Structural Units Important for Activity of a Novel-type Phosphoserine Phosphatase from Hydrogenobacter thermophilus TK-6 Revealed by Crystal Structure Analysis

Background: Sequence-based annotation of novel-type phosphoserine phosphatases (iPSPs) is difficult because of their diverse substrate preferences.

Results: Crystal structure and mutation analyses of iPSP1 from Hydrogenobacter thermophilus identified the residues involved in substrate recognition.

Conclusion: The His⁸⁵ side chain and C-terminal region of iPSP are important for its phosphoserine phosphatase activity.

Significance: These findings will help to identify iPSPs from sequence databases.

Abstract

Novel-type serine-synthesizing enzymes, termed metal-independent phosphoserine phosphatases (iPSPs), were recently identified and characterized from Hydrogenobacter thermophilus, a chemolithoautotrophic bacterium belonging to the order Aquificales. iPSPs are cofactor-dependent phosphoglycerate mutase (dPGM)-like phosphatases that have significant amino acid sequence similarity to dPGMs but lack phosphoglycerate mutase activity. Genes coding dPGM-like phosphatases have been identified in a broad range of organisms; however, predicting the function of the corresponding proteins based on sequence information alone is difficult due to their diverse substrate preferences. Here, we determined the crystal structure of iPSP1 from H. thermophilus in the apo-form and in complex with its substrate l-phosphoserine to find structural units important for its phosphatase activity toward l-phosphoserine. Structural and biochemical characterization of iPSP1 revealed that the side chains of His⁸⁵ and C-terminal region characteristic of iPSP1 are responsible for the PSP activity. The importance of these structural units for PSP activity was confirmed by high PSP activity observed in two novel dPGM-like proteins from Cyanobacteria and Chloroflexus in which the two structural units were conserved. We anticipate that our present findings will facilitate understanding of the serine biosynthesis pathways of organisms that lack gene(s) encoding conventional PSPs, as the structural information revealed here will help to identify iPSP from sequence databases.

Introduction

Novel-type serine-synthesizing enzymes, termed metal-independent phosphoserine phosphatases (iPSPs⁵; EC 3.1.3.3; l-O-phosphoserine + H₂O → l-serine + phosphate), were recently identified and characterized from Hydrogenobacter thermophilus TK-6, an obligately chemolithoautotrophic, thermophilic bacterium belonging to the order Aquificales (1–3). Although the kinetic parameters of iPSPs for l-phosphoserine are similar to those of conventional Mg²⁺-dependent phosphoserine phosphatases (dPSPs), iPSPs do not require divalent cations for their activity. In addition, iPSPs show no amino acid sequence homology to dPSPs. Therefore, the identification and properties of iPSPs suggest that these enzymes connect the missing link between phosphoserine and serine in the serine biosynthetic pathway of H. thermophilus, which lacks a gene encoding a conventional dPSP.

To date, two types of iPSPs have been identified in H. thermophilus, iPSP1 and iPSP2. The former is a homodimer of PspA subunits, and the latter is a heterodimer of PspA and PspB subunits. In contrast to dPSPs, which belong to the haloacid dehalogenase-like hydrolase superfamily, PspA and PspB belong to the histidine phosphatase superfamily, which is composed of a functionally diverse group of phosphatases and mutases, including cofactor-dependentphosphoglycerate mutases (dPGMs; EC 5.4.2.1), fructose-2,6-bisphosphatase (EC 3.1.3.46), and phytases (EC 3.1.3.–) (4). Members of the histidine phosphatase superfamily share four conserved catalytic core residues consisting of two histidine and two arginine residues (e.g. Arg⁹, His¹⁰, Arg⁵⁹, and His¹⁵¹ in Bacillus stearothermophilus PhoE). In PhoE, His¹⁰ acts as the base to extract the phosphate group from the substrate, whereas the other three core residues interact electrostatically with the phosphate group during the reaction. In addition to the four conserved residues, an aspartate or glutamate residue (e.g. Glu⁸³ in PhoE), which acts as a proton donor, is also required for the dephosphorylation reaction (4–6).

When compared with proteins with known function, PspA and PspB, which share 35% sequence identity, have the highest amino acid sequence similarity with dPGM (22 and 18% identities, respectively, to E. coli dPGM). In addition, the five catalytic core residues (four conserved residues and an additional aspartate/glutamate) that are characteristic of the histidine phosphatase superfamily are completely conserved in both iPSP subunits and dPGMs. For this reason, iPSPs were originally annotated as dPGMs; however, iPSPs lack the residues proposed to be important for mutase activity and show no phosphoglycerate mutase activity (1, 7, 8). As iPSPs exhibit substantial phosphatase activity specific to l-phosphoserine, these enzymes are now thought to function as PSPs in vivo.

Proteins with homology to dPGM, but without mutase activity, have been found in a broad range of organisms and are predicted to act as phosphatases, although their physiological roles remain unknown (8–10). For example, PhoE from B. stearothermophilus is a homodimeric phosphatase with broad substrate specificity, and it has the highest specificity toward 3-phosphoglyceric acid and α-naphthylphosphate (8). Our previous study revealed that the PspA and PspB subunits of iPSPs exhibit completely different substrate specificities from that of PhoE (1). Namely, PspA has strict substrate specificity toward l-phosphoserine, whereas PspB appears to have much lower phosphatase activity toward l-phosphoserine and higher activity toward p-nitrophenyl phosphate. We also found that PspA homologs from Thermus thermophilus and Arabidopsis thaliana had slight PSP activity. However, the T. thermophilus PspA homolog had higher phosphatase activity toward dl-phosphothreonine and 3-phosphoglycerate than toward l-phosphoserine (1). Taken together, these results indicate that dPGM-like phosphatases have diverse substrate preferences, and their function cannot be predicted based on the whole amino acid sequence similarity alone. Therefore, identification of the structural units in the PspA subunit of H. thermophilus iPSP1 that is important for the PSP activity are expected to enable the identification of iPSPs among dPGM-like proteins with increased accuracy.

