A practical approach for computing the active site of the ribonucleoside hydrolase of E. coli encoded by rihC.

We predict the potential active and catalytic sites, the transition state and how it is stabilized, and the mechanism of rihC ribonucleoside hydrolase of E. coli. Our approach is based on well-known primary sequence analysis techniques. A canonically associated extreme value distribution is used to assess the significance of the prediction. Parameters for the extreme value distribution are computed directly from data. Our practical approach is consistent with known results in the literature. We obtain BLOSUM matrices in a way that is intrinsically tied to the data base, and we employ user-friendly techniques that should be applicable to a range of medically significant scenarios.

Preparative enzymatic synthesis of trypanothione and trypanothione analogues.

Trypanosomatids, the causative agents of several tropical diseases, have a unique thiol metabolism based on trypanothione [bis(glutathionyl)spermidine]. Enzymes of the pathway are attractive drug target molecules but the availability of trypanothione remains an obstacle. Here, we present a convenient method for the production of trypanothione and trypanothione disulfide in >200mg quantities using a mutant of Crithidia fasciculata trypanothione synthetase in which Cys59 has been replaced by an alanine residue. The reagent costs less than 1% of the commercial price of trypanothione disulfide. The protocol also allows the synthesis of related glutathione conjugates. It will greatly facilitate the thorough analysis of this parasite's metabolism and drug screening approaches against trypanothione-dependent enzymes.

ATP-dependent ligases in trypanothione biosynthesis--kinetics of catalysis and inhibition by phosphinic acid pseudopeptides.

Glutathionylspermidine is an intermediate formed in the biosynthesis of trypanothione, an essential metabolite in defence against chemical and oxidative stress in the Kinetoplastida. The kinetic mechanism for glutathionylspermidine synthetase (EC 6.3.1.8) from Crithidia fasciculata (CfGspS) obeys a rapid equilibrium random ter-ter model with kinetic constants K(GSH) = 609 microM, K(Spd) = 157 microM and K(ATP) = 215 microM. Phosphonate and phosphinate analogues of glutathionylspermidine, previously shown to be potent inhibitors of GspS from Escherichia coli, are equally potent against CfGspS. The tetrahedral phosphonate acts as a simple ground state analogue of glutathione (GSH) (K(i) approximately 156 microM), whereas the phosphinate behaves as a stable mimic of the postulated unstable tetrahedral intermediate. Kinetic studies showed that the phosphinate behaves as a slow-binding bisubstrate inhibitor [competitive with respect to GSH and spermidine (Spd)] with rate constants k(3) (on rate) = 6.98 x 10(4) M(-1) x s(-1) and k(4) (off rate) = 1.3 x 10(-3) s(-1), providing a dissociation constant K(i) = 18.6 nM. The phosphinate analogue also inhibited recombinant trypanothione synthetase (EC 6.3.1.9) from C. fasciculata, Leishmania major, Trypanosoma cruzi and Trypanosoma brucei with K(i)(app) values 20-40-fold greater than that of CfGspS. This phosphinate analogue remains the most potent enzyme inhibitor identified to date, and represents a good starting point for drug discovery for trypanosomiasis and leishmaniasis.

Expression, purification and characterization of recombinant mitochondrial topoisomerase II of kinetoplastid Crithidia fasciculata in High-five insect cells.

Topoisomerase II of kinetoplastid parasites plays an important role in the replication of unusual networks of kinetoplast DNA (kDNA) and is a very useful target for antiparasitic drugs. In this study, we cloned full-length Crithidia fasciculata mitochondrial topoisomerase II gene into pFastBac-HTc vector and successfully expressed an active recombinant full-length mitochondrial topoisomerase II in Bac-to-Bac baculovirus expression system. A rapid and simple purification strategy was established by incorporating a FLAG-tag at the C-terminus of the protein. The purified recombinant topoisomerase II showed a major single band on SDS-PAGE (>96% purity) and was verified through Western blot analysis. The recombinant full-length mitochondrial topoisomerase II exhibited decatenation, catenation and relaxation activity of type II topoisomerase as well as various sensitivities to a series of known topoisomerase inhibitors. These studies explore new way and lay groundwork to express all other similar full-length kinetoplastid topoisomerases, it will also facilitate further elucidation of X-ray structure, catalysis mechanism of kinetoplastid topoisomerases and design of new antiparasitic drugs targeting kinetoplastid topoisomerases.

