Domain Shuffling and Site-Saturation Mutagenesis for the Enhanced Inhibitory Potential of Amaranthaceae α-Amylase Inhibitors

IF 1.9 4区 生物学 Q4 BIOCHEMISTRY & MOLECULAR BIOLOGY The Protein Journal Pub Date : 2023-08-19 DOI:10.1007/s10930-023-10148-y
Ashwini S. Rane, Vineetkumar S. Nair, Rakesh S. Joshi, Ashok P. Giri
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Abstract

Amaranthaceae α-amylase inhibitors (AAIs) are knottin-type proteins with selective inhibitory potential against coleopteran α-amylases. Their small size and remarkable stability make them exciting molecules for protein engineering to achieve superior selectivity and efficacy. In this report, we have designed a set of AAI pro- and mature peptides chimeras. Based on in silico analysis, stable AAI chimeras having a stronger affinity with target amylases were selected for characterization. In vitro studies validated that chimera of the propeptide from Chenopodium quinoa α-AI and mature peptide from Beta vulgaris α-AI possess 3, 7.6, and 4.26 fold higher inhibition potential than parental counterparts. Importantly, recombinant AAI chimera retained specificity towards target coleopteran α-amylases. In addition, to improve the inhibitory potential of AAI, we performed in silico site-saturation mutagenesis. Computational analysis followed by experimental data showed that substituting Asparagine at the 6th position with Methionine had a remarkable increase in the specific inhibition potential of Amaranthus hypochondriacus α-AI. These results provide structural–functional insights into the vitality of AAI propeptide and a potential hotspot for mutagenesis to enhance the AAI activity. Our investigation will be a toolkit for AAI’s optimization and functional differentiation for future biotechnological applications.

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区域洗牌和位点饱和诱变增强苋科α-淀粉酶抑制剂的抑制潜力
苋科α-淀粉酶抑制剂(AAIs)是一种对鞘翅目α-淀粉酶具有选择性抑制潜力的结蛋白型蛋白。它们的小尺寸和显著的稳定性使它们成为蛋白质工程中令人兴奋的分子,以实现优越的选择性和功效。在本报告中,我们设计了一套AAI前肽和成熟肽嵌合体。基于硅分析,选择与目标淀粉酶亲和力较强的稳定AAI嵌合体进行表征。体外实验证实,藜麦前肽α-AI与甜菜成熟肽α-AI嵌合体的抑制潜能分别比亲本高3倍、7.6倍和4.26倍。重要的是,重组AAI嵌合体保留了对目标鞘翅目α-淀粉酶的特异性。此外,为了提高AAI的抑制潜能,我们进行了硅位点饱和诱变。计算分析和实验数据表明,用蛋氨酸取代第6位的天冬酰胺可显著提高苋α-AI的特异性抑制电位。这些结果为了解AAI前肽的活性提供了结构-功能方面的见解,并为增强AAI活性提供了潜在的诱变热点。我们的研究将成为未来生物技术应用中AAI优化和功能分化的工具包。
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来源期刊
The Protein Journal
The Protein Journal 生物-生化与分子生物学
CiteScore
5.20
自引率
0.00%
发文量
57
审稿时长
12 months
期刊介绍: The Protein Journal (formerly the Journal of Protein Chemistry) publishes original research work on all aspects of proteins and peptides. These include studies concerned with covalent or three-dimensional structure determination (X-ray, NMR, cryoEM, EPR/ESR, optical methods, etc.), computational aspects of protein structure and function, protein folding and misfolding, assembly, genetics, evolution, proteomics, molecular biology, protein engineering, protein nanotechnology, protein purification and analysis and peptide synthesis, as well as the elucidation and interpretation of the molecular bases of biological activities of proteins and peptides. We accept original research papers, reviews, mini-reviews, hypotheses, opinion papers, and letters to the editor.
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