Glutathionylspermidine synthetase and processes for recovery...

Details Glutathionylspermidine synthetase and processes for recovery... Glutathionylspermidine synthetase and processes for recovery...

1999-06-11

2001-09-18

Stucker, Jeffrey (Department: 1648)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

C435S188000, C435S243000, C435S258100, C435S258300, C435S947000, C530S350000, C536S023200

Reexamination Certificate

active

06291217

ABSTRACT:

Glutathionylspermidine synthetase (GspS) catalyzes the first of two steps of trypanothione biosynthesis, the synthesis of glutathionylspermidine (Gsp) from glutathione (GSH) and spermidine with the consumption of ATP (1). Trypanothione (N
1
, N
8
-bis(glutatlhionyl)spermidine, TSH) is a metabolite unique to trypanosomatids such as Trypanosoma species, Leishmania species, and
Crithidia fasciculata
(2). These parasites comprise pathogens causing widespread and difficult-to-treat tropical diseases such as African sleeping sickness (
T. brucei gambiense
or
T. brucei rhodesiense
), Chagas disease (
T. cruzi
), kala azar (
L. donovani
), oriental sore (
L. tropica
) and mucocutaneous leishmaniasis (
L. braziliensis
). Others (e.g.
T. congolense
) affect domestic animals, whereas
C. fasciculata
is pathogenic to insects only.
Since the discovery of TSH in 1985 (3, 4), the pathways for its synthesis and utilization have attracted considerable interest as potential targets for selective therapeutic intervention (5, 6). In all trypanosomatids TSH substitutes for GSH in the defense against hydroperoxides and derived reactive oxygen species because of its ability to reduce peroxides either enzymatically (7-9) or spontaneously (10). It thereby protects the parasitic trypanosomatids, which apparently are deficient in catalase and glutathione peroxidases (11), against oxidative stress for instance during host-defense reactions (9, 12, 13). Trypanothione disulfide thereby formed is reduced by the NADPH-dependent trypanothione reductase (14, 15), a flavoprotein homologous to glutathione reductase which together with glutathione peroxidases (16, 17) constitutes a major part of the defense system of the host (18, 19). The precursor of TSH, Gsp, may have a distinct biological role. It was first identified in
Escherichia coli
(20), where it remains unprocessed to TSH due to the apparent lack of TSH synthetase. In
E. coli
GspS, and consequently Gsp, is prominent in the stationary phase (20, 21). Similarly, in
C. fasciculata
Gsp increases substantially during the transition of growth phase to stationary phase, while TSH simultaneously drops (22). These fluctuations of GSH conjugates or the associated variations in cellular spermidine levels have tentatively been implicated in growth regulation (2, 20, 21). The major biological function of TSH in trypanosomatids is to serve as a reducing substrate for thioredoxin-like proteins called tryparedoxins (23). Tryparedoxins in turn may have a variety of functions in replacing thioredoxin which so far could never be identified in trypanosomatids. A prominent role of tryparedoxin consists in the regeneration of tryparedoxin peroxidase after reaction thereof with a hydroperoxide such as H
2
O
2
, a fatty acid hydroperoxide, or a hydroperoxide of a complete lipid (24). Thereby, GspS together with trypanothione synthetase, trypanothione reductase, tryparedoxin, and tryparedoxin peroxidase constitutes the most complex system to protect trypanosomatids against oxidative damage. By analogy, reduced tryparedoxin may also substitue for thioredoxin in other pathways, e.g. in the reduction of ribonucleotides thereby becoming essential for the entire nucleic acid metabolism in trypanosomatids. The first enzyme catalyzing synthesis of Gsp has once been isolated in trace amounts from
C. fasciculata
(0.5 mg from 500 g wet cell mass) and characterized in terms of the apparent MW, kinetic parameters, and substrate specificity (1).
Another deduced amino acid sequence obtained from
C. fasciculata
first claimed to represent a trypanothione-synthetase-like protein(acc. number U66520) was submitted to Genbank on Aug. 9, 1996, became available to the public in February 1997 and wvas reported to be the sequence of glutathionylspermidine synthetase without, however, providing any experimental data supporting this assignment. In terms of size and sequence, this putative (GspS of
C. fasciculata
is not identical with the GspS of
C. fasciculata
described in the present invention. This protein differs substantially from GspS described here in molecular mass (90 (1) versus 78-79 kDa, respectively) and pH optimum (6.5 (1) versus 7.5). Also, the previously described enzyme reportedly hydrolyzed ATP in the absence of spermidine (1), whereas such activity was not detectable in GspS as characterized here. Taken together, these discrepancies demonstrate that the two preparations can not be considered identical or equivalent.
An enzyme catalyzing the analogous reaction in
E. coli
has recently been cloned. Surprisingly, this GspS also exhibits a substantial amidase activity with Gsp as substrate. The simultaneous catalysis of Gsp synthesis and breakdown results in an apparently futile ATP consumption, the biological role of which remains speculative (25, 26). Since
E. coli
does not produce TSH, its GspS has obviously to be seen in a biological context distinct from trypanosomal TSH metabolism, and also the structural and phylogenetic relationship of bacterial and trypanosomal GspS remains to be investigated.
Most importantly, we here describe a method for purification of GspS from
C. fasciculata
yielding an enzyme pur enough to be sequenced. The partial amino acid sequence enables the identification of the pertinent gene and the heterologous expression therof by methods known per se, thereby making GspS available for the identification of specific inhibitors useful as trypanocidal drugs. Further, we have invented a simple and convenient method to partially purify GspS from
C. fasciculata
. Such preparation does not catalyze any ATP hydrolysis in the absence of further GspS substrates and cofactors, i. e. spermidine and magnesium. This implies that GspS activity and inhibition thereof can be specifically measured simply by liberation of inorganic phosphate from ATP in such partially purified GspS. This test system is easily automatized for large scale inhibitor screening.
Thus, one embodiment of the invention concerns a protein characterized by its ability to catalyze the synthesis of glutathionylspermidine with a pH optimum of about 7.5.
The protein according to the invention is further characterized by an apparent molecular weight of 78,000±3000 Da.
The protein is further characterized by comprising partial sequences shown in FIG.
1
and being homologous to glutathionylspermidine synthetase/amidase of
Escherichia coli
. The protein according to the invention is further characterized by being isolated from a species of the family trypanosomatidae or produced in any other species by recombinant DNA techniques making use of the partial amino acid sequences shown in
FIG. 1
, genetic probes or primers derived thereof or encoding nucleic acid sequences thus obtained, e. g. SEQ ID NO1 (depicted in FIG.
2
A through
FIG. 2G
) or any useful part thereof.
The protein according to the invention is further characterized by comprising the partial amino acid sequence deduced from the nucleic acid sequence SEQ ID NO1.
The protein according to the invention is further characterized by comprising or having a sequence which is at least 70%, preferntially 75% identical to that deduced from SEQ ID NO1, respectively.
The protein according to the invention is also any modification thereof genetically designed for facilitaded purification such as e. g. an carboxyterminal polyhistidine extension.
Another embodiment of the invention is a simple process to purify GspS to an extent that its activity as well as the inhibition thereof can be conveniently but specifically tested by liberation of inorganic phosphate from adenosyltriphosphate (ATP) in the presence of spermidine, glutathione and magnesium ions.
The process of the invention is characterized by making use of aqueous two phase systems containing polyethyleneglycol.
Another embodiment of the invention is the specific determinateion of GspS activity by means of the detection of inorganic phosphate from ATP by partially purified GspS.
This analytical process is characterized by not being disturbed by any other ATP hydrolyzing ac

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