The purine salvage pathway of parasitic protozoa is currently considered as a target for drug development because these organisms cannot synthesize purines de novo. Insight into the structure and mechanism of the involved enzymes can aid in the development of potent inhibitors, leading to new curative drugs. Nucleoside hydrolases are key enzymes in the purine salvage pathway of Trypanosomatidae, and they are especially attractive because they have no equivalent in mammalian cells. We cloned, expressed and purified a nucleoside hydrolase from Trypanosoma vivax. The substrate activity profile establishes the enzyme to be a member of the inosine-adenosine-guanosine-preferring nucleoside hydrolases (IAG-NH). We solved the crystal structure of the enzyme at 1.6 A resolution using MAD techniques. The complex of the enzyme with the substrate analogue 3-deaza-adenosine is presented. These are the first structures of an IAG-NH reported in the literature. The T. vivax IAG-NH is a homodimer, with each subunit consisting of ten beta-strands, 12 alpha-helices and three small 3(10)-helices. Six of the eight strands of the central beta-sheet form a motif resembling the Rossmann fold. Superposition of the active sites of this IAG-NH and the inosine-uridine-preferring nucleoside hydrolase (IU-NH) of Crithidia fasciculata shows the molecular basis of the different substrate specificity distinguishing these two classes of nucleoside hydrolases. An "aromatic stacking network" in the active site of the IAG-NH, absent from the IU-NH, imposes the purine specificity. Asp10 is the proposed general base in the reaction mechanism, abstracting a proton from a nucleophilic water molecule. Asp40 (replaced by Asn39 in the IU-NH) is positioned appropriately to act as a general acid and to protonate the purine leaving group. The second general acid, needed for full enzymatic activity, is probably part of a flexible loop located in the vicinity of the active site.

The mechanism of pI258 arsenate reductase (ArsC) catalyzed arsenate reduction, involving its P-loop structural motif and three redox active cysteines, has been unraveled. All essential intermediates are visualized with x-ray crystallography, and NMR is used to map dynamic regions in a key disulfide intermediate. Steadystate kinetics of ArsC mutants gives a view of the crucial residues for catalysis. ArsC combines a phosphatase-like nucleophilic displacement reaction with a unique intramolecular disulfide bond cascade. Within this cascade, the formation of a disulfide bond triggers a reversible ''conformational switch'' that transfers the oxidative equivalents to the surface of the protein, while releasing the reduced substrate.

Arsenate reductase (ArsC) encoded by Staphylococcus aureus arsenic-resistance plasmid pI258 reduces intracellular arsenate(V) to the more toxic arsenite(III), which is subsequently extruded from the cell. It couples to thioredoxin, thioredoxin reductase and NADPH to be enzymatically active. ArsC is extremely sensitive to oxidative inactivation, has a very dynamic character hampering resonance assignments in NMR and produces peculiar biphasic Michaelis-Menten curves with two V(max) plateaus. In this study, methods to control ArsC oxidation during purification have been optimized. Next, application of Selwyn's test of enzyme inactivation was applied to progress curves and reveals that the addition of tetrahedral oxyanions (50 mM sulfate, phosphate or perchlorate) allows the control of ArsC stability and essentially eliminates the biphasic character of the Michaelis-Menten curves. Finally, 1H-15N HSQC NMR spectroscopy was used to establish that these oxyanions, including the arsenate substrate, exert their stabilizing effect on ArsC through binding with residues located within a C-X5-R sequence motif, characteristic for phosphotyrosine phosphatases. In view of this need for a tetrahedral oxyanion to structure its substrate binding site in its active conformation, a reappraisal of basic kinetic parameters of ArsC was necessary. Under these new conditions and in contrast to previous observations, ArsC has a high substrate specificity, as only arsenate could be reduced ( Km=68 microM, k(cat)/ Km =5.2 x 10(4 )M-1s-1), while its product, arsenite, was identified as a mixed inhibitor ( K*iu=534 microM, K*ic=377 microM).

