Formiminoglutamase from Trypanosoma cruzi is an arginase-like manganese metalloenzyme

Abstract

The crystal structure of formiminoglutamase from Trypanosoma cruzi (TcFIGase) is reported at 1.85 Å resolution. Although the structure of this enzyme was previously determined by the Structural Genomics of Pathogenic Protozoa Consortium (PDB accession code 2A0M), this structure was determined at low pH and lacked bound metal ions; accordingly, the protein was simply annotated as "arginase superfamily protein" with undetermined function. We show that reconstitution of this protein with Mn²⁺ confers maximal catalytic activity in the hydrolysis of formiminoglutamate to yield glutamate and formamide, thereby demonstrating that this protein is a metal-dependent formiminoglutamase. Equilibration of TcFIGase crystals with MnCl₂ at higher pH yields a binuclear manganese cluster similar to that observed in arginase, except that the histidine ligand to the Mn²⁺(A) ion of arginase is an asparagine ligand (N114) to the Mn²⁺(A) ion of TcFIGase. The crystal structure of N114H TcFIGase reveals a binuclear manganese cluster essentially identical to that of arginase, but the mutant exhibits a modest 35% loss of catalytic efficiency (k(cat)/K(M)). Interestingly, when TcFIGase is prepared and crystallized in the absence of reducing agents at low pH, a disulfide linkage forms between C35 and C242 in the active site. When reconstituted with Mn²⁺ at higher pH, this oxidized enzyme exhibits a modest 33% loss of catalytic efficiency. Structure determinations of the metal-free and metal-bound forms of oxidized TcFIGase reveal that although disulfide formation constricts the main entrance to the active site, other structural changes open alternative channels to the active site that may help sustain catalytic activity.

Figure 1

Pathways I–III for L-histidine catabolism. Formiminoglutamase catalyzes the final step in histidine utilization pathway I.

Figure 2

(a) Stereoview of the Mn²⁺₂-TcFIGase monomer. Secondary structure elements are defined by DSSP; α-helices are red and β-strands are blue (β strands 1–8) or purple (β-strands βA and βB). The Mn²⁺ ions are shown as white spheres. (b) Top view (left) and side view (right) of the Mn²⁺₂-TcFIGase trimer.

Figure 3

(a) Bijvoet difference Fourier map (grey, contoured at 5σ) of Mn²⁺ ions and simulated annealing omit map (blue, contoured at 4σ) of Mn²⁺-bound solvent molecules in the active site of Mn²⁺₂-TcFIGase (pH 8.0). Atoms are color-coded as follows: C = white, N = blue, O = red, Mn²⁺ = pink spheres, solvent = red spheres. Metal coordination and hydrogen bond interactions are represented by blue and green dashed lines, respectively. Note that the side chain thiol group of C242 is disordered between two positions. (b) Superposition of Mn²⁺₂-TcFIGase (color coded as in (a)) and apo-TcFIGase (PDB entry 2A0M, color coded as in (a) except that C = yellow). (c) Superposition of Mn²⁺₂-TcFIGase (color coded as in (a)) and H101N rat arginase I (PDB entry 1P8P, tan). TcFIGase and rat arginase I residue labels are black and red, respectively.

Figure 4

(a) Simulated annealing omit maps of Mn²⁺ ions (grey, contoured at 3σ) and metal-bound solvent molecules (purple, contoured at 5σ) in the active site of N114H Mn²⁺₂-TcFIGase (pH 7.5). Atoms are color-coded as follows: C = white, N = blue, O = red, Mn²⁺ = pink spheres, solvent = red spheres. Metal coordination and hydrogen bond interactions are represented by blue and green dashed lines, respectively. Note that the side chain thiol group of C242 is disordered between two positions. (b) Superposition of N114H Mn²⁺₂-TcFIGase (color coded as in (a)) and wild-type Mn²⁺₂-TcFIGase (color coded as in (a) except that C, Mn²⁺ = cyan).

Figure 5

(a) Bijvoet difference Fourier map (grey, contoured at 3σ) of Mn²⁺ ions and simulated annealing omit map (purple, contoured at 3σ) of Mn²⁺-bound solvent molecules in the active site of wild-type Mn²⁺₂-TcFIGase_ox (pH 6.0). Atoms are color-coded as follows: C = white, N = blue, O = red, Mn²⁺ = pink spheres, solvent = red spheres. Metal coordination and hydrogen bond interactions are represented by blue and green dashed lines, respectively. (b) Superposition of the Mn²⁺₂-TcFIGase_ox monomer (pH 6.0, purple) and the Mn²⁺₂-TcFIGase monomer (white); dotted lines indicate disordered polypeptide segments. Major conformational changes occur for helix A1 (dark pink (oxidized state), cyan (reduced state)) and the P146-S154 loop in response to disulfide bond formation.

