The TM1246 gene (smPurL) of Thermotoga maritima encodes a phosphoribosylformylglycinamidine synthase II (FGAM; EC 22.214.171.124) with a molecular weight of 65,834 Da (residues 1–603) and a calculated isoelectric point of 5.38. This enzyme is part of the de novo purine biosynthesis subsystem, where it forms a complex with purS (TM1244) and purQ (TM1245) to form formylglycinamide ribonucleotide amidotransferase (FGAR-AT). FGAR-AT catalyzes the adenosine 5′-triphosphate (ATP)-dependent conversion of FGAR and glutamine to formylglycinamidine ribonucleotide (FGAM), adenosine 5′-diphosphate (ADP), Pi, and glutamate in the fourth step of the purine biosynthetic pathway (EC 126.96.36.199).1–3 In Gram-positive bacteria, archaebacteria, and the Gram-negative T. maritima, FGAR-AT is a complex of three proteins: PurS, PurL (designated as smPurL), and PurQ. In eukaryotes and other Gram-negative bacteria, FGAR-AT is a multidomain protein with a molecular mass of about 140 kDa (designated as lgPurL). Based on iterative sequence similarity searches, it has been proposed that smPurL together with lgPurL, aminoimidazole ribonucleotide synthetase (PurM), Ni-Fe hydrogenase maturation protein (HypE), selenophosphate synthetase (SelD), and thiamine monophosphate kinase (ThiL) form a new structural superfamily of ATP-dependent enzymes.4 The structures of FGAR-AT (lgPurL) from Salmonella typhimurium [Protein Data Bank (PDB) 1t3t], PurM protein from Escherichia coli (PDB 1cli), and ThiL from Aquifex aeolicus (PDB 1vqv) are known.4–6 Herein, we report the crystal structure of TM1246 (smPurL), determined using the semiautomated, high-throughput pipeline of the Joint Center for Structural Genomics (JCSG).7
The crystal structure of TM1246 [Fig. 1(A)] was determined to 2.15 Å resolution using the multi-wavelength anomalous dispersion (MAD) method. Data collection, model, and refinement statistics are summarized in Table I. The final model includes one monomer (residues 2–186, 203–603), one chloride ion, and 221 water molecules in the asymmetric unit. The Matthews' coefficient (Vm)8 for TM1246 is 2.14 Å3/Da and the estimated solvent content is 42.0%. A main-chain torsion angle analysis by the program MolProbity9 shows that 97% and 100% of the residues are in the favored and allowed regions of the Ramachandran plot, respectively.
|Unit cell parameters||a = 59.78 Å, b = 72.69 Å, c = 128.42 Å|
|Resolution range (Å)||63.26–2.15||30.18–2.20||30.18–2.20|
|No. of observations||91,420||93,064||89,540|
|No. of reflections||28,272||27,791||27,151|
|Completeness (%)||90.8 (56.8)a||95.4 (72.7)a||93.2 (64.4)a|
|Mean I/σ(I)||10.2 (1.3)a||(8.9)(1.4)a||10.3 (1.6)a|
|Rsym on I||0.09 (0.55)a||0.11 (0.49)a||0.09 (0.46)a|
|Highest resolution shell (Å)||2.21–2.15||2.26–2.20||2.26–2.20|
|Model and refinement statistics|
|Resolution range (Å)||63.26–2.15||Data set used in refinement||λ0MADSe|
|No. of reflections (total)||28,237||Cutoff criteria|||F| > 0|
|No. of reflections (test)||1,368||Rcryst||0.186|
|Completeness (% total)||90.6||Rfree||0.254|
|Deviation from ideal geometry (rms)|
|Bond length||0.012 Å|
|Average isotropic B-value protein||24.9 Å2|
|Average isotropic B-value ions||51.1 Å2|
|Average isotropic B-value water||32.4 Å2|
|ESU based on R value||0.31 Å|
The TM1246 monomer contains 25 β-strands (β1–β25) in four β-sheets (A, B, C, E), one β-hairpin (D), 18 α-helices (H1–H7, H10–H11, H13, H15–H17, H19–H21, H23–H24), and seven 310-helices (H8–H9, H12, H14, H18, H22–H23′) [Fig. 1(A,B)]. The total β-strand, α-helical, and 310-helical content is 27.6, 31.7, and 3.1%, respectively. TM1246 comprises four α+β domains [Figs. 1(A) and 2(A)]. The first (A1: 2–166) and third domains (A2: 362–507) are related by pseudo-twofold symmetry and pack against each other to form the central structural unit of TM1246 [Fig. 2(A)]. These domains adopt a two-layered α+β fold whose core is composed of a mixed four-stranded β-sheet (strand order 1423) and two α-helices arranged in a βαβαββ motif. These domains resemble the N-terminal domain of PurM and belong to the “Bacillus chorismate mutase-like” fold.10 The other domains of smPurL (B1: 167–345 and B2: 508–603) have a curved β-sheet and adopt a three-layered α+β fold composed of a four-stranded antiparallel β-sheet and three α-helices arranged in a βαβαβαβ motif. This fold has some resemblance to the ferredoxin fold and has been classified in the SCOP database10 under the “PurM C-terminal domain-like” fold. A linker peptide (residues 346–361) connects the two PurM-like units [Figs. 1(A) and 2(A)]. The domains of smPurL are likely a result of a tandem duplication of an ancestral PurM-like subunit and are arranged like the PurM dimer (A1B1-A2B2) [Figs. 1(A) and 2(A)].
