Convenient Isolation and Kinetic Mechanism of Glutathionylspermidine Synthetase from Crithidia fasciculata
48
Citation
33
Reference
10
Related Paper
Citation Trend
Abstract:
Trypanothione, the essential metabolite in the oxidant defense system of trypanosomatids, is synthesized by two distinct proteins, glutathionylspermidine synthetase and trypanothione synthetase. Glutathionylspermidine synthetase was purified to homogeneity from the trypanosomatid Crithidia fasciculataby aqueous two-phase systems and chromatography. The enzyme showed a specific activity of 38 μmol of glutathionylspermidine formed per min per mg of protein. Its molecular mass was 78 kDa in SDS-polyacrylamide gel electrophoresis, and it appeared predominantly monomeric in native polyacrylamide gel electrophoresis and gel filtration. The isoelectric point was at pH 4.6, and the pH optimum was near 7.6. Partial amino acid sequencing revealed homology with, but low similarity to, the glutathionylspermidine synthetase/amidase of Escherichia coli, and amidase activity was not detected in glutathionylspermidine synthetase of C. fasciculata. The kinetics of trypanosomatid glutathionylspermidine synthetase revealed a rapid equilibrium random mechanism with limiting K m values for Mg2+-ATP, GSH, and spermidine of 0.25 ± 0.02, 2.51 ± 0.33, and 0.47 ± 0.09 mm, respectively, and a k cat of 415 ± 78 min−1. Partial reactions at restricted cosubstrate supply were not detected by 31P NMR, supporting the necessity of a quarternary complex formation for catalysis. ADP inhibited competitively with respect to ATP (K i = 0.08 mm) and trypanothione exerted a feedback inhibition competitive with GSH (K i = 0.48 mm).Keywords:
Crithidia fasciculata
Isolation
Crithidia
Crithidia
Cite
Citations (5)
Crithidia
Crithidia fasciculata
Bombus terrestris
Cite
Citations (4)
Crithidia fasciculata
Crithidia
Cite
Citations (104)
Kinetoplastida
Crithidia
Southern blot
Crithidia fasciculata
Cite
Citations (21)
Crithidia fasciculata
Castanospermine
Glucosidases
Crithidia
Oligosaccharide
Mannosidase
N-linked glycosylation
Cite
Citations (15)
Since the discovery of trypanothione (N1,N8-bis(glutathionyl)-spermidine), it has been debated whether its biosynthesis in Crithidia fasciculata requires one or two enzymes. After two related genes implicated in trypanothione synthesis had become available, the correct assignment of sequence to function remained controversial because heterologous expression did not yield active proteins (Tetaud, E., Manai, F., Barrett, M. P., Nadeau, K., Walsh, K. T., and Fairlamb, A. H. (1998) J. Biol. Chem.273, 19383–19390). In 2002, however, Oza et al. (Oza, S. L., Ariyanayagam, M. R., and Fairlamb, A. H. (2002) Biochem. J.364, 679–686) reported the functional expression of a gsps DNA sequence of C. fasciculata (GenBank™ accession number U66520.2) that did not comply with the partial amino acid sequences of glutathionylspermidine synthetase (GspS), as published in our above article. A deep-frozen sample of the GspS preparation that had been isolated from C. fasciculata and functionally characterized by us was therefore reinvestigated. It still displayed GspS activity and no trypanothione synthetase (TryS) activity, as originally stated. Matrix-assisted laser desorption ionization time-of-flight analysis of a tryptic digest, however, did not reveal the presence of any of the peptide sequences previously reported but instead revealed many mass peaks complying with the GspS sequence. Coverage was 45% of the deduced GspS sequence and 70% of theoretically detectable peptide masses. Major mass signals that could not be attributed to the GspS sequence were absent. Therefore, the sequence assignment to GspS by Oza et al. is the correct one. The partial sequences previously reported by us are contained in the expression product of trys (GenBank™ accession number AY603101), the homologous trypanothione synthetase that catalyzes both steps of trypanothione biosynthesis (Comini, M. A., Menge, U., Wissing, J., and Flohé, L. (2005) J. Biol. Chem.280, 6850–6860). Thus C. fasciculata is equipped with two enzymes, one catalyzing the formation of glutathionylspermidine only and the other one being competent to catalyze both steps of the trypanothione synthesis. How the wrong assignment of partial TryS sequences to GspS occurred could no longer be assessed with certainty. According to our laboratory notes, peptide sequencing was not performed with the same batch that had been functionally characterized. The sequenced sample had been purified to electrophoretic homogeneity by means of the chromatographic scheme described for the isolation of GspS but had not been subjected to the initial liquid/liquid extraction that separates and denatures most of the TryS. This may explain how ultimately an inactive TryS protein was obtained and sequenced instead of the presumed GspS. Leopold Flohé, who supervised this work, takes the full and sole responsibility for the erroneous interpretation of the sequence information.
Crithidia fasciculata
Sequence (biology)
Cite
Citations (3)
Dihydrofolate reductase
Crithidia fasciculata
Biopterin
Crithidia
Cite
Citations (20)
Crithidia fasciculata
Dihydrofolate reductase
Crithidia
Cite
Citations (33)
Crithidia fasciculata
Tyrosine aminotransferase
Crithidia
Cite
Citations (19)
Trypanosomatids are a very diverse group composed of monoxenous and dixenous parasites belonging to the excavate class Kinetoplastea. Here we studied the respiration of five monoxenous species (Blechomonas ayalai, Herpetomonas muscarum, H. samuelpessoai, Leptomonas pyrrhocoris and Sergeia podlipaevi) introduced into culture, each representing a novel yet globally distributed and/or species-rich clade, and compare them with well-studied flagellates Trypanosoma brucei, Phytomonas serpens, Crithidia fasciculata and Leishmania tarentolae. Differences in structure and activities of respiratory chain complexes, respiration and other biochemical parameters recorded under laboratory conditions reveal their substantial diversity, likely a reflection of different host environments. Phylogenetic relationships of the analysed trypanosomatids do not correlate with their biochemical parameters, with the differences within clades by far exceeding those among clades. As the S. podlipaevi canonical respiratory chain complexes have very low activities, we believe that its mitochondrion is utilised for purposes other than oxidative phosphorylation. Hence, the single reticulated mitochondrion of diverse trypanosomatids seems to retain multipotency, with the capacity to activate its individual components based on the host environment.
Crithidia fasciculata
Crithidia
Lineage (genetic)
Cite
Citations (15)