Structural Analysis and Construction of a Thermostable Antifungal Chitinase
Structural Analysis and Construction of a Thermostable Antifungal Chitinase
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DOI：
10.1128/aem.00652-22
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发表时间：
2022-06-02
影响因子：
4.4
通讯作者：
Atomi, Haruyuki
中科院分区：
文献类型：
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作者：
Kozome, Dan;Uechi, Keiko;Atomi, Haruyuki
研究了具有抗真菌活性的几丁质酶的晶体结构。此外，我们证明了通过10个氨基酸取代，包括5个脯氨酸残基取代，建立二硫键，并在酶中建立盐桥网络，构建了熔化温度（T-m）比野生型（WT）高6.9℃，60℃半衰期比野生型（WT）长15倍的耐热突变酶。几丁质是一种具有-1,4键的n -乙酰-d-氨基葡萄糖的生物聚合物，是节肢动物外骨骼和许多真菌细胞壁的主要成分。几丁质酶（EC 3.2.1.14）是一种水解几丁质中的-1,4键并将几丁质降解为低聚物的酶。它存在于多种生物体中。几丁质酶通过降解真菌细胞壁中的几丁质而具有抗真菌活性，有望在医疗和农业领域得到广泛应用。然而，酶的热稳定性是一个重要因素；几丁质酶在实际应用条件下，其耐热性不足以维持其活性。耐热几丁质酶除了具有抗真菌活性，可以在各种条件下使用外，在生产工艺和长期保存方面也有一定的优势，这在工业应用中是非常需要的。通过分析几丁质酶的晶体结构，探索其热稳定性的靶点。我们基于其他几丁质酶的晶体结构和序列比对，采用蛋白质工程方法在几丁质酶中合理引入脯氨酸残基、二硫键和盐桥。结果表明，我们成功构建了具有较高抗真菌活性和特异活性的耐热几丁质酶突变体。该结果为提高该酶家族的热稳定性提供了一种有用的策略。重要意义：研究了具有抗真菌活性的几丁质酶的晶体结构。此外，我们证明了通过10个氨基酸取代，包括5个脯氨酸残基取代，建立二硫键，并在酶中建立盐桥网络，构建了熔化温度（T-m）比野生型（WT）高6.9℃，60℃半衰期比野生型（WT）长15倍的耐热突变酶。这些突变不影响其抗真菌活性和几丁质酶活性，其晶体结构很好地解释了耐热几丁质酶的构建原理。我们的研究结果为提高该酶家族的热稳定性提供了一个有用的策略，并将热稳定性突变体作为抗真菌药物的种子用于实际应用。
We solved the crystal structure of the chitinase from Gazyumaru (Ficus microcarpa) latex exhibiting antifungal activity. Furthermore, we demonstrated that the thermostable mutant enzyme with a melting temperature (T-m) 6.9 degrees C higher than wild type (WT) and a half-life at 60 degrees C that is 15 times longer than WT was constructed through 10 amino acid substitutions, including 5 proline residues substitutions, making disulfide bonding, and building a salt bridge network in the enzyme.Chitin is a biopolymer of N-acetyl-d-glucosamine with beta-1,4-bond and is the main component of arthropod exoskeletons and the cell walls of many fungi. Chitinase (EC 3.2.1.14) is an enzyme that hydrolyzes the beta-1,4-bond in chitin and degrades chitin into oligomers. It has been found in a wide range of organisms. Chitinase from Gazyumaru (Ficus microcarpa) latex exhibits antifungal activity by degrading chitin in the cell wall of fungi and is expected to be used in medical and agricultural fields. However, the enzyme's thermostability is an important factor; chitinase is not thermostable enough to maintain its activity under the actual application conditions. In addition to the fact that thermostable chitinases exhibiting antifungal activity can be used under various conditions, they have some advantages for the production process and long-term preservation, which are highly demanded in industrial use. We solved the crystal structure of chitinase to explore the target sites to improve its thermostability. We rationally introduced proline residues, a disulfide bond, and salt bridges in the chitinase using protein-engineering methods based on the crystal structure and sequence alignment among other chitinases. As a result, we successfully constructed the thermostable mutant chitinases rationally with high antifungal and specific activities. The results provide a useful strategy to enhance the thermostability of this enzyme family. IMPORTANCE We solved the crystal structure of the chitinase from Gazyumaru (Ficus microcarpa) latex exhibiting antifungal activity. Furthermore, we demonstrated that the thermostable mutant enzyme with a melting temperature (T-m) 6.9 degrees C higher than wild type (WT) and a half-life at 60 degrees C that is 15 times longer than WT was constructed through 10 amino acid substitutions, including 5 proline residues substitutions, making disulfide bonding, and building a salt bridge network in the enzyme. These mutations do not affect its high antifungal activity and chitinase activity, and the principle for the construction of the thermostable chitinase was well explained by its crystal structure. Our results provide a useful strategy to enhance the thermostability of this enzyme family and to use the thermostable mutant as a seed for antifungal agents for practical use.