The crystallographic packing of the TM1246 structure, as well as analytical size exclusion chromatography in combination with static light scattering, indicates that a monomer is the biologically-relevant form. A search performed with the coordinates of TM1246 using the DALI server11 showed structural similarity to residues 183–960 of formylglycinamide synthetase from S. typhimurium (PDB: 1t3t) (Z = 28.6).6 The root-mean-square deviation (RMSD) for this structural alignment is 2.8 Å over 553 aligned Cα atoms with 21% sequence identity [Fig. 2(B)]. The smPurL is also homologous to the structures of PurM and ThiL.4–6, 12 The structural alignment of PurM from E. coli4 (PDB: 1cli) with the N-terminal domains of smPurL superimposes 229 Cα atoms with an RMSD of 3.2 Å, whereas the ThiL protein from Aquifex aeolicus (PDB: 1vqv) can be aligned over 210 Cα atoms with an RMSD of 3.6 Å. Because TM1246 represents the first structure of a smPurL, it is interesting to compare it with the lgPurL structure. Although the overall fold between TM1246 and the FGAM synthetase domain of lgPurL is very similar, two large insertions are found in the lgPurL structure [Fig. 2(A,B)]. These insertions, which occur on A1 and B2, are placed in close structural proximity, creating a new surface on one side of the enzyme [Fig. 2(A,B)]. The very compact nature of the T. maritima enzyme structure seems to suggest that this represents a minimal version of the FGAM synthetase domain.
The active site of the smPurL from T. maritima was inferred from sequence and structural comparison to the lgPurL from S. typhimurium.6 The putative active site is located in the cleft formed by the “Bacillus chorismate mutase-like” fold and the succeeding “PurM C-terminal domain-like” fold [Figs. 2(B) and 3(A)]. Unlike lgPurL, which binds two sulfate ions in its putative active site, the smPurL structure does not contain any bound ligands. The secondary structural elements around the active site of smPurL superimpose very well with the lgPurL structure. Seven of the nine residues that interact with the sulfate ions in the active site region of lgPurL are conserved in smPurL and adopt similar side-chain conformations [Fig. 3(A)].
The lgPurL structure contains an auxiliary ADP-binding site that is related to the active site by pseudo- twofold symmetry [Figs. 2(B) and 3(B)]. The sequence and structural conservation at this region is less pronounced than for the putative active site. Recent biochemical studies on the Bacillus subtilis smPurL have indicated that ADP is required for the assembly of the PurSLQ complex.5 However, in the TM1246 structure, no ADP or bound anions are observed at this site. Of the five highly conserved residues of the lgPurL protein family that are involved in interactions with the ADP moiety and the Mg2+ ions (K649, E718, N722, D884, and D887 of PDB 1t3t), only E425 (E718 in lgPurL) is conserved in TM1246 [Fig. 3(B)]. None of the residues that are involved in forming the hydrophobic pocket in lgPurL are conserved in TM1246, although these regions are structurally similar. The structural differences occur mainly around the regions that accommodate the base ring and the sugar moiety of ADP.
smPurL contains a glycine-rich loop that is structurally disordered (residues 187–202) and is located close to the active site [Fig. 3(A)]. Interestingly, the equivalent region of lgPurL (448–466) is also disordered and is positioned to cover the active site upon binding of the ATP moiety. It is likely that this loop will become ordered in the ATP-bound form of the enzyme.
As noted before, the lgPurL structure has two extra domains as compared with the smPurL structure: an N-terminal domain of unknown function [Fig. 2(A,B), colored green] and a C-terminal glutaminase domain [Fig. 2(A,B), colored red]. The structural study of lgPurL revealed that the N-terminal domain of unknown function is structurally homologous to a dimer of PurS.6 It is likely that the PurS and PurQ domains of T. maritima (TM1244 and TM1245) occupy similar positions to the additional N- and C-terminal domains of lgPurL [Fig. 2(A,B)].
smPurL is the last remaining enzyme in the purine biosynthetic pathway to have its structure determined. The smPurL family contains hundreds of sequence homologs. Models for TM1246 homologs can be accessed at http://www1.jcsg.org/cgi-bin/models/get_mor.pl?key=TM1246.
The crystal structure of TM1246 represents a smPurL protein. The information reported herein, in combination with the structure of lgPurL and further biochemical and biophysical studies, will yield valuable insights regarding the role of this protein in purine biosynthesis.