✍ Scribed by Peter K. Nielsen; Birgit C. Bønsager; Kenji Fukuda; Birte Svensson
No coin nor oath required. For personal study only.
Bifunctional a-amylase/subtilisin inhibitors have been implicated in plant defence and regulation of endogenous a-amylase action. The barley a-amylase/subtilisin inhibitor (BASI) inhibits the barley a-amylase 2 (AMY2) and subtilisin-type serine proteases. BASI belongs to the Kunitz-type trypsin inhibitor family of the h-trefoil fold proteins. Diverse approaches including site-directed mutagenesis, hybrid constructions, and crystallography have been used to characterise the structures and contact residues in the AMY2/BASI complex. The threedimensional structure of the AMY2/BASI complex is characterised by a completely hydrated Ca 2 + situated at the protein interface that connects the three catalytic carboxyl groups in AMY2 with side chains in BASI via water molecules. Using surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC), we have recently demonstrated Ca 2 + -modulated kinetics of the AMY2/BASI interaction and found that the complex formation involves minimal structural changes. The modulation of the interaction by calcium ions makes it unique among the currently known binding mechanisms of proteinaceous a-amylase inhibitors.
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We have cloned and sequenced a full-length cDNA from barley (Hordeum vulgate L.) seeds encoding the bifunctional ~-amylase/subtilisin inhibitor (BASI). The nucleotide sequence predicts an open reading frame coding for a protein of 203 amino acids. The first 22 amino acids exhibit the sequence charac