Structure of the Escherichia coli malate synthase G:pyruvate:acetyl-coenzyme A abortive ternary complex at 1.95 A resolution

Abstract

Malate synthase, an enzyme of the glyoxylate pathway, catalyzes the condensation and subsequent hydrolysis of acetyl-coenzyme A (acetyl-CoA) and glyoxylate to form malate and CoA. In the present study, we present the 1.95 A-resolution crystal structure of Escherichia coli malate synthase isoform G in complex with magnesium, pyruvate, and acetyl-CoA, and we compare it with previously determined structures of substrate and product complexes. The results reveal how the enzyme recognizes and activates the substrate acetyl-CoA, as well as conformational changes associated with substrate binding, which may be important for catalysis. On the basis of these results and mutagenesis of active site residues, Asp 631 and Arg 338 are proposed to act in concert to form the enolate anion of acetyl-CoA in the rate-limiting step. The highly conserved Cys 617, which is immediately adjacent to the presumed catalytic base Asp 631, appears to be oxidized to cysteine-sulfenic acid. This can explain earlier observations of the susceptibility of the enzyme to inactivation and aggregation upon X-ray irradiation and indicates that cysteine oxidation may play a role in redox regulation of malate synthase activity in vivo. There is mounting evidence that enzymes of the glyoxylate pathway are virulence factors in several pathogenic organisms, notably Mycobacterium tuberculosis and Candida albicans. The results described in this study add insight into the mechanism of catalysis and may be useful for the design of inhibitory compounds as possible antimicrobial agents.

Figure 1.

Ribbon diagram of malate synthase. Domains are indicated by color: N-terminal α-helical clasp (blue), extended surface loop linker (turquoise), TIM barrel (red), α/β domain (yellow), and C-terminal plug (purple). Magnesium, pyruvate, and acetyl-coenzyme A are shown in green ball-and-stick form.

Figure 2.

(A) Portion of an F_o-F_c electron density omit map, contoured at 2 σ. The refined model of acetyl-coenzyme A (acetyl-CoA), which was omitted for the purpose of map calculation, is superimposed and shown as a ball-and-stick model. (B) Portion of an F_o-F_c electron density omit map, contoured at 2 σ, showing bound pyruvate and the acetyl group of acetyl-CoA.

Figure 3.

(A) Stereo view of the adenosine portion of the acetyl-coenzyme A (acetyl-CoA) binding site. Hydrogen bonds are shown as dashed cylinders. Green spheres represent carbon atoms of acetyl-CoA, and carbon atoms of the protein are shown in gray. (B) Schematic diagram of the acetyl-CoA binding site. For simplicity, the three-dimensional structure has been projected onto a plane. Acetyl-CoA is shown in blue, waters are shown as black W’s, and protein bonds are black except for the catalytic Asp 631, shown in red. Heavily shaded curves indicate hydrophobic interactions. Hydrogen bonds are shown as dashed lines, except for unfavorable contacts between Asp 631 and the terminal atoms of acetyl-CoA, which are represented with dashed red lines. (C) Stereo view of active site. As in A, but with unfavorable interaction as black dashed lines.

Figure 4.

Proposed catalytic mechanism of malate synthase G, adapted from Howard et al. (2000).

Figure 5.

Stereo view comparison of loop 616–632 between structure with (yellow) and without (blue) bound acetyl-coenzyme A.

Figure 6.

Stereo view of a portion of an F_o-F_comit map contoured at 2.5 σ. The map shows additional electron density associated with Cys 617, interpreted to result from partial oxidation of Cys 617 to cysteine sulfenic acid. Asp 631 and Cys 617 were omitted for the purpose of phase calculation. Portions of the final refined model, including the terminal portion of acetyl-coenzyme A are superimposed. Ribbon and tube segments represent portions of the protein backbone.