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One of the last unsolved problems of molecular biology is how the sequential amino acid information leads to a functional protein. Correct disulfide formation within a protein is hereby essential. We present periplasmic ribonuclease I (RNase I) from Escherichia coli as a new endogenous substrate for the study of oxidative protein folding. One of its four disulfides is between nonconsecutive cysteines. In general view, the folding of proteins with nonconsecutive disulfides requires the protein disulfide isomerase DsbC. In contrast, our study with RNase I shows that DsbA is a sufficient catalyst for correct disulfide formation in vivo and in vitro. DsbA is therefore more specific than generally assumed. Further, we show that the redox potential of the periplasm depends on the presence of glutathione and the Dsb proteins to maintain it at ؊165 mV. We determined the influence of this redox potential on the folding of RNase I. Under the more oxidizing conditions of dsb ؊ strains, DsbC becomes necessary to correct non-native disulfides, but it cannot substitute for DsbA. Altogether, DsbA folds a protein with a nonconsecutive disulfide as long as no incorrect disulfides are formed.

The reduction of arsenate to arsenite by pI258 arsenate reductase (ArsC) combines a nucleophilic displacement reaction with a unique intramolecular disulfide cascade. Within this reaction mechanism, the oxidative equivalents are translocated from the active site to the surface of ArsC. The first reaction step in the reduction of arsenate by pI258 ArsC consists of a nucleophilic displacement reaction carried out by Cys10 on dianionic arsenate. The second step involves the nucleophilic attack of Cys82 on the Cys10-arseno intermediate formed during the first reaction step. The onset of the second step is studied here by using quantum chemical calculations in a density functional theory context. The optimised geometry of the Cys10-arseno adduct in the ArsC catalytic site (sequence motif: Cys10-Thr11-Gly12-Asn13-Ser14-Cys15-Arg16-Ser17) forms the starting point for all subsequent calculations. Thermodynamic data and a hard and soft acids and bases (HSAB) reactivity analysis show a preferential nucleophilic attack on a monoanionic Cys10-arseno adduct, which is stabilised by Ser17. The P-loop active site of pI258 ArsC activates first a hydroxy group and subsequently arsenite as the leaving group, as is clear from an increase in the calculated nucleofugality of these groups upon going from the gas phase to the solvent phase to the enzymatic environment. Furthermore, the enzymatic environment stabilises the thiolate form of the nucleophile Cys82 by 3.3 pH units through the presence of the eight-residue alpha helix flanked by Cys82 and Cys89 (redox helix) and through a hydrogen bond with Thr11. The importance of Thr11 in the pKa regulation of Cys82 was confirmed by the observed decrease in the kcat value of the Thr11Ala mutant as compared to that of wild-type ArsC. During the final reaction step, Cys89 is activated as a nucleophile by structural alterations of the redox helix that functions as a pKa control switch for Cys89; this final step is necessary to expose a Cys82-Cys89 disulfide.

Transfer ribonucleic acid (tRNA) modifications, especially at the wobble position, are crucial for proper and efficient protein translation. MnmE and MnmG form a protein complex that is implicated in the carboxymethylaminomethyl modification of wobble uridine (cmnm5U34) of certain tRNAs. MnmE is a G protein activated by dimerization (GAD), and active guanosine-5'-triphosphate (GTP) hydrolysis is required for the tRNA modification to occur. Although crystal structures of MnmE and MnmG are available, the structure of the MnmE/MnmG complex (MnmEG) and the nature of the nucleotide-induced conformational changes and their relevance for the tRNA modification reaction remain unknown. In this study, we mainly used small-angle X-ray scattering to characterize these conformational changes in solution and to unravel the mode of interaction between MnmE, MnmG and tRNA. In the nucleotide-free state MnmE and MnmG form an unanticipated asymmetric α2β2 complex. Unexpectedly, GTP binding promotes further oligomerization of the MnmEG complex leading to an α4β2 complex. The transition from the α2β2 to the α4β2 complex is fast, reversible and coupled to GTP binding and hydrolysis. We propose a model in which the nucleotide-induced changes in conformation and oligomerization of MnmEG form an integral part of the tRNA modification reaction cycle.