Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.
Date of Award
Bachelor of Science (BS)
Department of Biology
In the chloroplasts of leaf mesophyll cells, β-amylase proteins (BAMs) are responsible for breaking down starch into maltose when the plant cannot undergo photosynthesis. BAM2, which was previously considered inactive, was recently shown to be active under stromal-like levels of salt and has optimal activity at 80mM KCl. In addition, BAM2 is active as a tetramer in vivo and displays sigmoidal kinetics due to a secondary binding site that is responsible for activating BAM2 when bound to starch. A hypothesized tetramer model was created using a homology model of a BAM2 monomer and the configuration of a crystallized sweet potato BAM5. This model was supported by mutations, made by other members of the lab, in specific interfaces, which disrupted tetramerization and activity. This model along with sequence alignments of BAM2 orthologs revealed a potential acidic domain, containing 2-fold more acidic residues than the catalytic domain and 10-fold more acidic residues than the chloroplast transit peptide. This acidic domain is adjacent to the N-terminal end of the catalytic domain and is just upstream from a short peptide of conserved residues (ERDF). Further, the acidic domain and ERDF peptide are located very close to the starch-binding groove, which is an area of the enzyme where two secondary binding sites face each other. Hypothesizing that this acidic domain and ERDF peptide could interact with KCl and the starch binding groove, we created two mutant BAM2 proteins—one lacking just the acidic domain (B2-NDel1), and one lacking both the acidic domain and the ERDF peptide (B2-NDel2). We hypothesized that by removing the acidic domain and the ERDF peptide, the enzyme would not require salt to function. When the activity of these enzymes were compared to the activity of wild-type BAM2, we found that neither of the mutants required salt to function, while BAM2 had negligible activity without KCl. Additionally, both of these mutants still functioned as a tetramer, even though the activity of B2-NDel2 was about 5-fold lower than NDel1. Together, these data indicate that the acidic domain and ERDF peptide might be of importance to the salt dependency of BAM2.
Breault, Jillian, "An N-terminal acidic domain in β-amylase2 is required for K+ regulation." (2018). Senior Honors Projects, 2010-current. 585.
Available for download on Thursday, April 09, 2020