Sequence elements essential for the rapid turnover of Crithidia fasciculata ornithine decarboxylase.

Ornithine decarboxylase (ODC) has a very fast turnover in mammalian cells, but is a stable enzyme in T. brucei and other trypanosmatid parasites like Leishmania donovani. However, Crithidia fasciculata, which is a phylogenetically closely related trypanosomatid to L. donovani, has an ODC with a rapid turnover. Interestingly, C. fasciculata ODC, but not L. donovani ODC, is rapidly degraded also in mammalian systems. In order to obtain information on what sequences are important for the rapid degradation of C. fasciculata ODC, we produced a variety of C. fasciculata/L. donovani ODC hybrid proteins and characterized their turnover using two different mammalian expression systems. The results obtained indicate that C. fasciculata ODC contains several sequence elements essential for the rapid turnover of the protein and that these regions are mainly located in the central part of the enzyme.

Genetic selection for a highly functional cysteine-less membrane protein using site saturation mutagenesis.

We describe an efficient method for generating highly functional membrane proteins with variant amino acids at defined positions that couples a modified site saturation strategy with functional genetic selection. We applied this method to the production of a cysteine-less variant of the Crithidia fasciculata inosine-guanosine permease CfNT2 to facilitate biochemical studies using thiol-specific modifying reagents. Of 10 endogenous cysteine residues in CfNT2, two cannot be replaced with serine or alanine without loss of function. High-quality single- and double-mutant libraries were produced by combining a previously reported site saturation mutagenesis scheme based on the Stratagene Quikchange method with a novel gel purification step that effectively eliminated template DNA from the products. Following selection for functional complementation in Saccharomyces cerevisiae cells auxotrophic for purines, several highly functional noncysteine substitutions were efficiently identified at each desired position, allowing the construction of cysteine-less variants of CfNT2 that retained wild-type affinity for inosine. This combination of an improved site saturation mutagenesis technique and positive genetic selection provides a simple and efficient means to identify functional and perhaps unexpected amino acid variants at a desired position.

Inhibitory activity of plant stilbene oligomers against DNA topoisomerase II.

The inhibitory activity of 40 stilbene oligomers isolated from six plant species against topoisomerase II was evaluated, of which nine compounds showed a potent inhibitory effect, stronger than daunorubicin, a topoisomerase II inhibitor, used as an anti-cancer drug. The specificity of active stilbene oligomers on topoisomerase II was assessed by their effect on DNA restriction enzyme. In particular, specific inhibitory activity was observed in alpha-viniferin 13-O-beta-glucopryranoside (2) and hemsleyanol C (13).

Cell cycle-dependent localization and properties of a second mitochondrial DNA ligase in Crithidia fasciculata.

The mitochondrial DNA in kinetoplastid protozoa is contained in a single highly condensed structure consisting of thousands of minicircles and approximately 25 maxicircles. The disk-shaped structure is termed kinetoplast DNA (kDNA) and is located in the mitochondrial matrix near the basal body. We have previously identified a mitochondrial DNA ligase (LIG kbeta) in the trypanosomatid Crithidia fasciculata that localizes to antipodal sites flanking the kDNA disk where several other replication proteins are localized. We describe here a second mitochondrial DNA ligase (LIG kalpha). LIG kalpha localizes to the kinetoplast primarily in cells that have completed mitosis and contain either a dividing kinetoplast or two newly divided kinetoplasts. Essentially all dividing or newly divided kinetoplasts show localization of LIG kalpha. The ligase is present on both faces of the kDNA disk and at a high level in the kinetoflagellar zone of the mitochondrial matrix. Cells containing a single nucleus show localization of the LIG kalpha to the kDNA but at a much lower frequency. The mRNA level of LIG kalpha varies during the cell cycle out of phase with that of LIG kbeta. LIG kalpha transcript levels are maximal during the phase when cells contain two nuclei, whereas LIG kbeta transcript levels are maximal during S phase. The LIG kalpha protein decays with a half-life of 100 min in the absence of protein synthesis. The periodic expression of the LIG kalpha transcript and the instability of the LIG kalpha protein suggest a possible role of the ligase in regulating minicircle replication.

