N5-CAIR mutase: role of a CO2 binding site and substrate movement in catalysis

Abstract

N5-Carboxyaminoimidazole ribonucleotide mutase (N5-CAIR mutase or PurE) from Escherichia coli catalyzes the reversible interconversion of N5-CAIR to carboxyaminoimidazole ribonucleotide (CAIR) with direct CO2 transfer. Site-directed mutagenesis, a pH-rate profile, DFT calculations, and X-ray crystallography together provide new insight into the mechanism of this unusual transformation. These studies suggest that a conserved, protonated histidine (His45) plays an essential role in catalysis. The importance of proton transfers is supported by DFT calculations on CAIR and N5-CAIR analogues in which the ribose 5'-phosphate is replaced with a methyl group. The calculations suggest that the nonaromatic tautomer of CAIR (isoCAIR) is only 3.1 kcal/mol higher in energy than its aromatic counterpart, implicating this species as a potential intermediate in the PurE-catalyzed reaction. A structure of wild-type PurE cocrystallized with 4-nitroaminoimidazole ribonucleotide (NO2-AIR, a CAIR analogue) and structures of H45N and H45Q PurEs soaked with CAIR have been determined and provide the first insight into the binding of an intact PurE substrate. A comparison of 19 available structures of PurE and PurE mutants in apo and nucleotide-bound forms reveals a common, buried carboxylate or CO2 binding site for CAIR and N5-CAIR in a hydrophobic pocket in which the carboxylate or CO2 interacts with backbone amides. This work has led to a mechanistic proposal in which the carboxylate orients the substrate for proton transfer from His45 to N5-CAIR to form an enzyme-bound aminoimidazole ribonucleotide (AIR) and CO2 intermediate. Subsequent movement of the aminoimidazole moiety of AIR reorients it for addition of CO2 at C4 to generate isoCAIR. His45 is now in a position to remove a C4 proton to produce CAIR.

Figure 1

A possible mechanism for PurE-catalyzed conversion of N⁵-CAIR to CAIR. Note the proposed intermediates AIR/CO₂ and isoCAIR. The role of the protein is based on examination of 19 structures of PurEs from different sources and with different nucleotides bound. The aminoimidazole numbering scheme is shown on N⁵-CAIR. R = ribose-5’-phosphate.

Figure 2

Fluorescence titrations of PurE with NO₂-AIR and CAIR (A) Observed changes in fluorescence at 335 nm for H45N (●) and for H45Q (○) PurE upon titration with CAIR (K_d = 20.9 ± 1.9 μM, R² = 0.99 and K_d = 16.3 ± 2.6 μM, R² = 0.98 for H45N and H45Q, respectively). (B) Observed changes in fluorescence at 335 nm upon titration of wt PurE with NO₂-AIR (K_d =86 ± 31 nM, R² = 0.96).

Figure 3

pH-rate profiles for wt EcPurE monitoring the decarboxylation of CAIR. The solid line represents the best fit to Equation 2. The error bars for each point are shown. The k_cat vs. pH profile (●) was fit to pK₁ = 5.9 ± 0.4 and pK₂ = 8.6 ± 0.4 (R² = 0.94). The k_cat/K_m vs. pH profile (▼) was fit to pK₁ = 6.7 ± 1.6 and pK₂ = 7.5 ± 1.5 (R² = 0.95).

Figure 4

Structures of the AIR, N⁵-CAIR, and CAIR derivatives used in the computational studies. The calculated relative energies of the derivatives and the non-aromatic tautomers are shown.

Figure 5

Computational studies on the effects of protonation of compounds 1 (A) and 3 (B) on loss of CO₂ and bond lengths. Protonation of 1 at C4 or 3 at N5 results in bond cleavage and CO₂ dissociation.

Figure 6

(A) Stereoview of the wt EcPurE active site bound to NO₂-AIR with F_o-F_c density (2σ) shown. (B) Stereoview of H45Q EcPurE active site bound to CAIR with F_o-F_c density (2σ) shown.

Figure 7

(A) Modeling of N⁵-CAIR into the EcPurE-AIRx structure (1D7A) (3) after use of four-fold symmetry averaging to improve the signal. The initial F_o-F_c density (3.5σ) is shown. (B) Superposition of wt PurE with NO₂-AIR (blue), AIR/CO₂ from the re-interpreted AaPurE-isoCAIR structure described in panel C (red), and N⁵-CAIR modeled from the PurE-AIRx structure described in panel A (grey). The positions of the carboxylate groups stay the same in all structures and are located in an amide backbone pocket. (C) Re-interpretation of the AaPurE-isoCAIR complex structure (2FWP) (12). Initial F_o-F_c density (3σ) after exclusion of isoCAIR is shown with the final refined model of AIR and CO₂ in place of isoCAIR.