Figure 6

(a) Structural changes in active site access triggered by disulfide bond formation in Mn²⁺₂-TcFIGase. The original active site entrance is indicated by a green surface (left image). Following disulfide bond formation in Mn²⁺₂-TcFIGase_ox, this entrance becomes constricted and two new entrances (blue and red surfaces) become accessible (right image). Selected residues lining the entrance surface are shown as stick figures and Mn²⁺ ions are shown as pink spheres. Active site entrance surfaces were calculated using MOLE. (b) Cartoon representation of Mn²⁺₂-TcFIGase (pH 8.0, left) and Mn²⁺₂-TcFIGase_ox (pH 8.5, right) showing the atomic displacement parameters coded by thickness of the main chain and a color gradient from blue (low disorder) to red (high disorder). The red dotted line indicates the disordered P146-S154 loop.

Figure 7

(a) Close-up view of active site from the superposition of Mn²⁺₂-TcFIGase_ox (C = white) with Mn²⁺₂-TcFIGase (C = cyan). Metal coordination and hydrogen bond interactions in Mn²⁺₂-TcFIGase_ox are represented by red and green dashed lines, respectively. (b) Simulated annealing omit maps (purple, contoured at 3σ) of Mn²⁺ ions and Mn²⁺-bound solvent molecules observed in Mn²⁺₂-TcFIGase_ox soaked in a buffer solution containing 20 mM MnCl₂ at pH 8.5. Atoms are color-coded as follows: C = white, N = blue, O = red, Mn²⁺ = pink spheres, solvent = red spheres. Metal coordination and hydrogen bond interactions are represented by blue and green dashed lines, respectively. Note the reduced Mn²⁺_A occupancy and conformational change of former Mn²⁺_A ligand D142.

Figure 8

Sequence alignment of TcFIGase with FIGase enzymes identified in different bacteria. Sequences were aligned using Clustal Omega and displayed with Jalview. Highly conserved active site residues (identity ≥ 90%) are highlighted in red. Moderately conserved active site residues (identity < 90%) are highlighted in green. C35 and C242 (which can form a disulfide linkage in T. cruzi) and their equivalent residues in other species are highlighted in cyan. Other conserved residues (identity > 90%) are highlighted in blue. Uniprot accession codes for aligned sequences are as follows: Trypanosoma cruzi (Q4DSA0), Nocardia farcinica (Q5Z0G1), Macrococcus caseolyticus, (B9E7J8), Pseudomonas aeruginosa (Q9HZ59), Staphylococcus aureus (P99158), Salmonella typhi (Q8Z899), Klebsiella pneumonia (B5XZ82), Bacillus subtilis (P42068), Rhodococcus opacus (C1BBD1), Ralstonia solanacearum (Q8XW30), Vibrio cholera (Q9KSQ2), Psychrobacter cryohalolentis (Q1Q9E3), Photobacterium profundum (Q6LQ58).

Figure 9

Dependence of TcFIGase activity on metal ions. Metal-free apoenzyme was prepared by depleting the metal ions at low pH using wild-type enzyme expressed and purified from LB media. Metal-substituted TcFIGase was reconstituted with two equivalents metal ions and assayed for activity using 10 mM substrate. Activity was compared in terms of initial velocity.

Figure 10

Dependence of TcFIGase catalysis on metal ion stoichiometry. Metal-free enzyme (20 μM) was incubated with increasing molar equivalents (eq) of divalent metal ions in assay buffer at 4 °C for 30 minutes. The reaction was initiated by addition of 1 mM substrate and monitored as described in the text. Maximal catalysis requires ca. 2 Mn²⁺ ions per monomer.

Figure 11

Hypothetical model of substrate binding in TcFIGase. One water molecule originally in the active site is deleted and the conformations of R144 and C242 are manually adjusted to accommodate the substrate. Metal coordination interactions are shown as blue dashed lines and hydrogen bonds are indicated by green dashed lines. The Bürgi–Dunitz trajectory for nucleophilic attack by the metal-bridging hydroxide ion at the substrate imino group is indicated by a black dashed line.

Figure 12

Proposed catalytic mechanism for Mn²⁺₂-TcFIGase.