Transition state analysis of adenosine nucleosidase from yellow lupin (Lupinus luteus).

The transition state of adenosine nucleosidase (EC 3.2.2.7) isolated from yellow lupin (Lupinus luteus) was determined based upon a series of heavy atom kinetic isotope effects. Adenosine labeled with 13C, 2H, and 15N was analyzed by liquid chromatography/electrospray mass spectrometry to determine kinetic isotope effects. Values of 1.024+/-0.004, 1.121+/-0.005, 1.093+/-0.004, 0.993+/-0.006, and 1.028+/-0.005 were found for [1'-13C], [1'-2H], [2'-2H], [5'-2H], and [9-15N] adenosine, respectively. Using a bond order bond energy vibrational analysis, a transition state consisting of a significantly broken C-N bond, formation of an oxocarbenium ion in the ribose ring, a conformation of C3-exo for the ribose ring, and protonation of the heterocyclic base was proposed. This transition state was found to be very similar to the transition state for nucleoside hydrolase, another purine metabolizing enzyme, isolated from Crithidia fasciculata.

Kinetic isotope effects of nucleoside hydrolase from Escherichia coli.

rihC is one of a group of three ribonucleoside hydrolases found in Escherichia coli (E. coli). The enzyme catalyzes the hydrolysis of selected nucleosides to ribose and the corresponding base. A family of Vmax/Km kinetic isotope effects using uridine labeled with stable isotopes, such as 2H, 13C, and 15N, were determined by liquid chromatography/mass spectrometry (LC/MS). The kinetic isotope effects were 1.012+/-0.006, 1.027+/-0.005, 1.134+/-0.007, 1.122+/-0.008, and 1.002+/-0.004 for [1'-13C], [1-15N], [1'-2H], [2'-2H], and [5'-2H2] uridine, respectively. A transition state based upon a bond-energy bond-order vibrational analysis (BEBOVIB) of the observed kinetic isotope effects is proposed. The main features of this transition state are activation of the heterocyclic base by protonation of/or hydrogen bonding to O2, an extensively broken C-N glycosidic bond, formation of an oxocarbenium ion in the ribose ring, C3'-exo ribose ring conformation, and almost no bond formation to the attacking nucleophile. The proposed transition state for the prokaryotic E. coli nucleoside hydrolase is compared to that of a similar enzyme isolated from Crithidia fasciculata (C. fasciculata).

Trypanothione biosynthesis in Leishmania major.

Trypanothione plays a crucial role in regulation of intracellular thiol redox balance and in defence against chemical and oxidant stress. Crithidia fasciculata requires two enzymes for the formation of trypanothione, namely glutathionylspermidine synthetase (GspS; EC 6.3.1.8) and a glutathionylspermidine-dependent trypanothione synthetase (TryS; EC 6.3.1.9), whereas Trypanosoma cruzi and Trypanosoma brucei use a broad-specificity trypanothione synthetase to make trypanothione from glutathione (GSH) and spermidine. Here, we report the identification of two genes in Leishmania major with similarity to previously identified GSPS and TRYS. GSPS is an apparent pseudogene containing two frame shift mutations and two stop codons, whereas TRYS is in a single open-reading frame. The enzyme encoded by TRYS was expressed and found to catalyse formation of trypanothione with GSH and either spermidine or glutathionylspermidine. When GSH is varied as substrate the enzyme displays substrate inhibition (apparent Km=89 microM, Ki(s)=1mM, k(cat)=2s-1). At a fixed GSH concentration, the enzyme obeys simple hyperbolic kinetics with the other substrates with apparent Km values for spermidine, glutathionylspermidine and MgATP of 940, 40 and 63 microM, respectively. Immunofluorescence and sub-cellular fractionation studies indicate that TryS localises to the cytosol of L. major promastigotes. Phylogenetic analysis of the GspS and TryS amino acid sequences suggest that in the trypanosomatids, TryS has evolved to replace the GspS/TryS complex in C. fasciculata. It also appears that the L. major still harbours a redundant GSPS pseudogene that may be currently in the process of being lost from its genome.

Trypanothione S-transferase activity in a trypanosomatid ribosomal elongation factor 1B.

