Structure-based molecular design for thermostabilization of N-acetyltransferase Mpr1 involved in a novel pathway of l-arginine synthesis in yeast.
Structure-based molecular design for thermostabilization of N-acetyltransferase Mpr1 involved in a novel pathway of l-arginine synthesis in yeast.
复制标题
基于结构的分子设计，用于 N-乙酰转移酶 Mpr1 的热稳定性，参与酵母中 L-精氨酸合成的新途径。
DOI：
10.1093/jb/mvv101
复制
发表时间：
2016
期刊：
影响因子：
--
通讯作者：
Takagi H.
中科院分区：
文献类型：
--
作者：
Nasuno R;Hirase S;Norifune S;Watanabe D;Takagi H.
以前，N-乙酰转移酶Mpr 1被认为参与酵母中精氨酸生物合成的新途径。我们最近的晶体学分析表明，Mpr 1的整体结构是一个典型的折叠蛋白在Gcn 5-relatedN-乙酰转移酶超家族，也提供了线索的突变设计，以改善酶的功能。在这里，我们构建了新的稳定的变体，Asn 203 Lys-和Asn 203 Arg-Mpr 1，其活性半衰期分别比野生型Mpr 1长2.4倍和2.2倍，通过基于结构的分子设计。建议用碱性氨基酸取代Asn 203以稳定α-螺旋2，这对Mpr 1结构很重要，可能是通过中和其偶极来实现的。此外，Mpr 1的65和203位的两个氨基酸取代，Phe 65 Leu，这是先前通过PCR随机诱变文库筛选MPR 1分离的，以及Asn 203 Lys或Asn 203 Arg的组合，导致Mpr 1的进一步稳定。我们的生长测定表明，稳定的Mpr 1变体的过表达增加了酵母细胞中精氨酸的合成。我们的发现是第一次报告的合理工程Mpr 1的热稳定性，并可能是有用的，在建设新的酵母菌株具有较高的L-精氨酸合成活性，也提高了发酵能力。
Previously,N-Acetyltransferase Mpr1 was suggested to be involved in a novel pathway ofl-arginine biosynthesis in yeast. Our recent crystallographic analysis demonstrated that the overall structure of Mpr1 is a typical folding among proteins in the Gcn5-relatedN-acetyltransferase superfamily, and also provided clues to the design of mutations for improvement of the enzymatic functions. Here, we constructed new stable variants, Asn203Lys- and Asn203Arg-Mpr1, which exhibited 2.4-fold and 2.2-fold longer activity half-lives than wild-type Mpr1, respectively, by structure-based molecular design. The replacement of Asn203 with a basic amino acid was suggested to stabilize α-helix 2, which is important for the Mpr1 structure, probably by neutralizing its dipole. In addition, the combination of two amino acid substitutions at positions 65 and 203 in Mpr1, Phe65Leu, which was previously isolated by the screening from PCR random mutagenesis library ofMPR1, and Asn203Lys or Asn203Arg, led to further stabilization of Mpr1. Our growth assay suggests that overexpression of the stable Mpr1 variants increasel-arginine synthesis in yeast cells. Our finding is the first report on the rational engineering of Mpr1 for thermostabilization and could be useful in the construction of new yeast strains with higherl-arginine synthetic activity and also improved fermentation ability.