CRYSTAL-STRUCTURE OF THE TERNARY COMPLEX OF ESCHERICHIA-COLI PURINE NUCLEOSIDE PHOSPHORYLASE WITH FORMYCIN-B, A STRUCTURAL ANALOG OF THE SUBSTRATE INOSINE, AND PHOSPHATE (SULFATE) AT 2.1 ANGSTROM RESOLUTION

The ternary complex of purine nucleoside phosphorylase from E. coil wi th formycin B and a sulphate or phosphate ion crystallized in the hexa gonal space group P6(1)22 with unit cell dimensions a = 123.11, c = 24 1.22 Angstrom and three monomers per asymmetric unit. The biologically active hexamer is formed through 2-fold crystallographic symmetry, co nstituting a trimer of dimers. High-resolution X-ray diffraction data were collected using synchrotron radiation (Daresbury, En,oland). The crystal structure was determined by molecular replacement and refined at 2.1 Angstrom resolution to an R-value of 0.196. There is one active centre per monomer, composed of residues belonging to two subunits Of one dimer. The phosphate binding site is strongly positively charged and consists of three arginine residues (Arg24, Arg87 and Arg43 from a neighbouring subunit), Ser90 and Gly20. It is occupied by a sulphate or phosphate anion, each oxygen atom of which accepts at least two hyd rogen bonds or salt-bridges. The sulphate or phosphate anion is also i n direct contact with the ribose moiety of formycin B. The ribose bind ing site is composed of Ser90, Met180, Glu181 and His4, the latter bel onging to the neighbouring subunit. The base binding site is exposed t o solvent, and the base is unspecifically bound through a chain of wat er molecules and aromatic-aromatic interactions. Ln all monomers the n ucleosides are in the high syn conformation about the glycosidic bonds with chi in the range 100 to 130 degrees. The architecture of the act ive centre is in line with the known broad specificity and the kinetic properties of E. coli PNP. (C) 1998 Academic Press.