Author(s): SILVA, M. C. M. da; MELLO, L. V.; COUTINHO, M. V.; RIGDEN, D. J.; NESHICH, G.; CHRISPEELS, M. J.; GROSSI-DE-SÁ, M. F.

Summary: Despite the presence of a family of defense proteins, Phaseolus vulgaris can be attacked by bruchid insects resulting in serious damage to stored grains. The two distinct active forms of a-amylase inhibitors, a-AI1 and a-AI2, in P. vulgaris show different specificity toward a-amylases. Zabrotes subfasciatus a-amylase is inhibited by a-AI2 but not by a-AI1. In contrast, porcine a-amylase is inhibited by a-AI1 but not by a-AI2. The objective of this work was to understand the molecular basis of the specificity of two inhibitors in P. vulgaris (a-AI1 and a-AI2) in relation to a-amylases. Mutants of a-AI2 were made and expressed in tobacco plants. The results showed that all the a-AI2 mutant inhibitors lost their activity against the insect a-amylases but none exhibited activity toward the mammalian a-amylase. The replacement of His33 of a-AI2 with the a-AI1-like sequence Ser-Tyr-Asn abolished inhibition of Z. subfasciatus a-amylase. From structural modeling, the conclusion is that the size and complexity of the amylase-inhibitor interface explain why mutation of the N-terminal loop and resultant abolition of Z. subfasciatus a-amylase inhibition are not accompanied by gain of inhibitory activity against porcine a-amylase.

Publication year: 2004

Types of publication: Journal article

Unit: Embrapa Digital Agriculture

Keywords: Alfa amilase, Especificidade de interação, Inibidor de Enzima, Inibidores de a-amilases, Modelagem molecular, Phaseolus Vulgaris, mutagênese sítio-dirigida