Crystal structure of glutamine amidotransferase from <i>Thermotoga maritima</i>

Glutamine amidotransferases (GATases) are ubiquitous enzymes that transfer the amide nitrogen of glutamine to a variety of substrates.1 GATases catalyze two separate reactions at two active sites, which are located either on a single polypeptide chain or on different subunits. In the glutaminase reaction, glutamine is hydrolyzed to glutamate and ammonia, which is added to an acceptor substrate in the synthase reaction. Imidazole glycerol phosphate synthase (IGPS) is a GATase that catalyzes the bifurcation step of the histidine and de novo purine biosynthetic pathways. In yeast, IGPS is a single polypeptide,2 whereas in bacteria IGPS activity is effected by a complex of a glutaminase subunit, HisH, and a synthase subunit, HisF.3, 4 IGPS is a class I GATase, which is characterized by a Cys–His–Glu catalytic triad. IGPS is a potential target for antibiotic and herbicide development because the histidine pathway does not occur in mammals. Here, we present the crystal structure of the HisH protein from Thermotoga maritima (TmHisH), which complements the known structure of HisF from the same organism (TmHisF)5 [Protein Data Bank (PDB) ID 1THF] and HisF from Pyrobaculum aerophilum6 (PDB ID 1H5Y) and shows high structural similarity with the glutaminase domain of the recently determined structure of IGSP from yeast7 (HIS7) (PDB ID 1JVN). TmHisH was subcloned and the recombinant protein expressed, purified, and the intitial crystal trials performed as previously described.8 Diffraction-quality crystals of selenomethionine-labeled protein were obtained from sitting drop vapor diffusion set-up containing 2 μL protein plus 2 μL precipitant in 2.0 M monoammonium dihydrogen phosphate and 100 mM Tris HCl (pH 8.5) over 2–5 days at 21°C. The crystals were flash-frozen with the crystallization buffer with increased concentration of precipitant (2.6 M) and 10% ethylene glycol. TmHisH crystallized in the space group P3221 with unit cell parameters a = b = 82.0 Å and c = 176.4 Å and contains two subunits (46 kDa) in the asymmetrical unit. multiwavelength anomalous diffraction (MAD) data was collected at beam line 19ID of the Structural Biology Center (SBC) at the Advanced Photon Source (APS), Argonne National Laboratory (ANL). Ten Se peaks were found using the Shake-and-Bake program,9 phases were calculated with SOLVE at 2.8-Å resolution and subjected to the program RESOLVE,10 which automatically detected the non-crystallographic symmetry (NCS) matrix, and density modification was performed with NCS averaging and autotraced 79% of the main chain. The rest of the structure was built manually using the program O.11 The final model includes all residues together with two extra residues left at the N-terminus after His-tag cleavage. There was no density for two C-terminal residues of chain B and they were omitted from the final model. Six phosphate molecules were modeled corresponding to the strongest peaks in the electron density map. No water molecules were modeled because of the limited resolution. Statistics of data collection and refinement are shown in Table I. Atomic coordinates and structure factors have been deposited into the PDB with ID 1KXJ. The TmHisH structure has an α-β-α fold typical for other glutaminase structures. The two molecules in the asymmetrical unit have similar structures; the root mean square (RMS) deviation between 200 Cα atoms is 0.45 Å. The structures were almost identical near the active site part of the globule. The loop joining residues 39–41 was in particular flexible, with different conformations in two subunits (maximum distance of 4.4 Å between Cα atoms of N40). The TmHisH structure is similar to that of the glutaminase domain of yeast HIS7 protein. The two proteins exhibit an RMS deviation of 1.9 Å over 192 Cα atoms, with a high degree of structural similarity in the vicinity of the active center. The residues comprising the catalytic triad in TmHisH, C84–H178–E180, correspond to the catalytic triad of His7, C83–H193–E195. The TmHisH residues G52, Q88, E96, T142, and Y142 have similar conformation with corresponding residues G51, Q87, E95, S148, and F149 of His7, which are involved in interaction with acivicin, a glutamine analog covalently attached to C83 (Fig. 1). The catalytic C84 has well-defined electron density in both subunits of TmHisH. It has a backbone conformation in the forbidden region of Ramachandran plot, and this is identical to the conformation of C84 of His7. Unlike in His7, C83 Sγ is rotated toward Hi78 and is in similar orientation with Sγ of S86 of GMP synthase.12 There is no analog of His7 α-helix 153–165 in TmHisH. The N-terminal parts of the first two β-strands are twisted in a way that N-terminus of His7 hβ1 overlaps with the N-terminus of TmHisH β2. Close-up stereo view of active sites residues of TmHisH and TmHisF superimposed with His7. Residues are shown in stick representation with nitrogens, oxygens, and sulfurs shown in blue, red, and yellow, respectively; carbons of His7 residues are shown in gray, carbons of TmHisH are shown in cyan, and carbons of TmHisF are shown in green. TmHisH C84 and His7 C83 with covalent adduct and phosphate (phosphor is shown in orange) are shown in thicker sticks. Labels are shown only for TmHisH and TmHisF residues. 3D structural alignment and all figures were performed with the program ICM.14 The phosphate molecules were found in active sites of each subunit and their positions correspond to the position of the α-COO− group of acivicin in His7. They are coordinated by interactions with amide nitrogens of T142, Y143, and NE2 of Q88. Superposition of TmHisH and TmHisF5 with the His7 structure (Fig. 2) also brings Q123 NE2 of TmHisF in contact with the phosphate similarly to interaction of Q397 of His7 cyclase domain with the glutaminase active center. Stereo view of structural comparison of TmHisH (blue ribbon) and TmHisF (orange ribbon) with His7 (yellow ribbon). One of the features among enzymes with catalytic triads is an “oxyanion hole,” which stabilizes the negative charge during the hydrolytic reaction. In triad amidotransferases, an oxyanion hole is composed of the two amide nitrogens, one from the residue following nucleophile and the second from an adjacent β-strand, referred to as the “oxyanion strand.” In TmHisH, this strand (residues 45–52) is in identical conformation in both subunits and in the His7 structure. Interestingly, the carbonyl (but not amide) group of G50 is oriented toward the active site; thus, the oxyanion hole is lacking one of the amide nitrogens near the active center. HisH is activated by complex formation with HisF, to which either the product imidazole glycerol phosphate or a substrate analog must be bound. The structure of HisH revealed a high degree of structural similarity with the glutaminase domain of the yeast IGPS (His7), which also was crystallized in an inactive state. Presumably, considerable conformation changes must be transmitted through the cyclase domain of His7 (or HisF) upon substrate binding. A model of the TmHisH–TmHisF complex based on superposition of both structures with His7 revealed that the intersubunit interactions and therefore the mechanism of signaling between the two prokaryotic enzymes (HisH and HisF) is likely similar to the interactions between two subunits of single polypeptide chain of yeast IGPS. The authors thank all members of the SBC at ANL for their help in conducting experiments and Lindy Keller for help in preparation of this article. This work was supported by the Ontario Research and Development Challenge Fund.

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