Here, we report the crystal structures of the apo- and the substrate-bound forms of iPSP1. Based on these crystal structures and mutagenesis data, we determined the structural units that would aid in the identification of iPSPs among dPGM-like proteins.

EXPERIMENTAL PROCEDURES

Gene Cloning and Protein Expression and Purification

The genes encoding the PspA and PspB subunits of H. thermophilus TK-6 (IAM 12695, DSM 6534), pspA (YP_003431771, HTH0103), and pspB (YP_003431851, HTH0183) were cloned into the expression vectors pCDFDuet-1 and pET21c (Novagen, Darmstadt, Germany), respectively, as described previously (1). The constructed plasmids were then mutated for expressing H9A mutants of the PspA and PspB subunits in which His⁹ was converted to Ala, and H85A, ΔC1, and ΔC7 mutants of PspA, in which His⁸⁵ was converted to Ala (iPSP1 (H85A)); Ile²¹¹ was deleted (iPSP1 (ΔC1)), and Val²⁰⁵⁰–Ile²¹¹ were deleted (iPSP1 (ΔC7)), using PrimeSTAR mutagenesis basal kit (Takara Bio, Otsu, Japan) according to the manufacturer's instructions. The genes encoding iPSP homologs from Roseiflexus castenholzii DSM 13941 (YP_001432425, RCAS2324; YP_001430323, RCAS0171; and YP_001433513, RCAS3445) and Thermosynechococcus elongatus BP-1 (NP_682322, TLR1532) were amplified by PCR from the genomic DNAs of respective species. The amplified fragments were inserted into the NdeI/XhoI (R. castenholzii ones) or NdeI/EcoRI (T. elongatus one) sites of pET-21c. The nucleotide sequences of the above-mentioned genes were verified by sequencing. The primers used in this study are listed in Table 1.

TABLE 1.

Primers used in this study

Name	DNA sequence
PspA H9A F	5′-GTGCGAGCTGCGGAGAGCGAGTGGAA-3′
PspA H9A R	5′-CTCCGCAGCTCGCACCAGTATAAGCTT-3′
PspB H9A F	5′-GGTCAGGGCTGCGCAGAGCGAGTATAA-3′
PspB H9A R	5′-TGCGCAGCCCTGACCAAATACAAACG-3′
PspA H85A F	5′-ATAGACGCCGGTATGTGGTCGGGC-3′
PspA H85A R	5′-CATACCGGCGTCTATTTCTATTATC-3′
PspA ΔC1 F	5′-AAGGCTTAGTAGCTTGCTGGCATG-3′
PspA ΔC1 R	5′-AAGCTACTAAGCCTTGTGAGCTTC-3′
PspA ΔC7 F	5′-GTTTTATTAGCTTGCTGGCATGTAC-3′
PspA ΔC7 R	5′-GCAAGCTAATAAAACTCTCCCAGATG-3′
TLR1532 F	5′-GTCCATATGGCACAGTGGG-3′
TLR1532 R	5′-ACAGAATTCCCTATAGCGCTCC-3′
RCAS2324 F	5′-ATGCATATGCGCCTGATCATC-3′
RCAS2324 R	5′-CGTCTCGAGTTCAACCTTTAC-3′
RCAS0171 F	5′-TATCCATATGCGCACCGCTC-3′
RCAS0171 R	5′-GGTCTCGAGGGAAGGTCAAAG-3′
RCAS3445 F	5′-AACCATATGACCACCTTCTACATC-3′
RCAS3445 R	5′-TCGCTCGAGTTCTCATACCC-3′

Escherichia coli BL21-Codon Plus (DE3)-RIL was used for the individual and co-expression of the PspA and PspB subunits, and their mutants. E. coli BL21 (DE3) was used for the expression of iPSP homologs from R. castenholzii and T. elongatus. The transformed E. coli cells harboring the expression plasmids were inoculated into Luria-Bertani medium containing the appropriate antibiotic(s) as follows: 50 μg ml⁻¹ streptomycin, 50 μg ml⁻¹ ampicillin, and/or 34 μg ml⁻¹ chloramphenicol. After cultivating the cells aerobically at 37 °C until the absorbance at 600 nm reached ∼0.6, the expressions of iPSP1, iPSP2, and their mutants were induced by the addition of 1 mm (final concentration) isopropyl thio-β-d-galactopyranoside to the medium, followed by further cultivation for 3 h at 37 °C. The expressions of the R. castenholzii and T. elongatus iPSP homologs were induced with 0.5 mm isopropyl thio-β-d-galactopyranoside for 4 h at 37 °C. The cells were then harvested by centrifugation, resuspended in 20 mm Tris-HCl buffer (pH 8.0) containing 1 mm EDTA (4 ml/g of wet cells), sonicated, and subjected to centrifugation (100,000 × g for 1 h) to obtain supernatants. iPSP1s from H. thermophilus and its mutants were purified from the supernatants using butyl-Toyopearl (Tosoh, Tokyo, Japan) and Mono Q columns (GE Healthcare) after heat treatment at 80 °C for 10 min, as described previously (1). For the purification of iPSP homologs from R. castenholzii and T. elongatus, nonheat-treated supernatants were loaded onto a Q-Sepharose fast-flow column (GE Healthcare) and partially purified, as described previously (11).

Enzyme Assays

PSP activity was assayed by measuring the production of inorganic phosphate, as described previously with minor modifications (1). Briefly, the reaction mixture contained 10 μl of 1.0 m HEPES-NaOH (pH 8.0 at room temperature), 5 μl of 50 mm l-phosphoserine, 0.5 μl of 100 mm EDTA (pH 8.0), 5 μl of enzyme solution in 20 mm Tris-HCl (pH 8.0), and 29.5 μl of water (total volume = 50 μl). For the determination of kinetic parameters, reaction mixtures containing 0.5, 1.0, 3.0, 5.0, 10.0, and 20.0 mm l-phosphoserine were used. Note that in our previous study, the reaction mixture contained 0.5–10 mm l-phosphoserine for the determination of kinetic parameters (1) because of the limited amount of purified native enzymes. Here, we used up to 20 mm l-phosphoserine to more accurately determine the kinetic parameters. Therefore, the final buffer concentration of the reaction mixture was increased from 20 to 200 mm to maintain a constant pH. The reaction mixture was incubated for 5–20 min at 70 °C for H. thermophilus proteins and 55 °C for R. castenholzii and T. elongatus proteins, which were close to the optimal growth temperatures of the host organisms. One unit of PSP activity was defined as the amount of enzyme producing 1 μmol of inorganic phosphate/min.

