Conversion of feedback regulation in aspartate kinase by domain exchange.
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 alpha2beta2-type tetrameric structure. A chimeric AK, named BTT, composed of the chimeric alpha subunit that comprises of the N-terminal catalytic region from B. subtilis AK II and the C-terminal region from T. flavus, and the beta 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 beta subunit, having alpha2-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 beta subunit in AK. BTT showed extremely high thermostability comparable to that of T. flavus, suggesting that the beta subunit also contributed to the stability of the AK.
Kato
Chiaki
C
Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Kurihara
Takeshi
T
Kobashi
Nobuyuki
N
Yamane
Hisakazu
H
Nishiyama
Makoto
M
D000595:Amino Acid Sequence; D001222:Aspartate Kinase / Q000737:chemistry*; D001412:Bacillus subtilis / Q000201:enzymology; D020134:Catalytic Domain; D002850:Chromatography, Gel; D019281:Dimerization; D004926:Escherichia coli / Q000378:metabolism; D025461:Feedback, Physiological; D007700:Kinetics; D008239:Lysine / Q000494:pharmacology; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D013824:Thermus / Q000201:enzymology; D013912:Threonine / Q000737:chemistry / Q000494:pharmacology; D013997:Time Factors
D013912:Threonine; D001222:Aspartate Kinase; D008239:Lysine