Recombinant putative glutathione reductase of Plasmodium falciparum exhibits thioredoxin reductase activity

MOLECULAR

ii&HEMIcAL PARAsIToLoGy

ELSEVIER

Molecular

and Biochemical

Parasitology

80 (1996) 215-219

Short communication

putative glutathione reductase of Plasmodium falciparum exhibits thioredoxin reductase activity

Recombinant

Sylke Miiller”**, Tim-Wolf Gilberger”, Petra M. F&-herb, Katja Beckerb, R. Heiner Schirmerb, Rolf D. Walter” “Bernhard Nocht Institute for Tropical Medicine, Bernhard-Nocht-Str. 74, D-20359 Hamburg, Germany blnstitute of Biochemistry II, Heidelberg University, D-69120 Heidelberg, German) Received

Keywords: Plasmodium falciparum;

11 April 1996; revised 10 June 1996; accepted 17 June 1996

Disulfide

oxidoreductase;

Recently, the cDNA of a disulfide reductase from Plasmodium falciparum was cloned and sequenced [l]. The deduced amino acid sequence and the predicted three-dimensional structure revealed a high degree of similarity with human glutathione reductase (GR). However, marked differences between the plasmodial protein and human GR were found to occur at the ligandbinding sites suggesting a different mode of substrate binding [2]. As reported here, the Abbreviations: GR, Glutathione reductase; Trx-S,, thioredoxin disulfide; Trx-(SH),, thioredoxin; TrxR, thioredoxin reductase; PfTrxR, Plasmodium falciparum thioredoxin reductase; rPfTrxR, recombinant PffrxR; DTNB, 5,5’-dithiobis(2-nitrobenzoate); TNB, 5-thio 2-nitrobenzoate; IPTG, isopropyl-P-D-thiogalactopyranoside; PCR, polymerase chain reaction. * Corresponding author. Tel: +49 40 31182416; fax: + 49 40 31182418.

0166-6851/96/$15.00

C 1996 Elsevier

Pff S 166-685 1{96)02694- 1

Science

Thioredoxin

reductase

recombinant P. falciparum protein was expressed in Escherichia coli in order to obtain information about the substrate specificity and kinetic properties of the enzyme and to produce sufficient amounts for crystallographic analyses. The coding region of the cDNA was amplified by polymerase chain reaction (PCR) using oiigonucleotides encoding the first and last eight amino acids of the polypeptide sequence. The S-sense primer contained a Hind111 and the 3’-antisense primer a BamHI restriction site. The amplified PCR fragment was digested with Hind111 and BarnHI, purified and subcloned into the expression vector pJC40 previously cleaved with HindIII/Bam HI [3]. The vector encodes 10 histidine residues preceding the N-terminus of the recombinant protein which offered the possibility to purify the fusion protein by Ni*+ -chelating sepharose (Novagen) (Fig. 1).

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S. Miller et al. 1 Molecular and Biochemical Parasitology 80 (1996) 215-219

In order to identify the nature of the recombinantly expressed and purified P. falciparum protein, several disulfide compounds were tested as substrates. The enzyme was active with E. coli thioredoxin and with dithiobisnitrobenzoate (DTNB) but not with glutathione disulfide [4]. The catalyzed reactions were found to be: NADPH+Trx-S,+H+ + NADP + + Trx - (SH), NADPH + DTNB + H + -+ NADP + + 2TNB With respect to the reducing substrate, the enzyme showed a strong preference for NADPH over NADH. According to these results, the plasmodial enzyme is a thioredoxin reductase (TrxR) [5,6] and not a GR as was originally proposed on the basis of the gene sequence [l]. Consequently, we shall refer to it henceforth as recombinant P. falciparum thioredoxin reductase (rPff rxR). Since this enzyme and human TrxR are the only Zarge TrxRs for which kinetic as well as sequence data are available, we compared the molecular properties of these proteins (Table 1). Using the k,,,/K, criterion, human TrxR appears to be the more competent enzyme but a detailed kinetic comparison has to be deferred until the mature enzymes can be tested with their respective cognate substrate [5,7]. According to gel filtration on a calibrated Sephadex-S-200 FPLC column, rPffrxR is active as a dimer of 130 kDa. The subunit size is about 64 kDa as determined by SDS-PAGE (Fig. 1) and thus comparable to mammalian TrxR [5,6]. By contrast, the subunit M, of E. coli TrxR, a typical small TrxR, is only 35 kDa [5,8,9]. Large type TrxRs have a broad substrate specificity: they reduce Trx-S, from other species as well as a variety of important electrophilic metabolites, glutathione disulfide being a notable exception [57,101. Other functions of TrxR such as deoxyribonucleotide production, thiol redox control of metabolic pathways and promotion of cell proliferation [7,11- 131 - are mediated by TrxRreduced thioredoxin. The degree of identity between the amino acid sequences of PfTrxR and human TrxR is as high as 44%. PffrxR and human GR are still identical

