Structure of 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase from Escherichia coli: comparison of the Mn2+∗2-phosphoglycolate and the Pb2+∗2-Phosphoenolpyruvate complexes and implications for catalysis

The crystal structure of the phenylalanine-regulated 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (DAHPS) from Escherichia coli in complex with Mn2+ and the substrate analog, 2-phosphoglycolate (PGL), was determined by molecular replacement using X-ray diffraction data to 2.0 Å resolution. DAHPS∗Mn∗PGL crystallizes in space group C2 (a=210.4 Å, b=53.2 Å, c=149.4 Å, β=116.1 °) with its four (β/α)8 barrel subunits related by non-crystallographic 222 symmetry. The refinement was carried out without non-crystallographic symmetry restraints and yielded agreement factors of R=20.9 % and Rfree=23.9 %. Mn2+, the most efficient metal activator, is coordinated by the same four side-chains (Cys61, His268, Glu302 and Asp326) as is the poorly activating Pb2+. A fifth ligand is a well-defined water molecule, which is within hydrogen bonding distance to an essential lysine residue (Lys97). The distorted octahedral coordination sphere of the metal is completed by PGL, which replaces the substrate, 2-phosphoenolpyruvate (PEP), in the active site. However, unlike PEP in the Pb∗PEP complex, PGL binds the Mn2+ via one of its carboxylate oxygen atoms. A model of the active site is discussed in which PEP binds in the same orientation as does PGL in the DAHPS∗Mn∗PGL structure and the phosphate of E4P is tethered at the site of a bound sulfate anion. The re face of E4P can be positioned to interact with the si face of PEP with only small movement of the protein.