Archives of Microbiology

, Volume 174, Issue 6, pp 406–414 | Cite as

A stable archaeal pyruvate carboxylase from the hyperthermophile Methanococcus jannaschii

  • Biswarup Mukhopadhyay
  • Vipool J. Patel
  • Ralph S. Wolfe
Original Paper

Abstract.

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, ≥80 °C, and a KCl 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 °C. However, it was rapidly inactivated at 80 °C. The enzyme was insensitive to aspartate and glutamate, mildly inhibited by α-ketoglutarate, and was strongly inhibited by ATP and ADP (apparent Km for ATP, 0.374±0.039 mM; apparent Ki for ATP, 5.34±2.14 mM; Ki 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 α4β4-type PYC composed of a non-biotinylated 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 α-subunit of ion-pumping oxaloacetate decarboxylase (OADα), respectively.

Methanarchaea Methanococcus jannaschii Pyruvate carboxylase Extreme thermophile Storage stable Thermolability pyc genes 

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Copyright information

© Springer-Verlag 2000

Authors and Affiliations

  • Biswarup Mukhopadhyay
    • 1
  • Vipool J. Patel
    • 1
  • Ralph S. Wolfe
    • 1
  1. 1.Department of Microbiology, University of Illinois at Urbana-Champaign, B103 Chemical and Life Sciences Laboratory, 601 S. Goodwin Avenue, Urbana, IL 61801, USA

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