A stable archaeal pyruvate carboxylase from the hyperthermophile Methanococcus jannaschii

The pyruvate carboxylase (PYC) of the hyperthermophilic. strictly hydrogeno trophic, autotrophic and marine methanarchaeon Methanococcus jannaschii was purified to homogeneity. Optimal activity was at pH 8.5, greater than or e qual to 80 degreesC, and a KCI concentration of 0.175 M. This enzyme is the most thermophilic PYC so far studied. Unlike the Methanobacterium thermoau totrophicum enzyme, Mc. jannaschii PYC was expressed in cells grown without an external source of biotin and in the purified form was stable during st orage at 4, -20 and -80 degreesC. However, it was rapidly inactivated at 80 degreesC. The enzyme was insensitive to aspartate and glutamate, mildly in hibited by alpha -ketoglutarate, and was strongly inhibited by ATP and ADP (apparent K-m for ATP, 0.374+/-0.039 mM; apparent K-i for ATP, 5.34+/-2.14 mM; K-i for ADP, 0.89+/-0.18 mM). It was also strongly inhibited when the M g2+ concentration in the assay exceeded that of ATP. Thus, this stable PYC could serve as a model for mechanistic studies on archaeal PYCs. It was app arently an alpha (4)beta (4)-type PYC composed of a nonbiotinylated 55.5-kD a subunit (PYCA) and a 64.2-kDa biotinylated subunit (PYCB). The determined NH2-terminal sequences for these subunits provided additional support for our earlier proposal to rename the ORFs MJ1229, and MJ1231 in the NCBI Mc. jannaschii genome sequence database as PYCA and PYCB, respectively; even ve ry recently, these have been misidentified as a subunit of acetyl-CoA carbx oylase (AccC) and the alpha -subunit of ion-pumping oxaloacetate decarboxyl ase (OAD alpha), respectively.