Molecular cloning and characterization of the genes encoding two isoforms of cysteine synthase in the enteric protozoan parasite Entamoeba histolytica.
The enteric protozoan parasite Entamoeba histolytica was shown to possess cysteine synthase (CS) activity. The cDNA and genomic clones that encode two isoforms of the E. histolytica CS were isolated and characterized from a clonal strain of E. histolytica by genetic complementation of the cysteine-auxotrophic Escherichia coli NK3 with an E. histolytica cDNA library. The two types of the E. histolytica CS genes differed from each other by three nucleotides, two of which resulted in amino acid substitution. Deduced amino acid sequences of the E. histolytica CS, with a calculated molecular mass of 36721 Da and an isoelectric point of 6.39, exhibited 38-48% identity with CS of bacterial and plant origins. The absence of the amino-terminal transit peptide in the deduced protein sequences and the presence of the CS protein mainly in the supernatant fraction of the amoebic lysate after cellular fractionation suggested that the identified E. histolytica CS genes encoded cytosolic isoforms. Substrate specificity of the recombinant E. histolytica CS was similar to that of plant CS. Phylogenetic analysis indicates that the amoebic CS, first described in Protozoa, does not belong to any families of the CS superfamily, and represents a new family.
Nozaki
T
T
Department of Tropical Medicine and Parasitology, Keio University, School of Medicine, Tokyo, Japan. nozakit@med.keio.ac.jp
Asai
T
T
Kobayashi
S
S
Ikegami
F
F
Noji
M
M
Saito
K
K
Takeuchi
T
T
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D006224:Cricetinae; D003545:Cysteine / Q000096:biosynthesis; D003547:Cysteine Synthase / Q000032:analysis / Q000145:classification / Q000235:genetics*; D018076:DNA, Complementary; D004748:Entamoeba histolytica / Q000201:enzymology / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D017125:Genes, Protozoan; D015151:Immunoblotting; D008969:Molecular Sequence Data; D010802:Phylogeny; D015800:Protozoan Proteins / Q000032:analysis / Q000145:classification / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis; D016415:Sequence Alignment; D013379:Substrate Specificity
D018076:DNA, Complementary; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D003547:Cysteine Synthase; D003545:Cysteine