PURIFICATION, REGULATION, AND MOLECULAR AND BIOCHEMICAL-CHARACTERIZATION OF PYRUVATE-CARBOXYLASE FROM METHANOBACTERIUM-THERMOAUTOTROPHICUM STRAIN DELTA-H

We discovered that Methanobacterium thermoautotrophicum strain Delta H possessed pyruvate carboxylase (PYC), and this biotin prototroph requ ired exogenously supplied biotin to exhibit detectable amounts of PYC activity, The enzyme was highly labile and was stabilized by 10% inosi tol in buffers to an extent that allowed purification to homogeneity a nd characterization. The purified enzyme was absolutely dependent on A TP, Mg2+ (or Mn2+ or Co2+), pyruvate, and bicarbonate for activity; ph osphoenolpyruvate could not replace pyruvate, and acetyl-CoA was not r equired, The enzyme was inhibited by ADP and alpha-ketoglutarate but n ot by aspartate or glutamate. ATP was inhibitory at high concentration s, The enzyme, unlike other PYCs, exhibited non-linear kinetics with r espect to bicarbonate and was inhibited by excess Mg2+, Mn2+, or Co2+, Th, 540-kDa enzyme of A(4)B(4) composition contained a non-biotinylat ed 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 72 7 kilobase pairs away from pycB on the chromosome and was probably co- transcribed with the biotin ligase gene (birA), PYCA and PYCB showed s ubstantial sequence identities (33-62%) to, respectively, the biotin c arboxylase and biotin carboxyl carrier + carboxyltransferase domains o r subunits of known biotin-dependent carboxylases/decarboxylases. We d iscovered that PYCB and probably the equivalent domains or subunits of all biotin-dependent carboxylases harbored the serine/threonine dehyd ratase types of pyridoxal-phosphate attachment site, Our results and t he existence of an alternative oxaloacetate synthesizing enzyme phosph oenolpyruvate carboxylase in M. thermoautotrophicum strain Delta H (Ke nealy, W. R., and Zeikus, J. G. (1982) FEMS Microbiol. Lett, 14, 7-10) raise several questions for future investigations.