Improvement of ST0452 N-Acetylglucosamine-1-Phosphate Uridyltransferase Activity by the Cooperative Effect of Two Single Mutations Identified through Structure-Based Protein Engineering
Improvement of ST0452 N-Acetylglucosamine-1-Phosphate Uridyltransferase Activity by the Cooperative Effect of Two Single Mutations Identified through Structure-Based Protein Engineering
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DOI：
10.1128/aem.02213-18
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发表时间：
2018-10
影响因子：
4.4
通讯作者：
Y. Honda;S. Nakano;S. Ito;M. Dadashipour;Zilian Zhang;Y. Kawarabayasi
中科院分区：
文献类型：
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作者：
Y. Honda;S. Nakano;S. Ito;M. Dadashipour;Zilian Zhang;Y. Kawarabayasi
我们证明，一个热稳定蛋白的酶活性是超过4倍高于野生型蛋白的单个氨基酸取代后，而不影响其热稳定性。确定了改进的突变蛋白与底物复合的三维结构。与在充分表征的细菌酶中观察到的相同的整体结构和取代的残基与GlcNAc底物之间的相互作用表明，位置97处的Tyr被Asn取代可能略微改变相互作用。这种相互作用的微妙变化可能潜在地增加突变蛋白的GlcNAc-1-P UTase活性。这些观察结果表明，天然热稳定酶的结构的急剧变化并不是增加其活性所必需的;与底物相互作用的细微变化可能就足够了。在适当的双突变蛋白中观察到协同效应。本工作为今后天然酶的工程化提供了有用的信息。我们以前发现，从东京硫化叶菌分离的ST 0452蛋白的Y 97 N突变体与野生型ST 0452蛋白相比，表现出超过4倍的N-乙酰葡糖胺-1-磷酸（GlcNAc-1-P）尿苷酰转移酶（UTase）活性。我们确定了Y 97 N蛋白的三维结构，以探索这种活性增加的详细机制。整体结构与野生型ST 0452蛋白（PDB ID 2GGO）几乎相同，其中残基97（Asn）与复合物中N-乙酰葡糖胺（GlcNAc）的O-5原子相互作用，而无金属离子。在不存在金属离子的情况下，观察到大肠杆菌GlmU的相同的相互作用。这些观察结果表明，Y 97 N蛋白的三维结构没有被这种取代改变，但与底物的相互作用略有改变，这可能导致活性增加。Y 97 N蛋白的晶体结构还显示，位置146（Glu）和80（Thr）与GlcNAc形成相互作用，并且将工程化策略应用于这些残基以增加活性。与野生型蛋白质相比，在位置146处取代的所有蛋白质具有显著降低的活性，而在位置80处取代的几种蛋白质显示出更高的GlcNAc-1-P UTase活性。在位置80和97处的取代的氨基酸可能导致与底物的优化的相互作用;因此，我们预测这两个取代的组合可能协同增加GlcNAc-1-P UTase活性。在产生的四个双突变ST 0452蛋白中，T80 S/Y 97 N显示出比野生型ST 0452蛋白高6.5倍的活性，这表明这两个取代的残基协同作用以增加GlcNAc-1-P UTase活性。重要性我们证明，热稳定蛋白质的酶活性比野生型蛋白质的酶活性高4倍以上，在单个氨基酸取代后，不影响其热稳定性。确定了改进的突变蛋白与底物复合的三维结构。与在充分表征的细菌酶中观察到的相同的整体结构和取代的残基与GlcNAc底物之间的相互作用表明，位置97处的Tyr被Asn取代可能略微改变相互作用。这种相互作用的微妙变化可能潜在地增加突变蛋白的GlcNAc-1-P UTase活性。这些观察结果表明，天然热稳定酶的结构的急剧变化并不是增加其活性所必需的;与底物相互作用的细微变化可能就足够了。在适当的双突变蛋白中观察到协同效应。本工作为今后天然酶的工程化提供了有用的信息。
We demonstrated that the enzymatic activity of a thermostable protein was over 4 times higher than that of the wild-type protein following substitution of a single amino acid, without affecting its thermostability. The three-dimensional structure of the improved mutant protein complexed with substrate was determined. The same overall structure and interaction between the substituted residue and the GlcNAc substrate as observed in the well-characterized bacterial enzyme suggested that the substitution of Tyr at position 97 by Asn might slightly change the interaction. This subtle change in the interaction might potentially increase the GlcNAc-1-P UTase activity of the mutant protein. These observations indicated that a drastic change in the structure of a natural thermostable enzyme is not necessary to increase its activity; a subtle change in the interaction with the substrate might be sufficient. Cooperative effects were observed in the appropriate double mutant protein. This work provides useful information for the future engineering of natural enzymes. ABSTRACT We showed previously that the Y97N mutant of the ST0452 protein, isolated from Sulfolobus tokodaii, exhibited over 4 times higher N-acetylglucosamine-1-phosphate (GlcNAc-1-P) uridyltransferase (UTase) activity, compared with that of the wild-type ST0452 protein. We determined the three-dimensional structure of the Y97N protein to explore the detailed mechanism underlying this increased activity. The overall structure was almost identical to that of the wild-type ST0452 protein (PDB ID 2GGO), with residue 97 (Asn) interacting with the O-5 atom of N-acetylglucosamine (GlcNAc) in the complex without metal ions. The same interaction was observed for Escherichia coli GlmU in the absence of metal ions. These observations indicated that the three-dimensional structure of the Y97N protein was not changed by this substitution but the interactions with the substrate were slightly modified, which might cause the activity to increase. The crystal structure of the Y97N protein also showed that positions 146 (Glu) and 80 (Thr) formed interactions with GlcNAc, and an engineering strategy was applied to these residues to increase activity. All proteins substituted at position 146 had drastically decreased activities, whereas several proteins substituted at position 80 showed higher GlcNAc-1-P UTase activity, compared to that of the wild-type protein. The substituted amino acids at positions 80 and 97 might result in optimized interactions with the substrate; therefore, we predicted that the combination of these two substitutions might cooperatively increase GlcNAc-1-P UTase activity. Of the four double mutant ST0452 proteins generated, T80S/Y97N showed 6.5-times-higher activity, compared to that of the wild-type ST0452 protein, revealing that these two substituted residues functioned cooperatively to increase GlcNAc-1-P UTase activity. IMPORTANCE We demonstrated that the enzymatic activity of a thermostable protein was over 4 times higher than that of the wild-type protein following substitution of a single amino acid, without affecting its thermostability. The three-dimensional structure of the improved mutant protein complexed with substrate was determined. The same overall structure and interaction between the substituted residue and the GlcNAc substrate as observed in the well-characterized bacterial enzyme suggested that the substitution of Tyr at position 97 by Asn might slightly change the interaction. This subtle change in the interaction might potentially increase the GlcNAc-1-P UTase activity of the mutant protein. These observations indicated that a drastic change in the structure of a natural thermostable enzyme is not necessary to increase its activity; a subtle change in the interaction with the substrate might be sufficient. Cooperative effects were observed in the appropriate double mutant protein. This work provides useful information for the future engineering of natural enzymes.