Trypanothione is a thiol unique to the Kinetoplastida and has been shown to be a vital component of their antioxidant defenses. However, little is known as to the role of trypanothione in xenobiotic metabolism. A trypanothione S-transferase activity was detected in extracts of Leishmania major, L. infantum, L. tarentolae, Trypanosoma brucei, and Crithidia fasciculata, but not Trypanosoma cruzi. No glutathione S-transferase activity was detected in any of these parasites. Trypanothione S-transferase was purified from C. fasciculata and shown to be a hexadecameric complex of three subunits with a relative molecular weight of 650,000. This enzyme complex was specific for the thiols trypanothione and glutathionylspermidine and only used 1-chloro-2,4-dinitrobenzene from a range of glutathione S-transferase substrates. Peptide sequencing revealed that the three components were the alpha, beta, and gamma subunits of ribosomal eukaryotic elongation factor 1B (eEF1B). Partial dissociation of the complex suggested that the S-transferase activity was associated with the gamma subunit. Moreover, Cibacron blue was found to be a tight binding inhibitor and reactive blue 4 an irreversible time-dependent inhibitor that covalently modified only the gamma subunit. The rate of inactivation by reactive blue 4 was increased more than 600-fold in the presence of trypanothione, and Cibacron blue protected the enzyme from inactivation by 1-chloro-2,4-dinitrobenzene, confirming that these dyes interact with the active site region. Two eEF1Bgamma genes were cloned from C. fasciculata, but recombinant C. fasciculata eEF1Bgamma had no S-transferase activity, suggesting that eEF1Bgamma is unstable in the absence of the other subunits.

Purification and properties of poly(ADP-ribose)polymerase from Crithidia fasciculata. Automodification and poly(ADP-ribosyl)ation of DNA topoisomerase I.

Poly(ADP-ribose)polymerase has been purified more than 160000-fold from Crithidia fasciculata. This is the first PARP isolated to apparent homogeneity from trypanosomatids. The purified enzyme absolutely required DNA for catalytic activity and histones enhanced it 2.5-fold, when the DNA:histone ratio was 1:1.3. The enzyme required no magnesium or any other metal ion cofactor. The apparent molecular mass of 111 kDa, determined by gel filtration would correspond to a dimer of two identical 55-kDa subunits. Activity was inhibited by nicotinamide, 3-aminobenzamide, theophylline, thymidine, xanthine and hypoxanthine but not by adenosine. The enzyme was localized to the cell nucleus. Our findings suggest that covalent poly(ADP-ribosyl)ation of PARP itself or DNA topoisomerase I resulted in the inhibition of their activities and provide an initial biochemical characterization of this covalent post-translational modification in trypanosomatids.

Mitochondrial DNA ligase in Crithidia fasciculata.

Kinetoplast DNA (kDNA), the form of mitochondrial DNA in trypanosomatids, consists of thousands of interlocked circular DNAs organized into a compact disk structure. A type II DNA topoisomerase, a DNA polymerase beta, and a structure-specific endonuclease have been localized to antipodal sites flanking the kDNA disk along with nascent DNA minicircles. We have cloned a gene (LIG k) encoding a mitochondrial DNA ligase in the trypanosomatid Crithidia fasciculata, and we show that an epitope-tagged form of the ligase colocalizes with the other replication proteins at the antipodal sites and also at the two faces of the kDNA disk. DNA LIG k becomes adenylated in reactions with ATP, and the adenylate moiety is removed by incubation with pyrophosphate or nicked DNA. The ligase interacts physically with the beta polymerase and is proposed to be involved in the repair of gaps in the newly synthesized minicircles. In yeast and mammals, a single gene encodes both nuclear and mitochondrial forms of DNA ligase. The LIG K protein sequence has low similarity to mitochondrial DNA ligases in other eukaryotes and is distinct from the C. fasciculata nuclear DNA ligase (LIG I).

Extremely rapid turnover of S-adenosylmethionine decarboxylase in Crithidia fasciculata.

