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 structure of GMP synthetase (GMPS) from Escherichia coli. GMPS catalyzes the conversion of XMP to GMP in the final step of de novo guanine nucleotide biosynthesis, and is a member of the glutamine amidotransferase family: a group of enzymes responsible for the assimilation of nitrogen into compounds such as amino acids, purine and pyrimidine bases, amino sugars, and antibiotics. The E. coli guaA gene encoding GMPS was cloned into a tac expression vector, overexpressed, and its gene product purified. Conditions for the growth of protein crystals were developed using recombinant GMPS in the presence of MgCl2, ATP, and XMP. The crystals are monoclinic, space group P2(1), with cell parameters of a = 156.0 A, b = 102.0 A, c = 78.8 A, beta = 96.7 degrees. Diffraction data to 2.8 A spacings were collected on a Xuong-Hamlin area detector with an overall Rsym of 5.2%. Both the volume of the unit cell and the peaks in the self-rotation function are consistent with one GMPS tetramer of D2 symmetry in the crystallographic asymmetric unit. Previously, GMPS has been observed only as a dimer in solution. GMPS was covalently modified with p-chloromercuribenzylsulfonic acid (PCMBS), and its X-ray fluorescence spectrum was measured through the LIII absorption edge of mercury. Anomalous scattering factors for cysteinyl mercury were derived from this spectrum, and the feasibility of structure determination by multi-wavelength anomalous diffraction was evaluated. The optimal MAD dispersive signal is 4.5% of magnitude of F, and the optimal MAD Bijvoet signal is 7.5% of magnitude of F at a concentration of approximately 1 mercury per 10-kDa protein. The anomalous scattering factors tabulated here should be transferable to cysteinyl mercury in other proteins.
Tesmer
J J
JJ
Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907.
Stemmler
T L
TL
Penner-Hahn
J E
JE
Davisson
V J
VJ
Smith
J L
JL
D001483:Base Sequence; D019731:Carbon-Nitrogen Ligases; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000031:analogs & derivatives* / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology*; D008025:Ligases / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry
D011994:Recombinant Proteins; D008025:Ligases; D019731:Carbon-Nitrogen Ligases; C020828:GMP synthase (glutamine-hydrolyzing); D003545:Cysteine