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Date of Award
Bachelor of Science (BS)
Department of Biology
Jonathan D. Monroe
Plants store starch during the day for use at night. This process of transitory starch degradation is mostly attributable to the β-Amylase (BAM) family, which are starch exohydrolases that cleave the penultimate α-1,4 glycosidic bonds of starch to release maltose. BAM2 was recently characterized as a catalytically active, K+-requiring tetramer with sigmoidal kinetics and cooperativity. All other catalytically active BAMs display Michaelis-Menten kinetics, no cooperativity, and do not require salt, making BAM2’s characteristics intriguing. Due to a lack of a crystal structure, a monomeric homology model of BAM2 was generated using I-TASSER based on a BAM5 from soybean. The monomer model was then used to make a tetramer model based on the homotetrameric crystal structure of sweet potato BAM5 with YASARA. The tetramer model was tested through site directed mutagenesis of conserved residues that were hypothesized to form interfaces between subunits. Two residues, D490 and W456, are perfectly conserved and located in one interface of the tetramer model. Residue F238 is a perfectly conserved residue that would be in another interface of the tetramer model if the model were slightly shifted and more compact. Site directed mutagenesis was used to swap these residues with a different amino acid, and the mutant proteins were purified for use in enzyme activity assays and size analysis by Size Exclusion Chromatography-Multi Angle Light Scattering. BAM2 D490R and BAM2 W456A disrupted tetramerization and catalytic activity. The F238A mutant is dimeric and maintains catalytic activity resembling BAM2 WT, but with a higher Km. The results of these mutagenesis experiments revealed that activity of BAM2, unlike other active BAMs, is dependent on its quaternary structure and that the starch-binding groove must be held together with residues in interface A for BAM2 to be active.
Pope, Lauren Elizabeth, "Tetrameric structure of beta-amylase 2 (BAM2) in Arabidopsis thaliana" (2017). Senior Honors Projects, 2010-current. 506.
Available for download on Saturday, November 30, 2019