Structural insights into the prereaction state of pyruvate decarboxylase from Zymomonas mobilis

Abstract

Pyruvate decarboxylase (PDC) uses thiamine diphosphate as an essential cofactor to catalyze the formation of acetaldehyde on the pathway of ethanol synthesis. Here we report the crystallographic image of a prereaction intermediate of a bacterial pyruvate decarboxylase prepared by cocrystallizing the enzyme with pyruvate and a stable analogue of the cofactor's activated ylid form. A second crystal structure of PDC in complex with fluoride shows that the ion organizes a water molecule that occludes the pyruvate binding site, accounting for the inhibitory effect of the halide. Also reported is a structure of the cofactor-free apo form, which when compared to the structure of the holo form indicates how thiamine diphosphate organizes the active site pocket of pyruvate decarboxylase to support catalysis. Guided by the structural and enzymatic data, we propose roles for several key residues in the catalytic mechanism.

Figure 1

(a) Catalytic cycle of pyruvate decarboxylation by PDC. Protonation at N-1′ and deprotonation at 4′-NH₂ of ThDP cofactor (1) give the imino tautomer (2) which promotes the deprotonation of C2 on the thiazolium ring to form the active ylid (3). The ylid attacks C2 of pyruvate to give the lactyl adduct (4). Decarboxylation then gives the enamine intermediate (5); protonation gives hydroxyethyl-ThDP (6), and then release of the product acetaldehyde regenerates the ylid (3). (b) Structure of the intermediate analogue, triazole-ThDP 7. The C2 and S atoms of ThDP are replaced with nitrogen atoms in the triazole derivative.

Figure 2

(a) Overall structure of homotetrameric ZmPDC with triazole-ThDP (shown in stick representation) bound. The four protomers are shown in different colors. (b) View of ZmPDC rotated by 90° compared to the view in panel a. (c) Cartoon representation of one protomer of ZmPDC from panel a. The PYR domain (residues 1−188), R domain (residues 189−354), and PP domain (residues 355−566) are colored brown, yellow, and cyan, respectively. The termini of the peptide are labeled with N and C. The color scheme for stick representations in all figures is as follows: red for oxygen, blue for nitrogen, purple for magnesium, and orange for phosphate.

Figure 3

Active site of the holo structure in stick representation with the triazole derivative of thiamine diphosphate (yellow), a fluoride ion (cyan sphere), and a water molecule (red sphere). Residues from two different protomers of the tetramer are colored wheat and cyan. Black dashed lines represent hydrogen bonds. The electron density is a difference map contoured at 4.5σ using phases that do not include the contribution of the fluoride ion that was subsequently incorporated into the model.

Figure 4

Cartoon and stick representation of one of the active sites of the holo structure in a complex with the substrate pyruvate and the triazole-ThDP cofactor. The two protomers of the enzyme are colored cyan and yellow. The omit map of the triazole-ThDP molecule (blue) was calculated from a difference map at the 3σ level at 2.2 Å resolution. The phases were derived from simulated annealing using a model with triazole-ThDP molecules removed. The omit map of pyruvate (green) was calculated from the difference map at the 3σ level at 2.2 Å resolution. The phases were derived from simulated annealing with pyruvate molecules removed.

Figure 5

Cartoon and stick representation comparing the cofactor engaging loop (CE loop) conformations in the apo structure (orange), holo structure (cyan and yellow for the two protomers), and pyruvate-bound holo structure (blue). Tyr^b290 is not shown for clarity.

Figure 6

Possible mechanism for the enzymic reaction, involving multiple proton transfers between Asp^a27/Glu^b473 and the substrate/cofactor.

Figure 7

Water tunnel between active sites that is conserved in both Z. mobilis and yeast PDC. PDC from Z. mobilis is colored gray for protomer A and green for protomer B, and waters are represented as red spheres. The hydrogen bonding network of the water tunnel from Z. mobilis is presented as dashed lines. Yeast PDC with pyruvamide (PDB entry 1QPB) is colored cyan and yeast E477Q variant (PDB entry 2VK8) blue. Residues from Z. mobilis involved in the aqueous tunnel are labeled.

Figure 8

Possible two-step binding process of the triazole-ThDP analogue (I) to the enzyme (E).