Eur. J. Biochem.

228, 745-752 (1995)

0 FEBS 1995

Site-directed mutagenesis of the redox-active cysteines

of TYypanosoma cruzi trypanothione reductase
Adolfo BORGES, Mark L. CUNNINGHAM, Jorge TOVAR and Alan H. FAIRLAMB
Biochemistry and Chemotherapy Unit, Department of Medical Parasitology, London School of Hygiene and Tropical Medicine, England
(Received 17 October 1994) - EJB 94 157814

The gene for trypanothione reductase from the Silvio strain of Trypanosoma cruzi has been cloned,
sequenced and overexpressed in Escherichia coli using the constitutive lpp promoter on the expression
plasmid pBSTNAV. Up to 13 % of the total soluble protein is enzymically active trypanothione reductase
with kinetic properties similar to the enzyme purified from T cruzi. In order to assess the catalytic role
of the putative active-site cysteine residues (C53 and C58), three mutant proteins have been constructed
by site-directed mutagenesis substituting alanine or serine residues for cysteine ; [C53A]trypanothione
reductase, [C53S]trypanothione reductase and [C58S]trypanothione reductase. Although the purified, recombinant mutant proteins were catalytically inactive with NADPH and trypanothione disulphide as
substrates, all showed comparable levels of transhydrogenase activity between NADPH and thio-NADP',
suggesting that the mutant proteins had correctly folded in vivo. All three mutants showed substantially
different catalytic parameters for thio-NADP' than the wild-type enzyme, presumably as a consequence
of modifying the environment of the enzyme-bound flavin, thereby altering its chemical reactivity. The
purified [C58S]trypanothione reductase showed spectral properties similar to the oxidised wild-type enzyme but, unlike the wild-type enzyme, did not acquire the characteristic charge-transfer complex of the
EH, form on addition of NADPH. In contrast, in the absence of NADPH both [C53A]trypanothione
reductase and [C53S]trypanothione reductase showed spectral properties similar to the EH, form of the
wild-type enzyme. These data indicate that both C53 and C58 are essential for overall catalysis, with the
thiolate anion of C58 interacting with the enzyme-bound FAD and C53 interacting with the disulphide
substrate. These mutants should be useful in crystallographic studies of reaction intermediates which
cannot be obtained with the catalytically active native enzyme.
Keywords. Trypanothione reductase ; expression ; reaction mechanism ; flavoprotein disulphide oxidoreductase.

In most eukaryotic cells, the thiol-containing tripeptide glutathione has key roles in the antioxidant defence process, cell
division, amino acid transport through membranes, regulation of
enzymic activity, damage repair and drug metabolism (for a review, see [l]).In protozoa of the order Kinetoplastida, which
includes parasites responsible for Chagas' disease, African
sleeping sickness and Leishmaniasis, the majority of glutathione
is conjugated to spermidine, principally as NL,PP-bis(glutathiony1)spermidine (trypanothione) [2]. Many of the functions ascribed to glutathione in mammalian cells appear to have been
taken over by trypanothione in these parasites (for a recent review, see [ 3 ] ) .
In most cells, glutathione disulphide is maintained as the free
thiol by means of the flavoprotein disulphide oxidoreductase
Correspondence to A. H. Fairlamb, Biochemistry and Chemotherapy
Unit, Department of Medical Parasitology, London School of Hygiene
and Tropical Medicine, Keppel Street, London, England WClE 7HT
Fax: +44 171 636 8739.
Abbreviations. T(S),, trypanothione disulphide ; thio-NADP',
thionicotinamide adenine dinucleotide phosphate ; ECPCR, expressioncassette polymerase chain reaction; WT, wild-type.
Enzymes. Trypanothione reductase (EC 1.6.4.8)
; glutathione reductase (EC 1.6.4.2).
Note. The novel amino acid sequence data published here has been
deposited with the EMBL, GenBank and DDJ sequence data banks and
is available under accession number 213958.

glutathione reductase. Trypanosomatids do not contain glutathione reductase [4] and reduce glutathione disulphide and other
disulphides by means of the concerted action of trypanothione
and trypanothione reductase, another member of the flavoprotein
disulphide oxidoreductases :
(Trypanothione reductase)
T(S),

+ NADPH + H'

* T(SH),

+ NADP';

(Non enzymic)
T(SH),

+ GSSG

T(S),

+ 2 GSH,

where T(S), and T(SH), refer to trypanothione disulphide and

dihydrotrypanothione, respectively, and GSSG and GSH refer to
glutathione disulphide and glutathione, respectively.
The enzymic properties and gene sequences of trypanothione
reductases from a number of different species have been reported [5- 101. In spite of their approximately 40 % identity at
the amino acid level, the trypanothione reductases and human
glutathione reductase show a remarkable degree of specificity
towards their respective substrates, making selective inhibition
of the parasite enzyme a possibility [3, 111.
In glutathione reductases, all available evidence supports a
ping-pong mechanism in which the flow of electrons is from
NADPH to the FAD, subsequently from the reduced FAD to

