title: Production and characterization of bifunctional enzymes. Domain swapping to produce new bifunctional enzymes in the aspartate pathway.
abstract: The bifunctional enzyme aspartokinase-homoserine dehydrogenase I from Escherichia coli catalyzes non-consecutive reactions in the aspartate pathway of amino acid biosynthesis. Both catalytic activities are subject to allosteric regulation by the end product amino acid L-threonine. To examine the kinetics and regulation of the enzymes in this pathway, each of these catalytic domains were separately expressed and purified. The separated catalytic domains remain active, with each of their catalytic activities enhanced in comparison to the native enzyme. The allosteric regulation of the kinase activity is lost, and regulation of the dehydrogenase activity is dramatically decreased in these separate domains. To create a new bifunctional enzyme that can catalyze consecutive metabolic reactions, the aspartokinase I domain was fused to the enzyme that catalyzes the intervening reaction in the pathway, aspartate semialdehyde dehydrogenase. A hybrid bifunctional enzyme was also created between the native monofunctional aspartokinase III, an allosteric enzyme regulated by lysine, and the catalytic domain of homoserine dehydrogenase I with its regulatory interface domain still attached. In this hybrid the kinase activity remains sensitive to lysine, while the dehydrogenase activity is now regulated by both threonine and lysine. The dehydrogenase domain is less thermally stable than the kinase domain and becomes further destabilized upon removal of the regulatory domain. The more stable aspartokinase III is further stabilized against thermal denaturation in the hybrid bifunctional enzyme and was found to retain some catalytic activity even at temperatures approaching 100 degrees C.
authors.affiliation: Department of Chemistry, University of Toledo, Toledo, Ohio 43606, USA.
authors.forename: Cindy L
authors.initials: CL
authors.lastname: James
authors.forename: Ronald E
authors.initials: RE
authors.lastname: Viola
mesh_terms: D000494:Allosteric Regulation; D001224:Aspartic Acid / Q000378:metabolism*; D020134:Catalytic Domain; D002460:Cell Line; D004795:Enzyme Stability; D009097:Multienzyme Complexes / Q000138:chemical synthesis* / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D009097:Multienzyme Complexes; D001224:Aspartic Acid