PRELIMINARY-X-RAY ANALYSIS OF ESCHERICHIA-COLI GMP SYNTHETASE - DETERMINATION OF ANOMALOUS SCATTERING FACTORS FOR A CYSTEINYL MERCURY DERIVATIVE

We have initiated a project to determine the three-dimensional structu re of GMP synthetase (GMPS) from Escherichia coli. GMPS catalyzes the conversion of XMP to GMP in the final step of de novo guanine nucleoti de biosynthesis, and is a member of the glutamine amidotransferase fam ily: a group of enzymes responsible for the assimilation of nitrogen i nto compounds such as amino acids, purine and pyrimidine bases, amino sugars, and antibiotics. The E. coli guaA gene encoding GMPS was clone d into a tac expression vector, overexpressed, and its gene product pu rified. Conditions for the growth of protein crystals were developed u sing recombinant GMPS in the presence of MgCl2, ATP, and XMP. The crys tals are monoclinic, space group P2(1), with cell parameters of a = 15 6.0 angstrom, b = 102.0 angstrom, c = 78.8 angstrom, beta = 96.7-degre es. Diffraction data to 2.8 angstrom spacings were collected on a Xuon g-Hamlin area detector with an overall R(sym) of 5.2%. Both the volume of the unit cell and the peaks in the self-rotation function are cons istent with one GMPS tetramer of D2 symmetry in the crystallographic a symmetric unit. Previously, GMPS has been observed only as a dimer in solution. GMPS was covalently modified with p-chloromercuribenzylsulfo nic acid (PCMBS), and its X-ray fluorescence spectrum was measured thr ough the L(III) absorption edge of mercury. Anomalous scattering facto rs for cysteinyl mercury were derived from this spectrum, and the feas ibility of structure determination by multi-wavelength anomalous diffr action was evaluated. The optimal MAD dispersive signal is 4.5% of Abs olute value of F, and the optimal MAD Bijvoet signal is 7.5% of Absolu te value of F at a concentration of approximately 1 mercury per 10-kDa protein. The anomalous scattering factors tabulated here should be tr ansferable to cysteinyl mercury in other proteins. (C) 1994 Wiley-Liss , Inc.