Protein Assay

Protein concentrations were measured using Bradford protein assay (Bio-Rad) with bovine serum albumin as the standard.

Crystallization and Data Collection for iPSP1 and iPSP1 (H9A)-l-Phosphoserine

The crystallization and x-ray diffraction data collection of wild-type iPSP1 (iPSP1 (WT)) were performed as described previously (12). In brief, iPSP1 (WT) was crystallized by the sitting-drop vapor diffusion method. A 1.5-μl drop was first prepared by mixing 0.75 μl of an iPSP1 (WT) solution (10 mg ml⁻¹ in 5 mm Tris-HCl (pH 8.0)) and a 0.75-μl reservoir solution (100 mm HEPES-NaOH (pH 7.5), 10% (v/v) 2-propanol, and 20% (w/v) polyethylene glycol 4000). Obtained crystals were cryoprotected by soaking in a 3:1 (v/v) mixture of reservoir solution and ethylene glycol. Two x-ray diffraction data sets from a single crystal of iPSP1 (WT) were collected as described previously (12). The obtained data sets were indexed, integrated, scaled, and merged with the XDS program (13).

iPSP1 (H9A) complexed with l-phosphoserine was crystallized by the sitting-drop vapor diffusion method. A 1.5-μl drop was first prepared by mixing 0.75 μl of an iPSP1 (H9A) solution (10 mg/ml in 5 mm Tris-HCl (pH 8.0) containing 12-molar excess of l-phosphoserine) and 0.75 μl of reservoir solution (100 mm sodium acetate trihydrate (pH 4.6), 20 mm calcium chloride dihydrate, and 30% (v/v) (±)-2-methyl-2,4-pentanediol). Obtained crystals were cryoprotected by soaking in a 4:1 (v/v) mixture of reservoir solution and ethylene glycol. X-ray diffraction experiments for iPSP1 (H9A)·l-phosphoserine crystals were performed at beamline NE3A at Photon Factory-Advanced Ring (Ibaraki, Japan). A data set for the iPSP1 (H9A)·l-phosphoserine crystal was collected using an ADSC Quantum 270 detector under the following conditions: wavelength, 1.0000 Å; distance between the crystal and the detector, 213.9 mm; oscillation angle, 1.0°; and exposure time, 1 s per image. The collected dataset was indexed, integrated, scaled, and merged with the programs XDS (13).

Structural Solution and Refinement of iPSP1 (WT) and iPSP1 (H9A)-l-Phosphoserine

The initial structure of iPSP1 (WT) was obtained by the molecular replacement method using the program Molrep (14) in the CCP4 suite. The crystal structure of PhoE from B. stearothermophilus (25% amino acid sequence identity with iPSP1; PDB code 1H2E) was used as the search model. The initial structure was refined using Coot (15) and Refmac5 in the CCP4 suite (16). The stereochemistry of the structure was checked by the program RAMPAGE (17). The initial structure of the iPSP1 (H9A)·l-phosphoserine complex was obtained by the molecular replacement method using the program Molrep in the CCP4 suite using iPSP1 (WT) as a template. l-Phosphoserine and water molecules were modeled in the final stages of refinement based on the F_obs − F_calc electron density map. The refined structures were visualized with PyMOL (Version 1.5.0.1, Schrödinger, LLC.). The structural similarity between any two protomers was evaluated with the root mean square deviation of C^α atoms calculated with the DaliLite server (18). Interface analysis was performed using the Protein Interfaces, Surfaces, and Assemblies (PISA) server of the European Bioinformatics Institute (19–21).

Homology Modeling

The tertiary structure of the PspB subunit was modeled using Swiss-Model (automated mode) (22–24) and the crystal structure of chain A of iPSP1 (WT) as the template. The tertiary structures of TLR1532 and RCAS2324 were modeled using Modeler 9.10 and the same template structure as above. Here, the pairwise sequence alignments obtained with MUSCLE (25) were used (see Fig. 1). The residues after Thr²⁴⁹ and entire amino acid sequence were used for the modeling of TLR1532 and RCAS2324, respectively.

FIGURE 1.

Multiple sequence alignment of PspA and its homologs constructed using the MUSCLE program. A, gaps after Gly²⁰¹ (PspA subunit numbering) were manually removed. Amino acids surrounded by a black box and shaded indicate residues that can form hydrogen bonds with the phospho group and other parts of l-phosphoserine in PspA, respectively, and their corresponding amino acids conserved in other sequences. Residue numbers of PspA are shown above the sequences. B, alignment used for structure modeling. The alignments shown here, other than that of the C terminus, are the same as those shown in A.

Data Deposition

The atomic coordinates and structural factors of H. thermophilus iPSP1 have been deposited in the Protein Data Bank under PDB codes 4IJ5 and 4IJ6.

RESULTS

PSP Activity of H9A Mutants

Our previous studies suggested that the PSP activities of H. thermophilus iPSP1 (PspA-PspA) and iPSP2 (PspA-PspB) are mainly catalyzed by the PspA subunit, because the V_max of iPSP2 is nearly half that of iPSP1, whereas their K_m values are similar (1). To determine whether the PspB subunit had detectable PSP activity, we constructed H9A mutants of the PspA and PspB subunits, where His⁹, a putative active-site residue predicted to act as a base, was mutated to Ala.

