Department of Chemical Biology and State Key Laboratory of Elemento-organic Chemistry, Nankai University, Weijin 94, Tianjin 300071, China.
Chembiochem. 2013 Apr 15;14(6):746-52. doi: 10.1002/cbic.201200680. Epub 2013 Mar 19.
Acetohydroxyacid synthases (AHASs), which catalyze the first step in the biosynthesis of branched-chain amino acids, are composed of a catalytic subunit (CSU) and a regulatory subunit (RSU). The CSU harbors the catalytic site, and the RSU is responsible for the activation and feedback regulation of the CSU. Previous results from Chipman and co-workers and our lab have shown that heterologous activation can be achieved among isozymes of Escherichia coli AHAS. It would be interesting to find the minimum peptide of ilvH (the RSU of E. coli AHAS III) that could activate other E. coli CSUs, or even those of ## species. In this paper, C-terminal, N-terminal, and C- and N-terminal truncation mutants of ilvH were constructed. The minimum peptide to activate ilvI (the CSU of E. coli AHAS III) was found to be ΔN 14-ΔC 89. Moreover, this peptide could not only activate its homologous ilvI and heterologous ilvB (CSU of E. coli AHAS I), but also heterologously activate the CSUs of AHAS from Saccharomyces cerevisiae, Arabidopsis thaliana, and Nicotiana plumbaginifolia. However, this peptide totally lost its ability for feedback regulation by valine, thus suggesting different elements for enzymatic activation and feedback regulation. Additionally, the apparent dissociation constant (Kd ) of ΔN 14-ΔC 89 when binding CSUs of different species was found to be 9.3-66.5 μM by using microscale thermophoresis. The ability of this peptide to activate different CSUs does not correlate well with its binding ability (Kd ) to these CSUs, thus implying that key interactions by specific residues is more important than binding ability in promoting enzymatic reactions. The high sequence similarity of the peptide ΔN 14-ΔC 89 to RSUs across species hints that this peptide represents the minimum activation motif in RSU and that it regulates all AHASs.
乙酰羟酸合酶（AHASs）催化支链氨基酸生物合成的第一步，由一个催化亚基（CSU）和一个调节亚基（RSU）组成。CSU 含有催化位点，RSU 负责 CSU 的激活和反馈调节。Chippman 及其同事和我们实验室的先前结果表明，大肠杆菌 AHAS 同工酶之间可以实现异源激活。有趣的是，找到能够激活其他大肠杆菌 CSU 的最小 ilvH 肽（大肠杆菌 AHAS III 的 RSU），甚至是 ## 种的最小肽。在本文中，构建了 ilvH 的 C 端、N 端和 C、N 端截断突变体。发现激活 ilvI（大肠杆菌 AHAS III 的 CSU）的最小肽为 ΔN 14-ΔC 89。此外，该肽不仅可以激活其同源的 ilvI 和异源的 ilvB（大肠杆菌 AHAS I 的 CSU），还可以异源激活酿酒酵母、拟南芥和烟草的 AHAS 的 CSU。然而，该肽完全失去了对缬氨酸的反馈调节能力，因此暗示了酶激活和反馈调节的不同因素。此外，通过微量热泳动发现，不同物种的 CSUs 与 ΔN 14-ΔC 89 结合的表观解离常数（Kd）为 9.3-66.5 μM。该肽激活不同 CSU 的能力与其与这些 CSU 的结合能力（Kd）没有很好的相关性，这表明特定残基的关键相互作用比促进酶反应的结合能力更重要。该肽 ΔN 14-ΔC 89 与跨物种的 RSU 具有很高的序列相似性，这暗示该肽代表了 RSU 中的最小激活基序，并且它调节所有的 AHAS。
