MSG

The Crystal Structure of Malate Synthase G

PDB ID 1D8C

Crystal structure of Escherichia coli malate synthase G complexed with magnesium and glyoxylate at 2.0 Angstroms resolution: mechanistic implications.

Howard BR, Endrizzi JA, Remington SJ.

Biochemistry. 2000 Mar 21;39(11):3156-68.

The Active Site of Malate Synthase

The active site of E. coli malate synthase G contains a Magnesium ion coordinated to two carboxylate groups --

Aspartate 455 and Glutamate 427 (numbering of the E.coli isozyme G; aka MSG). In the crystal structure solved to 2.0 Angstroms resolution, a molecule of glyoxylate was observed coordinated to this Mg²⁺, as shown below. The glyoxylate molecule is also held in place by two hydrogen bonds to the protonated backbone amide nitrogen atoms at the N-terminus of an a-helix (green), and by another hydrogen bond donated from Arginine 338 to the aldehyde carbonyl oxygen. Two water molecules complete the octahedral coordination sphere of the magnesium ion. (Hydrogen atoms are not shown)

We favor a model in which a protonated, free terminal nitrogen of the guanidinium group of Arg338, activates the carbonyl oxygen of the thioester bond within the second substrate, acetyl-CoA, while Aspartate 631 acts as the catalytic base, deprotonating the terminal methyl group to form the enolate ion, which can then collapse to attack the carbonyl carbon of glyoxylate to form a carbon-carbon bond, resulting in a malyl-Coenzyme A intermediate. Arg338 also activates the gloxylate aldehyde carbonyl with the other protonated terminal nitrogen, thus bridging the two subtrates for the condensation reaction. The malyl-CoA intermediate is then hydrolyzed to form the products: malate and coenzyme A. For details, see the biochemistry paper and pdb links above. Below, the electron density for the magnesium binding site is shown in an "omit map".

An Omit Map of the Active Site of Malate Synthase

In this figure (below), one can see the electron density surrounding the magnesium coordination sphere. This electron-density map was calculated after refinement of the structure in the absence of these atoms. A difference map or an Fo-Fc map shows the electron density in regions where the density is stronger than what would be expected from the model alone. Positive peaks in a difference map show where there is additional electron density that needs to be explained. This technique is used in model building to obtain a relatively unbiased view of the electron density in regions where the model has been omitted and to verify the occupancy of atoms in the model. The electon density is contoured at 3 sigma as a blue wire cage surrounding the atomic coordinates of the final refined model. The magnesium is purple, carbons green and oxygens red. Metal-oxygen bonds are shown in black, while other bonds are shown in yellow. Glyoxylate is on the left, Aspartate 455 on the right and Glutamate 427 below. The oxygen atom in front and the one at the top represent water molecules. Hydrogen atoms are not included due to the resolution limits of the observed data.