Cloning of a cDNA encoding phosphoenolpyruvate carboxykinase from Haemonchus contortus.
Biochemical and metabolic data have led to the conclusion that the enzyme phosphoenolpyruvate carboxykinase (PEPCK; EC 4.1.1.32) contributes to a critical point of divergence in energy conservation pathways between mammals and nematodes. To facilitate the determination of the molecular basis for host vs parasite differences in PEPCK, we have cloned a cDNA encoding this enzyme from a parasitic nematode of ruminants, Haemonchus contortus. H. contortus PEPCK was cloned by functional complementation of a PEPCK-, malic enzyme- strain of Escherichia coli (E1786) using an egg stage H. contortus cDNA library in lambda ZAPII. Selection was for growth on malate as the sole carbon source (malate+ phenotype). We isolated a plasmid, pPEPCK, which reproducibly confers a malate+ phenotype in E1786. The sequence of the 2.0-kb EcoRI insert of pPEPCK predicts a 612-amino acid protein which shows about 74% similarity to Drosophila melanogaster and chicken PEPCK. Extracts of E1786[pPEPCK], but not E1786, contain IDP- or GDP-dependent PEPCK enzyme activity. Sequence analysis revealed that the open reading frame (ORF) in pPEPCK lacked a 5' initiation codon and was probably expressed as an in-frame fusion protein with beta-galactosidase. A strategy combining library screening with PCR analysis of positive clones led to the identification of a clone encoding 6 additional NH2-terminal amino acids, including a Met, which, by comparison with known PEPCK amino acid sequences, is likely to be the translation initiation site.
Klein
R D
RD
Molecular Biology Research, Upjohn Laboratories, Kalamazoo, MI 49001.
Winterrowd
C A
CA
Hatzenbuhler
N T
NT
Shea
M H
MH
Favreau
M A
MA
Nulf
S C
SC
Geary
T G
TG
D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D004560:Electricity; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D005816:Genetic Complementation Test; D006189:Haemonchus / Q000235:genetics*; D008969:Molecular Sequence Data; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000235:genetics* / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D051381:Rats; D012689:Sequence Homology, Nucleic Acid; D014169:Transformation, Bacterial
D010729:Phosphoenolpyruvate Carboxykinase (GTP)