Molecular Microbiology (1992) 6(21), 3089-3099

Molecular characterization of the trypanothione reductase gene from Crithidia fasciculata and Trypanosoma brucei: comparison with other flavoprotein disulphide oxidoreductases with respect to substrate specificity and catalytic machanism T. Aboagye-Kwarteng, K. Smith and A. H. Fairlamb* Department of Medical Parasitology, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT. UK. Summary Trypanothione reductase belongs to the family of flavoprotein disulphide oxidoreductases that include gtutathione reductases, dihydroiipoamide dehydrogenases and mercuric reductases. Trypanothione reductase and its substrate, trypanothione disulphide, are unique to parasitic trypanosomatids responsible for several tropical diseases. The crystal structure of the enzyme from Crithidia fasciculata is currently under investigation as an aid in the design of selective inhibitors with a view to producing new drugs. We report here the cloning and sequencing of the genes for trypanothione reductase from C. fasciculata and Trypanosoma brucei. Alignment of the deduced amino acid sequences with 21 other members of this family provides insight into the role of certain amino acid residues with respect to substrate specificity and catalytic mechanism as well as conservation of certain elements of secondary structure.

Introduction Many parasitic protozoans belonging to the order Kinetoplastida (suborder Trypanosomatina) are human pathogens causing tropical diseases such as Chagas' disease (Trypanosoma cruzi), sleeping sickness {Trypanosoma brucei rhodesiense and T. brucei gambiense) and visceral leishmaniasis {Leishmania donovani). Current treatment for these life-threatening diseases is inadequate and new and more effective drugs are urgently needed. Given the overall lack of interest in this area by the pharmaceutical industry, much effort in academic

Received 11 November, 1991; revised and accepted 24 July. 1992. *For correspondence, Tel. (071) 927 2455; Fax (071) 636 8739.

institutions Is now directed towards drug design by the socalled rational approach, which involves the molecular characterization of novel biochemical targets that are essential for parasite survival, but absent from the host (Hoi etal., 1989: Fairlamb, 1989; 1982). A lot of current interest surrounds the unique dithiol, trypanothione (A/\W^-bis(glutathionyl)spermidine, Fairlamb etal., 1985) and its metabolism (Fairlamb, 1991; 1989; Docampo, 1990). Most aerobic organisms contain millimolar concentrations of the tripeptide glutathione (L-yglutamyl-L-cysteinylglycine). which, in concert with glutathione reductase (EC 1.6.4.2), serves an important role in the regulation of intracellular thiol-redox and in defence against oxidant or other chemical induced damage (Dolphin etal., 1989; Meister and Anderson, 1983). Although trypanosomatids contain glutathione, they are uniquely different in that glutathione reductase and glutathione peroxidase are absent (Fairiamb and Cerami, 1985; Henderson et al., 1987a) and have evolved an analogous defence system for involving trypanothione reductase (EC 1.6.4.8) (Shames etal., 1986). trypanothione peroxidase (Henderson et al., 1987a) and their substrates trypanothione disulphide and dihydrotrypanothione, respectively. Trypanothione biosynthesis and function also appear to be implicated in the mode of action of a number of existing drugs (e.g. difluoromethylornithine (Fairlamb et al., 1987; Bellofatto et al., 1987) and trivalent arsenicals (Fairlamb ef al., 1989)) and therefore trypanothione represents a promising candidate for rationally based drug design (Fairlamb, 1990a,b; Smith etal., 1991a; Walsh et at., 1991; Fairlamb and Cerami, 1992). To date, the bestcharacterized drug target is the enzyme trypanothione reductase, which is physically and mechanistically similar to human glutathione reductase (Shames et al.. 1986; Krauth-Siegel et al., 1987; Ghisia and Massey. 1989). Significantly, the two enzymes are highly discriminatory for their cognate disulphide substrates (Shames et al., 1986; Henderson et al., 1987b) and should therefore be susceptible to selective inhibition. Initial attempts at selective inhibitor design have already yielded encouraging results (Henderson et al., 1988; Jockers-Scherubl et al..