746

Borges et al. ( E m J. Biochern. 228)

expression vector pBSTNAV [161 to generate the expression

plasmid pBTR.
Site-directed mutagenesis. The mutagenic primers used to
modify cysteine residues C53 and C58 are shown in Table 1.
Residue C53 was changed to either an alanine (C53A) or a serine residue (C53S) by the phosphorothioate method [20]. Residue C58 was converted into a serine residue (C58S) by the overlap extension method using plasmid pBTR as template, essentially as described in [21] with the following modifications: (a)
recombinant Pyrococcus furious DNA polymerase (Stratagene)
was used in all DNA amplifications and (b) the overlap extension reaction consisted of two rounds of denaturation (94"C,
1.5 min), annealing (50"C, 2 min) and extension (72"C, 3 min)
in the absence of primers, followed by addition of flanking oligonucleotides (5' and 3' ECPCR primers, 0.4 @
each)
I and 30
cycles of denaturation (93"C, 1 rnin), annealing (60"C, 2 min)
and extension (72"C, 2.5 min). Mutant sequences were subcloned into pBSTNAV to produce expression plasmids pC53A,
pC53S
and pC58S. Sequencing of the entire trypanothione-reMATERIALS AND METHODS
ductase-coding region in these plasmids using 12 internal synMaterials. T(S), and Ar-glutathionylspermidine disulphide thetic oligonucleotides confirmed the absence of other unwanted
were custom synthesised by Bachem. All other chemicals were mutations.
Purification of recombinant WT and mutant trypanothiof analytical grade. Kits for PCR and site-directed mutagenesis
were purchased from Perkin-Elmer/Cetus and Amersham In- one reductases. 1-1 bacterial cultures harbouring recombinant
ternational, respectively. Plasmid pBSTNAV [161 was supplied plasmids were grown at 37C to an A , of 1.8 in Luria-Bertani
by Dr F. Dardel, Ecole Polytechnique, Paris, France. Escherichia medium containing ampicillin (75 pg/ml). All subsequent procoli strain TG1 was obtained from Amersham International and cedures were carried out at 4C. Cells were harvested by centrifugation (6000 g, 20 rnin), washed free of medium with 0.9 %
JMI 09 from Boehringer-Mannheim.
Preparation of genomic DNA from T. cruzi cells. 1: cruzi (masshol.) NaC1, re-centrifuged and finally resuspended in
promastigotes (Brazilian Silvio strain, XlO/l) were cultured at buffer A (20 mM potassium dihydrogen phosphate, pH 7.2,
28C in RPMI 1640 liquid medium (Gibco) supplemented with 1 mM EDTA and 1 mM dithiothreitol), supplemented with
10% (by vol.) foetal calf serum (heat inactivated), 0.5 % (mass/ 5 mM benzamidine, 5 mM phenanthroline and 100 pM
vol.) Hepes, 0.5 (% (mass/vol.) trypticase and 0.03 mM haemin. phenylmethylsulphonyl fluoride. Cells were lysed by repeated
Genomic DNA was extracted from parasite cells using standard sonication and, following centrifugation (I4 000 g, 30 rnin), the
protocols [17].
resulting cell-free extract was adjusted to 0.4 % (mass/vol.) with
Amplification and cloning of the trypanothione reductase protamine sulphate, then to 35% saturation with solid ammogene. PCR oligonucleotide primers (Table 1) were designed ac- nium sulphate. After centrifugation (14000 g, 30 min), the sucording to the ECPCR protocol of MacFerrin et al. [15] so as to pernatant was adjusted to 70 % saturation ammonium sulphate
include suitable restriction sites at each end of the amplified and the precipitate isolated by centrifugation as described above.
fragment (EcoRI and PstI, not represented within the coding The resulting pellet was resuspended and dialysed extensively
frame of the published 1: cruzi sequence for the CL strain [8]), against buffer A followed by application to 2',5'-ADP-Sephartogether with the Shine-Dalgarno sequence and the AfT-rich ose affinity resin previously equilibrated with buffer A (radius =
region which precede the translation start signal for the lipoam0.6 cm, height = 6.0 cm). Columns were washed extensively
ide dehydrogenase gene (pdhD) of Bacillus stearothermophilus with buff& A supplemented with 20 mM KC1 until negligible
(base pairs 4871 -4884 in the original sequence [18]). These protein could be detected in the eluate. The WT and [C58S]trysignal sequences are known to allow constitutive expression of panothione reductase enzymes are visible as bright yellow
the pdhD gene in E. coli under the control of the lpp promoter bands, while [C53A]trypanothione reductase and [C53S]trypanothione reductase are deep red in colour (WT trypanothione
[I 81.
The PCR mixture (50 pl) contained 10 mM Tris, pH 8.0, reductase was detected by its activity, while the mutant proteins
50 mM KCI, 1.5 mM MgCl,, 5 mM sodium citrate, all four were detected by their colour). Following elution with 5 mM
dNTPs (each at 200 pM), 5' and 3' primers (each at 1 pM), NADP', fractions containing trypanothione reductase were
0.2 pg i? cruzi genomic DNA and 2.5 U Thermus aquaticus pooled, dialysed extensively against buffer B (20 mM bistrisDNA polymerase. PCR used the following protocol: 94C propane, pH 7.4, 1 mM EDTA and 1 mM dithiothreitol) prior to
(2.5 min), 55C (1 min), 72C (3 min; 1 cycle); 94C (45 s), application to Q-Sepharose anion-exchange resin (Pharmacia
55C (1 min), 72C (3 min; 34 cycles); 94C (45 s), 55C LKB Biotechnology Inc) previously equilibrated with buffer B
(1 rnin), 72C (10 min; 1 cycle). At the end of the 36th cycle, (radius = 0.8 cm, height = 8.0 cm). Following extensive washthe reaction mixture was extracted with phenollchloroform, and ing with buffer B, the coloured bands were eluted with a linear
the DNA was precipitated with 0.5 vol. 7.5 M ammonium ace- gradient of KCl (0-0.3 M). Fractions containing trypanothione
tate plus 3 vol. ethanol. The PCR fragment (1500 bp) was puri- reductase were pooled, concentrated to 2 ml using Amicon P-30
fied from agarose gels using the GenecleanTMmethod (BiolOl Centricons (molecular mass cut off 30000 Da), then purified
Inc.), digested with EcoRI and PstI and cloned into EcoWPstI- using a gel-sizing column (Pharmacia Hiload 16/60 Sephadex
restricted M13mp18 and M13mp19. The nucleotide sequence of 200) previously equilibrated against buffer B plus 0.15 M KCI.
the wild-type (WT) trypanothione reductase was determined Aliquots of fractions containing trypanothione reductase were
using the dideoxynucleotide-termination method [191 with the analysed by SDSPAGE and those without visible contamination
SequenaseTMkit (United States Biochemicals). The EcoRI-PstI
pooled. Protein concentration was determined using Coomassie
fragment was then ligated into the EcoRI-PstI-restricted shuttle blue [22] with bovine serum albumin as a standard. The N-ter-

