X-ray crystal structure of aminoimidazole ribonucleotide synthetase (PurM), from the Escherichia coli purine biosynthetic pathway at 2.5 A resolution.

BACKGROUND: The purine biosynthetic pathway in procaryotes enlists eleven enzymes, six of which use ATP. Enzymes 5 and 6 of this pathway, formylglycinamide ribonucleotide (FGAR) amidotransferase (PurL) and aminoimidazole ribonucleotide (AIR) synthetase (PurM) utilize ATP to activate the oxygen of an amide within their substrate toward nucleophilic attack by a nitrogen. AIR synthetase uses the product of PurL, formylglycinamidine ribonucleotide (FGAM) and ATP to make AIR, ADP and P(i). RESULTS: The structure of a hexahistidine-tagged PurM has been solved by multiwavelength anomalous diffraction phasing techniques using protein containing 28 selenomethionines per asymmetric unit. The final model of PurM consists of two crystallographically independent dimers and four sulfates. The overall R factor at 2.5 A resolution is 19.2%, with an R(free) of 26.4%. The active site, identified in part by conserved residues, is proposed to be a long groove generated by the interaction of two monomers. A search of the sequence databases suggests that the ATP-binding sites between PurM and PurL may be structurally conserved. CONCLUSIONS: The first structure of a new class of ATP-binding enzyme, PurM, has been solved and a model for the active site has been proposed. The structure is unprecedented, with an extensive and unusual sheet-mediated intersubunit interaction defining the active-site grooves. Sequence searches suggest that two successive enzymes in the purine biosynthetic pathway, proposed to use similar chemistries, will have similar ATP-binding domains.

Purification and crystallization of fumarase C from Escherichia coli.

Fumarase C purified from Escherichia coli has been crystallized in the presence of polyethylene glycol in both a citrate buffer at pH 5.3 and a 3-(4-morpholino)-propanesulfonic acid buffer at pH 7.5 yielding two crystal forms. An orthorhombic C222(1) form was obtained in citrate at pH 5.3 and an orthorhombic I222 form was obtained in 3-(4-morpholino)-propanesulfonic acid (pH 7.5). Complete native data sets have been collected on both crystal forms: the C222(1) form is complete to 2.10 A and the I222 form is complete to 2.20 A.

The pi-helix translates structure into function.

A search for the occurrence of the rare pi-helix was performed with Iditis from the Oxford Molecular Group upon the Protein Data Bank. In 8 of the 10 confirmed crystal structures that harbor the pi-helix, its unique conformation has been linked directly to the formation or stabilization of a specific binding site within the protein. In the discussion to follow, the role for each of these eight pi-helices will be addressed in regard to protein function. It is clear upon closer examination that the conformation of the pi-helix has evolved to provide unique structural features within a variety of proteins.

Purification, crystallization and preliminary X-ray diffraction data from selenomethionine glycinamide ribonucleotide synthetase.

In this study, the overexpression, purification and crystallization of selenomethionine (SeMet) incorporated glycinamide ribonucleotide synthetase (GAR-syn) from Escherichia coli are reported. The overexpression of SeMet GAR-syn was placed under the control of the isopropylthio-beta-galactoside (IPTG) inducible T7 RNA-polymerase system. The newly developed construct contained a removable histidine tag on the amino terminus of GAR-syn, which allowed rapid purification using metal-chelate chromatography techniques. The SeMet GAR-syn crystals were grown by hanging-drop vapor diffusion and belong to the space group P212121 with unit-cell parameters a = 56.2, b = 62.4 and c = 129.8 A and a single monomer in the asymmetric unit. The crystals diffract to 1.6 A resolution and have led to the determination of multiple-wavelength anomalous diffraction phases to 2.2 A resolution.

The multisubunit active site of fumarase C from Escherichia coli.

The crystal structure of the tetrameric enzyme, fumarase C from Escherichia coli, has been determined to a resolution of 2.0 A. A tungstate derivative used in the X-ray analysis is a competitive inhibitor and places the active site of fumarase in a region which includes atoms from three of the four subunits. The polypeptide conformation is similar to that of delta-crystallin and is comprised of three domains. The central domain, D2, is a unique five-helix bundle. The association of the D2 domains results in a tetramer which has a core of 20 alpha-helices. The other two domains, D1 and D3, cap the helical bundle on opposite ends giving both the single subunit and the tetramer a dumbbell-like appearance. Fumarase C has sequence homology to the eukaryotic fumarases, aspartase, arginosuccinate lyase, adenylosuccinate lyase and delta-crystallin.