3090

T. Aboagye-Kwarteng, K. Smith and A. H. Fairlamb

1989), but could be markedly improved if the threedimensional structure of the active site were known. especially since the crystal structure of human glutathione reductase, bound to its substrate, glutathlone, is already available to high resolution (Karplus ef al., 1989), Crystallographic analyses of two entirely different crystal forms of trypanothione reductase from Crithidia fascicu/afa are already underway (Hunter ef a/,, 1990; Kuriyan ef al., 1990; 1991; Hunter etal.. 1992). We report here the primary sequence of trypanothione reductase used in our crystallographic studies (Hunter etal., 1990; 1992) and also the sequence of cattle parasite T. brucei brucei trypanothione reductase and compare these sequences with other members of this family, namely glutathione reductases (GRs), dihydrolipoamide dehydrogenases (DLDs) and mercury reductases (MRs), with respect to recent advances in knowledge of their structure and function.

domains, particularly those involved in catalysis in the disulphide-binding site. Accordingly, residues NVGCVPKK (residues 54-61), and TIGVHPT (residues 457463) were selected for the design of the degenerate oligonucleotide primers (see the Experimental procedures and Fig, 1), Using these primers, polymerase chain reaction (PCR) amplification of C. fasciculata genomic DNA prepared from a cloned line (HS6) produced a single 1,2 kb fragment of the predicted size, consisting of approximately 80% of the TR gene. This was inserted into pUC19 and a recombinant plasmid (pHS6AB3) sequenoed on both strands. The nucleotide sequence of pHS6AB3 and the predicted amino acid sequence showed high homology to the Trypanosoma congolense TRgene (Shames etal., 1988) and confirmed the authenticity of the 1.2 kb clone as part of the C. fasciculata TR gene. Using a similar approach PCR amplification of T. iiruce/genomic DNA also produced as expected a single 1.2 kb fragment. This was inserted into pUCi9 and a clone (pTR1.2) sequenced on both strands.

Results and Discussion Selection of oligonucleotide primers and PCR cloning of the trypanothione reductase gene

Isolation of a genomic DNA clone encoding the TR genes A 1200 bp BamH\-Hind\\\ fragment of pHS6AB3 was then used to screen a size-selected C. fasciculata EMBL3 genomic library. Four positive plaques were obtained and plaque-purified. A 5.5 kb Sail fragment from one clone

Trypanothione reductase (TR) belongs to the family of FAD-containing pyridine nucleotide disulphide oxidoreductases, all of which contain highly conserved catalytic

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+ + + + + f + QHL GIEGDDLCITSNEAFYLDEAPKRALCVGGGYISIEFAGIFNAYKARGGQVDLAY QMP---AIPGVEHCISSNEAFYLPEPPRRVLTVGGGFISVEFAGIFNAYKPPGGKVTLCY QHL KIPGIEHCISSNEAFYLEEPPRRVLTVGGGFISVEFAGIFNAYKPVGGKVTLCY HMP NIPGIEHCISSNEAFYLPEPPRRVLTVGGGFISVEFAGIFNAYKPKDGQVTLCY STPHESQIPGASLGITSDGFFQLEELPGRSVIVGAGYIAVEMAGILSA LGSKTSLMI SHP DIPGVEYGIDSDGFFALPALPERVAVVGAGYIAVELAGVING LGAKTHLFV QVP DIPGKEHAITSNEAFFLERLPRRVLVVGGGYIAVEFASIFHG LGAETTLLY

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RGDMILRGFDSELRKQLTEQLRANGINVRTHENPAKVTKNADGTRHVVFESGAE RNNLILRGFDETIREEVTKQLTANGIEIMTNENPAKVSLNTDGSKHVTFESGKT RNNPILRGFDYTLRQELTKQLVANGIDIMTNENPSKIELNPDGSKHVTFESGKT RGEMILRGFDHTLREELTKQLTANGIQILTKENPAKVELNADGSKSVTFESGKK RHDKVLRSFDSMISTNCTEELENAGVEVLKFSQVKEVKKTLSGLEVSMVTAVPGRLPVMT RKHAPLRSFDPMISETLVEVMNAEGPQLHTNAIPKAVVKNTDGSLTLELED--GRSET— RRDLFLRGFDRSVREHLRDELGKKGLDLQFNSDIARIDKQADGSLAATLKD--GR

