Dynamic gating of substrate binding in β‐amylase2 from Arabidopsis thaliana

Abstract

β‐Amylases (BAMs) in plants are hydrolases involved in starch degradation, which is important for providing sugar when current photosynthate is unavailable. Plant genomes contain multiple BAM genes some of which have unknown functions. BAM2 in Arabidopsis thaliana is an unusual b‐amylase in that it is tetrameric with sigmoidal kinetics. Other catalytically active BAMs are monomeric with hyperbolic kinetics. A deep groove formed between BAM2 dimers forms a secondary binding site (SBS) for starch and this binding is necessary for activity. To investigate the nature of this catalytic mechanism we aligned BAM2 orthologs with other BAMs and noticed several regions that are uniquely conserved. One of these regions, a surface loop between the active site and the SBS, which we refer to as the HED loop, is three‐amino acids longer in BAM2 than in other BAMs. The loop is proximal to the conserved substrate binding sites suggesting a role for these amino acids in substrate binding. When this loop is shortened, the KM for starch is about 2‐fold lower with a sigmoidal kinetics curve. Using molecular docking, we compared the affinity of WT and mutated BAM2 models for maltose and maltodextrin substrates and found that the mutant shows higher affinities. A second unique site is a Ser residue adjacent to a catalytic Glu that when mutated to Gly, the residue conserved in typical BAMs, results in a 7.5‐fold lower KM for starch. Simulation of the dynamics of BAM2 with the Ser to Gly substitution shows increased tendency for the HED loop to be away from the active site. These data suggest the presence of an active site gate in BAM2 which controls substrate binding and is unique to BAM2.