The crystal structure of a paradigmatic protein: GMP synthetase from Escherichia coli at 2.2 A resolution
Abstract
The primary focus of the present study was to determine the structure of a Class I glutamine amidotransferase (GAT) domain. The Class I GAT domain is found in at least seven key biosynthetic enzymes, and catalyzes the hydrolysis of glutamine to glutamate and the transfer of the amide nitrogen of glutamine to a specific carbon substrate. A high resolution structure of a Class I GAT domain would help define the molecular mechanism of glutamine hydrolysis and amide transfer determine these enzymes. E. coli GMP synthetase, one such amidotransferase, was picked as a likely candidate for a structure determination because the enzyme had already been extensively characterized and purified to homogeneity. An efficient overexpression system and purification protocol were developed in order to produce the large quantities of GMP synthetase required for a crystallographic structure determination. The purified, recombinant enzyme was crystallized, and diffraction intensities from these crystals were collected to 2.2 A spacings. The phases for GMP synthetase were derived from three heavy atom derivatives using a combination of multiple isomorphous replacement, multiwavelength anomalous diffraction and density modification. The resulting electron density revealed that the GMP synthetase dimer has five structural domains: two GAT domains, two ATP pyrophosphatase domains, and one dimerization domain. The GAT domain of GMP synthetase contains a catalytic triad and oxyanion hole similar to those of cysteine proteases and the $\alpha/\beta$ hydrolases. Therefore, the hydrolysis of glutamine is expected to follow a mechanism that is similar to peptide hydrolysis in these enzymes. The second domain contains a nucleotide-binding P-loop whose amino acid fingerprint was used to identify a novel family of homologous "N-type" ATP pyrophosphatases. However, in the crystal structure the GAT and ATP pyrophosphatase active sites are 30 A apart, leaving the mechanism of amide transfer an intriguing mystery.
Degree
Ph.D.
Advisors
Smith, Purdue University.
Subject Area
Biophysics|Chemistry|Molecular biology
Off-Campus Purdue Users:
To access this dissertation, please log in to our
proxy server.