Location of the active site of the bean α-amylase inhibitor and involvement of a Trp, Arg, Tyr triad

Mirkov, T. Erik; Evans, Stephen V.; Wahlstrom, Jan L.; Gomez, Lizabeth; Young, Neville; Chrispeels, M. J.

doi:10.1093/glycob/5.1.45

Cited by 39 publications

(30 citation statements)

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“…Phaseolus coccineus (cv Tecomari) and Phaseolus acutifolius (cv White) were obtained from Native Seeds/ Search (Tucson, AZ). Tobacco seeds were obtained from plants transformed with the aAI-1 gene or a mutant aAI-1 (WSY188-190GNV) gene as described (Mirkov et al, 1995).…”

Section: Materials a N D Methods Lnsects And Seedsmentioning

confidence: 99%

Protective Mechanism of the Mexican Bean Weevil against High Levels of α--Amylase Inhibitor in the Common Bean

1996

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~The seeds of starchy grain legumes are an important staple in many countries, but as with other crops, insect pests can cause considerable crop losses. The postharvest damage done by larvae of bruchids (Co1eoptera:Bruchidae) can be quite extensive. Crops of the common bean (Pkaseolus vulgaris), the scarlet runner bean (Phaseoltis coccineus), and the tepary bean (Phaseolus acutifolius) can be severely damaged by the bean weevil (Acanthoscelides obtectus) and the Mexican bean weevil (Zabrotes subfasciatus) . This damage occurs ín spite of the presence in the seeds of a family of plant defense proteins that comprises PHA and aAI (see Chrispeels and Raikhel, 1991). The three Phaseolus species are not equally sensitive to the bruchids. For example, tepary bean is more resistant than common bean to the bean weevil (Shade et al., 1987).

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Section: Materials a N D Methods Lnsects And Seedsmentioning

confidence: 99%

Protective Mechanism of the Mexican Bean Weevil against High Levels of α--Amylase Inhibitor in the Common Bean

1996

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“…A comparison of the energetic contribution of various contact points of these inhibitors with their targets awaits detailed mutational studies. For instance, it has been reported on the basis of homology modeling that mutating Arg 74 , Trp 188 , and Tyr 190 abolished the inhibitory activity of ␣-AI on PPA (26). Inspection of the complex structure, however, shows that only Tyr 190 is in contact with PPA, implying that the loss of activity observed for the two other residues is probably caused by the perturbation of intramolecular interactions rather than loss of crucial contacts to PPA.…”

Section: Table IV Spr-derived Binding Parameters For Amy2 and Basi Mumentioning

confidence: 99%

“…In this context, the AMY2-BASI system presents a different example of how inhibition can be attained even though no direct bonds between any of the catalytic groups of the enzyme and the inhibitor are present. Although some binding kinetics and mutagenesis were reported for lectin-like (25)(26)(27), cereal-type (28,29), and tendamistat inhibitors (30), thorough analysis is lacking for most systems. An exception is AMY2-BASI, which has been the subject of extensive studies with respect to binding mechanisms and structure/function relationships (2,5,6,9).…”

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confidence: 99%

Mutational Analysis of Target Enzyme Recognition of the β-Trefoil Fold Barley α-Amylase/Subtilisin Inhibitor

Bønsager

Nielsen

Hachem

et al. 2005

Journal of Biological Chemistry

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The barley ␣-amylase/subtilisin inhibitor (BASI) inhibits ␣-amylase 2 (AMY2) with subnanomolar affinity. The contribution of selected side chains of BASI to this high affinity is discerned in this study, and binding to other targets is investigated. Seven BASI residues along the AMY2-BASI interface and four residues in the putative protease-binding loop on the opposite side of the inhibitor were mutated. A total of 15 variants were compared with the wild type by monitoring the ␣-amylase and protease inhibitory activities using Blue Starch and azoalbumin, respectively, and the kinetics of binding to target enzymes by surface plasmon resonance. Generally, the mutations had little effect on k on , whereas the k off values were increased up to 67-fold. The effects on the inhibitory activity, however, were far more pronounced, and the K i values of some mutants on the AMY2-binding side increased 2-3 orders of magnitude, whereas mutations on the other side of the inhibitor had virtually no effect. The mutants K140L, D150N, and E168T lost inhibitory activity, revealing the pivotal role of charge interactions for BASI activity on AMY2. A fully hydrated Ca 2؉ at the AMY2-BASI interface mediates contacts to the catalytic residues of AMY2. Mutations involving residues contacting the solvent ligands of this Ca 2؉ had weaker affinity for AMY2 and reduced sensitivity to the Ca 2؉ modulation of the affinity. These results suggest that the Ca 2؉ and its solvation sphere are integral components of the AMY2-BASI complex, thus illuminating a novel mode of inhibition and a novel role for calcium in relation to glycoside hydrolases.The double-headed barley ␣-amylase/subtilisin inhibitor (BASI) 1 of the Kunitz soybean trypsin inhibitor family acts on proteases of the subtilisin family and the endogenous high pI ␣-amylase (AMY2) but has no effect on the minor isozyme AMY1 that shares 80% sequence identity with AMY2