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—ADYDVVMLAIGRVPRSQTLQLDKAGVEVAKNGAIKVDAYSKTNVDNIYAIGDVTDRVM --LDVDVVHHAIGRIPRTNDLQLGNVGVKLTPKGGVQVDEFSRTNVPNIYAIGDITDRLH --LDVDVVHMAIGRLPRTGYLQLQTVGVNLTDKGAIQVDEFSRTNVPNIYAIGDVTGRIH --MDFDLVHMAIGRSPRTKDLQLQNAGV-MIKNGGVQVDEYSRTNVSNIYAIGDVTNRVM MIPDVDCLLWAIGRVPNTKDLSLNKLGIQTDDKGHIIVDEFQNTNVKGIYAVGDVCGKAL VDCLIWAIGREPANDNINLEAAGVKTNEKGYIVVDKYQNTNIEGIYAVGDNTGAVE -VLEADCVFYATGRRPMLDDLGLENTAVKLTDKGFIAVDEHYOTSEPSILALGDVIGRVO

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Fig. 1. Alignment of the C fasciculata JH sequence with other TRs and GRs. The deduced amino acid sequence of C. fesc;cu/ara was aligned with TR sequences for T. brucei, T. congolense {Shames etal.. 1988) and T. cruzi (Sullivan and Walsh, 1991) as well as GR sequences for human (Krauth-Siegel et al., 1982). E. CO//(Greer and Perham, 1986) and P. aeruginosa {Perry et al.. 1991) All the sequence data (except the C. fasciculata and T. brucei sequences] were obtained from the OWL composite protein sequence database. The nucleotide sequences of the C. tasciculata and 7. brucei TR gene sequences appear in the EMBLyGsnBank/DDBJ Nucleotide Sequence Data Libraries under the accession numbers Z12618 and X63188 respectively. Alignment was made by the ALIEN multiple sequence alignment program (Daresbury Computer Centre). Identities are indicated by hash symbols (#] and homologies by wavy lines (-). For human GR. residues forming the disulphide-binding site are shown in bold and residues involved in binding FAD and NADPH are overlined and underlined respectively. The figure is based on crystallographic data from Karplus eO/. (1989), Karplus and Schuiz (1987] and PaieraA(l988).

(2B1) was subcloned into pUCi 9 (pKSTR) and the region containing TR was sequenced on both strands using a family of custom made oiigonucieotide primers. Using a similar approach a 1200bp Acd\-Kpn\ fragment of pTR1.2 was used to screen a size-selected T. brucei Xgtii genomic library. One positive clone (TRd3) was found to contain a 4.2 kb insert (determined by PCR of phage DNA using >.gt11 primers). The region containing TR was sequenced as described in the Experimental procedures secWon. The translated C. fasciculata and T. brucei sequences encode for proteins of 491 and 492 residues respectively. The predicted subunit molecular mass of C. fasciculata TR (53 235 Da) is in good agreement with the subunit M,

of 53 800 Da determined by SDS-PAGE and amino acid composition of the pure protein (Shames et al., 1986). The derived C. fasciculata TR amino acid sequence Is also identical to the tryptic peptide sequences H39 to Kg,, K335 to L399, I415 to M429 and U51 to A4e5 reported by Shames et al.. (1988). One other tryptic peptide sequence (E75 to RQS) showed only partial identity (82%). The reason for this discrepancy is not clear. However, the original TR protein was isolated from a wild-type C. fasciculata from which our clone HS6 was derived, so this may represent sequence polymorphisms within a mixed population. It is not the result of DNA sequencing errors since our entire sequence has been confirmed by crystallographic analysis (Hunter et ai, 1992).

