Yun Mi-Kyung, Subramanian Chitra, Miller Karen, Jackson Pamela, Radka Christopher D, Rock Charles O
Department of Host-Microbe Interactions, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, United States.
Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky, Lexington, Kentucky 40536, United States.
Biochemistry. 2025 Jul 1;64(13):2793-2810. doi: 10.1021/acs.biochem.5c00168. Epub 2025 Jun 10.
In , the branched-chain amino acid biosynthetic pathway provides essential intermediates for membrane biosynthesis. Threonine deaminase (IlvA) is the first enzyme in the pathway, and isoleucine feedback regulates the enzyme in . These studies on IlvA (EcIlvA) introduced the concept of allosteric regulation. To investigate the regulation of IlvA (SaIlvA), we first conducted additional studies on EcIlvA. The previously determined crystal structure of EcIlvA revealed a tetrameric assembly of protomers, each with catalytic and regulatory domains, but the structural basis of isoleucine regulation was not characterized. Here, we present the crystal structure of the EcIlvA regulatory domain bound to isoleucine, which reveals the isoleucine binding site and conformational changes that initiate at Phe352 and propagate 23 Å across the domain. This suggests an allosteric pathway that extends to the active site of the adjacent protomer, mediating regulation across the protomer-protomer interface. The EcIlvA(F352A) mutant binds isoleucine but is feedback-resistant due to the absence of the initiating Phe352. In contrast, SaIlvA is not feedback-regulated by isoleucine and does not bind it. The structure of the SaIlvA regulatory domain reveals a different organization that lacks the isoleucine binding site. Other potential allosteric inhibitors of SaIlvA, including phospholipid intermediates, do not affect enzyme activity. We propose that the absence of feedback inhibition in SaIlvA is due to its role in membrane biosynthesis. These findings enhance our understanding of IlvA's allosteric regulation and offer opportunities for engineering feedback-resistant IlvA variants for biotechnological use.
在[具体生物名称]中，支链氨基酸生物合成途径为膜生物合成提供必需的中间体。苏氨酸脱氨酶（IlvA）是该途径中的第一个酶，在[具体生物名称]中异亮氨酸对该酶进行反馈调节。这些对IlvA（EcIlvA）的研究引入了变构调节的概念。为了研究IlvA（SaIlvA）的调节机制，我们首先对EcIlvA进行了更多研究。先前确定的EcIlvA晶体结构揭示了原体的四聚体组装，每个原体都有催化结构域和调节结构域，但异亮氨酸调节的结构基础尚未明确。在这里，我们展示了与异亮氨酸结合的EcIlvA调节结构域的晶体结构，该结构揭示了异亮氨酸结合位点以及从苯丙氨酸352起始并在整个结构域中传播23埃的构象变化。这表明存在一条延伸至相邻原体活性位点的变构途径，介导跨原体 - 原体界面的调节。EcIlvA（F352A）突变体结合异亮氨酸，但由于缺乏起始的苯丙氨酸352而对反馈具有抗性。相比之下，SaIlvA不受异亮氨酸的反馈调节，也不与之结合。SaIlvA调节结构域的结构揭示了一种不同的组织形式，缺乏异亮氨酸结合位点。SaIlvA的其他潜在变构抑制剂，包括磷脂中间体，均不影响酶活性。我们认为SaIlvA中不存在反馈抑制是由于其在膜生物合成中的作用。这些发现加深了我们对IlvA变构调节的理解，并为工程改造用于生物技术用途的抗反馈IlvA变体提供了机会。
