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

• Published in: The protein journal 2023-10, Vol.42 (5), p.519
• Main Authors: Rane, Ashwini S, Nair, Vineetkumar S, Joshi, Rakesh S, Giri, Ashok P
• Format: Article
• Language: English
• Subjects: Amino acids; Amylases; Proteins; Quinoa
• ISSN: 1572-3887
• DOI: 10.1007/s10930-023-10148-y

Amaranthaceae [alpha]-amylase inhibitors (AAIs) are knottin-type proteins with selective inhibitory potential against coleopteran [alpha]-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 [alpha]-AI and mature peptide from Beta vulgaris [alpha]-AI possess 3, 7.6, and 4.26 fold higher inhibition potential than parental counterparts. Importantly, recombinant AAI chimera retained specificity towards target coleopteran [alpha]-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 [alpha]-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.