3092

T. Aboagye-Kwarteng, K. Smith and A. H. Fairiamb

Homoiogy of primary amino acid sequences ofC. fasciculata andJ. brucei TRs with other fiavoprotein disulphide oxidoreductases The predicted amino acid sequences of the C. fascicuiata and T. brucei TR gene products show marked identity with TRs from T. congoiense (68 and 85% respectively) (Shames et ai, 1988), and T. cruzi (69 and 83% respectively) (Sullivan and Walsh, 1991), C. fasciculata and T. brucei JBs show lesser identity with human, Escherichia ccli and Pseudomonas aeruginosa GRs (in the range 35-45%). Multiple alignment of these seven proteins using the program ALIEN reveals an overall identity of 23% and homoiogy of 43% (Fig. 1). However, the similarity increases markedly when one considers only the regions of human GR that comprise the NADPH-, FAD- and disulphide-binding sites which constitute 28% of the alignment (underlined, overlined, and bold respectively in Fig. 1). Within these regions the similarity rises considerably from 23 to 39% and from 43 to 6 1 % for identity and homoiogy, respectively. Although all TRs show low overall identity to 9 DLDs and 7 MRs (26-31% and 26-28% respectively), these enzymes still share many common structural features within these regions as will be described below. Absence of an N -terminai extension found in human GR Another interesting feature arising from this alignment is that, of all TRs and GRs, only the human enzyme contains an W-terminal extension of 16 residues. Mammalian GRs are thought to have an intracellular location both in the cytosol and in the mitochondrion (Mbemba et ai., 1985; Taniguohi etai., 1986), whereas TR from 7. brucei has been shown to be exclusively cytosolic (Smith etal., 1991b). Thus in concert with DLDs and other proteins that are synthesized cytosolically and imported into the mitochondrion, this extension could represent such a signal sequence. Although mitochondrial targeting peptides do not share any simple consensus signal, they tend to be rich in basic, hydrophobic and hydroxylated residues and lack acidic amino acid residues (von Heijne etal.. 1989). In addition, the DLDs from pig, human and yeast all contain cleavable W-terminal extensions with an arginine two residues upstream of the mature A/-terminus in agreement with the cleavage motifs described by Gavel and von Heijne, (1990). However, human GR fits neither of these consensus motifs. Indeed, GR isolated from ratliver mitochondria appears to be identical to the cytosolic enzyme in every respect, suggesting that proteolytic cleavage is not involved in the import mechanism (Taniguchiera/,, 1986). Sequence and structural homoiogy in the redox-active site The most highly conserved sequence observed in Fig. 1

is the 14-amino-acid segment containing the two redoxactive cysteine residues involved in the final electron transfer from GR to giutathione disulphide or from TR to trypanothione disulphide (Krohne-Ehrich et ai., 1977; Shames et ai.. 1986). This segment forms an a-helix at the base and one side of the active site in human GR (Karplus and Schuiz, 1989) as well as in C. fasciculata TR (Kuriyan etal., 1991; Hunter etal., 1992). A similar a-helix containing these redox-active cysteines has recently been reported for the crystal structures of DLD from Azotobacter vineiandii (Mattevi et al., 1991) and the MR from Bacillus spp. (Schiering etal., 1991). As shown in Fig. 2, despite the low overall identity mentioned above, primary sequence alignment of all 23 flavoproteins shows that this sequence is highly conserved within this family, yielding an overall consensus of GGXCXNXQCXPXK. A search of the OWL composite database confirmed that this is unique to the fiavoprotein disulphide oxidoreductase family. Conservation of 0 and P in the above consensus sequence has been suggested to be important in distorting the helix, thereby determining the correct conformation of the turn carrying the disulphide bridge (Mattevi et a/,, 1991). The opposite side of the active site comprises two strands from the C-terminal region of the opposite subunii (the interface domain in GR). one of which contains a histidine residue {H467. in GR) which is thought to be involved as a proton donor/acceptor in the enzyme reaction. The imidazolium ring is oriented in the active site of GR, TR and DLDs by means of a glutamate residue located five residues C-terminal to the histidine (Fig. 2, shown in bold). This sequence is also highly conserved in all TRs, GRs and DLDs with the consensus HP(T/A)XXE. The conservation of the proline is also noteworthy, since this residue forms a cis peptide bond which allows a H-bond between the carbonyl oxygen of the histidine and A/-3 of the flavin ring in GR and DLD (Karplus and Schuiz, 1987; Mattevi et al., 1991). In contrast, MRs have substituted YL for HP, while retaining the glutamate residue (Fig. 2, shown in bold). A recent report on the crystal log rap hie structure of the active site of MR from Bacillus sp. strain RC607 has noted that the inhibitor Cd^' is liganded to this tyroslne (Ygos') and that this residue may have a similar mechanistic role in binding the substrate Hg^* (Schiering et al.. 1991). MRs also contain a highly conserved vicinal cysteine pair in the C-terminal extension (Fig. 2). Chemical modification and site-directed mutagenesis have provided strong support for a role for these cysteines in the enzyme mechanism (Miller et al., 1989; Moore and Walsh, 1989). Although DLDs contain a C-terminal extension relative to GRs, there is no obvious homoiogy between the nine sequences, suggesting that they play no role in catalysis (Fig. 2). Indeed, removal of the last five amino acid residues of DLD from A. vineiandii has no

