Structures of Trypanosoma cruzi dihydroorotate dehydrogenase complexed with substrates and products: atomic resolution insights into mechanisms of dihydroorotate oxidation and fumarate reduction

Dihydroorotate dehydrogenase (DHOD) from Trypanosoma cruzi (TcDHOD) is a member of family 1A DHOD that catalyzes the oxidation of dihydroorotate to orotate (first half-reaction) and then the reduction of fumarate to succinate (second half-reaction) in the de novo pyrimidine biosynthesis pathway. The oxidation of dihydroorotate is coupled with the reduction of FMN, and the reduced FMN converts fumarate to succinate in the second half-reaction. TcDHOD are known to be essential for survival and growth of T. cruzi and a validated drug target. The first-half reaction mechanism of the family 1A DHOD from Lactococcus lactis has been extensively investigated on the basis of kinetic isotope effects, mutagenesis and X-ray structures determined for ligand-free form and in complex with orotate, the product of the first half-reaction. In this report, we present crystal structures of TcDHOD in the ligand-free form and in complexes with an inhibitor, physiological substrates and products of the first and second half-reactions. These ligands bind to the same active site of TcDHOD, which is consistent with the one-site ping-pong Bi-Bi mechanism demonstrated by kinetic studies for family 1A DHODs. The binding of ligands to TcDHOD does not cause any significant structural changes to TcDHOD, and both reduced and oxidized FMN cofactors are in planar conformation, which indicates that the reduction of the FMN cofactor with dihydroorotate produces anionic reduced FMN. Therefore, they should be good models for the enzymatic reaction pathway of TcDHOD, although orotate and fumarate bind to TcDHOD with the oxidized FMN and dihydroorotate with the reduced FMN in the structures determined here. Cys130, which was identified as the active site base for family 1A DHOD (Fagan, R. L., Jensen, K. F., Bjornberg, O., and Palfey, B. A. (2007) Biochemistry 46, 4028-4036.), is well located for abstracting a proton from dihydroorotate C5 and transferring it to outside water molecules. The bound fumarate is in a twisted conformation, which induces partial charge separation represented as C 2 (delta-) and C 3 (delta+). Because of this partial charge separation, the thermodynamically favorable reduction of fumarate with reduced FMN seems to proceed in the way that C 2 (delta-) accepts a proton from Cys130 and C 3 (delta+) a hydride (or a hydride equivalent) from reduced FMN N 5 in TcDHOD.     

Characterization of phytochelatin synthase produced by the primitive red alga Cyanidioschyzon merolae

Phytochelatins (PCs), non-protein peptides with the general structure [(γ-Glu-Cys)n-Gly (n≥ 2)], are involved in the detoxification of toxic heavy metals mainly in higher plants. The synthesis of the peptides is mediated by phytochelatin synthase (PCS), which is activated by a range of heavy metals. CmPCS, a PCS-like gene found in the genomic DNA of the primitive red alga Cyanidioschyzon merolae, was isolated and a recombinant protein (rCmPCS) fused with a hexahistidine tag at the N-terminus of CmPCS was produced. The finding that this protein mediated PC synthesis from glutathione in a metal-dependent way clearly establishes that rCmPCS is functional. The maximum activity was attained at a reaction temperature of 50 °C, considerably higher than the temperature required for the maximal activity of PCS isolated from the higher plant Silene cucubalus, probably due to the alga being a thermophile. CmPCS showed optimal pH in a slightly higher region than higher plant PCSs, probably due to the less effective charge relay network in the catalytic triad. In addition, the pattern of enzyme activation by metal ions was specific to rCmPCS, with Ag+, Cu2+, and Hg2+ showing only limited activation. In contrast to other eukaryotic PCSs, CmPCS has an extra domain in the N-terminal region from residues 1 to 109, and contains fewer cysteine residues in the C-terminal domain. These differences may be responsible for the metal specificity of the activation of CmPCS. Although the enzyme preparation lost PCS activity progressively when stored at 4 °C, the inclusion of Cd2+ in the preparation effectively prevented the reduction of activity. Furthermore, Cd2+ effectively restored the activity of the inactivated enzyme. These results indicate that Cd2+ ions bind the enzyme to maintain the structural integrity of the peptides.     

Isolation of a new potent cytotoxic pigment along with indigotin from the pathogenic basidiomycetous fungus Schizophyllum commune

An indole derivative, schizocommunin, was isolated along with indigotin (indigo), indirubin, isatin, and tryptanthrin, from the liquid culture medium in which a culture of Schizophyllum commune, isolated from the bronchus of a human patient with allergic bronchopulmonary mycosis, had been grown. The structure of schizocommunin was established by spectroscopic investigation. Schizocommunin showed the strong cytotoxicity against murine lymphoma cells. The assignments of the ¹H- and ¹³C-NMR signals of indigotin were also listed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Synthesis and absolute configuration of hormone alpha1

An important biological event in phytopathogens of the genus Phytophthora is sexual reproduction, which is conducted by two mating types, A1 and A2. A factor known as hormone alpha1 is secreted by the A1 mating type and induces the formation of sexual spores (oospores) in the A2 mating type. Here we describe the asymmetric synthesis and assignment of the absolute configuration of hormone alpha1 by oospore-inducing assays of the synthesized isomers.     