The heat-treated culture supernatant of an E. coli strain overexpressing a homodimer of PspA (H9A)-PspA (H9A) showed almost no PSP activity (≤0.01 units mg⁻¹, Table 2). We confirmed by SDS-PAGE analysis that the expression level of the mutant protein was similar to that of PspA (WT) (data not shown). This result strongly suggests that His⁹ of the PspA subunit acts as a base, as found in other histidine phosphatase superfamily proteins. PSP activity was also not detected in culture supernatant containing the PspA (H9A)-PspB (WT) heterodimer; however, culture supernatant from E. coli cells overexpressing the PspA (WT)-PspB (H9A) heterodimer (Fig. 2) exhibited PSP activity (39 ± 0 units mg⁻¹; Table 2). These results indicate that the PspB subunit has no or undetectable PSP activity.

TABLE 2.

PSP activity of H9A mutants

Component	PSP activity
	units mg−1
PspA (H9A)-PspA (H9A)	≤0.01
PspA (WT)-PspB (H9A)	39 ± 0
PspA (H9A)-PspB (WT)	≤0.01
PspA (H9A)-PspB (H9A)	≤0.01

FIGURE 2.

12% SDS-PAGE analysis of partially purified iPSPs used for the enzymatic activity measurements. Lane 1, Q-Sepharose fraction of RCAS2324; lane 2, Q-Sepharose fraction of TLR1532; lanes 3–5, supernatant of iPSP2-expressed and heat-treated CFE. Lane 3, PspA (WT)·PspB (H9A); lane 4, PspA (H9A)·PspB (WT); lane 5, PspA (H9A)·PspB (H9A). M, molecular mass markers. The arrowheads indicate the bands corresponding to the expressed proteins.

Structural Determination of iPSP1 (WT) without a Ligand and iPSP1 (H9A) Complexed with l-Phosphoserine

The crystal structure of iPSP1 (WT) with no ligand was solved at 1.5-Å resolution by molecular replacement using the atomic coordinates of PhoE from B. stearothermophilus as a search model. The iPSP1 crystal belonged to the orthorhombic space group P2₁2₁2₁ with unit cell parameters a = 49.8 Å, b = 73.6 Å, and c = 124.3 Å, and it contained two PspA subunits in an asymmetric unit. The final R and R_free values of iPSP1 (WT) were 18.4 and 19.7%, respectively. The Ramachandran plot indicated that 98.6 and 1.4% of the residues were in the most favored and allowed regions, respectively. The data collection and refinement statistics are presented in Table 3.

TABLE 3.

Crystallographic data collection and refinement statistics

Protein name	iPSP1 (WT)	iPSP1 (H9A)·l-phosphoserine
X-ray data collection
Beamline	SPring-8 BL-32XU	PF-AR NE3A
Wavelength	1.0000 Å	1.0000 Å
Resolutiona	45.0 to 1.50 Å (1.54 to 1.50 Å)	38.2 to 1.80 Å (1.85 to 1.80 Å)
Space group	P212121	P212121
Unit cell parameter	a 49.8 Å, b 73.6 Å, c 124.3 Å	a 49.3 Å, b 68.9 Å, c 123.6 Å
VM	2.32 Å3 Da−1	2.14 Å3 Da−1
Total reflectionsa	630,969 (32,454)	277,086 (16,180)
Unique reflectionsa	73,612 (5300)	39,566 (2740)
Redundancya	8.6 (6.1)	7.0 (5.9)
Completenessa	99.9% (99.0%)	99.3% (94.1%)
Rsyma,b	9.1% (58.7%)	4.2% (16.6%)
〈I/σI〉a	14.8 (3.3)	33.6 (10.4)
Refinement
Protein Data Bank code	4IJ5	4IJ6
Resolution	36.1 to 1.50 Å	38.2 to 1.80 Å
Rwork/Rfreec	18.4/19.7%	19.0/21.8%
Root mean square deviations
Bonds	0.0084 Å	0.0053 Å
Angles	1.2716°	1.0986°
No. of atoms (average B-factors (Å2))
Protein	3481 (16.0)	3365 (17.3)
l-Phosphoserine		22 (11.5)
Water	242 (23.2)	147 (23.9)
Ethylene glycol	16 (22.3)	28 (23.1)
Chloride	1 (13.0)	2 (19.8)
Observed residue range(s)
Chain A	1 to 211	1 to 207
Chain B	1 to 211	1 to 203
Ramachandran plot
Most favored region	98.6%	98.0%
Additionally favored regions	1.4%	2.0%

^a Values for the highest resolution shells are shown in parentheses for x-ray data collection.

^b R_sym = Σ_hkl ((Σ_i|I_i − 〈I〉|)/Σ_i|I_i|).

^c R_work = (Σ_hkl‖F_o| − |F_c‖)/Σ_hkl|F_o|. R_work was calculated with 95% of the data used for refinement, and R_free was calculated with the remaining 5% of the data.

The crystal structure of the iPSP1 (H9A)·l-phosphoserine complex was solved at 1.8-Å resolution by molecular replacement using the atomic coordinates of iPSP1 (WT) as a template. The iPSP1 (H9A)·l-phosphoserine complex crystal belonged to the orthorhombic space group P2₁2₁2₁ with unit cell parameters a = 49.3 Å, b = 68.9 Å, and c = 123.6 Å, and it consisted of two PspA (H9A) subunit l-phosphoserine complexes in an asymmetric unit. The final R and R_free values of iPSP1 (H9A)·l-phosphoserine were 19.0 and 21.8%, respectively. The Ramachandran plot indicated that 98.0 and 2.0% of the residues were in the most favored and allowed regions, respectively. The data collection and refinement statistics are shown in Table 3.

Overall Structures of iPSP1s

The final model of iPSP1 (WT) contained all of the amino acid residues (residues 1–211) for the two PspA subunits (chains A and B) in an asymmetric unit, and it consisted of 3481 protein atoms, one chloride ion, four ethylene glycol molecules, and 242 water molecules. The final model of the iPSP1 (H9A)·l-phosphoserine complex contained all of the amino acid residues, with the exception of several C-terminal residues (residues 1–207 and 1–203 for chains A and B, respectively), and it consisted of 3365 protein atoms, two chloride ions, seven ethylene glycol molecules, and 147 water molecules.

