3-deoxy-D-manno-octulosonate-8-phosphate synthase from Escherichia coli. Model of binding of phosphoenolpyruvate and D-arabinose-5-phosphate

The crystal structure of 3-deoxy-D-manno-octulosonate-8-phosphate synthase (KDOPS) from Escherichia coil was determined by molecular replacement using coordinates given to us by Radaev and co-workers prior to publication. The KDOPS crystals reported by Radaev et al. were grown in the presence of 1.4 M (NH4)(2)SO4 and 0.4 M (K/H)(3)PO4. They are in the cubic space group I23 (a = 228.6 Angstrom) with a tetramer in the asymmetric unit; the structure has been refined with data to 2.4 Pi. Our crystals of E. coil KDOPS, grown in 24% (w/v) polyethylene glycol (PEG) 1500 in the presence of the substra tes, 2-phosphoenolpyruvate (PEP) and D-arabinose-5-phosphate (A5P), are als o in space group 123 (a = 118.2 Angstrom), with one subunit in the asymmetr ic unit. The medium of crystallization, 1.8 M SO4/PO4 versus 24% PEG, does not signi ficantly affect the conformation of KDOPS. The inter-monomer contacts in bo th structures are the same. The beta(8)/alpha(8) loop (residues 246 to 251) situated near the entrance to the active site is not seen in the 229 Angst rom structure but can be traced in the 118 Angstrom structure. Most significantly, Radaev et nl. interpreted two SO4/PO4 sites in the 229 A structure as marking the phosphate positions of the substrates, PEP and A 5P, after the precedent of DAMPS. In the 118 Angstrom structure the inner o f these two SO4/PO4 peaks is present at the same position as in the 229 A s tructure of KDOPS. The outer phosphate peak in the 118 A KDOPS is 3.7 A fro m the outer SO4/PO4 peak in the 229 A structure and is within hydrogen bond ing distance of Arg63 of the same subunit and Arg120 of another subunit. Ba sed on the precedent of the D-erythrose-4-phosphate (E4P) modeled in the ac tive site of DAMPS, we have modeled PEP and A5P in KDOPS and compared the c oordination of PEP and A5P in KDOPS with that of PEP and E4P in DAMPS. (C) 2000 Academic Press.