Substrate specificity of the flavoprotein trypanothione disulfide reductase from Crithidia fasciculata

The substrate specificity of the trypanosomatid enzyme trypanothione reductase has been studied by measuring the ability of the enzyme to reduce a series of chemically synthesized cyclic and acyclic derivatives of N1,N8-bis(glutathionyl)spermidine disulfide (trypanothione). Kinetic analysis of the enzymatic reduction of these synthetic substrates indicates that the mutually exclusive substrate specificity observed by the NADPH-dependent trypanothione disulfide reductase and the related flavoprotein glutathione disulfide reductase is due to the presence of a spermidine binding site in the substrate binding domain of trypanothione reductase. Trypanothione reductase will reduce the disulfide form of N1-monoglutathionylspermidine and also the mixed disulfide of N1-monoglutathionylspermidine and glutathione. The Michaelis constants for these reactions are 149 microM and 379 microM, respectively. Since the disulfide form of N1-monoglutathionylspermidine and the mixed disulfide of N1-monoglutathionylspermidine and glutathione could be formed in trypanosomatids, the binding constants and turnover numbers for the enzymatic reduction of these acyclic disulfides are consistent with these being potential alternative substrates for trypanothione reductase in vivo.     

Minicircle-encoded guide RNAs from Crithidia fasciculata.

Although the mitochondrial uridine insertion/deletion, guide RNA (gRNA)-mediated type of RNA editing has been described in Crithidia fasciculata, no evidence for the encoding of gRNAs in the kinetoplast minicircle DNA has been presented. There has also been a question as to the capacity of the minicircle DNA in this species to encode the required variety of gRNAs, because the kinetoplast DNA from the C1 strain has been reported as essentially containing a single minicircle sequence class. To address this problem, the genomic and mature edited sequences of the MURF4 and RPS12 cryptogenes were determined and a gRNA library was constructed from mitochondrial RNA. Five specific gRNAs were identified, two of which edit blocks within the MURF4 mRNA, and three of which edit blocks within the RPS12 mRNA. The genes for these gRNAs are all localized with identical polarity within one of the two variable regions of specific minicircle molecules, approximately 60 bp from the "bend" region. These minicircles were found to represent minor sequence classes representing approximately 2% of the minicircle DNA population in the network. The major minicircle sequence class also encodes a gRNA at the same relative genomic location, but the editing role of this gRNA was not determined. These results confirm that kinetoplast minicircle DNA molecules in this species encode gRNAs, as is the case in other trypanosomatids, and suggest that the copy number of specific minicircle sequence classes can vary dramatically without an overall effect on the RNA editing system.     

Partial purification and properties of a nucleoside hydrolase from Crithidia

An enzyme catalyzing the hydrolysis of nucleosides was found to occur in Crithidia fasciculata and was partially purified (30- to 40-fold) by treatment with either streptomycin sulfate or MnCl₂, ammonium sulfate fractionation, acidification and neutralization, passage through Sephadex G-200, and isoelectric focusing. The specific activity of these preparations was about 6 μmnoles of uridine hydrolyzed per mg protein per min. Specificity for the puriue or pyrimidine base was very broad; uridine gave the maximum rate of hydrolysis. Deoxyribosides were not hydrolyzed. The enzyme is relatively stable to heat and to acidification and can be stored frozen. Hydrolysis of uridine is inhibited by borate ions and by adenosine, inosine, and guanosine, but not by cytidine or xanthosine.     

A DNase from the trypanosomatid Crithidia fasciculata.

We have purified to homogeneity a DNase from a Crithidia fasciculata crude mitochondrial lysate. The enzyme is present in two forms, either as a 32 kDa polypeptide or as a multimer containing the 32 kDa polypeptide in association with a 56 kDa polypeptide. Native molecular weight measurements indicate that these forms are a monomer and possibly an alpha 2 beta 2 tetramer, respectively. The monomeric and multimeric forms of the enzyme are similar in their catalytic activities. Both digest double-stranded DNA about twice as efficiently as single-stranded DNA. They introduce single-strand breaks into a supercoiled plasmid but do not efficiently make double-strand breaks. They degrade a linearized plasmid more efficiently than a nickel plasmid. Both enzymes degrade a 5'-32P-labeled double-stranded oligonucleotide to completion, with the 5'-terminal nucleotide ultimately being released as a 5'-mononucleotide. One difference between the monomeric and multimeric forms of the enzyme, demonstrated by a band shift assay, is that the multimeric form binds tightly to double-stranded DNA, possibly aggregating it.     

Characterization of a novel endonuclease from Crithidia fasciculata.