The PspA subunit adopted a dPGM-like fold (6, 26) containing six α-helices and six β-strands that formed an α/β/α sandwich of the central mixed β-sheet flanked by α-helices on both sides (Fig. 3, A and B). An additional smaller domain was predominantly composed of two α-helices (α3 and α4). Approximately 11% of the surface area of PspA (1,082 Ų per protomer) was buried in the dimer interface, 40% of which was occupied by hydrophobic residues. At the dimer interfaces that were mainly formed by the β6 strand and a loop connecting α6 and β5, 24 hydrogen bonds, eight salt bridges, and a disulfide bond were predicted to exist as well as hydrophobic interactions (Fig. 3A and Table 4). These findings were consistent with the gel filtration data of iPSP1 indicating that the protein behaves as a homodimer of the PspA subunit in solution (1). The dimerization behavior of the PspA subunit in iPSP1 was similar to that of the dPGM-like protein Rv3124 from Mycobacterium tuberculosis (PDB code 2A6P) (27).

FIGURE 3.

Structure of iPSP1 and its homologs. Overall structure of homodimeric iPSP1 (WT; A) and its protomer (B). The conserved catalytic core residues (Arg⁸, His⁹, Arg⁵⁸, Glu⁸², and His¹⁵⁰) are shown as sticks. C, superimposition of active-site residues of iPSP1 (WT) (gray) and PhoE (PDB code 1H2E) (cyan). The PspA subunit numberings are shown. D, F_obs − F_calc omit map of iPSP1 (H9A)·l-phosphoserine. E, superimposition of iPSP1 (WT) (gray) and iPSP1 (H9A)·l-phosphoserine (pink). F, 2F_obs − F_calc electron density map contoured at 1.0σ (blue mesh) of the C-terminal residues region (residues 205–211) of iPSP1 (WT) (shown as sticks). l-Phosphoserine, also shown as sticks, is modeled by superposing iPSP1 (WT) and iPSP1 (H9A) complexed with l-phosphoserine. G, superimposition of d-phosphoserine onto the structure of PspA (H9A)·l-phosphoserine. Carbon atoms of iPSP1 (WT), iPSP1 (H9A), and d- and l-phosphoserine are shown in gray, pink, cyan, and light green, respectively. Phosphorus, oxygen, and nitrogen atoms are shown in orange, red, and blue, respectively. Images were generated with PyMOL.

TABLE 4.

Interface of iPSP1 (WT) homodimer

Chain A	Chain B	Distance
		Å
Disulfide bond
Cys198/Sγ	Cys198/Sγ	2.07
Hydrogen bonds
Val108/O	Arg188/Nη2	2.85
Glu109/O	Arg188/Nη2	3.82
Leu162/O	Lys168/Nζ	2.75
Ser171/O	Ile191/N	2.81
Asp175/Oδ2	Lys193/Nζ	2.93
Asn189/O	Ser171/Oγ	2.80
Ile191/O	Gly173/N	2.87
Leu192/O	Asn195/Nδ2	2.79
Leu194/O	Leu194/N	3.12
Asn195/O	Lys193/Nζ	2.82
Thr197/Oγ1	Lys193/Nζ	2.87
Arg188/Nη2	Val108/O	2.74
Arg187/Nη2	Glu109/Oϵ2	3.08
Arg188/Nη2	Glu109/O	3.71
Lys168/Nζ	Leu162/O	2.79
Ile191/N	Ser171/O	2.82
Lys193/Nζ	Asp175/Oδ2	2.90
Ser171/Oγ	Asn189/O	2.77
Gly173/N	Ile191/O	2.83
Asn195/Nδ2	Leu192/O	2.83
Leu194/N	Leu194/O	3.09
Lys193/Nζ	Asn195/O	2.74
Lys193/Nζ	Thr197/Oγ1	2.84
Salt bridges
Glu109/Oϵ1	Arg187/Nη2	3.74
Glu109/Oϵ1	Arg187/Nη1	3.81
Glu109/Oϵ2	Arg187/Nη1	3.64
Glu109/Oϵ2	Arg187/Nϵ	3.98
Asp175/Oδ2	Lys193/Nζ	2.93
Arg187/Nη2	Glu109/Oϵ2	3.08
Lys193/Nζ	Asp175/Oδ2	2.90

The root mean square deviations between PspA (WT), PspA (H9A), and PhoE ranged from 0.2 to 1.6 Å, suggesting that their overall structures were nearly identical. The spatial positions and orientations were highly conserved between the essential residues of the PspA subunit (Arg⁸, His⁹, Asn¹⁵, Gln²¹, Gly²², Arg⁵⁸, Glu⁸², and His¹⁵⁰) and those of PhoE (Arg⁹, His¹⁰, Asn¹⁶, Gln²², Gly²³, Arg⁵⁹, Glu⁸³, and His¹⁵¹) (Fig. 3C).

Binding Manner of l-Phosphoserine to iPSP1

Electron densities of l-phosphoserine were observed at the catalytic pocket when the inactive iPSP 1 (H9A) mutant was crystallized in the presence of this substrate (Fig. 3D). In the crystal structure of the enzyme·substrate complex, oxygen atoms of the carboxyl group of l-phosphoserine formed hydrogen bonds with main-chain amides of Gln²¹ and Gly²², and N^ϵ2 of Gln²¹, and partly formed a hydrogen or an ionic bond with N^ϵ2 of His⁸⁵. Nitrogen atom of amino group of l-phosphoserine formed a hydrogen or an ionic bond with oxygen atoms of the side-chain carboxyl group of Glu⁸², and formed hydrogen bonds with N^ϵ2 of His⁸⁵ and O^γ1 of Thr¹⁵¹. As the phosphate group of l-phosphoserine was considered to be negatively charged, the crystal structure of the iPSP1 (H9A)·l-phosphoserine complex indicated that this group interacted with iPSP1 residues as follows: (i) O1P of the phosphate group formed an ionic bond with N^η2 of Arg⁸, and hydrogen bonds with side chains N^δ2 of Asn¹⁵ and N^ϵ2 of Gln²¹; (ii) O2P of the phosphate group formed an ionic bond with N^ϵ and N^η1 of Arg⁵⁸ and formed a hydrogen or an ionic bond with N^ϵ2 of Glu²¹ and N^δ1 of His¹⁵⁰; and (iii) O3P of the phosphate group formed an ionic bond with N^ϵ of Arg⁸ and a hydrogen bond with main-chain amide of Thr¹⁵¹ (Fig. 3E and Table 5).

