The genes from the thermophilic archaeabacterium Methanococcus jannaschii that code for the putative catalytic and regulatory chains of aspartate transcarbamoylase were expressed at high levels in Escherichia coli. Only the M. jannaschii PyrB (Mj-PyrB) gene product exhibited catalytic activity. A purification protocol was devised for the Mj-PyrB and M. jannaschii PyrI (Mj-PyrI) gene products. Molecular weight measurements of the Mj-PyrB and Mj-PyrI 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 aspartate transcarbamoylase from M. jannaschii cell-free extract revealed that the 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 cooperativity and little if any regulatory properties. The purified Mj-catalytic trimer exhibited hyperbolic kinetics, with an activation energy similar 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-dimensional structures of the homologous E. coli proteins. The residues known to be critical for catalysis, regulation, and formation of the quaternary structure from the well characterized E. coli aspartate transcarbamoylase were compared.Organisms from the archaea, prokarya, and eukarya kingdoms all produce aspartate transcarbamoylase, the enzyme that catalyzes the committed step of the pyrimidine biosynthetic pathway, the reaction of carbamoyl phosphate and Laspartate to form N-carbamoyl-L-aspartate and inorganic phosphate (1). There are four major classes or forms of quaternary structures known for aspartate transcarbamoylases. In prokaryotes, aspartate transcarbamoylase is known to exist in three classes. The simplest is class C, a homotrimer of catalytic chains each with a molecular mass of approximately 34 kDa. The aspartate transcarbamoylase from Bacillus subtilis, which lacks both homotropic and heterotropic properties, is an example of this class (2). A second form of aspartate transcarbamoylase, class A, is a dodecamer of six 34-kDa and six 45-kDa polypeptides. Catalytic and regulatory functions of this enzyme are both located on the 34-kDa polypeptides, whereas the function of the 45-kDa polypeptides is unknown. There are several species of Pseudomonas that produce this type of aspartate transcarbamoylase, including Pseudomonas fluorescens (3, 4). The third and best characterized class of aspartate transcarbamoylase is class B, comprised of two trimeric catalytic subunits of 34-kDa polypeptides and three dimeric regulatory subunits of 17-kDa polypeptides. The class B form is an allosteric enzyme, exhibiting both homotropic and heterotropic interactions. Escherichia coli, Salmonella typhimurium, Erwinia herbicola, Serratia marcescens, and other members of the family Enterobacteriaceae produce class B aspartate transcarbamoylase ...