in 35% of the positions whereas the corresponding value for PffrxR and E. coli TrxR is at best 25% (Table 1). The close relationship between large TrxRs and GRs is as unexpected as the great difference between the large and small TrxRs [2,9,14]. Consequently several putative GR genes - for instance of the nematode Caenorhabditis may actually encode TrxR-like enelegans zymes (Fig. 2). Up to now, dimeric TrxR species with Mr 2 110 kDa were designated mammalian TrxRs [5,6]. In view of the data for the P. falciparum enzyme we prefer the term large TrxR in order to facilitate the identification of similar proteins in non-mammalian organisms [15]. On the basis of the sequence comparisons, the large TrxRs are members of an oxidoreductase

kDa 97.466 45 -

Fig. 1. SDS-PAGE of recombinant Plasmodium falciparum thioredoxin reductase. Lanes l-4 represent proteins which were extracted from E. coli cells after IPTG-induction. The proteins were separated by SDS-PAGE on a 7.5% separation gel and stained with Coomassie blue [19]. Lane I, Iysate of E. cofi BL21 containing pJC40. Lane 2, pellet fraction (after sonification and subsequent centrifugation for 60 min at 100000 x g) of a lysate of E. co/i BL21 containing the expression construct pJC40-PffrxR. Lane 3, supernatant fraction matching the pellet fraction shown in Lane 2. Lane 4, purified rPffrxR after Ni’ * -chelating sepharose (Novagen).

S. Miiller et al. I Molecular and Biochemical Parasitology 80 (1996) 215-219 Table 1 Properties of recombinant in comparison to human

P. falciparum thioredoxin thioredoxin reductase rPffrxR study)

Parameter

K,

4.1 pM 1090 PM 66 /IM

for NADPH” for DTNB” for E.coli Trxb

k,,, with DTNB’ with E. (*o/i Trx’ with GSSG mM

(this

at O.lL20

M, of active dimer of monomeric apoenzyme from amino acid sequence

425 min- ’ >300 min-’ ~0.1

min-’

reductase

Human TrxR [6,14] 3.6 PM 365 FM 20 PM 1600 min-’ Not determined No substrate

130 kDa 64 kDa 59 kDa

130 kDa 58 kDa 54.2 kDa

541

495

44% 37%

100% 44%

35% 25%

35% 24%

u.21 Number

of amino

acids [I,21

Degree of sequence identity with human TrxR with putative CR of Caenorhabditis elegans with human CR [1,2] with E. coli TrxR