TABLE 5.

Hydrogen bonds formed between iPSP1 (H9A) and l-phosphoserine

Molecule/atom name	Molecule/residue/Atom name	Distance
		Å
l-Phosphoserine/O1P	PspA/Arg8/Nη2	2.99
l-Phosphoserine/O1P	PspA/Asn15/Nδ2	2.88
l-Phosphoserine/O1P	PspA/Gln21/Nϵ2	2.99
l-Phosphoserine/O2P	PspA/Gln21/Nϵ2	3.44
l-Phosphoserine/O2P	PspA/Arg58/Nη1	3.01
l-Phosphoserine/O2P	PspA/Arg58/Nϵ	3.40
l-Phosphoserine/O2P	PspA/His150/Nδ1	3.70
l-Phosphoserine/O3P	PspA/Arg8/Nϵ	3.12
l-Phosphoserine/O3P	PspA/Thr151/N	2.97
l-Phosphoserine/Oγ	PspA/Thr151/Oγ1	2.84
l-Phosphoserine/N	PspA/His85/Nϵ2	3.09
l-Phosphoserine/N	PspA/Glu82/Oϵ2	2.84
l-Phosphoserine/N	PspA/Thr151/Oγ1	3.23
l-Phosphoserine/OXT	PspA/Gln21/N	2.92
l-Phosphoserine/OXT	PspA/Gln21/Nϵ2	2.83
l-Phosphoserine/O	PspA/Gly22/N	2.96
l-Phosphoserine/O	PspA/His85/Nϵ2	3.40
l-Phosphoserine/O	PspA/Arg58/Nη1	3.95

To predict the substrate-binding manner of iPSP1 (WT), we modeled the structure of iPSP (WT) in complex with l-phosphoserine by superposing the crystal structure of iPSP1 (WT) onto that of iPSP1 (H9A) (Fig. 3E). The modeled structure of the protein·ligand complex suggested that hydrogen bonds could be formed by oxygen atom of the phosphate group of l-phosphoserine and N^ϵ2 of His⁹, in addition to the interactions observed in the crystal structure of the iPSP1 (H9A)·l-phosphoserine complex. The iPSP1 (WT)·substrate model suggested that the invariant catalytic residues (Arg⁸, His⁹, Arg⁵⁸, and His¹⁵⁰; PspA subunit numbering) and the proton donor Glu (Glu⁸²) in dPGMs and their relatives are also essential for the phosphatase activity of iPSP1.

Multiple amino acid sequence alignment indicated that the PspA subunit residues that interact with l-phosphoserine were highly conserved in the PspB subunit and PhoE (Fig. 1A). Among the PspA residues involved in forming hydrogen and/or ionic bonds with the phosphate group of l-phosphoserine (Arg⁸, His⁹, Asn¹⁵, Gln²¹, Arg⁵⁸, His¹⁵⁰, and Thr¹⁵¹), only Thr¹⁵¹ was replaced by Gly in both the PspB subunit and PhoE. These findings suggest that the phosphate group binding and dephosphorylation mechanisms of the PspA subunit in iPSP1 are similar to those of the PspB subunit, PhoE, and other proteins in the histidine phosphatase superfamily. The alignment analysis also indicated that the residues forming hydrogen and/or ionic bonds with the l-serine moiety of the substrate were also conserved in PspB and PhoE, with the exception of His⁸⁵, which was replaced by Phe⁸⁵ and Leu⁸⁶ in PspB and PhoE, respectively. Thus, His⁸⁵ was suggested to be important for the substrate specificity of iPSP1.

The crystal structure-based modeling also revealed that several C-terminal residues in iPSP1 (WT) formed a lid covering the active site cleft. In the iPSP1 (WT)·l-phosphoserine model, the C-terminal Ile²¹¹ residue in iPSP1 (WT) was found to be close enough to form a hydrophobic interaction with l-phosphoserine (Fig. 3F), whereas the orientation of the side chain of Ile²¹¹ in iPSP1 (H9A) complexed with l-phosphoserine was unclear because no electron density was observed for Ile²¹¹ in iPSP1 (H9A). The residue corresponding to Ile²¹¹ was absent in the PspB subunit and PhoE, whose C termini were seven and five residues shorter, respectively, than that of PspA (Fig. 1A), suggesting that the C-terminal residues of PspA, PspB, and PhoE interact differently with the bound substrate. Thus, the C-terminal residues of the PspA subunit were also suggested to be important for the PSP activity of iPSP1.

Kinetic Analyses of iPSP1 Mutants

The kinetic parameters of several iPSP1 mutants were determined and compared with those of iPSP1 (WT) to confirm the validity of the residues suggested to be important for the PSP activity based on the crystal structure analyses. The iPSP1 mutants, iPSP1 (H85A), iPSP1 (ΔC1), and iPSP1 (ΔC7), were purified by the same method as used for iPSP1 (WT), and their activities were then assayed using 0.5–20 mm l-phosphoserine (Table 6 and Fig. 4). iPSP1 (H85A) exhibited a markedly decreased affinity to l-phosphoserine (K_m > 20 mm) when compared with iPSP1 (WT) (K_m = 1.5 mm). In contrast, iPSP1 (ΔC1) displayed a drastically decreased V_max (9.5 units mg protein⁻¹) when compared with iPSP1 (WT) (217 units mg protein⁻¹), whereas its K_m was almost the same level as that of iPSP (WT). iPSP1 (ΔC7) showed the lowest PSP activity of the three examined mutants, displaying higher K_m and lower V_max values than iPSP (WT). The PSP activities of iPSP1 (H85A), iPSP1 (ΔC1), and iPSP1 (ΔC7) in the presence of 5 mm l-phosphoserine (15 ± 0.1, 5.7 ± 0.1, and 1.3 ± 0.1 units mg protein⁻¹, respectively) were markedly lower than that of iPSP1 (WT) (173 ± 9 units mg protein⁻¹). These results confirmed that both His⁸⁵ and the C-terminal residues were critical for the PSP activity of iPSP1.

