Molecular characterization and expression of Onchocerca volvulus glutathione reductase.
Glutathione metabolism represents a potential target for anti-parasite drug design. The central role of glutathione reductase (GR) in maintenance of the thiol redox state and in anti-oxidative defence has to be evaluated in more detail in order to establish the essential function of this enzyme for the survival of the filarial parasite Onchocerca volvulus. The O. volvulus GR (OvGR) gene was cloned and sequenced. The gene is composed of 13 exons and 12 introns and spans 4065 bp. The first intron is located within the 5'-untranslated region of the gene, 16 nucleotides upstream of the translation initiation codon. Southern-blot analysis and structural characterization of the genomic sequence indicate that OvGR is encoded by a single-copy gene. Isolation of various cDNA clones revealed a polymorphism of polyadenylation initiation with no consensus polyadenylation sites in any of the cDNAs analysed. The entire cDNA is 1977 bp long and carries the nematode-specific spliced leader sequence SL1 at its 5' end, 236 nucleotides upstream of the first in-frame methionine. The cDNA codes for a polypeptide of 462 amino acids with 53.5% sequence identity with human GR (HsGR). A total of 18 out of 19 residues contributing to glutathione binding are identical in OvGR and HsGR. However, one of the arginine residues (Arg-224 in HsGR) involved in discrimination between NADPH and NADH in all known GRs is substituted by tryptophan (Trp-207 in OvGR). The coding region of OvGR was expressed in Escherichia coli as a histidine-fusion protein, and it was established that the parasite protein still favours the binding of NADPH (Km 10.9 microM) over NADH (Km 108 microM). The histidine-fusion protein has a subunit size of 54 kDa and is active as a homodimer of 110 kDa.
Müller
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S
Bernhard Nocht Institute of Tropical Medicine, Department of Biochemistry, D-20359 Hamburg, Germany.
Gilberger
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TW
Fairlamb
A H
AH
Walter
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RD
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary; D005980:Glutathione Reductase / Q000235:genetics*; D008969:Molecular Sequence Data; D017181:Onchocerca volvulus / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D005980:Glutathione Reductase