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

We discovered that Methanobacterium thermoautotrophicum strain Delta H 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 non-linear kinetics with respect to bicarbonate and was inhibited by excess Mg2+, Mn2+, or Co2+, Th, 540-kDa enzyme of A(4)B(4) 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 Delta H (Kenealy, W. R., and Zeikus, J. G. (1982) FEMS Microbiol. Lett, 14, 7-10) raise several questions for future investigations.