TABLE 6.

Kinetic parameters for wild-type and mutant iPSP1

iPSP1	Km	Vmax	kcat/Km
	mm	units mg−1	s−1 mm−1
WT	1.5 ± 0.1	217 ± 5	98
H85A	≥20	173 ± 52
ΔC1	3.3 ± 0.1	9.5 ± 0.1	2.0
ΔC7	15.2 ± 1.4	5.2 ± 0.3	0.24

FIGURE 4.

Kinetic plots of iPSP1 and its mutants for l-phosphoserine.

Detection of PSP Activity in iPSP Homologs

Proteins with high amino acid sequence similarity to the PspA subunit of iPSP1, namely TLR1532 from T. elongatus and RCAS0170, RCAS2324, and RCAS3445 from R. castenholzii (Fig. 1A), were expressed in E. coli BL21 (DE3) and subjected to PSP activity assays. TLR1532, RCAS2324, and RCAS3445 were obtained in the soluble fraction, whereas RCAS0170 formed inclusion bodies. PSP activity was detected in the cell-free extracts of TLR1532- and RCAS2324-expressing E. coli but was not observed in that of RCAS3445-expressing E. coli. Partially purified TLR1532 and RCAS2324 (8.9- and 2.3-fold purified, respectively, from E. coli cell-free extract; Fig. 2) showed PSP activities of 63 ± 1 and 9.8 ± 1 units mg protein⁻¹, respectively, suggesting that these proteins possessed substantial PSP activities.

DISCUSSION

In this study, we solved the crystal structures of the substrate-free and -bound forms of iPSP1 from H. thermophilus. An inactive mutant iPSP1 (H9A) was used for the structural analysis of the bound form. Moreover, we identified that two structural units are necessary for the PSP activity of iPSP1 based on both structural and functional data.

His⁸⁵ in the PspA subunit was not conserved in the PspB subunit nor in PhoE and was found to be critical for the high PSP activity of iPSP1, because iPSP1 (H85A) possesses only 8.7% of the PSP activity of iPSP1 (WT) in the presence of 5 mm l-phosphoserine. His⁸⁵ very likely contributes to substrate affinity, as iPSP1 (H85A) has a markedly increased K_m value compared with that of iPSP1 (WT). The successful determination of the crystal structure of iPSP1 (H9A)·l-phosphoserine has demonstrated that the N^ϵ2 atom of His⁸⁵ forms a hydrogen bond with the nitrogen of l-phosphoserine and allows us to speculate on the molecular mechanism of l-phosphoserine recognition by iPSP1. The imidazole group of His⁸⁵ is perpendicular to the aromatic ring of Phe¹¹⁶, which is suggested to form an aromatic hydrogen bond with hydrogenated N^δ1 of His⁸⁵ (28). In addition, as the amino group of l-phosphoserine was positively charged (−NH₃⁺) around physiological pH values (29, 30), the nonhydrogenated N^ϵ2 of His⁸⁵ may be appropriately oriented for recognizing the amino group of l-phosphoserine by the interaction between the hydrogenated N^δ1 of His⁸⁵ and the side chain of Phe¹¹⁶. It is possible that the PspB subunit in iPSP2 lacks iPSP activity due to the replacement of His⁸⁵ with Phe⁸⁵ in this subunit, because the side chain of Phe⁸⁵ cannot form a hydrogen bond with the amino group of l-phosphoserine.

The importance of His⁸⁵ for substrate affinity also sheds light on the stereo selectivity of iPSP1. Our previous study revealed that iPSP1 has extremely low activity toward d-phosphoserine (1). This property can be explained by the formation of hydrogen bonds between the amino group of l-phosphoserine and the side chain of His⁸⁵, and the carboxyl group of l-phosphoserine and the main-chain amides in Gln²¹ and Gly²², which are considered to contribute to substrate binding. When d-phosphoserine is superimposed onto the PspA (H9A)·l-phosphoserine structure by fitting the α-carbon and carboxyl groups of l- and d-phosphoserine, the amino group of d-phosphoserine cannot form a hydrogen bond with the side chain of His⁸⁵ or any other residue (Fig. 3G). Therefore, the affinity of PspA to d-phosphoserine is expected to be lower than that to l-phosphoserine, resulting in a lower phosphatase activity to d-phosphoserine. This explanation is supported by the fact that iPSP1 also shows no activity toward 3-phosphoglyceric acid, in which the amino group of d-phosphoserine is substituted by a hydroxyl group (1).

The C-terminal region of the PspA subunit was also revealed to affect the PSP activity of iPSP1, because ΔC1 and ΔC7 mutants of iPSP1 have drastically reduced activities toward l-phosphoserine as a target substrate. The deletion of the C-terminal Ile²¹¹ residue in iPSP1 (ΔC1), which could interact with l-phosphoserine in the active cleft, clearly reduced V_max but did not significantly affect K_m values. These results suggest that Ile²¹¹ contributes to the PSP activity of iPSP1 by stabilizing the transition state or increasing the pre-exponential factor of the reaction. However, it remains unclear whether the deletion of a single C-terminal residue at position 211 or the substitution of it by a residue without a hydrophobic side chain can cause the drastic decrease of PSP activity. Although further study is needed, we speculate that not only the length of the C terminus but the hydrophobicity of the side chain may influence the activity of iPSPs because the C-terminal residues of all proteins from which substantial levels of PSP activity have been detected and iPSP1 orthologs from Aquificae (1) are Ile or Leu.

