In vivo formation of hybrid aspartate transcarbamoylases from native subunits of divergent members of the family Enterobacteriaceae.
The genes encoding the catalytic (pyrB) and regulatory (pyrI) polypeptides of aspartate transcarbamoylase (ATCase, EC 2.1.3.2) from several members of the family Enterobacteriaceae appear to be organized as bicistronic operons. The pyrBI gene regions from several enteric sources were cloned into selected plasmid vectors and expressed in Escherichia coli. Subsequently, the catalytic cistrons were subcloned and expressed independently from the regulatory cistrons from several of these sources. The regulatory cistron of E. coli was cloned separately and expressed from lac promoter-operator vectors. By utilizing plasmids from different incompatibility groups, it was possible to express catalytic and regulatory cistrons from different bacterial sources in the same cell. In all cases examined, the regulatory and catalytic polypeptides spontaneously assembled to form stable functional hybrid holoenzymes. This hybrid enzyme formation indicates that the r:c domains of interaction, as well as the dodecameric architecture, are conserved within the Enterobacteriaceae. The catalytic subunits of the hybrid ATCases originated from native enzymes possessing varied responses to allosteric effectors (CTP inhibition, CTP activation, or very slight responses; and ATP activation or no ATP response). However, each of the hybrid ATCases formed with regulatory subunits from E. coli demonstrated ATP activation and CTP inhibition, which suggests that the allosteric control characteristics are determined by the regulatory subunits.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000494:Allosteric Regulation; D001221:Aspartate Carbamoyltransferase / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D003570:Cytidine Triphosphate / Q000494:pharmacology; D004755:Enterobacteriaceae / Q000201:enzymology* / Q000235:genetics; D004789:Enzyme Activation; D005796:Genes; D009876:Operon; D010957:Plasmids
D003570:Cytidine Triphosphate; D000255:Adenosine Triphosphate; D001221:Aspartate Carbamoyltransferase