the active-site disulphide bridge, and finally from the active-site

dithiol to oxidised glutathione by two sequential thiol-disulphide
interchange reactions (see reviews [12, 131). Although the reaction mechanism of trypanothione reductase has not been studied
as extensively as that of glutathione reductase, current evidence
suggests it to be similar to that of glutathione reductase [14].
To gain insights into the reaction mechanisms of trypanothione reductase and to provide enzyme-substrate reaction intermediates for crystallographic studies, we have expressed the enzyme from Trypanosoma cruzi using the expression cassette
polymerase chain reaction (ECPCR) method [15] and performed
site-directed mutagenesis experiments on the amplified gene. We
describe here a series of mutants used to assess the catalytic
role of the two cysteine residues in the putative redox-active
disulphide bridge of the enzyme. The spectral and kinetic analyses of these altered proteins are reported herein.

747

Borges et al. (EUKJ. Biochem. 228)

116
97 84 66 -

55 45-

36 -

29
24 -

Fig. 1. SDSffAGE of purified WT and mutant trypanothione reductases. Lane 1, cell-free extract prepared from E. coli TG1 cells (40 fig);
lane 2, cell-free extract prepared from E. coli JM109 cells harbouring
the pBTR plasmid (40 pg); lanes 3-6, purified enzymes (2 fig), WT,
C53A, C53S and C58S, respectively. Molecular-mass markers are shown
on the left in kDa.

minal sequence of WT recombinant trypanothione reductase was

determined by A. Harris at the Protein Sequencing Facility,
NIMR, Mill Hill, London, England. Isolated enzymes were
stored at 4C in 80 % saturation ammonium sulphate.
Enzyme assays and kinetics. Trypanothione reductase activity was routinely assayed spectrophotometrically at 340 nm,
as previously described [23]. For comparative kinetic studies,
trypanothione reductase was also assayed in 40mM Hepes,
pH 7.5, 1 mM EDTA, 100 pM NADPH and varying T(S), concentrations [24]. Transhydrogenase activity was measured at
25C in 60 mM sodium phosphate, 0.5 mM EDTA, pH 7.6, by
observing the initial rate of formation of thio-NADPH at 395 nm
as described [25], using an absorption coefficient of 1.13X104
M-' cm-'. NADPH stock solutions were determined spectrophotometrically at 340 nm using an absorption coefficient of
6.22X103 M-' cm-' [25] ; the thio-NADP' concentration was
determined at 260 nm using an absorption coefficient of
1.95X104M-' cm-' [26]. Trypanothione disulphide and Rr-glutathionylspermidine disulphide stock solutions were assayed by
the oxidation of NADPH in the presence of excess WT trypanothione reductase. Initial velocities were fitted to the Michaelis-Menten equation using the non-linear regression data analysis
programmes Enzfitter (Elsevier-Biosoft, Cambridge, England)
and Grafit (Erithacus Software Ltd, Staines, England). Standard
errors were typically G 10 % of the mean.
Determination of absorption coefficients. Enzyme-bound
flavin was liberated by thermal denaturation at 100C for 20 min
in the presence of IOmM MgC1,. Denatured protein was removed by micro-centrifugation and the concentration of free flavin determined from its absorption coefficient at 450 nm of
11.3X103 M-' cm-'. Values are means of five determinations.
Absorption spectra. Absorption spectra were collected on a
Beckman DU-70 temperature-regulated spectrophotometer using
1-cm path length cells. All proteins were equilibrated against
0.1 M Hepes, pH 7.8,O.l mM EDTA, prior to use. Samples were
placed into a round-necked cuvette fitted with a Subaseal rubber
bung and oxygen-free nitrogen gently bubbled through for
20 min. Enzyme concentrations were calculated from previously
determined absorption coefficients. Additions were made
through the rubber septum using a gas-tight Hamilton syringe.

Borges et al. ( E m J. Biochern. 228)

748

Table 2. Purification of recombinant T. cruzi WT trypanothione reductase from E. coli. See methods for assay conditions.
Step