The C-terminal regions of histidine phosphatase superfamily proteins, including dPGM and fructose-2,6-bisphosphatase, have been studied because these regions protrude over the active-site cleft and are presumed to affect substrate selectivity (8, 31–33). Several C-terminal residues in iPSP1 also form a lid that covers the active site cleft, as shown in the wild-type structure (Fig. 5). This lid is expected to have a certain degree of flexibility, as in the case of dPGM (34), because the entrance to the active site of iPSP1 (WT) is too small for l-phosphoserine (Fig. 5) and the B-factor of the C terminus is relatively high. The C-terminal lid of iPSP1 likely functions to hold l-phosphoserine in the active site, as the K_m value of iPSP1 (ΔC7), in which the lid is completely lost and the active site cleft is fully exposed to the solvent (Fig. 5), was increased ∼10-fold when compared with that of iPSP1 (WT). It has been reported that deletion of the C-terminal region (residues 235–246) of dPGM from Saccharomyces cerevisiae reduces the mutase activity but has no effect on phosphatase activity (31, 32). However, the C-terminal length of yeast dPGM was found to be 12 residues longer than that of PspA (data not shown). In addition, the phosphatase activity of dPGM is only a side reaction and is considerably lower than that of iPSP. Therefore, there is no conflict between the contribution of the C termini of dPGMs and iPSPs on their phosphatase activities.

FIGURE 5.

Entrance to the catalytic site of iPSP1 and its homologs. The structures of the PspA subunit (chain A of iPSP1 (WT)), modeled PspB, and PhoE were aligned and compared from the same angle. l-Phosphoserine was superimposed on each structure at the orientation determined using iPSP1 (H9A)·l-phosphoserine (chain A). The structures of PspA (ΔC1) and PspA (ΔC7) were constructed using the structure of PspA (WT). Images were generated with PyMOL.

The active site of the PspB subunit in iPSP2 is predicted to be wide open and to lack a lid based on the homology model constructed here using the crystal structure of the PspA subunit in iPSP1 (WT) as the template, because the C terminus of the PspB subunit is seven residues shorter than that of the PspA subunit (Fig. 5). The molecular model of the PspB subunit helps explain why PspB lacks PSP activity and has stronger phosphatase activity toward p-nitrophenyl phosphate than PspA (1); the wider entrance of the active-site pocket of PspB enables access to larger substrates, as in the case of PhoE (Fig. 5) (8).

Interestingly, dPGM-like proteins from both Cyanobacteria and Chloroflexi (TLR1532 and RCAS2324, respectively, which contain conserved histidine residues corresponding to His⁸⁵ of iPSP1) exhibit substantial PSP activity. In contrast, dPGM-like proteins that lack this conserved histidine residue (PspB from H. thermophilus, RCAS3445, TTHA0368, At5g04120, and At3g05170 (1)) possess no activity or very weak PSP activity. Moreover, the substrate selectivity of PhoE, in which His⁸⁵ is not conserved, markedly differs from that of iPSP1, although the PSP activity of PhoE has not been tested (1, 8). Although PhoE and dPGM-like proteins with low PSP activity (TTHA0368 and At5g04120) have a histidine residue at the position corresponding to Asp⁸⁴ (adjacent to His⁸⁵) of iPSP1, the crystal structure of PhoE (PDB code 1H2E) shows that the imidazole ring of this histidine residue is oriented in the opposite direction of the active core. Based on this finding, it is unlikely that the histidine residue at position 84 of PhoE, or other similar dPGM-like proteins, can form hydrogen bonds with the substrate.

We also determined that the C-terminal regions of TLR1532 and RCAS2324, which have high PSP activities, are similar to that of iPSP1 (Fig. 1B). These proteins contain conserved “-¹⁹⁹HLG²⁰¹-” and “-²¹⁰A(I/L)²¹¹-COOH” (PspA subunit numbering) sequences that are connected by a loop consisting of 8–10 residues that are not conserved (Fig. 1). Homology models of TLR1532 and RCAS2324 suggest that the interactions between these conserved sequences and neighboring residues are similar to those that occur in iPSP1 (Fig. 6). In addition, the connecting loops are bound to the entrance region of the active sites by a comparable number of interactions as those identified in iPSP1, even though the sequences of the loops are not conserved. Therefore, the C-terminal Leu residue of TLR1532 and RCAS2324 will also be able to thrust into the active-site cleft in the same manner as the Ile residue of iPSP1. These observations strongly suggest that the C termini of dPGM-like proteins from Cyanobacteria and Chloroflexi also interact with the substrate, product, and/or the reaction intermediates in the active site and are important for PSP activity.

FIGURE 6.

C-terminal lids of iPSP1 (A), TLR1532 (B), and RCAS2324 (C).

The amino acid sequence identities of TLR1532 and RCAS2324 with the PspA subunit in iPSP1 are 32 and 36%, respectively, although that between the PspA and PspB subunits is 35%. The similar levels of sequence identity despite the differences in substrate specificity highlight the difficulty in predicting high PSP activity from the primary structure of a dPGM-like protein. However, our present findings indicate that the presence of His⁸⁵ and the characteristic C-terminal sequence and/or length are useful indicators of high PSP activity of a dPGM-like protein. Using these indicators, we can predict that several Firmicutes species, in addition to most Cyanobacteria and Chloroflexi species, which lack conventional dPSP gene(s) (1), possess iPSP gene(s).

The identification of iPSP gene(s) is important for understanding the serine and glycine anabolic pathways that function in organisms lacking dPSP gene(s) (1). For example, nearly all Cyanobacteria species have an iPSP gene instead of a dPSP gene and synthesize serine from l-phosphoserine as indicated by ¹⁴C-labeling analysis (35). Therefore, iPSP may actually function as a serine-synthesizing enzyme in Cyanobacteria.

In conclusion, the crystal structures of iPSP1 (WT) and (H9A)·l-phosphoserine from H. thermophilus determined in this study and the kinetic analyses of mutated proteins have revealed that the presence of a His residue at position 85 and the sequence and/or length of the C-terminal region are both necessary for the PSP activity of iPSP1. These two structural features can be used as indicators to identify dPGM-like proteins with high PSP activity.