells showed an OmpC ϩ constitutive phenotype. The OmpC ϩ constitutive phenotype observed in the other seven Taz1-1(T247X) mutants (other than Taz1-1(T247S)) is to be expected since they have negligible phosphatase activity leading to accumulation of OmpR-P in the cell, resulting in the constitutive induction of ompC-lacZ expression. It has been proposed that ligand binding to the receptor increases the ratio of the kinase to phosphatase activity, thereby regulating porin expression (24). We compared the kinase/phosphatase activity ratios of the wild-type EnvZc and EnvZc(T247S) and EnvZc(T247N) mutant proteins. EnvZc(T247S) exhibits 88% and EnvZc(T247N) exhibits 60% of the wild-type EnvZc autokinase activity (estimated by densitometric analysis of maximum levels of incorporation of the phosphoryl group into respective proteins). Again, EnvZc(T247S) shows a higher phosphatase activity (40% of the wild-type EnvZc level) than EnvZc(T247N), which shows only 16.7% of the wild-type level. If the kinase/phosphatase activity ratio of wild-type EnvZc is considered to be 1, then that of EnvZc-(T247S) is 88:40 (i.e. 2.2), and that of EnvZc(T247N) is 60:18 (i.e. 3.3). The higher kinase/phosphatase activity ratio observed for EnvZc(T247N) may account for the inability of Taz1-1(T247N) to be aspartate-responsive. This notion is further supported by results from the kinase/phosphatase assay of the wild-type and mutant EnvZc proteins, where mutants unresponsive to aspartate showed a much higher accumulation of OmpR-P compared with the wild-type EnvZc and EnvZc-(T247S) mutant proteins (Fig. 9). Note that all the mutants showing a kinase/phosphatase activity ratio Ն3.3 display OmpC ϩ constitutive phenotypes in Taz1-1, supporting the previous proposal (24). In the nitrogen regulator NtrB, in which the highly conserved Thr residue is replaced with Asn, dephosphorylation of the response regulator NtrC is dependent on the presence of protein PII. It would be worthwhile to examine the consequence of substituting Asn with Thr on the phosphatase activity of NtrB.
The ATP-binding domains of histidine kinases are being targeted for the development of potential antibiotics. The evidence presented in this study suggests that the histidine phosphotransfer domain may be another possible target. In EnvZ, both the conserved His 243 and Thr 247 residues are in domain A. They are together critical for determining the kinase and phosphatase activities of EnvZ. Domain A is also the specificitydetermining domain in this class of enzymes (30). On the other hand, as the nucleotide-binding CA domain (domain B) shares extensive homology with the Hsp90, Mut1, and gyrase families of ATPases (25), antibiotics developed against domain B may have some cross-reactivities with mammalian enzymes.