The 1.30 Å resolution structure of theBacillus subtilischorismate mutase catalytic homotrimer

Abstract

The crystal structure of the Bacillus subtilis chorismate mutase, an enzyme of the aromatic amino acids biosynthetic pathway, was determined to 1.30 A resolution. The structure of the homotrimer was determined by molecular replacement using orthorhombic crystals of space group P2(1)2(1)2(1) with unit-cell parameters a = 52.2, b = 83. 8, c = 86.0 A. The ABC trimer of the monoclinic crystal structure [Chook et al. (1994), J. Mol. Biol. 240, 476-500] was used as the starting model. The final coordinates are composed of three complete polypeptide chains of 127 amino-acid residues. In addition, there are nine sulfate ions, five glycerol molecules and 424 water molecules clearly visible in the structure. This structure was refined with aniosotropic temperature factors, has excellent geometry and a crystallographic R factor of 0.169 with an R(free) of 0.236. The three active sites of the macromolecule are at the subunit interfaces, with residues from two subunits contributing to each site. This orthorhombic crystal form was grown using ammonium sulfate as the precipitant; glycerol was used as a cryoprotectant during data collection. A glycerol molecule and sulfate ion in each of the active sites was found mimicking a transition-state analog. In this structure, the C-terminal tails of the subunits of the trimer are hydrogen bonded to residues of the active site of neighboring trimers in the crystal and thus cross-link the molecules in the crystal lattice.

Chorismate‐Mutase‐Catalyzed Claisen Rearrangement

“…EcCM and BsCM were also unable to catalyze the rearrangement of 6 (which lacks the ring carboxylic acid group) [22,23], even though 6 proved to be a weak to modest competitive inhibitor (K i of 6 is 0.4 mM and 0.5 mM for EcCM and BsCM, repectively; for chorismate K m is 0.32 mM and 0.28 mM, respectively). Thus, the existence of the ring carboxyl group is also essential for the catalysis, but may not [6,7,12,34]; the structures without TSA bound were also obtained for BsCM and yeast CM as well as for some of their mutants [8,10,12,31]. The X-ray structures for the monofunctional amino-terminal chorismate mutase domain engineered from the P-protein (EcCM) and a less active catalytic antibody 1F7 complexed with TSA have been determined by Lee et al [4] and Haynes et al [33], respectively.…”

“…Arg63 was not visible in the electron-density map in an earlier X-ray structure determination [7]. But a more recent X-ray structure [8] of higher resolution (1.3 Å) without TSA bound showed that Arg63 is turned inward toward the active site and may therefore interact with the ring carboxylate group of TSA. Another interaction that exists in all the three CMs is the hydrogen bond between the C 4 -hydroxyl group of TSA and a Glu residue (Glu52 in EcCM, Glu198 in yeast CM and Glu78 in BsCM).…”

“…However, for BsCM the corresponding residue has not been clearly identified. A recent X-ray structure [8] for BsCM suggested that Arg63 may interact with the ring carboxylate group, but the mutagenesis study for the Arg63 mutants has not been available.…”

Biochemical and Structural Characterization of the Secreted Chorismate Mutase (Rv1885c) from Mycobacterium tuberculosis H 37 R v : an *AroQ Enzyme Not Regulated by the Aromatic Amino Acids

“…CM accelerates the rearrangement by a factor of 10 6 compared to the uncatalyzed rearrangement at 25°C (1). Several biochemical and structural studies on CM have been performed due to the diverse nature of CMs (6,24,26,29,51,58). Besides CM, the other enzymes that utilize chorismate are chorismate lyase, isochorismate synthase, anthranilate synthase, and p-aminobenzoate synthase (16).…”

Allosteric Regulation of Catalytic Activity: Escherichia coli Aspartate Transcarbamoylase versus Yeast Chorismate Mutase

“…The X-ray structure of this enzyme was determined at 1.9-Å resolution ( Fig. 3A and B) (12) and more recently at 1.3-Å resolution (55). The aroH gene product is a nonallosteric CM of 127 amino acids per monomer.…”