A new endonuclease activity has been identified in whole cell lysates of the trypanosomatid Crithidia fasciculata. This activity, termed endonuclease A (Endo A), introduces single-strand breaks at highly preferred sites in double stranded DNA substrates Physical analysis of this enzyme indicates that it has a sedimentation coefficient S20,W of 4.9 and a Stokes radius of 59A and thus, a native molecular weight of 125,000 and a frictional coefficient of 1.8. A monomeric structure is suggested for the enzyme based on the recovery of Endo A activity associated with a polypeptide with a molecular weight of 116,000-120,000, following electrophoresis on sodium dodecyl sulfate polyacrylamide gels. Endo A shows an absolute requirement for Mg2+ or Mn2+ and exhibits activity over a broad pH and temperature range, with optimal conditions for activity at pH 8.0 and 30 degrees C.     

Purification of glutathionylspermidine and trypanothione synthetases from Crithidia fasciculata.

Two enzymes involved in the biosynthesis of the trypanosomatid-specific dithiol trypanothione-glutathionylspermidine (Gsp) synthetase and trypanothione (TSH) synthetase--have been identified and purified individually from Crithidia fasciculata. The Gsp synthetase has been purified 93-fold and the TSH synthetase 52-fold to apparent homogeneity from a single DEAE fraction that contained both activities. This constitutes the first indication that the enzymatic conversion of two glutathione molecules and one spermidine to the N1,N8-bis(glutathionyl)spermidine (TSH) occurs in two discrete enzymatic steps. Gsp synthetase, which has a kcat of 600/min, shows no detectable TSH synthetase activity, whereas TSH synthetase does not make any detectable Gsp and has a kcat of 75/min. The 90-kDa Gsp synthetase and 82-kDa TSH synthetase are separable on phenyl Superose and remain separated on gel filtration columns in high salt (0.8 M NaCl). Active complexes can be formed under low to moderate salt conditions (0.0-0.15 M NaCl), consistent with a functional complex in vivo.     

Novobiocin affinity purification of a mitochondrial type II topoisomerase from the trypanosomatid Crithidia fasciculata

A mitochondrial type II DNA topoisomerase (topoIImt) has been purified to near homogeneity from the trypanosomatid Crithidia fasciculata. A rapid purification procedure has been developed based on the affinity of the enzyme for novobiocin, a competitive inhibitor of the ATP-binding moiety of type II topoisomerases. The purified enzyme is capable of ATP-dependent catenation and decatenation of kinetoplast DNA networks as well as catalyzing the relaxation of supercoiled DNA. topoIImt exists as a dimer of a 132-kDa polypeptide. Immunoblots of whole cell lysates show a single predominant band that comigrates with the 132-kDa polypeptide, indicating that the 264-kDa homodimer represents the intact form of the enzyme. Localization of the enzyme within the single mitochondrion of C. fasciculata (Melendy, T., Sheline, C., and Ray, D. S. (1988) Cell, in press) suggests an important role for topoIImt in kinetoplast DNA replication.     

Purification of a mitochondrial DNA polymerase from Crithidia fasciculata.

The mitochondrial DNA polymerase from Crithidia fasciculata has been purified to near homogeneity. SDS-PAGE analysis of the purified enzyme reveals a single polypeptide with a molecular weight of approximately 43,000. The protein is basic, with an isoelectric point between 7.6-8.0. Its Stokes radius of 22 A and its sedimentation coefficient of 4.1 S suggest a native molecular weight of 38,000, indicating that the protein is a monomer under our experimental conditions. Western blots and immunoprecipitations of crude extracts reveal a cross-reacting protein of 48 kDa, suggesting that the purified enzyme may be an enzymatically active proteolytic product. The mitochondrial origin of the polymerase was confirmed by cell fractionation. Our results indicate that the C. fasciculata enzyme may be among the smallest known mitochondrial polymerases.     

Cell surface charge and sugar residues of Crithidia fasciculata and Crithidia luciliae.

The surface charge of Crithidia fasciculata and Crithidia luciliae was analysed by measurement of the zeta-potential and labelling of the protozoan surface with cationized ferritin particles. Both trypanosomatids have a net negative surface charge, with a zeta-potential of -10.39 mV and -11.12 mV for C. luciliae and C. fasciculata, respectively. Enzyme treatment showed that phosphate groups, but not sialic acid, significantly contributed to the negative surface charge. Lectin-induced agglutination was used to analyse the presence of surface-exposed carbohydrates in C. fasciculata and C. luciliae. The cells did not agglutinate when incubated in the presence of lectins which recognized L-fucose, N-acetyl-D-glucosamine and sialic acid. However, lectins which bind to N-acetyl-D-galactosamine, D-galactose and D-mannose agglutinated both protozoa.     

Sequence, heterologous expression and functional characterization of tryparedoxin1 from Crithidia fasciculata.