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in human GR by I in E. coHGB and in all TRs. The most striking difference occurs at the Giu-t site, where R347 in GR that interacts with the carboxylate of Glu-I of the substrate is replaced by an alanine in all TRs or a methionine in P. aeruginosa. Mutating this alanine to arginine in TR (A434R} does not markedly affect the catalytic efficiency {KcJK^) for either trypanothione disulphide or glutathione disulphide, indicating that it Is not important for recognition of the substrate {Sullivan et al.. 1991), Taken together, the above results suggest that the orientation of the Y-glutamylcysteinyl disulphide moiety is likely to be similar in both enzyme types, Trypanothione and glutathione differ markedly at the Gly-I and Gly-ll sites, where the free carboxylate in glutathione is in covalent linkage to spermidine in trypanothione (Fig. 4). In these regions four out of five residues are different (the conserved residue Yi,4 also interacts with Cys-I and Cys-ll). Attempts to engineer a reversal of the substrate specificity between GR and TR and vice versa have been partially successful, particularly where (GR) A34E (TR) and (GR) R37W (TR) have been mutated in combination (Sullivan et al., 1991; Bradley et al.. 1991). Although these combinational mutations have resulted in quite spectacular alterations in the relative substrate specificity of these enzymes, they are nontheless several orders of magnitude less efficient catalytically than the native proteins. Presumably this is the result of more subtle changes in the relative disposition of the amino acid segments comprising the active site due to more distant amino acid substitutions, deletions or insertions that are not so readily detected by this approach. Crystallographic studies on TR will ultimately be able to resolve these questions and allow a truly rational approach to drug design.

Experimental procedures Materials Chemicals purchased from Sigma or BDH Ltd were of the higfiest purity available. Restriction enzymes were from

Boehringer; other DNA-modifying enzymes were obtained from Northumbria Biologicals Ltd. Taq polymerase was from Perkin Elmer/Cetus, Oligonucleotides were synthesized In the Department of Clinical Sciences, London School of Hygiene and Tropical Medicine and the Department of Biochemistry and Pathology, University of Cambridge, UK; Ml 3 universal and M13 reverse sequencing primers were from United States Biochemicals. The C. tascicutata clone HS6 was cultured as described previously (Shim and Fairlamb, 1988), T. brucei brucei ILTat 1,1 (Aboagye-Kwarteng et al., 1991) was grown in adult lethaily irradiated Spraque-Dawley rats; trypanosomes were purified from blood components as described (Lanham, 1968),