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“…In the last ten years, several studies of the interaction between α-amylase inhibitors and their target enzymes have been carried out (Pueyo et al, 1993;Mirkov et al, 1995;Grossi-de-Sá et al, 1997;Takahashi et al, 1999). Based on the pea lectin structure (Einspahar et al, 1986) and their own sequence alignments, Mirkov et al (1995) constructed an α-AI1 mutant by replacement of the 188 WSY 190 triplet with GNV producing an inactive inhibitor.…”

Section: Introductionmentioning

confidence: 99%

“…Based on the pea lectin structure (Einspahar et al, 1986) and their own sequence alignments, Mirkov et al (1995) constructed an α-AI1 mutant by replacement of the 188 WSY 190 triplet with GNV producing an inactive inhibitor. The same triplet was considered later by Grossi-de-Sá et al (1997) in substitutions using the corresponding α-AI2 sequence.…”

Section: Introductionmentioning

confidence: 99%

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

Silva

Mello

Coutinho

et al. 2004

Pesq. agropec. bras.

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-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 α-amylase inhibitors, α-AI1 and α-AI2, in P. vulgaris show different specificity toward α-amylases. Zabrotes subfasciatus α-amylase is inhibited by α-AI2 but not by α-AI1. In contrast, porcine α-amylase is inhibited by α-AI1 but not by α-AI2. The objective of this work was to understand the molecular basis of the specificity of two inhibitors in P. vulgaris (α-AI1 and α-AI2) in relation to α-amylases. Mutants of α-AI2 were made and expressed in tobacco plants. The results showed that all the α-AI2 mutant inhibitors lost their activity against the insect α-amylases but none exhibited activity toward the mammalian α-amylase. The replacement of His33 of α-AI2 with the α-AI1-like sequence Ser-Tyr-Asn abolished inhibition of Z. subfasciatus α-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 α-amylase inhibition are not accompanied by gain of inhibitory activity against porcine α-amylase.Index terms: Phaseolus vulgaris, α-amylase inhibitors, inhibitor specificity, site directed mutagenesis, structural modeling. Mutantes do inibidor-2 de alfa-amilase do feijão-comum para investigação da especificidade de ligação a alfa-amilasesResumo -Apesar de possuir uma família de proteínas de defesa, o feijão-comum (Phaseolus vulgaris L.) pode ser atacado por insetos bruquídeos causando sérios danos aos grãos armazenados. O P. vulgaris possui duas formas ativas de inibidores de α-amilases, denominadas α-AI1 e α-AI2, que apresentam diferentes especificidades em relação às α-amilases. A α-amilase de Zabrotes subfasciatus é inibida por α-AI2 mas não por α-AI1. Em contraste, a α-amilase pancreática de porco é inibida por α-AI1 mas não é por α-AI2. O objetivo deste trabalho foi entender as bases moleculares da especificidade desses inibidores em relação às α-amilases. Para tanto, foram construídos mutantes do α-AI2, os quais foram expressados em plantas de fumo. Todos os inibidores mutantes deixaram de inibir a α-amilase de inseto sem, contudo, passar a exibir atividade contra a α-amilase de mamífero. Os modelos estruturais explicam por que a substituição de His33 do α-AI2 pela seqüência correspondente do α-AI1 (Ser-Tyr-Asn) aboliu a inibição da α-amilase de Z. subfasciatus. Dos estudos de modelagem molecular pode-se concluir que o tamanho e a complexidade da interface α-amilase-inibidor explicam por que a mutação da alça N-terminal e a quebra da atividade inibitória para α-amilase de Z. subfasciatus não levam ao ganho de atividade inibitória do mutante em relação à α-amilase de porco.Termos para indexação: Phaseolus vulgaris, inibidores de α-amilases, especificidade de interação, mutagênese sítio-dirigida, modelagem molecular.

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Location of the active site of the bean α-amylase inhibitor and involvement of a Trp, Arg, Tyr triad

Cited by 39 publications

References 0 publications

Protective Mechanism of the Mexican Bean Weevil against High Levels of α--Amylase Inhibitor in the Common Bean

Protective Mechanism of the Mexican Bean Weevil against High Levels of α--Amylase Inhibitor in the Common Bean

Mutational Analysis of Target Enzyme Recognition of the β-Trefoil Fold Barley α-Amylase/Subtilisin Inhibitor

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

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