The activity of S-adenosylmethionine decarboxylase (AdoMetDC) in Crithidia fasciculata was shown to be correlated to the growth of the parasite. An increase in activity was observed during exponential growth. Inhibition of protein synthesis induced an extremely rapid decay of AdoMetDC activity. The half-life of the enzyme was estimated to be about 3 min, which is the shortest half-life ever recorded for an eukaryotic AdoMetDC. The reduction in AdoMetDC activity was correlated with a decrease in AdoMetDC protein content, demonstrating a rapid turnover of the enzyme. No polyamine-mediated feedback regulation of AdoMetDC was observed in the parasite.

Proteasomal degradation of a trypanosomal ornithine decarboxylase.

Mammalian ornithine decarboxylase (ODC), which catalyses the first step in polyamine biosynthesis, has a very fast turnover. It is degraded by the 26S proteasome in an ubiquitin-independent process and the degradation is stimulated by polyamines in a feedback control of the enzyme. Interestingly, there is a major difference in the metabolic stability between ODCs from various trypanosomatids. Trypanosoma brucei and Leishmania donovani both contain stable ODCs, whereas Crithidia fasciculata has an ODC with a rapid turnover. In spite of the difference in stability there is a high degree of sequence homology between C. fasciculata ODC and L. donovani ODC. In the present study we demonstrate that C. fasciculata ODC is rapidly degraded also in mammalian systems like CHO cells and rabbit reticulocyte lysate, suggesting that the degradation signals of the enzyme are recognised by the mammalian systems. L. donovani ODC, on the other hand, is degraded very slowly in the same systems. The degradation of C. fasciculata ODC in the mammalian systems is markedly reduced by inhibition of the 26S proteasome. However, unlike mammalian ODC, C. fasciculata ODC is not down-regulated by polyamines. Thus, the turnover of C. fasciculata ODC and L. donovani ODC in the mammalian systems reflects the degradation of the enzyme in the parasites, making such systems potentially useful as complements to parasitic knockout models for further analysis of the mechanisms involved in the rapid degradation of C. fasciculata ODC.

[Inhibitory action of Fenton systems on topoisomerase I from Trypanosoma cruzi and Crithidia fasciculata]]. / Acción inhibitoria de sistemas Fenton sobre la topoisomerasa I de Trypanosoma cruzi y Crithidia fasciculata.

Fenton systems (H2O2/Fe(II) or H2O2/Cu(II)) inhibited Trypanosoma cruzi and Crithidia fasciculata topoisomerase I activity. About 61-71% inactivation was produced by 25 mM Fe(II) or Cu(II) with 3 mM H2O2. Thiol compounds and free radicals scavengers prevented the Fenton systems effects, depending on the topoisomerase assayed. With the T. cruzi enzyme, reduced glutathione, DL-dithiothreitol, cysteine and N-acetyl-L-cysteine entirely prevented the effect of the H2O2/Fe(II) system, mannitol protected 37%, whereas histidine and ethanol were ineffective. With C. fasciculata topoisomerase, reduced glutathione, DL-dithiothreitol and N-acetyl-L-cysteine protected 100%, cysteine, histidine and mannitol protected 28, 34 and 48% respectively, whereas ethanol was ineffective. With the H2O2/Cu(II) system and T. cruzi topoisomerase, DL-dithiothreitol and histidine protected 100% and 60%, respectively but the other assayed protectors were less effective. Similar results were obtained with the C. fasciculata enzyme. Topoisomerase inactivation by H2O2/Fe(II) or H2O2/Cu(II) systems was irreversible since they were not reverted by the more effective enzyme protectors. It is suggested that topoisomerases could act either as scavengers of "reactive oxygen species" (ROS) generated by Fenton systems or bind the corresponding metal ions, whose redox cycling would generate reactive oxygen species "in situ".

Inactivation of Trypanosoma cruzi and Crithidia fasciculata topoisomerase I by Fenton systems.

