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Mutants of common bean alpha-amylase inhibitor-2 as an approach to investigate binding specificity to alpha-amylases.

By M.C.M. da SILVA, L.V. MELLO, M.V. COUTINHO, D.J. RIGDEN, G. NESHICH, M.J. CHRISPEELS and M.F. GROSSI-DE-SÁ

Abstract

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.200

Topics: Phaseolus vulgaris, a-amylase inhibitors, inhibitor specificity, site directed mutagenesis, structural modeling, inibidores de a-amilases, especificidade de interação, mutagênese sítio-dirigida, modelagem molecular
Publisher: Pesquisa Agropecuária Brasileira, Brasília, DF, v. 39, n. 3, p. 201-208, mar. 2004.
Year: 2004
OAI identifier: oai:www.alice.cnptia.embrapa.br:doc/110087
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