Energetic heavy ions accelerate differentiation in the descendants of irradiated normal human diploid fibroblasts

Ionizing radiation-induced genomic instability has been demonstrated in a variety of endpoints such as delayed reproductive death, chromosome instability and mutations, which occurs in the progeny of survivors many generations after the initial insult. Dependence of these effects on the linear energy transfer (LET) of the radiation is incompletely characterized; however, our previous work has shown that delayed reductions in clonogenicity can be most pronounced at LET of 108 keV/microm. To gain insight into potential cellular mechanisms involved in LET-dependent delayed loss of clonogenicity, we investigated morphological changes in colonies arising from normal human diploid fibroblasts exposed to gamma-rays or energetic carbon ions (108 keV/microm). Exposure of confluent cultures to carbon ions was 4-fold more effective at inactivating cellular clonogenic potential and produced more abortive colonies containing reduced number of cells per colony than gamma-rays. Second, colonies were assessed for clonal morphotypic heterogeneity. The yield of differentiated cells was elevated in a dose- and LET-dependent fashion in clonogenic colonies, whereas differentiated cells predominated to a comparable extent irrespective of radiation type or dose in abortive colonies. The incidence of giant or multinucleated cells was also increased but much less frequent than that of differentiated cells. Collectively, our results indicate that carbon ions facilitate differentiation more effectively than gamma-rays as a major response in the progeny of irradiated fibroblasts. Accelerated differentiation may account, at least in part, for dose- and LET-dependent delayed loss of clonogenicity in normal human diploid cells, and could be a defensive mechanism that minimizes further expansion of aberrant cells.     

Vacuolar processing enzymes are essential for proper processing of seed storage proteins in Arabidopsis thaliana

The proprotein precursors of storage proteins are post-translationally processed to produce their respective mature forms within the protein storage vacuoles of maturing seeds. To investigate the processing mechanism in vivo, we isolated Arabidopsis mutants that accumulate detectable amounts of the precursors of the storage proteins, 12 S globulins and 2 S albumins, in their seeds. All six mutants isolated have a defect in the beta VPE gene. VPE (vacuolar processing enzyme) is a cysteine proteinase with substrate specificity toward an asparagine residue. We further generated various mutants lacking different VPE isoforms: alpha VPE, beta VPE, and/or gamma VPE. More than 90% of VPE activity is abolished in the beta vpe-3 seeds, and no VPE activity is detected in the alpha vpe-1/beta vpe-3/gamma vpe-1 seeds. The triple mutant seeds accumulate no properly processed mature storage proteins. Instead, large amounts of storage protein precursors are found in the seeds of this mutant. In contrast to beta vpe-3 seeds, which accumulate both precursors and mature storage proteins, the other single (alpha vpe-1 and gamma vpe-1) and double (alpha vpe-1/gamma vpe-1) mutants accumulate no precursors in their seeds at all. Therefore, the vegetative VPEs, alpha VPE and gamma VPE, are not necessary for precursor processing in the presence of beta VPE, but partly compensates for the deficiency in beta VPE in beta vpe-3 seeds. In the absence of functional VPEs, a proportion of pro2S albumin molecules are alternatively cleaved by aspartic proteinase. This cleavage by aspartic proteinase is promoted by the initial processing of pro2S albumins by VPE. Our overall results suggest that seed-type beta VPE is most essential for the processing of storage proteins, and that the vegetative-type VPEs and aspartic proteinase complement beta VPE activity in this processing.     

Structure of acidic phospholipase A2 for the venom of Agkistrodon halys blomhoffii at 2.8 A resolution.

The crystal structure of acidic phospholipase A2 from the venom of Agkistrodon halys blomhoffii has been determined by molecular replacement methods based on the known structure of Crotalus atrox PLA2, a same group II enzyme. The overall structures, except the calcium-binding regions, are very similar to each other. A calcium ion is pentagonally ligated to two carboxylate oxygen atoms of Asp-49 and each carbonyl oxygen atoms of Tyr-28, Gly-30 and Ala-31. A reason why the former enzyme functions as monomeric form, while the latter one does as dimer, could be presumed by the structural comparison of these calcium-binding regions. Although Gly-32 is usually participated as a ligand in the coordination with calcium ion in group I PLA2, it is characteristically replaced to Ala-31 in the present structure, and thus the coordination geometry of calcium ion is rather different from the usually observed one.