Conversion of feedback regulation in aspartate kinase by domain exchange

Abstract To elucidate the mechanism for the regulation of aspartate kinase (AK) via feedback inhibition, we constructed several chimeric enzymes between Bacillus subtilis AK II, a lysine-sensitive mesophilic enzyme, and Thermus flavus AK, a threonine-sensitive thermostable enzyme, each having the same α 2 β 2 -type tetrameric structure. A chimeric AK, named BTT, composed of the chimeric α subunit that comprises of the N-terminal catalytic region from B. subtilis AK II and the C-terminal region from T. flavus , and the β subunit from T. flavus , was inhibited only by threonine. Another chimeric enzyme, BT, which has a similar structure to that of BTT but lacks the β subunit, having α 2 -type homo-dimeric structure, was also responsive only to threonine. However, the addition of threonine enhanced the activity of BT. These results indicate the regulatory function of C-terminal region and β subunit in AK. BTT showed extremely high thermostability comparable to that of T. flavus , suggesting that the β subunit also contributed to the stability of the AK.