Suppr超能文献

通过冷冻电子显微镜从原子结构角度揭示亮氨酸脱氢酶的热稳定性机制。

Structural insights into thermostabilization of leucine dehydrogenase from its atomic structure by cryo-electron microscopy.

机构信息

Institute for Innovation, Ajinomoto Co., Inc., 1-1 Suzuki-cho, Kawasaki 210-8681, Japan.

Cellular and Structural Physiology Institute, Nagoya University, Chikusa, Nagoya 464-8601, Japan; CeSPIA Inc., 2-1-1, Otemachi, Chiyoda, Tokyo 100-0004, Japan.

出版信息

J Struct Biol. 2019 Jan 1;205(1):11-21. doi: 10.1016/j.jsb.2018.12.001. Epub 2018 Dec 10.

Abstract

Leucine dehydrogenase (LDH, EC 1.4.1.9) is a NAD-dependent oxidoreductase that catalyzes the deamination of branched-chain l-amino acids (BCAAs). LDH of Geobacillus stearothermophilus (GstLDH) is a highly thermostable enzyme that has been applied for the quantification or production of BCAAs. Here the cryo-electron microscopy (cryo-EM) structures of apo and NAD-bound LDH are reported at 3.0 and 3.2 Å resolution, respectively. On comparing the structures, the two overall structures are almost identical, but it was observed that the partial conformational change was triggered by the interaction between Ser147 and the nicotinamide moiety of NAD. NAD binding also enhanced the strength of oligomerization interfaces formed by the core domains. Such additional interdomain interaction is in good agreement with our experimental results showing that the residual activity of NAD-bound form was approximately three times higher than that of the apo form after incubation at 80 °C. In addition, sequence comparison of three structurally known LDHs indicated a set of candidates for site-directed mutagenesis to improve thermostability. Subsequent mutation analysis actually revealed that non-conserved residues, including Ala94, Tyr127, and the C-terminal region, are crucial for oligomeric thermostability.

摘要

亮氨酸脱氢酶(LDH,EC 1.4.1.9)是一种 NAD 依赖性氧化还原酶,可催化支链 L-氨基酸(BCAAs)的脱氨作用。地芽孢杆菌(Geobacillus stearothermophilus)的 LDH(GstLDH)是一种高度耐热的酶,已被用于 BCAAs 的定量或生产。本文报道了 apo 和 NAD 结合的 LDH 在 3.0 和 3.2Å 分辨率下的低温电子显微镜(cryo-EM)结构。通过比较结构,发现两种整体结构几乎相同,但观察到 Ser147 与 NAD 的烟酰胺部分之间的相互作用引发了部分构象变化。NAD 结合还增强了由核心结构域形成的寡聚化界面的强度。这种额外的结构域间相互作用与我们的实验结果非常吻合,表明在 80°C 孵育后,NAD 结合形式的残留活性约为 apo 形式的三倍。此外,对三种结构已知的 LDH 的序列比较表明了一组用于定点突变的候选物,以提高耐热性。随后的突变分析实际上表明,非保守残基,包括 Ala94、Tyr127 和 C 末端区域,对寡聚体耐热性至关重要。

文献AI研究员

20分钟写一篇综述,助力文献阅读效率提升50倍。

立即体验

用中文搜PubMed

大模型驱动的PubMed中文搜索引擎

马上搜索

文档翻译

学术文献翻译模型,支持多种主流文档格式。

立即体验