Tryparedoxin (TXN) has recently been discovered as a constituent of the complex peroxidase system in the trypanosomatid Crithidia fasciculata [Nogoceke et al. (1997) Biol. Chem. 378, 827-836] where it catalyzes the reduction of a peroxiredoxin-type peroxidase by trypanothione. Here we report on the full-length DNA sequence of the TXN previously isolated from C. fasciculata (TXN1). The deduced amino acid sequence comprises 147 residues and matches with all the peptide sequences of fragments obtained from TXN1. It shares a characteristic sequence motif YFSAxWCPPCR with some thioredoxin-related proteins of unknown function. This motif is homologous with the CXXC motif, which characterizes the thioredoxin superfamily of proteins and is known to catalyze disulfide reductions. Sequence conservations between TXNs and the typical thioredoxins are restricted to the intimate environment of the CXXC motif and three more remote residues presumed to contribute to the folding pattern of the thioredoxin-type proteins. The TXNs thus form a distinct molecular clade within the thioredoxin superfamily. TXN1 was expressed in Escherichia coli BL21 (DE3)pLysS as a C-terminally extended and His-tagged protein, isolated by chelate chromatography and characterized functionally. The recombinant product exhibited a kinetic pattern identical with, and kinetic parameters similar to those of the authentic enzyme in the trypanothione/peroxiredoxin oxidoreductase assay. The recombinant TXN1 can therefore be considered a valuable tool for the screening of specific inhibitors as potential trypanocidal agents.     

Pyrimidine biosynthesis in parasitic protozoa: purification of a monofunctional dihydroorotase from Plasmodium berghei and Crithidia fasciculata

Dihydroorotase (DHOase) catalyzes the reversible cyclization of N-carbamoyl-L-aspartate (L-CA) to L-5,6-dihydroorotate (L-DHO), which is the third enzyme in de novo pyrimidine biosynthesis. The enzyme was purified from two parasitic protozoa, Crithidia fasciculata (about 16,000-fold) and Plasmodium berghei (about 790-fold). The C. fasciculata enzyme had a native molecular weight (Mr) of 42,000 +/- 5000, determined by gel filtration chromatography, and showed a single detectable protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with Mr 44,000 +/- 3000. The DHOase from P. berghei had a native molecular weight of 40,000 +/- 4000 and a subunit molecular weight on SDS-PAGE of 38,000 +/- 3000. The DHOase from both parasites, in contrast to the mammalian enzyme which resides on a trifunctional protein of the first two enzymes of the pathway, carbamoyl-phosphate synthase and aspartate transcarbamylase, is monomeric and has no oligomeric structure as studied by chemical cross-linking with dimethyl suberimidate. The rate of cyclization of L-CA by the C. fasciculata enzyme was relatively high at acidic pH, decreasing to a very low rate at alkaline pH. In contrast, the rate of ring cleavage of L-DHO was very low at acidic pH and increased to a higher rate at alkaline pH. These pH-activity profiles gave an intersection at pH 6.6. The Km and kcat for L-CA were 0.846 +/- 0.017 mM and 39.2 +/- 6.4 min-1, respectively; for L-DHO, they were 25.85 +/- 2.67 microM and 258.6 +/- 28.5 min-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of DNA ligase I from the trypanosomatid Crithidia fasciculata.

A DNA ligase has been purified approximately 5000-fold, to near homogeneity, from the trypanosomatid Crithidia fasciculata. The purified enzyme contains polypeptides with molecular masses of 84 and 80 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both polypeptides formed enzyme-adenylate complexes in the absence of DNA, contained an epitope that is highly conserved between human and bovine DNA ligase I and yeast and vaccinia virus DNA ligases, and were identified in fresh lysates of C. fasciculata by antibodies raised against the purified protein. Hydrodynamic measurements indicate that the enzyme is an asymmetric protein of approximately 80 kDa. The purified DNA ligase can join oligo(dT) annealed to poly(dA), but not oligo(dT) annealed to poly(rA), and can ligate blunt-ended DNA fragments. The enzyme has a low Km for ATP of 0.3 microM. The DNA ligase absolutely requires ATP and Mg2+, and is inhibited by N-ethylmaleimide and by KCI. Substrate specificity, Km for ATP, and the conserved epitope all suggest that the purified enzyme is the trypanosome homologue of DNA ligase I.     

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.     

Iron-containing superoxide dismutase from Crithidia fasciculata. Purification, characterization, and similarity to Leishmanial and trypanosomal enzymes

Leishmania tropica, Trypanosoma brucei, Trypanosoma cruzi, and Crithidia fasciculata have superoxide dismutases which are insensitive to cyanide and sensitive to peroxide and azide, properties characteristic of iron-containing superoxide dismutase. Studies on the superoxide dismutase of C. fasciculata have revealed that: 1) the enzyme is located in the cytosol; 2) isozymes exist; 3) the major superoxide dismutase isozyme (superoxide dismutase 2) has Mr approximately equal to 43,000 and consists of two equal-sized subunits, each of which contains 1.4 atoms of iron. Comparisons of the amino acid content of this crithidial superoxide dismutase with those of superoxide dismutases from other sources suggests that the crithidial enzyme is closely related to bacterial Fe-containing superoxide dismutases, and only distantly related to human Mn- and Cu,Zn-containing superoxide dismutases and to Euglena Fe-containing superoxide dismutase. Attempts are now underway to develop specific inhibitors of the trypanosomatid superoxide dismutase which may be of use in the treatment of leishmaniasis or trypanosomiasis.