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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.
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