A plant-seed inhibitor of two classes of α-amylases: X-ray analysis ofTenebrio molitorlarvae α-amylase in complex with the beanPhaseolus vulgarisinhibitor

Abstract

The alpha-amylase from Tenebrio molitor larvae (TMA) has been crystallized in complex with the alpha-amylase inhibitor (alpha-AI) from the bean Phaseolus vulgaris. A molecular-replacement solution of the structure was obtained using the refined pig pancreatic alpha-amylase (PPA) and alpha-AI atomic coordinates as starting models. The structural analysis showed that although TMA has the typical structure common to alpha-amylases, large deviations from the mammalian alpha-amylase models occur in the loops. Despite these differences in the interacting loops, the bean inhibitor is still able to inhibit both the insect and mammalian alpha-amylase.

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

“…In contrast, in the modeled complex with α-AI2, charge compensation is provided in the form of His33 (Figure 3). These results are consistent with the inhibition of TMA (but not ZSA) by α-AI1 and ZSA (but not TMA) by α-AI2 (Suzuki et al, 1993;Nahoum et al, 1999).…”

“…In the cases of the ZSA complexes, the present models should be significantly more accurate than those previously analysed since they were based on the TMA-α-AI1 (Nahoum et al, 1999) Figure 2. Activities of α-AI1and α-AI2(a), extracted from Phaseolus vulgaris, α-AI2(b) and mutants, extracted from transformed tobacco seeds, in relation to ZSA and PPA.…”

“…Since the patterns of inhibition of M2-4 were identical to that of M1, these other mutants were not studied further. The structure of PPA in complex with α-AI1, PDB code 1DHK (Bompard-Gilles et al, 1996) was used to construct the PPA-α-AI2 and PPA-M1 model complexes while the structure of TMA in complex with α-AI1, PDB code 1VIW (Nahoum et al, 1999) was used for modeling ZSA-α-AI2 and ZSA-M1. Alignments were adjusted so that insertions and deletions in the model proteins relative to the templates could be smoothly accommodated.…”

“…Research was further stimulated after the crystal structures of α-AI1 complexed with PPA (BompardGilles et al, 1996), Tenebrio molitor α-amylase (TMA) alone (Strobl et al, 1998), and in complex with α-AI1 (Nahoum et al, 1999), were determined.…”

α-Amylase Inhibition: Some Practical Considerations

“…Seven types of proteinaceous -amylase inhibitors are found in nature as defined by similarities in sequence and three-dimensional structures [4][5][6][7]. In addition, artificial inhibitors (or designer inhibitors) were recently obtained by *Address correspondence to this author at the Department of Biochemistry, Faculty of Science, Banaras Hindu University, Varanasi-221005, U.P., India; Tel: +91 0542 6702589; Fax: +91 0542 2368174; E-mail: nive_june29@yahoo.co.in applying an in vitro evolution strategy [8,9] ( Table 1).…”