Volume

Total protein

Total activity

Specific activity

Yield

mg
360
275
31
22.8
19.0

U/mg

Cell-free extract
35 -70% (NH,),SO,
2',5'-ADP-Sepharose

ml
61
37
14.4
6.5
5.1

3994
3746
3429
2910
2718

11.1

100

13.6
110.6
127.6
143.2

94
86
73
68

Mono Q
Gel filtration

RESULTS
ECPCR amplification of the trypanothione reductase gene
from T. cruzi.An efficient heterologous expression system in E.
coli was designed in order to make the gene encoding trypanothione reductase from 7: cruzi accessible to protein engineering
studies (see Materials and Methods). The ECPCR technique [I51
was used to amplify the trypanothione reductase gene from genomic DNA isolated from the Brazilian strain of 7: cruzi Silvio
X10/1. The amplified fragment was ligated into M13mp18 and
M13mpl9 and its nucleotide sequence determined. When compared to that reported for the 7: cruzi CL strain [8], they shared
98% identity. 20 differences were detected at the DNA level,
seven of which resulted in changes at the amino acid level (K95
to asparagine, El12 to asparagine, N156 to histidine, N353 to
threonine, N402 to lysine, I403 to valine and V441 to isoleucine). The differences observed at the DNA level between the
sequences from the Silvio X10/1 and CL strains may be the
result of the pronounced heterogenity within 7: cruzi species at
the antigenic, enzymological and pathological level (see reviews
[27, 281). The translated amino acid sequence from the Silvio
strain corresponds to a protein with a predicted molecular mass
of 53 870 Da and a PI of 6.57.
Heterologous overexpression and purification of recombinant proteins. The expression cassette trypanothione reductase
fragment was originally subcloned into pKK223-3 [29] allowing
expression in E. coli under the control of the inducible lac promoter. Following induction, enzymically active trypanothione
reductase accounted for 4-5 % of the total soluble cell protein
(data not shown). To optimise expression, the trypanothione reductase gene was subcloned into the expression plasmid
pBSTNAV [16] under the control of the constitutive promoter
of the E. coli lipoprotein gene, lpp [30]. This resulted in a further
2-3-fold increase in synthesis when expressed in E. coli strain
JM109, such that the recombinant trypanothione reductases constituted 8-13 % of the soluble cell protein, as assessed by enzymic activity (12-20 U/mg) and SDSPAGE (Fig. 1, Table 2).
High levels of expression were achieved for all the overproduced proteins, yielding 10-30 mg homogeneous trypanothione
reductase from 1-1 cultures. This lpp-driven system allows a 13fold higher expression level than that obtained for the 7: congolense WT enzyme overproduced under the control of the inducible tuc I1 promoter 171. The purified recombinant trypanothione reductases have subunit relative molecular masses of
54000, as estimated by SDSPAGE (Fig. l), in agreement with
the value predicted by the gene sequence (53870 for the WT
enzyme). N-terminal sequencing of the WT enzyme indicated
that 90 % of the protein contained the sequence MMSKIFDLVV-.
A second sequence, which accounted for 10 % of the expected
yield, was obtained where both the N-terminal methionine residues were missing, presumably cleaved post-translationally by
an E. coli methionine amino-peptidase.

3.5

7.0
12.0
20.0

50.0

-0.2

-0.1

0.0

0.1

l/[NADPH]

0.2

0.3

0.4

(PI,-')

Fig. 2. Double-reciprocal plot with respect to NADPH at fixed concentrations of trypanothione disulphide. Enzymic activity was determined using 40 m M Hepes, pH 7.5, 1 mM EDTA, as described in Materials and Methods. The concentrations of trypanothione disulphide are
0,
3.5 pM; 0 , 7.0 pM; V, 12.0 pM; V,20.0 pM; 0,
50.0 pM.

Kinetic and spectral properties of WT enzyme. The kinetic

mechanism for the NADPH-dependent reduction of T(S), under
steady-state conditions was examined by varying the concentration of NADPH at several fixed concentrations of T(S), (Fig. 2).
The results indicate that the reaction mechanism is ping-pong
with respect to the two substrates, in agreement with previous
results with 7: congolense trypanothione reductase [I41 and human glutathione reductase [31]. The WT enzyme is able to catalyse both trypanothione reduction and transhydrogenase reactions, with specific activities of 143 U mg-' and 6.49 U mg-',
respectively (Table 3). A comparison of the kinetic parameters
of the WT recombinant 7: cruzi trypanothione reductase with
those of the enzyme isolated from 7: cmzi cells [6] is shown in
Table 4. With the substrate trypanothione disulphide, the K , and
k,,/K,,, are in good agreement for the recombinant and native
enzymes. With the other physiological substrate, hr-glutathionylspermidine disulphide, there is also good agreement in the
values of kcJKm, despite the K , for the recombinant enzyme
being twofold higher than the native enzyme (46 pM and 22 pM,
respectively).
The absorption spectrum of the oxidised WT recombinant
trypanothione reductase (Fig. 3a, trace A), where the redoxactive cysteines residues (C53 and C58) form a disulphide
bridge, closely resembles that of trypanothione reductase purified from 7: cruzi cells [6],with a ,Immfor the oxidised enzyme
at 464 nm (E = 11.4X103 M-' cm-') and a shoulder at 486 nm.

Borges et al. (Eul: J. Biochem. 228)

Table 3. Specific catalytic activities of the WT and mutant T. cruzi
trypanothione reductases. Protein concentrations were determined
using absorption coefficients at the respective A, (see Table 5). Trypanothione reductase activities were determined in 0.1 M Hepes, pH 7.8,
0.1 mM EDTA, 150 pM NADPH with 50 pM T(S), (wild-type) or
100 pM T(S), (mutants). Transhydrogenase activities were determined
as described in Materials and Methods using saturating NADPH
(100 pM) and thio-NADP' at five times the respective K, value. C53A,
C53S and C58S are trypanothione reductase enzymes with mutations

C53A, C53S and C58S, respectively.

Reaction

Catalytic activity for

WT

C53A

c53s

749

Table 4. Kinetic parameters of WT and mutant trypanothione reductases. Rednctase activity was assayed in 40 mM Hepes, 1 mM
EDTA, pH 7.5, containing 100 pM NADPH and varying concentrations
of W-glutathionylspemidine disulphide [(GspdS),]. Values in parentheses are those reported by Jockers-Scherubl et al. [24] for native WT
enzyme purified from 7: cruzi. Transhydrogenase activity was assayed
as described in Materials and Methods. Where NADPH was the variable
substrate, assay mixtures contained 100 pM thio-NADP', except for the
WT which contained 800 pM thio-NADP'. Where thio-NADP' was the
variable substrate, all assays contained 100 pM NADPH. C53A, C53S,
C58S are trypanothione reductase enzymes with mutations C53A, C53S
and C58S, respectively.

