Influence of mutation in the regulatory domain of α-isopropylmalate synthase from <i>Saccharomyces cerevisiae</i> on its activity and feedback inhibition
Influence of mutation in the regulatory domain of α-isopropylmalate synthase from <i>Saccharomyces cerevisiae</i> on its activity and feedback inhibition
复制标题
酿酒酵母α-异丙基苹果酸合酶调控域突变对其活性及反馈抑制的影响
DOI：
10.1093/bbb/zbac045
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发表时间：
2022
期刊：
影响因子：
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通讯作者：
Uegaki Koichi
中科院分区：
文献类型：
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作者：
Takagi Hironobu;Yamamoto Kazuki;Matsuo Yoshifumi;Furuie Miki;Kasayuki Yasuha;Ohtani Rina;Shiotani Mizuki;Hasegawa Tetsuya;Ohnishi Toru;Ohashi Masataka;Johzuka Katsuki;Kurata Atsushi;Uegaki Koichi
异戊醇（i-AmOH）是由α-酮异己酸在酵母亮氨酸生物合成途径中合成的，并受α-异丙基苹果酸合成酶（IPMS）的负反馈调节，该酶能感知亮氨酸的积累。已知当调节结构域中的突变降低对反馈抑制的敏感性时，i-AmOH产生增加。然而，该结构域中的突变对IPMS活性的影响尚未被研究。在本研究中，我们获得了5个编码LEU 4基因的IPMS突变体，N515 D/S520 P/S542 F/A551 D/A551 V，它们对5，5，5-三氟-dl-亮氨酸具有耐受性。所有突变体蛋白均经纯化，并通过体外实验检测其IPMS活性和负反馈活性。结果表明，Leu 4突变体不仅基本丧失了L-亮氨酸的负反馈调节作用，而且IPMS活性也大大降低，且L-亮氨酸存在下Leu 4突变体间IPMS活性的差异与i-AmOH产量显著相关。
Isoamyl alcohol (i-AmOH) is produced from α-ketoisocaproate in thel-leucine biosynthetic pathway in yeast and controlled by the negative feedback regulation of α-isopropylmalate synthase (IPMS), which senses the accumulation ofl-leucine. It is known that i-AmOH production increases when mutations in the regulatory domain reduce the susceptibility to feedback inhibition. However, the impact of mutations in this domain on the IPMS activity has not been examined. In this study, we obtained 5 IPMS mutants, encoding theLEU4gene, N515D/S520P/S542F/A551D/A551V, that are tolerant to 5,5,5-trifluoro-dl-leucine. All mutant proteins were purified and examined for both IPMS activity and negative feedback activity byin vitroexperiments. The results showed that not only the negative-feedback regulation byl-leucine was almost lost in all mutants, but also the IPMS activity was greatly decreased and the difference in IPMS activity among Leu4 mutants in the presence ofl-leucine was significantly correlated with i-AmOH production.