Protein structures of common bean (Phaseolus vulgaris) α-amylase inhibitors

Two nucleotide sequences for genes that encode alpha-amylase inhibitor 4 (alphaAl-4) from white kidney bean (WKB) cv. 858, designated gene alphaAl-4 (Accession No. U84390), and alpha-amylase inhibitor 5 (alphaAl-5) from black bean (BB), designated gene alphaAl-5 (Accession No. U84391), were determined. Genes alphaAl-4 and alphaAl-5 encode 244 amino acid prepro-alphaAl-4 and prepro-alphaAl-5 polypeptides that are 93 and 95% identical with alpha-amylase inhibitor I (alpha-Al-1; Hoffman, L. M.; Ma, Y.; Barker, R, F. Nucleic Acids Res. 1982, 10, 7819-7828), 40 and 43% identical with red kidney bean lectin, and 52 and 55% identical with arcelin I of wild-type bean. The high degree of sequence similarity indicates the evolutionary relationship among these genes, PCR analysis of genomic DNA purified from six genotypes of Phaseolus vulgaris showed very similar band patterns in 2% agarose gel, another indication of the conserved size homology among these genes. Proteolytic processing sites were located between Asn77 and Ser78 for pro-alphaAl-4 and pro-alphaAl-5. A bend next to Asn77 in three-dimensional model structures of alphaAl-4 and alphaAl-5 proinhibitors indicates that the proteolytic cleavage is necessary to remove the conformational constraint for activation to the mature protein. Mature WKB alphaAl-4 was composed of four subunits (2alpha2beta) and had a molecular weight of 50000 determined by multiangle laser light scattering and 56714 determined by laser-assisted time-of-flight mass spectrometry.