Purification, regulation, and molecular and biochemical characterization of pyruvate carboxylase from Methanobacterium thermoautotrophicum strain deltaH

We discovered that Methanobacterium thermoautotrophicum strain DeltaH possessed pyruvate carboxylase (PYC), and this biotin prototroph required exogenously supplied biotin to exhibit detectable amounts of PYC activity. The enzyme was highly labile and was stabilized by 10% inositol in buffers to an extent that allowed purification to homogeneity and characterization. The purified enzyme was absolutely dependent on ATP, Mg2+ (or Mn2+ or Co2+), pyruvate, and bicarbonate for activity; phosphoenolpyruvate could not replace pyruvate, and acetyl-CoA was not required. The enzyme was inhibited by ADP and alpha-ketoglutarate but not by aspartate or glutamate. ATP was inhibitory at high concentrations. The enzyme, unlike other PYCs, exhibited nonlinear kinetics with respect to bicarbonate and was inhibited by excess Mg2+, Mn2+, or Co2+. The 540-kDa enzyme of A4B4 composition contained a non-biotinylated 52-kDa subunit (PYCA) and a 75-kDa biotinylated subunit (PYCB). The pycB gene was probably monocistronic and followed by a putative gene of a DNA-binding protein on the opposite strand. The pycA was about 727 kilobase pairs away from pycB on the chromosome and was probably co-transcribed with the biotin ligase gene (birA). PYCA and PYCB showed substantial sequence identities (33-62%) to, respectively, the biotin carboxylase and biotin carboxyl carrier + carboxyltransferase domains or subunits of known biotin-dependent carboxylases/decarboxylases. We discovered that PYCB and probably the equivalent domains or subunits of all biotin-dependent carboxylases harbored the serine/threonine dehydratase types of pyridoxal-phosphate attachment site. Our results and the existence of an alternative oxaloacetate synthesizing enzyme phosphoenolpyruvate carboxylase in M. thermoautotrophicum strain DeltaH (Kenealy, W. R., and Zeikus, J. G. (1982) FEMS Microbiol. Lett. 14, 7-10) raise several questions for future investigations.