Starch is a key energy-storage molecule in plants that requires controlled synthesis and breakdown for effective plant growth. -Amylases (BAMs) hydrolyze starch into maltose to help to meet the metabolic needs of the plant. In the model plant Arabidopsis thaliana there are nine BAMs, which have apparently distinct functional and domain structures, although the functions of only a few of the BAMs are known and there are no 3D structures of BAMs from this organism. Recently, AtBAM2 was proposed to form a tetramer based on chromatography and activity assays of mutants; however, there was no direct observation of this tetramer. Here, small-angle X-ray scattering data were collected from AtBAM2 and its N-terminal truncations to describe the structure and assembly of the tetramer. Comparison of the scattering of the AtBAM2 tetramer with data collected from sweet potato (Ipomoea batatas) BAM5, which is also reported to form a tetramer, showed there were differences in the overall assembly. Analysis of the N-terminal truncations of AtBAM2 identified a loop sequence found only in BAM2 orthologs that appears to be critical for AtBAM2 tetramer assembly as well as for activity.

Starch is a key energy‐storage molecule in plants that requires controlled synthesis and breakdown for effective plant growth. β‐Amylases (BAMs) hydrolyze starch into maltose to help to meet the metabolic needs of the plant. In the model plant Arabidopsis thaliana there are nine BAMs, which have apparently distinct functional and domain structures, although the functions of only a few of the BAMs are known and there are no 3D structures of BAMs from this organism. Recently, AtBAM2 was proposed to form a tetramer based on chromatography and activity assays of mutants; however, there was no direct observation of this tetramer. Here, small‐angle X‐ray scattering data were collected from AtBAM2 and its N‐terminal truncations to describe the structure and assembly of the tetramer. Comparison of the scattering of the AtBAM2 tetramer with data collected from sweet potato (Ipomoea batatas) BAM5, which is also reported to form a tetramer, showed there were differences in the overall assembly. Analysis of the N‐terminal truncations of AtBAM2 identified a loop sequence found only in BAM2 orthologs that appears to be critical for AtBAM2 tetramer assembly as well as for activity.

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The β‐amylase (BAM) family in Arabidopsis thaliana has nine members, some of which are known to hydrolyze starch to maltose. The members of this family show unique functional, regulatory, and catalytic behaviors due to changes in sequence or domain composition. However, the molecular basis for functional specificity and regulation is not always clear. Some of the inactive members of the lack the catalytic amino acids required for hydrolysis. However, this is not the case for all the enzymes such as BAM2, which appears to be allosterically regulated and requires KCl for activity. Given the lack of structural information on any member of this protein family, we homology modeled all 9 BAM proteins from Arabidopsis and performed molecular dynamics simulations to propose how these proteins may differ at a functional level. All simulations were equilibrated for at least 50 ns in explicit solvent, the equilibrated models were aligned structurally, and then analyzed to compare the structure, dynamics, and chemical properties. All 9 proteins modeled well as TIM barrel proteins with most of the structural deviations occurring in predicted loops. Comparison of the simulated dynamics shows that overall the BAM proteins show similar areas of high and low motion. Interestingly, BAM4 and BAM9 showed fluctuation profiles that were distinct from the other BAM proteins, which matches their predicted lack of catalytic activity and distinct active site sequences. BAM7 and BAM8 which also have distinct active sites however these enzymes show dynamics similar to those of the catalytically active BAM1 and BAM3 suggesting that BAM7 and BAM8 may also have activity, however the proper conditions are not known. We further modeled BAM2 as a tetramer based on recent biochemical information to identify how allostery and solution conditions may influence the activity of this protein. We identified predicted changes in the dynamics upon binding to a ligand in the starch‐binding site as well as changes in hydrogen bonding within the protein in potassium. Both findings provide testable proposals for the regulation of BAM2 activity.Support or Funding InformationThis work was supported by a National Science Foundation Research at Undergraduate Institutions Grant MCB‐1616467 and National Science Foundation Research Experience for Undergraduates Grant CHE‐1461175This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Starch is an important compound in the lives of both plants and humans, with roles in human nutrition and industry such as paper making. β‐amylases are a class of proteins that break down starch in plants to produce maltose. My focus of study is β‐amylase2 (BAM2); one of the β‐amylase genes Arabidopsis thaliana. While the physiological function of BAM2 is unclear, we know three key things about its properties: 1) BAM2 has a tetrameric structure, 2) the enzyme exhibits sigmoidal kinetics through secondary binding sites, and 3) potassium salts are required for catalytic activity. We investigated the molecular basis for potassium stimulating activity in BAM2. Using molecular dynamics, we simulated the effect of potassium on BAM2 finding that potassium increased the flexibility of the enzyme. We are purifying BAM2 wild type and BAM2 with point substitutions which we propose alter the effect of potassium activity. Using these enzyme variants, we compared the structural stability of the enzymes in the presence of potassium, lithium, and sodium using Differential Scanning Fluorimetry (DSF) and Circular Dichroism (CD). Results from DSF showed that the melting temperature decreased with increasing salt concentration, which we interpret as an increase in structural flexibility of BAM2. The CD results support this trend as the melting temperature for BAM2 in the presence of lithium is higher than that of BAM2 in the presence of potassium. These data support a mechanism where potassium ions enhance the flexibility of BAM2.