Plant inhibitors challenge insect digestion

Researchers are exploring the potential of plant α-amylase inhibitors for biotechnological pest control. These proteins, found in seeds and plant tissues, interfere with insects' starch digestion, reducing their ability to feed and develop. A study by an international consortium led by Embrapa detailed the molecular mechanisms of these compounds and identified their potential applications.

Phytophagous insects use α-amylase enzymes to degrade starch and glycogen present in plants. These enzymes belong to the GH13 family and act in the midgut or foregut, and in some cases also in saliva.

Alpha-amylase activity varies depending on the insect's pH and life stage. In many cases, the number of genes encoding the enzyme reflects dietary adaptations and co-evolution with host plants.

Attacks by pests such as weevils, borers, and stink bugs cause significant losses to grains and seeds. Plant-produced alpha-amylase inhibitors act by blocking these enzymes. The interaction occurs primarily through ionic and hydrogen bonds, directly affecting the enzyme's catalytic site or starch-binding subsites.

Classes of plant inhibitors

The study classified the inhibitors into seven families based on three-dimensional structure and function:

• Knottins (ICK): small, extremely stable proteins. Example: AAI from amaranth.

• gamma-Thionins: plant defensins with strong antienzymatic activity and toxicity against insects.

• CM-Proteins: common inhibitors in cereals, form stable structures with several disulfide bonds.

• Kunitz-type: bifunctional proteins that inhibit alpha-amylases and proteases in mammals and insects.

• Thaumatin-like: inhibitors with antifungal and entomotoxic activity, such as zeamatin from corn.

• Lectin-like: inhibitors such as alphaAI-1 from common bean, which block enzymes by mimicry of the substrate.

• Actinobacteria (Streptomyces): although they are not plants, they produce similar compounds, with potential application in biotechnology.

Molecular interaction

The effectiveness of inhibitors depends on the structural compatibility between the plant protein and the insect enzyme.

Small variations in the binding amino acids, structural loops, and degree of glycosylation determine the strength of inhibition. The inhibitor alphaAI-1, for example, efficiently blocks alpha-amylases from Callosobruchus chinensis e C. maculatus, but does not affect Zabrotes subfasciatus. AlphaAI-2 inhibits the latter species, but has low efficacy against the former.

Furthermore, factors such as pH, temperature, relative concentration between enzyme and inhibitor and exposure time influence the stability and efficiency of the complex formed.

Applications in plants

Genes encoding these proteins are being tested in genetically modified plants. Trials with beans and peas have shown significant increases in pest resistance. In some cases, larval mortality occurred in the early stages of development.

The researchers warn of the importance of correct gene selection, considering the target pest, protein stability, and the possibility of coevolution. They also emphasize the need to avoid antinutritional effects on humans and animals, common with some inhibitors.

Evolutionary conservation

Phylogenetic analysis revealed that functional domains are highly conserved within each inhibitor family. This suggests strong selective pressure for pest control efficiency. Despite this conservation, the inhibitors exhibit different specificities, allowing targeted applications depending on the crop and insect.

Modeling with AlphaFold2 shows strong structural similarity between plant inhibitors and their enzyme targets. These representations help predict interactions and guide the design of new, more effective proteins.

Further information at doi.org/10.1002/biot.70098