The bifunctional enzyme dihydrofolate reductase-thymidylate synthase catalyses both the reductive methylation of 2'-deoxyuridylate and the subsequent reduction of dihydrofolate to yield 2'-deoxythymidylate and tetrahydrofolate at two spacially discrete sites situated on different protein domains. The X-ray structure of dihydrofolate reductase-thymidylate synthase from Leishmania major indicates that transfer of dihydrofolate between these sites does not occur by transient binding at both sites but rather by movement of dihydrofolate across the surface of the protein. The enzyme has an unusual surface charge distribution that could account for this channelling of dihydrofolate between active sites.
The three-dimensional structure of phosphoribosylglycinamide formyltransferase (10-formyltetrahydrofolate:5'-phosphoribosylglycinamide formyltransferase, EC 2.1.2.2) has been solved both as an apoenzyme at 2.8-A resolution and as a ternary complex with the substrate glycinamide ribonucleotide and a folate inhibitor at 2.5-A resolution.The structure is a modified doubly wound a/S sheet with flexibility in the active site, including a disordered loop in the apo structure, which is ordered in the ternary complex structure. This enzyme is a target for anti-cancer therapy and now for structure-based drug design. (3) and is currently completing phase I clinical trials in a number of centers. This enzyme is, therefore, a good target for anti-cancer therapy and structure-based drug design. In prokaryotes, GART is found as a single protein but in most eukaryotes it is found as the C-terminal portion of a large multifunctional protein (Mr > 100,000) also containing GAR synthetase and aminoimidazole ribonucleotide synthetase activities. The sequences of GART from prokaryotes and eukaryotes are homologous (4, 5), and the structures are expected to be similar.Here we report the three-dimensional structure of Escherichia coli GART as an apoenzyme (2.8-A resolution) and also as a ternary complex (2.5-A resolution) with the substrate GAR and a folate-based inhibitor, 5-deaza-5,6,7,8-tetrahydrofolate (5dTHF) (Fig. 1). The GART structure is a modification of the classic doubly wound a/3 sheet with the active site located at the C-terminal edge, near the middle of the seven- stranded 83-sheet. We describe the major changes in the structure between the complexed and uncomplexed forms, interpret structure-activity relationships (SAR), and discuss the mechanism for catalysis based on our structural results.MATERIALS AND METHODS The coding region ofE. coli GART was amplified from E. coli K-12 chromosomal DNA with PCR method (6). Primer sequences were derived from E. coli purN gene (5). Amplified DNA was confirmed and placed downstream of the T7 bacteriophage gene 10 promoter (7) as the second cistron using modified E. coli DHFR translation initiation region as the first cistron (8,9). The E. coli strain AP401-harboring expression plasmid was induced as described (9).GART was purified by using a DEAE-Sephacel column elution with a 0.03-0.8 M NaCl gradient followed by a Sephadex G75 column and a fast protein liquid chromotography mono Q elution with a 0.03-1 M NaCl gradient. The specific activity of the purified enzyme was 10 Jumol/min per mg. All steps were done at 40C in 50 mM Tris, pH 7.5/30 mM NaCl/1 mM dithiothreitol. A similar purification has since been published (10).All crystals were grown at 20'C by hanging-drop vapor diffusion. Apo crystals grew from 30 mg of protein per ml in 50 mM Tris (pH 7.5), 1 mM dithiothreitol, and a reservoir of 0.8 M Na+,K+ phosphate (pH 6.75) similar to conditions in Abbreviations: GAR, glycinamide ribonucleotide; GART, phosphoribosylglycinamide formyltransferase; DHFR, dihydrofolate reduct...
The majority of the VEGFR2 KID residues are not necessary for kinase activity. The unique structure observed for the ends of the KID may also occur in other PDGFR family members and may serve to properly orient the KID for signal transduction. This VEGFR2 kinase structure provides a target for design of selective anti-angiogenic therapeutic agents.
Proteolytic processing of capsid assembly protein precursors by herpesvirus proteases is essential for virion maturation. A 2.5 A crystal structure of the human cytomegalovirus protease catalytic domain has been determined by X-ray diffraction. The structure defines a new class of serine protease with respect to global-fold topology and has a catalytic triad consisting of Ser-132, His-63, and His-157 in contrast with the Ser-His-Asp triads found in other serine proteases. However, catalytic machinery for activating the serine nucleophile and stabilizing a tetrahedral transition state is oriented similarly to that for members of the trypsin-like and subtilisin-like serine protease families. Formation of the active dimer is mediated primarily by burying a helix of one protomer into a deep cleft in the protein surface of the other.
The single-chain 28 kDa human cytomegalovirus (HCMV) protease catalytic domain containing the A143Q mutation has been kinetically and conformationally characterized. The specific activity of the HCMV A143Q protease (HCMVp) increases as the protease concentration increases, suggesting that this protease oligomerizes at high protein concentration to form a more active species. Both cross-linking and light-scattering studies of HCMVp show the existence of a homodimer with an apparent molecular mass of 56 kDa under low ionic strength and high protein concentration. The cosolvent and solute effects of glycerol, trisodium citrate, and NaCl as well as the temperature effects on the HCMVp activity and quaternary structure were investigated. The effects induced by cosolvents and temperature can largely be explained by their influences in the dimerization or oligomerization state of HCMVp. The dissociation constant (Kd) for the HCMVp homodimer was determined to be 8 +/- 1 microM with all activity attributed to the dimeric form. Monomeric HCMVp is inactive. This report demonstrates that in vitro, HCMV A143Q protease exists as an obligate catalytic homodimer. This protease dimerization may have regulatory significance during viral replication.
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