PCR cloning procedures Otigonucleotide primers were designed from regions found to be highly conserved between glutathione reductase (human and E. coli) and trypanothione reductase (7, congolense) sequences, encoding protein residues 54-61 and 457-463 (see Fig, 1), The degenerate oligonucleotides. 5'-CGCGCGGATCCAACGT(C/G)GG(C/G)TGCGT(C/G)CC(C/G)AA(A/G)AA-3' and 5'-GCGCAAGCTTGT(C/G)GGGTG(C/G)AC(C/G)CC(A/GAT)AT(A/C/G/T)GT-3' were used to amplify genomic DNA from C. fasciculata. PCR amplifications were performed with 0.5 nM primers for 30 cycles ( 9 4 X for 1 min 30 s; 60°C for 1 min; 72°C for 2 min), using the GeneAmp Kit (Perkin ElmerCetus). A PCR product of 1,2 kb was obtained, cloned into pUC19 predigested with eamHI-H/ndlll and sequenced, A similar strategy was used to isolate a 1.2 kb PCR product from T. brucei genomic DNA,

Genomic DNA library preparation and screening Size-selected C, fasciculata (9-23 kb) and T". brucei (2.5-7 kb) genomic DNA libraries were prepared in the EMBL3 (Promega) and Xgti 1 (Stratagene) according to the suppliers' instructions. The cloned C. fasciculata TR PCR product (HS6AB3) was excised from the plasmid vector by double digestion with SamHI and H/ndlll, radiolabelled with the Amersham Multiprime System according to the supplier's instructions and used to screen 30 000 plaques of the EMBL3 genomic library, using high-stringency hybridization and washing conditions. Four positive clones were Identified and plaque-purified, A 5,5 kb

Trypanothione reductase genes from Crithidia fasciculata and Trypanosoma brucei Sal\ fragment of one of these clones (2B1) contained the complete TR gene and was subcloned into pUCI 9 (pKSTR). The cloned T. brucei JH PCR product {TR1.2) was excised from the plasmid vector by digestion with Acd and Kpn\, radiolabelled and used to screen 10 000 plaques of the Xgtii library. Four positive clones were plaque purified. Digestion of one of these phage {TRd3) DNAs with Eco Rl produced two fragments of 3.8 and 0.4 kb. The 3.8 kb containing approximately 92% of the TR gene was subcloned into pUC19 (pTRd3UC19). Plasmid or phage DNAs were prepared from clones using standard methods (Sambrook e; al., 1989). All plasmids were sequenced on both strands as double-stranded DNA using Sequenase (US Biochemical) and custom-made oiigonucieotide primers. The final C-terminal 115 nucleotides of the T bruceiJR gene were determined by double-stranded sequencing of TRd3 phage DNA with a synthetic oligonucleotide primer 5'-GCCTTAGGCACACACCG-3'.

Sequence analysis Published protein sequences were obtained from the nonreductant composite database OWL (Bleasby and Wootton, 1990) at SERC, Daresbury Laboratory, UK. Sequence analysis was performed with the DNASTAR software package. Paired sequence alignments were carried out using the program AALiGN in DNASTAR, which is a derivative of the FASTP algorithm (Lipman and Pearson, 1985). A gap penalty of 4 and deletion penalty of 8 were found to give optimal alignments. Multiple sequence alignment used the program ALIEN and was carried out using the SEQNET service at SERC, Daresbury Laboratory, U.K. Separate alignments were carried out for TRs and GRs, DLDs and MRs. The alignments for the amino acid segments shown in Figs 2 and 3 were then made with reference to the results obtained with AALIGN. The programme ALIEN had some difficulty in correctly aligning the W-terminal regions of these proteins (due to N-terminal extensions in some sequences); these were corrected manually.

Notes added in proof Field, H., Cerami, A., and Henderson, G. B. {1992, Mol Biochem Parasitol 50: 47-56) have recently reported on the cloning, sequencing and demonstration of polymorphism in trypanothione reductase from an uncloned strain of C. fasciculata. Despite discrepancies between their reported DNA sequences and their amino acid sequences there is still about 99% identity with our amino acid sequence.

Acknowledgements We are extremely grateful to Dr S. M. Beverley, Department of Biological Chemistry and Molecular Pharmacology, Harvard University, for his advice on the initial strategy for isolating the trypanothione reductase gene. This work was supported by grants from the National Institutes of Health (Al 21429) and the Wellcome Trust.

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