C58S
-

U/mg
T(S), reduction

Transhydrogenase

143
6.49

<0.0005

0.21

<0.0011
0.44

24

<0.0007

46
(22)
Addition of 1.2 molar equivalents of NADPH to FAD under
anaerobic conditions leads to a shift in Amax of 26 nm towards
the blue end of the spectrum, accompanied by a decrease in
this absorbance. Concomitant formation of a long-wavelengthabsorption band centred on 530 nm is observed due to reduction
of the disulphide bridge and the accompanying formation of the
characteristic charge-transfer complex between the thiolate anion of C58 and the isoalloxazine ring of FAD [31]. The stoichiometry of reduction shows that the absorbance of the chargetransfer complex reaches a maximum after addition of one
equivalent of reductant to FAD (Fig. 3a, trace B). The twoelectron reduced enzyme shows isosbestic points at 507, 438,
398 and 357 nm. On addition of fractional equivalents of
NADPH, further spectral changes are impeded by a kinetic barrier, as noted by Shames et al. [5]. However, addition of excess
NADPH (2 mM) results in the complete bleaching of the flavin
after 30 min (Fig. 3a, trace C) suggestive of the formation of
the four-electron reduced enzyme where direct reduction of the
FAD to the colourless FADH, has occurred [31].

Characterization of mutant proteins. All three mutant proteins

were unable to catalyse the NADPH-dependent reduction of
T(S),, the apparent activities being of the same magnitude as the
T(S),-independent oxidation of NADPH, indicating that both
C53 and C58 residues are crucial in the catalytic mechanism
(Table 3). However, all three mutants catalysed the reduction of
thio-NADP+ using NADPH as a substrate, albeit at lower rates
than that of the WT enzyme, suggesting that the mutant proteins
had folded correctly (Table 3). The specific activities for
[C53A]-, [C53S]- and [C58S]trypanothione reductase, determined at concentrations of thio-NADP' equal to five-times the
respective K, values and saturating NADPH, were 3.2 %, 6.8 %
and 2.8 % of the WT specific activity, respectively.
The absorption spectra of [C53S]trypanothione reductase
(Fig. 3 b) and [C53A]trypanothione reductase (Fig. 3 c) in the
absence of reductant differ from that of the WT enzyme, but
closely resemble that of two-electron reduced form (see Table
5). Both [C53S]trypanothione reductase and [C53A]trypanothione reductase exhibit the broad absorbance band centred at
530 nm, indicative of the charge-transfer complex and suggesting that the C58 residue is in close proximity to the FAD and
hence able to share an electron with this moiety. The visible A,,,
for both enzymes is shifted towards the blue end of the spectrum
relative to the WT enzyme; [C53S]trypanothione reductase to
451 nm and [C53A]trypanothione reductase to 442 nm, with absorption coefficients of 13.9X103 M-' cm-' and 9.1X103 M-'

108.1

(18)

0.18

NADPH

WT

C53A
c53s
C58S
Thio-NADP'

WT

C53A
c53s
C58S

17
11
7
14

555
11

15
4

M-1 s-'

S-'

85.4
6.33
0.31
0.51
0.21
7.02
0.22
0.47
0.19

4.53 X106
(4.63 X106)
1.88 X106
(2.65 X106)
0.382 X lo6
0.029 X lo6
0.069 X 1O6
0.015 X lo6

0.013 X lo6
0.020 x lo6
0.031 X lo6
0.049 X lo6

cm-I, respectively. On addition of excess NADPH under anaerobic conditions, both mutants behave similarly, with absorbances
at their respective A,, values decreasing over time, until spectra
consistent with reduction of FAD to FADH, are observed
(Fig. 3 b and c, trace B). The absorption spectra of [C53A]trypanothione reductase and [C53S]trypanothione reductase oxidised
enzymes differ slightly in the charge-transfer region, the absorbance band of [C53A]trypanothione reductase being somewhat
broader than that of [C53S]trypanothione reductase. This may
be due to the influence of the mutant residue at position 53 on
the interaction between the thiolate anion of C58 and the flavin
in the charge-transfer complex as has been reported previously
1321.
In contrast, the [C58S]trypanothione reductase mutant
spectra in the absence of reductant very closely resembles that
of the WT enzyme (Fig. 3 d, trace A), though once again the A,,
at 446 nm is shifted towards the blue end of the spectrum, with
an absorption coefficient of 12.5X lo3 M-' cm-' (Table 5). Addition of excess NADPH under anaerobic conditions again results in bleaching of the absorbance at the visible A,,, but is
accompanied by the acquisition of a broad long-wavelength absorbance band centred around 600 nm (Fig. 3d, trace B). This
indicates that the FAD can still undergo reduction to FADH,.
The absence of absorbance in the 530-nm region indicates
firstly, that C53 is too distant to share an electron with the FAD
and secondly, that the oxygen atom of the S58 side chain is
unable to mimic the sulphur found in the WT enzyme.

Transhydrogenase activity of WT and mutant enzymes. The

flavoprotein disulphide oxidoreductases are known to catalyse a
pyridine nucleotide transhydrogenase reaction which consists of
the NADPH-dependent or NADH-dependent reduction of thioNADP+ in the absence of oxidised or reduced substrate [25].
For NADPH-dependent enzymes, such as trypanothione reductase, the reaction is :

750

Borges et al. (Eu,:J. Biochem. 228)

14

12

10

400

800

600

700

400

800

600

600

700

800

Wavelength (nm)

Wavelength (nm)

d
I

14

.i 12
h

-8
I

10

v
4

.%

.*
c
.3

.d

!?