“In the DTNB reduction assay [5], the mixture (1 ml; 20°C) consisted of 100 mM potassium phosphate buffer, pH 7.4, 0.2 mg ml-’ bovine serum albumin, 2 mM EDTA, 1.5 )“g ml-’ rPffrxR, 200 PM NADPH and 0.6-6 mM DTNB. The change in absorbance was monitored spectrophotometrically at 412 nm (Uvikon 932, Kontron). The activity was calculated using the molar absorption coefficient of thionitrobenzoate (TNB) at 412 nm (13 600 M-’ cm-’ per TNB-unit). bin the insulin reduction assay, E. coli Trx-S, was used as a substrate [5,6]. The mixture (I ml) contained 100 mM Hepess NaOH buffer. pH 7.6, 2 mM EDTA, 200 PM NADPH, 0.4 mg human insulin (Sigma), 20-120 PM E. coli Trx-S, (Calbiochem). 0.2 mg bovine serum albumin and 3 pg rPflYxR. The change in absorbance at 340 nm was monitored in a thermostated spectrophotometer at 37°C (Uvikon 932, Kontron). ‘Protein was determined using the Bradford procedure [18].

family which was so far represented by the homodimeric flavoenzymes GR, trypanothione redihydrolipoamide dehydrogenase, ductase, mercuric ion reductase and NADH peroxidase [8,16]. Small TrxR species like E. co/i TrxR are only distantly related (Fig. 2). This conclusion is supported by crystallographic studies on human GR [ 171 and E. coli

217

TrxR [8,9]. Both proteins consist of two closely interacting subunits; each shbunit contains three clearly distinguishable domains: FAD-binding domain, NADPH-binding domain, and central domain. In addition, GR has a fourth one, the so-called subunit interface domain. The respective tertiary structures of the first three domains are similar in E. coli TrxR and human GR, although the orientation of the domains as solid bodies is different. Since both P. falciparum and human TrxR have a C-terminal segment which is homologous to the interface domain in human GR [1] it is likely that this part of the protein is involved in dimer formation [16,17]. Taken together the results suggest that the quarternary structure of PfTrxR and human TrxR will turn out to be quite different from E. coli TrxR but similar to human GR [1,2]. Large and small TrxR species differ from each other also in the topology of the active site cysteines. In E. coli TrxR the two catalytic Cys residues are located in the NADPH-binding domain and they are separated by two residues in the sequence [5,7,9]. By contrast, in PfTrxR these residues (Cys88 and Cys93) belong to the FADbinding domain and they are four residues apart in the primary structure as is the case in human GR and human TrxR [1,2,14]. Work is in progress to substantiate these conclusions by crystallographic studies on rPfTrxR. The crystal structure of the enzyme is primarily needed as a basis for the development of specific inhibitors which are of interest as analytical tools and potential antiparasitic drugs [ 161. In addition, the biochemical and physiological characterization of plasmodial TrxR will be pursued in order to elucidate its functions.

Acknowledgements

The authors wish to thank Mrs B. Bergmann for excellent technical assistance. The study was supported by the Deutsche Forschungsgemeinschaft (Wa 395/8) and the Bundesministerium fiir Forschung und Technologie (Research Focus Tropical Medicine 01 IL4 9301).

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S. Miiller et al. I Molecular and Biochemical Parasitology 80 (1996) 215-219

C. bumettii -TrxR E. co/i - TrxR N. crassa

- TrxR

E. acidoaminophi/um H. sapiens

- TrxR

- TrxR

F! falciparum - TrxR C. elegans

- “GR”

E. coli - GR H. sapiens N. tabacum

- GR - GR

Fig. 2. Dendrogram of different thioredoxin reductases, glutathione reductases, and a putative oxidoreductase from C. elegans. The PILEUP programm (GCG, Wisconsin) scores the similarity between every possible pair of sequences creating clusters. The data strongly suggest (a) the existence of small TrxRs including e.g. Coxiella burnettii (AC: P39916), E. coli (AC: PO9625), Neurospora crassa (AC: D45049), and Eubacterium acidoaminophilum (AC: LO4500) and (b) a new group of large TrxRs including human TrxR [14], P. falciparum TrxR (AC: X87095), and possibly the oxidoreductase from C. elegans (AC: P30635), and that (c) both groups of TrxRs can clearly be distinguished from glutathione reductases from E. coli (AC: A24409), man (AC: SO8979), and Nicotiana tabacum (AC: X76293),

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