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-hept ulosonate-7-phosphate synthase (DAHPS) from Escherichia coli in complex wit h Mn2+ and the substrate analog, 2-phosphoglycolate (PGL), was determined b y molecular replacement using X-ray diffraction data to 2.0 Angstrom resolu tion. DAHPS*Mn*PGL crystallizes in space group C2 (a = 210.4 Angstrom, b = 53.2 Angstrom, c = 149.4 Angstrom, beta = 116.1 degrees) with its four (bet a/alpha)(8) barrel subunits related by non-crystallographic 222 symmetry. T he refinement was carried out without non-crystallographic symmetry restrai nts and yielded agreement factors of R = 20.9% and R-free = 23.9%. Mn2+, th e most efficient metal activator, is coordinated by the same four side-chai ns (Cys61, His268, Glu302 and Asp326) as is the poorly activating Pb2+. A f ifth ligand is a well-defined water molecule, which is within hydrogen bond ing distance to an essential lysine residue (Lys97). The distorted octahedr al coordination sphere of the metal is completed by PGL, which replaces the substrate, 2-phosphoenolpyruvate (PEP), in the active site. However, unlik e 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 pho sphate 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 sma ll movement of the protein. (C) 2000 Academic Press.