P
4

4
2

400

600

800

700

800

Wavelength (nm)

400

600

600

700

800

Wavelength (nm)

Fig. 3. Absorption spectra of WT and mutant trypanothione reductases under anaerobic conditions. Samples were deoxygenated and spectra
recorded as in Malerials and Methods. (a) WT protein; trace A, oxidised enzyme; trace B, plus 1.2 molar equivalents of NADPH to FAD; trace C,
30 min after addition of 2 mM NADPH. (b) [C53S]trypanothione reductase; trace A, oxidised enzyme; trace B, 80 min after addition of 2 mM
NADPH. (c) [C53A]trypanothione reductase; trace A, oxidised enzyme; trace B, 80 min after addition of 2 mM NADPH. (d) [C58S]trypanothione
reductase; trace A, oxidised enzyme; trace B, 80 min after addition of 2 mM NADPH.

NADPH + thio-NADP'
NADP' + thio-NADPH.
This reaction can be used to assess the structural integrity of
the mutant trypanothione reductases, since the catalytic site for
the transhydrogenase reaction is the pyridine-nucleotide-binding
site, known to be spatially independent of the active-site disulphide bridge according to the X-ray-derived structure of the
Crithidia fasciculata [33-351 and T cruzi [36] enzymes. It can
also shed light on the flavin environment of the mutant proteins.
It is known from mutations performed on the gene encoding
Pseudomonas aeruginosa mercuric reductase that the rate of the
transhydrogenase reaction is dependent on the redox potential of
the flavin, which is in turn determined by the nature of the side

chains located in close proximity to the FAD ring in the substrate-binding-site area [32].
The kinetic parameters K,,, and k,,,lK, for the transhydrogenase reaction catalysed by the WT and mutant trypanothione
reductases are presented in Table 4. The K,,, values for thioNADP' at saturating NADPH for the three mutant enzymes are
markedly lower than that of the WT enzyme, the C53 mutants
being approximately 40-fold lower, while [C58S]trypanothione
reductase is a further threefold lower. However, the catalytic
efficiencies (kJK,,,) of all the enzymes, including the WT, are
remarkably similar, suggesting that the respective mutations
have similarly affected both the magnitude of K,,, and the turn-

Borges et al. (EUKJ. Biochem. 228)

Table 5. Spectral properties of the WT and mutant T. cruzi trypanothione reductases. WT enzyme was reduced to the EH, form with
1.2 equivalents of NADPHPAD.

Enzyme

A,,

Absorption

Chargetransfer
A,,

coefficient
~~

WT (C53, C58)
WT EH, ((253, C58)
C53S (S53, C58)
C53A (A53, C58)
C58S ( 0 3 , ,558)

nm
464
452
451
442
446

mM-' cm-'

nm

11.4
8.8
13.9

none

9.1
12.5

530
530
530
none

over number (kcat) of the WT enzyme. The K , values for

NADPH at saturating thio-NADP' concentrations differ less
markedly among the mutant enzymes than in the case of thioNADP'. The catalytic efficiencies of the mutants are very similar but are approximately 10-fold lower than that of the WT
enzyme due mainly to the higher k,, value of the native enzyme.

DISCUSSION
This work describes the amplification by ECPCR of the gene
encoding trypanothione reductase from the Brazilian strain Silvio of 2: cruzi and the construction of a vector (pBTR) for constitutive expression of large amounts of protein in E. coli. The
kinetic and physical properties of the recombinant WT enzyme
agree well with those published previously for the enzyme isolated from 2: cruzi [6, 241. The steady-state kinetic parameters
determined here are consistent with a ping-pong mechanism, as
proposed for the T congolense enzyme [14]. Our heterologous
expression system has already proved useful for inhibitor design
[37] and for crystallographic studies [38].
Site-directed mutagenesis has allowed us to demonstrate the
essential roles of C53 and C58 residues in the catalytic mechanism. Mutation of the C-terminal residue (C58) to a serine residue abolishes disulphide-reductase activity, as do equivalent
mutations in E. coli glutathione reductase (391 and f! aeruginosa
mercuric reductase [40]. Similar to these other mutant flavoproteins, the spectrum of the oxidised [C58S]trypanothione reductase enzyme resembles that of the oxidised WT enzyme, where
addition of reductant does not produce a spectrum typical of the
EH, form of the WT enzyme. Conversely, in the absence of
reductant [C53A]trypanothione reductase and [C53S]trypanothione reductase possess the characteristic absorption band centred
at 530 nm due to C58 interacting with the isoalloxazine ring of
FAD. C58 can thus be ascribed as the participant in the thiolateFAD charge-transfer complex, in agreement with previous
studies [5-71.
The retention of transhydrogenase activity in the mutants indicates that expression of the amplified mutant genes results in
correctly folded protein. In contrast to the equivalent [C47S]
glutathione reductase of E. coli [39], [CSSSItrypanothione reductase mutant possesses low but significant transhydrogenase
activity, resembling the equivalent [C14OS]mercuric reductase
mutant [32, 401. The reason for this discrepancy is not known,
but could possibly reflect differences in flavin-redox chemistry
of the three enzymes, as proposed by Deonarain et al. [39].
Compared to the WT enzyme, the mutations at C53 and C58
decrease both K , and k,, for thio-NADP' in the transhydrogenase reaction. However, the ratio kc,/K,,, is similar in both WT
and mutant enzymes. Modification at these sites would not be
expected to directly affect the nicotinamide-binding site where

