Characterization of the aspartate transcarbamoylase from Methanococcus jannaschii

The genes from the thermophilic archaeabacterium Methanococcus jannaschii t hat code for the putative catalytic and regulatory chains of aspartate tran scarbamoylase were expressed at high levels in Escherichia coli, Only the M . jannaschii PyrB (Mj-PyrB) gene product exhibited catalytic activity. A pu rification protocol was devised for the Mj-PyrB and M. jannaschii PyrI (Mj- PyrI) gene products. Molecular weight measurements of the Mj-PyrB and Mj-Py rI gene products revealed that the Mj-PyrB gene product is a trimer and the Mj-PyrI gene product is a dimer. Preliminary characterization of the aspar tate transcarbamoylase from M. jannaschii cell-free extract revealed that t he enzyme has a similar molecular weight to that of the E. coli holoenzyme, Kinetic analysis of the M. jannaschii aspartate transcarbamoylase from the cell-free extract indicates that the enzyme exhibited limited homotropic c ooperativity and little if any regulatory properties. The purified Mj-catal ytic trimer exhibited hyperbolic kinetics, with an activation energy simila r to that observed for the E, coli catalytic trimer, Homology models of the Mj-PyrB and Mj-PyrI gene products were constructed based on the three-dime nsional structures of the homologous E. coli proteins. The residues known t o be critical for catalysis, regulation, and formation of the quaternary st ructure from the well characterized E. coli aspartate transcarbamoylase wer e compared.