TY - JOUR
T1 - Cloning and characterization of the gene encoding ATP-dependent phospho-enol-pyruvate carboxykinase in Trypanosoma cruzi
T2 - comparison of primary and predicted secondary structure with host GTP-dependent enzyme
AU - Linss, Jutta
AU - Goldenberg, Samuel
AU - Urbina, Julio A.
AU - Amzel, L. Mario
N1 - Funding Information:
This investigation received support from The UNDP/WORLD BANK/WHO SpecialP rogrammef or Researcha nd Training in Tropical DiseasesC, onselho National de DesenvolvimentoC ientifico e Tecnologico and the German DAAD to S.G.; the work of J.A.U. and L.M.A. was supported by a CONICIT (Venezuela)/NationaSl cience Foundation (USA) ex-changeg rant.W e thank Thomas Seebeck( Universityo f Bern, Switzerland) for kindly providing the clone pTBp60B that containsT . brucei PEPCK, Rainer Frank (ZBMH, University of Heidelberg, Germany) for his interesti n this work and Stenio P. Fragoso for helpful discussions.
PY - 1993/12/22
Y1 - 1993/12/22
N2 - The complete nucleotide (nt) sequence of the PEPCK gene encoding Trypanosoma cruzi phospho-enol-pyruvate carboxykinase (PEPCK; ATP dependent, EC 4.1.1.49) has been determined. The predicted primary sequence has 473 amino acids (aa) with a calculated molecular mass of 52.5 kDa. The ubiquitous spliced leader is present at nt position - 60 from the AUG start codon in PEPCK mRNA; the coding region is followed by a long 3'-non-coding region of 777 nt. Northern and Southern blot analysis showed that the PEPCK mRNA is 2.7 kb long and that the PEPCK gene is polymorphic in T. cruzi, with more than one copy in the genome of the epimastigote form. Comparison of the available aa sequences of ATP(protozoa, yeast and bacteria)- and GTP(vertebrates, insects, helminths and fungi)-dependent PEPCKs showed that the former lack two characteristic, highly conserved regions present in the GTP-dependent enzymes: one is associated with the binding of PEP while the second is frequently labeled as 'catalytic' and contains a conserved Cys residue of unusual reactivity. On the other hand, two consensus sequences with conserved predicted secondary structure were identified in all PEPCKs, independent of their nt specificity; one of them is a divalent metal-binding site previously identified in pyruvate kinase by X-ray crystallographic studies.
AB - The complete nucleotide (nt) sequence of the PEPCK gene encoding Trypanosoma cruzi phospho-enol-pyruvate carboxykinase (PEPCK; ATP dependent, EC 4.1.1.49) has been determined. The predicted primary sequence has 473 amino acids (aa) with a calculated molecular mass of 52.5 kDa. The ubiquitous spliced leader is present at nt position - 60 from the AUG start codon in PEPCK mRNA; the coding region is followed by a long 3'-non-coding region of 777 nt. Northern and Southern blot analysis showed that the PEPCK mRNA is 2.7 kb long and that the PEPCK gene is polymorphic in T. cruzi, with more than one copy in the genome of the epimastigote form. Comparison of the available aa sequences of ATP(protozoa, yeast and bacteria)- and GTP(vertebrates, insects, helminths and fungi)-dependent PEPCKs showed that the former lack two characteristic, highly conserved regions present in the GTP-dependent enzymes: one is associated with the binding of PEP while the second is frequently labeled as 'catalytic' and contains a conserved Cys residue of unusual reactivity. On the other hand, two consensus sequences with conserved predicted secondary structure were identified in all PEPCKs, independent of their nt specificity; one of them is a divalent metal-binding site previously identified in pyruvate kinase by X-ray crystallographic studies.
KW - Nucleotide sequence
KW - consensus sequences
KW - protein functional domains
KW - sequence homology
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U2 - 10.1016/0378-1119(93)90449-D
DO - 10.1016/0378-1119(93)90449-D
M3 - Article
C2 - 8294043
AN - SCOPUS:0027725284
SN - 0378-1119
VL - 136
SP - 69
EP - 77
JO - Gene
JF - Gene
IS - 1-2
ER -