751

transhydrogenation takes place, since it is physically separated

from the redox-active disulphide bridge and the trypanothionebinding site [35, 361. However, such mutations could either induce a conformational change that indirectly affects the
nicotinamide-binding site or alter the redox state of the FAD by
perturbation of the environment of the isoalloxazine ring system,
as proposed previously for disulphide-bridge mutants of mercuric reductase [32]. Transhydrogenation requires initial hydride
transfer from NADPH to generate bound FADH,, and presumably subsequent hydride-transfer back from FADH, to thioNADP', with the FADFADH, reduction potential possibly influencing both the turnover number [32] and nicotinamide binding. All the mutations performed on the Z cruzi trypanothione
reductase gene have most likely generated enzymes with a twoelectron equivalent capacity on destruction of the redox-active
disulphide and, therefore, they are restricted to a FADFADH,
redox chemistry. The presence of such an obligatory reduced
flavin intermediate may stabilize the binding of the thioNADP+-pyridine ring in the nicotinamide-binding site. However, interpretation of the results for the individual mutants must
await determination of the redox potential for the enzyme-bound
flavin in [C53A]-, [C53S]- and [C58S]trypanothione reductase.
By analogy to mercuric reductase, it can be speculated that mutation of C58 to a serine residue could have altered the flavin
redox properties in view of the possible hydrogen bonding between the serine hydroxyl and flavin 0 - 4 or N-5 acceptor sites.
Also, it has been demonstrated for the P: aeruginosa mercuric
reductase enzyme that the active-site disulphide-bridge residue
closer to the N-terminus (position 53 in Z cruzi trypanothione
reductase) plays a role in modulating the energy of the thiolateflavin charge-transfer complex by modifying the protonation
rate of the thiolate anion [32]. In this sense, it is possible for the
serine hydroxyl in [C53S]trypanothione reductase to hydrogenbond to the thiolate anion of C58, increasing its ionization potential, thereby altering the energy of the charge-transfer complex and, in turn, influencing the FADPADH, redox potential
and transhydrogenation rate. Although it is not possible for the
alanine residue in [C53A]trypanothione reductase to interact directly with C58, it could influence its environment by inductive
means.
This work was supported by the Wellcome Trust.

REFERENCES
1. Dolphin, D., Poulson, R.

& Avramovic, 0. (1989) Glutathione:

chemical, biochemical and medical aspects, parts A and B, Wiley
Interscience, New York.
2. Fairlamb, A. H., Blackburn, P., Ulrich, P., Chait, B. T. & Cerami,
A. (1985) Trypanothione: a novel bis(glutathiony1)spermidine cofactor for glutathione reductase in trypanosomatids, Science 227,

1485-1487.
3. Fairlamb, A. H. & Cerami, A. (1992) Metabolism and functions of
trypanothione in the Kinetoplastida, Annu. Rev. Microbiol. 46,
695 -729.
4. Fairlamb, A. H. & Cerami, A. (1985) Identification of a novel, thiolcontaining co-factor essential for glutathione reductase enzyme
activity in trypanosomatids, Mol. Biochem. Parasitol. 14, 187198.
5. Shames, S. L., Fairlamb, A. H., Cerami, A. & Walsh, C. T. (1986)
Purification and characterization of trypanothione reductase from
Crithidia fasciculatu, a newly discovered member of the family
of disulphide-containing flavoprotein reductases, Biochemistry

25,3519-3526.
6. Krauth-Siegel, R. L., Enders, B., Henderson, G . B., Fairlamb, A.
H. & Schirmer, R. H. (1987) Trypanothione reductase from Trypanosoma cruzi: purification and characterization of the crystalline
enzyme, Eul: J. Biochem. 164, 123-128.

752

Borges et al. (Eur. J. Biochem. 228)

7. Sullivan, F. X., Shames, S. L. & Walsh, C. T. (1989) Expression of

TrypanosomLi congolense trypanothione reductase in Escherichia
coli: overproduction, purification, and characterization, Biochemistry 28,4986-4992.
8. Sullivan, F. X. & Walsh, C. T. (1991) Cloning, sequencing, overproduction and purification of trypanothione reductase from Trypanosoma cruzi, Mol. Biochem. Parasitol. 44, 145- 148.
9. Aboagye-Kwarteng, T., Smith, K. & Fairlamb, A. H. (1992) 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 mechanism, Mol. Microbiol. 6,
3089-3099.
10. Taylor, M. C., Kelly, J. M., Chapman, C. J., Fairlamb, A. H. &
Miles, M. A . (1994) The structure, organization, and expression
of the Leishmania donovani gene encoding trypanothione reductase, Mol. Biochem. Parasitul. 64, 293 -301.
11. Walsh, C. T., Bradley, M. & Nadeau, K. (1991) Molecular studies
on trypanothione reductase, a target for antiparasitic drugs, Trends
Biochem. Sci. 16, 305-309.
12. Ghisla, S. & Massey, V. (1989) Mechanisms of flavoprotein-catalyzed reactions, Eul: J. Biochem. 181, 1-17.
13. Schirmer, R. H., Krauth-Siegel, R. L. & Schulz, G. E. (1989) Glutathione reductase, in Glutathione: chemical, biochemical and medical aspects.. part A (Dolphin, D., Poulson, R. & Avramovic, O.,
eds) pp. 553-596, John Wiley and Sons, New York.
14. Leichus, B. N., Bradley, M., Nadeau, K., Walsh, C. T. & Blanchard,
J. S. (1992) Kinetic isotope effect analysis of the reaction catalyzed by Trypanosoma congulense trypanothione reductase, Biochemistry 3.1, 6414-6420.
15. MacFerrin, K. D., Terranova, M. P., Schreiber, S. L. & Verdine,
G. L. (1990) Overproduction and dissection of proteins by the
expression-cassette polymerase chain reaction, Proc. Natl Acad.
Sci. USA 87, '1937-1941.
16. Meinnel, T., Mechulam, Y. & Fayat, G. (1988) Fast purification of
a functional elongator tRNA""' expressed from a synthetic gene
in vivo, Nucleic Acids Res. 16, 8095-8096.
17. Hyde, J. E. (1993) Methods in molecular biology, vol. 21, Humana
Press, Totowa, NJ.
18. Borges, A,, Hawkins, C. F., Packman, L. C. & Perham, R. N. (1990)
Cloning and sequence analysis of the genes encoding the
dihydrolipoamide acetyltransferase and dihydrolipoamide dehydrogenase components of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus, Eul: J. Biochem.
194, 95-102.
19. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) DNA sequencing
with chain-terminating inhibitors, Proc. Natl Acad. Sci. USA 74,
5463 -546-7.
20. Taylor, J. W., Ott, J. & Eckstein, F. (1985) The rapid generation of
oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA, Nucleic Acids Res. 13, 8765 - 8785.
21. Ho, S. N., Hunt, H. D., Horton, H. M., Pullen, J. K. & Pease, L. R.
(1989) Site-directed mutagenesis by overlap extension using the
polymerase chain reaction, Gene (Amst.) 77, 51 -59.
22. Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of
protein-dye binding, Anal. Biochem. 72, 248 - 254.
23. Cunningham, M. L., Zvelebil, M. J. J. M. & Fairlamb, A. H. (1994)
Mechanism of inhibition of trypanothione reductase and glutathione reductase by trivalent organic arsenicals, Eul: J. Biuchem.
221, 285-295.
24. Jockers-Scherubl, M. C., Schirmer, R. H. & Krauth-Siegel, R. L.
(1989) Trypanothione reductase from Trypanusoma cruzi: cata-