Fenton systems (H(2)O(2)/Fe(II) or H(2)O(2)/Cu(II)) inhibited Trypanosoma cruzi and Crithidia fasciculata topoisomerase I activity. About 61-71% inactivation was produced by 25 microM Fe(II) or Cu(II) with 3.0 mM H(2)O(2). Thiol compounds and free radical scavengers prevented Fenton system effects, depending on the topoisomerase assayed. With the T. cruzi enzyme, reduced glutathione (GSH), dithiothreitol (DTT), cysteine and N-acetyl-L-cysteine (NAC) entirely prevented the effect of the H(2)O(2)/Fe(II) system; mannitol protected 37%, whereas histidine and ethanol were ineffective. With C. fasciculata topoisomerase, GSH, DTT and NAC protected 100%, cysteine, histidine and mannitol protected 28%, 34% and 48%, respectively, whereas ethanol was ineffective. With the H(2)O(2)/Cu(II) system and T. cruzi topoisomerase, DTT and histidine protected 100% and 60%, respectively, but the other assayed protectors were less effective. Similar results were obtained with the C. fasciculata enzyme. Topoisomerase inactivation by the H(2)O(2)/Fe(II) or H(2)O(2)/Cu(II) systems proved to be irreversible since it was not reversed by the more effective enzyme protectors. It is suggested that topoisomerases could act either as targets of 'reactive oxygen species' (ROS) generated by Fenton systems or bind the corresponding metal ions, whose redox cycling would generate reactive oxygen species in situ.

Exploring nucleoside hydrolase catalysis in silico: molecular dynamics study of enzyme-bound substrate and transition state.

The mechanism of action of inosine-uridine nucleoside hydrolase has been investigated by long-term molecular dynamics (MD) simulation in TIP3P water using stochastic boundary conditions. Five MD studies have been performed with enzyme substrate complex (E.S), enzyme substrate complex with protonated His241 (EH.S), enzyme transition state complex (E.TS), enzyme transition state complex with protonated His241 (EH.TS), and His241Ala transition state complex E(H241A).TS. Special attention has been given to the role of His241, which has been considered as the general acid catalyst to assist departure of the leaving nucleobase on the basis of its location in the active site in the X-ray crystal structure (). Yet on the basis of the location in the active site, Tyr229 is closer to the aniline ring of pAPIR as compared to His241. On initiation of MD simulations, His241 does not approach the nucleobase in the structures of EH.S, E.S, EH.TS, and E.TS. In the solvated enzyme, Tyr229, which is a member of the hydrogen bonding network inosine O2'.Asp14.His241.Tyr229.inosine N7, serves as a proton source to the leaving nucleobase. The loss of significant activity of His241Ala mutant is shown to be related to the disruption of the above hydrogen bonded network and the distancing of Tyr229 from inosine N7. The structures of the enzyme complexes with substrate or TS are not visibly altered on protonation of His241, a most unusual outcome. The bell-shaped pH dependence upon pK(app)'s of 7.1 and 9.1 may be attributed to the necessity of the dissociation of Asp10 or Asp15 and the acid form of Tyr229, respectively. In TS, the residue Ile81 migrated closer, whereas Arg233 moved away from the nucleobase. The probability of ribooxocarbenium ion stabilization by Asn168 and Asp14 is discussed. The Asp14-CO(2)(-) is hydrogen bonded to the ribose 2'-OH for 96% of the MD simulation time. Nucleophilic addition of water138 to ribooxocarbenium ion is suggested to be assisted by the proton shuttle from water138 --> Asp10 --> Asp15 --> water pool. An anticorrelation motion between Tyr229-OH and Asn168-OD1 in EH.S and E.S is observed. The relationship of this anticorrelated motion to mechanism, if any, deserves further exploration, perhaps the formation of a near attack conformation.

The mitochondrial DNA polymerase beta from Crithidia fasciculata has 5'-deoxyribose phosphate (dRP) lyase activity but is deficient in the release of dRP.

DNA polymerase beta (pol beta) has long been described as a nuclear enzyme involved in DNA repair. A pol beta from the trypanosomatid parasite Crithidia fasciculata, however, is the first example of a mitochondrial enzyme of this type. The mammalian nuclear enzyme functions not only as a nucleotidyl transferase but also has a dRP lyase activity that cleaves 5'-deoxyribose phosphate (dRP) groups from DNA, thus contributing to two consecutive steps of the base excision repair pathway. We find that the mitochondrial pol beta also has dRP lyase activity. Interestingly, the K(m) of this enzyme for a dRP-containing substrate is similar to that for the rat enzyme, but its k(cat) is very low. This difference is due to a deficiency of the mitochondrial enzyme in the release of dRP from the enzyme following its cleavage from the DNA.