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

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.

Molecular determinant for specificity: Differential interaction of α-amylases with their proteinaceous inhibitors.

BACKGROUND: α-Amylase inhibitors (α-AIs) belong to the discrete classes, and exhibited differential specificities against α-amylases from various sources. Several α-amylases and their complexes with inhibitors at the molecular level have been studied in detail. Interestingly, some α-AIs depict specific and selective interactions amid different insect α-amylases. SCOPE OF REVIEW: There are studies to understand evolutionary variability and functional differentiation of insect α-amylases and their cognate inhibitors. We have examined sequence, structural, and interaction diversity between various α-amylases and α-AIs. Based on these analyses, we are providing a potential basis for the functional differentiation among certain insect α-amylases concerning mammalian counterparts and their interactions with different proteinaceous α-AIs. MAJOR CONCLUSIONS: Insect α-amylases have conserved domain architecture with differences in length, number of disulfide bonds, and secondary structure. Furthermore, few of them exhibit variable characteristics like chloride dependent activity, the presence of N-terminal glutamine residue to protect against proteolytic degradation, and loop variations near the enzyme active site. Conformation of α-AI protein could be an essential factor for their specificity and binding affinities towards target α-amylase(s). Furthermore, variation into the enzyme binding pocket residues might contribute to differential interactions with inhibitors. GENERAL SIGNIFICANCE: Molecular insights in the interactions between insect α-amylases and plant α-AI will provide the details of mechanisms assisting the inhibitor specificity. Furthermore, this information will help to design potent and effective α-AIs against specific α-amylase.

Molecular investigation of Coleopteran specific α-Amylase inhibitors from Amaranthaceae members.

α-Amylase inhibitors (α-AIs) target α-amylases and interfere with the carbohydrate digestion of insects. Among different classes of α-AIs, a knottin-type inhibitor from Amaranthus hypochondriacus (AhAI) was found to be specific against coleopteran storage pests. In this report, we have characterized three previously unidentified knottin-type α-AIs from various Amaranthaceae plants namely, Amaranthus hypochondriacus (AhAI2), Alternanthera sessilis (AsAI) and Chenopodium quinoa (CqAI). They contain a signal peptide, pro-peptide, and mature peptide. The mature peptides of the new α-AIs shared 68 to 78% identity with AhAI and have highly variable pro-peptide regions. Along with the cystine-knot fold, they showed conservation of reactive site residues. All recombinant α-AIs were successfully expressed in their active form and native state using an oxidative cytoplasmic environment. Inhibition studies against various amylases revealed that these inhibitors showed selective inhibition of coleopteran recombinant insect α-amylases viz., Tribolium castaneum, and Callosobruchus chinensis. Tribolium castaneum α-amylase inhibition potency was highest for AhAI2 (Ki ~ 15 µM) followed by AsAI (Ki ~ 43 µM) and CqAI (Ki ~ 61 µM). Interaction analysis of these inhibitors illustrated that the reactive site of inhibitors make several non-covalent interactions with the substrate-binding pocket of coleopteran α-amylases. The selectivity of these inhibitors against coleopteran α-amylases highlights their potential in storage grain pest control.