Journal
ARCHIVES OF MICROBIOLOGY
Volume 174, Issue 6, Pages 406-414Publisher
SPRINGER-VERLAG
DOI: 10.1007/s002030000225
Keywords
methanarchaea; Methanococcus jannaschii; pyruvate carboxylase; extreme thermophile; storage stable; thermolability; pvc genes
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Funding
- NIGMS NIH HHS [GM 51334] Funding Source: Medline
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The pyruvate carboxylase (PYC) of the hyperthermophilic. strictly hydrogenotrophic, autotrophic and marine methanarchaeon Methanococcus jannaschii was purified to homogeneity. Optimal activity was at pH 8.5, greater than or equal to 80 degreesC, and a KCI concentration of 0.175 M. This enzyme is the most thermophilic PYC so far studied. Unlike the Methanobacterium thermoautotrophicum enzyme, Mc. jannaschii PYC was expressed in cells grown without an external source of biotin and in the purified form was stable during storage at 4, -20 and -80 degreesC. However, it was rapidly inactivated at 80 degreesC. The enzyme was insensitive to aspartate and glutamate, mildly inhibited 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 Mg2+ 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 apparently an alpha (4)beta (4)-type PYC composed of a nonbiotinylated 55.5-kDa 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 very recently, these have been misidentified as a subunit of acetyl-CoA carbxoylase (AccC) and the alpha -subunit of ion-pumping oxaloacetate decarboxylase (OAD alpha), respectively.
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