Crystal structure of Escherichia coli malate synthase G complexed with magnesium and glyoxylate at 2.0 angstrom resolution: Mechanistic implications

The crystal structure of selenomethionine-substituted malate synthase G, an 81 kDa monomeric enzyme from Escherichia coli has been determined by MAD p hasing, model building, and crysrallographic refinement to a resolution of 2.0 Angstrom. The crystallographic R factor is 0.177 for 49 242 reflections observed at the incident wavelength of 1.008 Angstrom, and the model stere ochemistry is satisfactory. The basic fold of the enzyme is that of a beta 8/alpha 8 (TIM) barrel. The barrel is centrally located, with an N-terminal ex-helical domain flanking one side. An inserted beta-sheet domain folds a gainst the opposite side of the barrel, and an alpha-helical C-terminal dom ain forms a plug which caps the active site, Malate synthase catalyzes the condensation of glyoxylate and acetyl-coenzyme A and hydrolysis of the inte rmediate to yield malate and coenzyme A, requiring Mg2+ . The structure rev eals an enzyme-substrate complex with glyoxylate and Mg2+ which coordinates the aldehyde and carboxylate functions of the substrate, Two strictly cons erved residues, Asp631 and Arg338, are proposed to provide concerted acid-b ase chemistry for the generation of the enol(ate) intermediate of acetyl-co enzyme A, while main-chain hydrogen bonds and bound Mg2+ polarize glyoxylat e in preparation for nucleophilic attack. The catalytic strategy of malate synthase appears to be essentially the same as that of citrate synthase, wi th the electrophile activated for nucleophilic attack by nearby positive ch arges and hydrogen bonds, while concerted acid-base catalysis accomplishes the abstraction of a proton from the methyl group of acetyl-coenzyme A. An active site aspartate is, however, the only common feature of these two enz ymes, and the active sites of these enzymes are produced by quite different protein folds. Interesting similarities in the overall folds and modes of substrate recognition are discussed in comparisons of malate synthase with pyruvate kinase and pyruvate phosphate dikinase.