Oxaloacetate synthesis in the methanarchaeon Methanosarcina barkeri: Pyruvate carboxylase genes and a putative Escherichia coli-type bifunctional biotin protein ligase gene (bpl/birA) exhibit a unique organization

Evidence is presented that, in Methanosarcina barkeri oxaloacetate synthesi s, an essential and major CO, fixation reaction is catalyzed by an apparent alpha (4)beta (4)-type acetyl coenzyme A-independent pyruvate carboxylase (PYC), composed of 64.2-kDa biotinylated and 52.9-kDa ATP-binding subunits, The purified enzyme was most active at 70 degreesC, insensitive to asparta te and glutamate, mildly inhibited by alpha -ketoglutarate, and severely in hibited by ATP, ADP, and excess Mg2(+), It showed negative cooperativity to wards bicarbonate at 70 degreesC but not at 37 degreesC, The organism expre ssed holo-PYC without an external supply of biotin and, thus, synthesized b iotin, pycA, pycB, and a putative bpl gene formed a novel operon-like arran gement. Unlike other archaeal homologs, the putative biotin protein ligases (BPLs) of M. barkeri and the closely related euryarchaeon Archaeoglobus fu lgidus appeared to be of the Escherichia coli-type (bifunctional, with two activities: BirA or a repressor of the biotin operon and BPL), We found the element Tyr(Phe)PraX(5)Phe(Tyr) to be fully conserved in biotin-dependent enzymes; it might function as the hinge for their "swinging arms".