Identification and functional verification of archaeal-type phosphoenolpyruvate carboxylase, a missing link in archaeal central carbohydrate metabolism.
Despite the fact that phosphoenolpyruvate carboxylase (PEPC) activity has been measured and in some cases even purified from some Archaea, the gene responsible for this activity has not been elucidated. Using sensitive sequence comparison methods, we detected a highly conserved, uncharacterized archaeal gene family that is distantly related to the catalytic core of the canonical PEPC. To verify the predicted function of this archaeal gene family, we cloned a representative from the hyperthermophilic acidophile Sulfolobus solfataricus and functionally produced the corresponding enzyme as a fusion with the Escherichia coli maltose-binding protein. The purified fusion protein indeed displayed highly thermostable PEPC activity. The structural and biochemical properties of the characterized archaeal-type PEPC (atPEPC) from S. solfataricus are in good agreement with previously reported biochemical analyses of other archaeal PEPC enzymes. The newly identified atPEPC, with its distinct properties, constitutes yet another example of the versatility of the enzymes of the central carbon metabolic pathways in the archaeal domain.
Ettema
Thijs J G
TJ
Laboratory of Microbiology, Agrotechnology and Food Sciences, Wageningen University, Wageningen, The Netherlands. thijs.ettema@wur.nl
Makarova
Kira S
KS
Jellema
Gera L
GL
Gierman
Hinco J
HJ
Koonin
Eugene V
EV
Huynen
Martijn A
MA
de Vos
Willem M
WM
van der Oost
John
J
D000595:Amino Acid Sequence; D001105:Archaea / Q000201:enzymology; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019295:Computational Biology / Q000379:methods; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D020745:Genome, Archaeal; D008969:Molecular Sequence Data; D010730:Phosphoenolpyruvate Carboxylase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010802:Phylogeny; D017422:Sequence Analysis, DNA; D048229:Sulfolobus solfataricus / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development
D019843:Archaeal Proteins; D010730:Phosphoenolpyruvate Carboxylase