lytic properties of the enzyme and inhibition studies with trypanocidal compounds, Eur. J. Biochem. 180, 267-272.
25. Moroff, G., Ochs, R. S. & Brandt, K. G. (1976) Yeast glutathione
reductase. Steady-state kinetic studies of its transhydrogenase activity, Arch. Biochem. Biophys. 173, 42-49.
26. Stein, A. M., Lee, J. K., Anderson, C. D. & Anderson, B. M. (1963)
The thionicotinamide analogs of DPN and TPN. I. Preparation
and analysis, Biochemistry 2, 1015-1017.
27. Gibson, W. C. & Miles, M. A. (1985) Application of new technologies to epidemiology, Brit. Med. Bull. 41, 115-121.
28. Miles, M. A. (1983) The epidemiology of South American Trypanosomiasis - biochemical and immunological approaches and their
relevance to control, Trans. Roy. Soc. Trop. Med. Hyg. 77, 5-23.
29. Brosius, J . & Holy, A. (1984) Regulation of ribosomal RNA promoters with a synthetic lac operator, Proc. Nut1 Acad. Sci. USA 81,
6929 -6933.
30. Nakamura, K. & Inouye, M. (1979) DNA sequence of the gene for
the outer membrane lipoprotein of E. coli: An extremely AT-Rich
promoter, Cell 18, 1109-1117.
31. Williams, C. H. Jr (1992) Lipoamide dehydrogenase, glutathione
reductase, thioredoxin reductase, and mercuric ion reductase - a
family of flavoenzyme transhydrogenases, in Chemistry and biochemistry offlavoenzymes (Muller, F., ed.) vol. 3, pp. 121-211,
CRC Press, Boca Raton.
32. Distefano, M. D., Au, K. G. & Walsh, C. T. (1989) Mutagenesis of
the redox-active disulphide in mercuric ion reductase : catalysis
by mutant enzymes restricted to flavin redox chemistry, Biochemistry 28, 1168-1183.
33. Kuriyan, J., Kong, X.-P., Krishna, T. S. R., Sweet, R. M., Murgolo,
N. J., Field, H., Cerami, A. & Henderson, G. B. (1991) X-ray
strucpre of trypanothione reductase from Crithidia fusciculata at
2.4-A resolution, Proc. Natl Acad. Sci. USA 88, 8764-8767.
34. Hunter, W. N., Bailey, S., Habash, .I.
Harrop,
,
S. J., Helliwell, J. R.,
Aboagye-Kwarteng, T., Smith, K. & Fairlamb, A. H. (1992)
Active site of trypanothione reductase. A target for rational drug
design, J. Mul. Biol. 227, 322-333.
35. Bailey, S., Fairlamb, A. H. & Hunter, W. N. (1994) Structure of
trypanothione reductase from Crithidia fascicukata at 2.6 A resolution - enzyme-NADP interactions at 2.8 A resolution, Actu
Crystallogr. Sect. D Biol. Crystallugl: 50, 139 -154.
36. Lantwin, C. B., Schlichting, I., Kabsch, W., Pai, E. F. & KrauthSiegel, R. L. (1994) The structure of Trypanosoma cruzi trypanothione reductase in the oxidized and NADPH reduced state,
Proteins Struct., Funct, Genet. 18, 161- 173,
37. Benson, T. J., McKie, J. H., Garforth, J., Borges, A., Fairlamb, A.
H. & Douglas, K. T. (1992) Rationally designed selective inhibitors of trypanothione reductase : Phenothiazines and related tricyclics as lead structures, Biochem. J. 286, 9-11.
38. Zhang, Y., Bailey, S., Naismith, J. H., Bond, C. S., Habash, J.,
McLaughlin, P., Papiz, M. Z., Borges, A., Cunningham, M., Fairlamb, A. H. & Hunter, W. N. (1993) Trypanosoma cruzi trypanothione reductase: crystallization, unit cell dimensions and structure solution, J. Mol. Biol. 232, 1217-1220.
39. Deonarain, M. P., Scrutton, N. S., Berry, A. & Perham, R. N. (1990)
Directed mutagenesis of the redox-active disulphide bridge in glutathione rednctase from Escherichia coli, Proc. R. Soc. Land.
Biol. 241, 179-186.
40. Schultz, P. G., Au, K. G. & Walsh, C. T. (1985) Directed mutagenesis of the redox-active disulphide in the flavoenzyme mercuric
ion reductase, Biochemistry 24, 6840-6848.