Quaternary Structure, Salt Sensitivity, and Allosteric Regulation of β-AMYLASE2 From Arabidopsis thaliana

Abstract

The β-amylase family in Arabidopsis thaliana has nine members, four of which are both plastid-localized and, based on active-site sequence conservation, potentially capable of hydrolyzing starch to maltose. We recently reported that one of these enzymes, BAM2, is catalytically active in the presence of physiological levels of KCl, exhibits sigmoidal kinetics with a Hill coefficient of over 3, is tetrameric, has a putative secondary binding site (SBS) for starch, and is highly co-expressed with other starch metabolizing enzymes. Here we generated a tetrameric homology model of Arabidopsis BAM2 that is a dimer of dimers in which the putative SBSs of two subunits form a deep groove between the subunits. To validate this model and identify key residues, we generated a series of mutations and characterized the purified proteins. (1) Three point mutations in the putative subunit interfaces disrupted tetramerization; two that interfered with the formation of the starch-binding groove were largely inactive, whereas a third mutation prevented pairs of dimers from forming and was active. (2) The model revealed that a 30-residue N-terminal acidic region, not found in other BAMs, appears to form part of the putative starch-binding groove. A mutant lacking this acidic region was active and did not require KCl for activity. (3) A conserved tryptophan residue in the SBS is necessary for activation and may form π-bonds with sugars in starch. (4) Sequence alignments revealed a conserved serine residue next to one of the catalytic glutamic acid residues, that is a conserved glycine in all other active BAMs. The serine side chain points away from the active site and toward the putative starch-binding groove. Mutating the serine in BAM2 to a glycine resulted in an enzyme with a VMax similar to that of the wild type enzyme but with a 7.5-fold lower KM for soluble starch. Interestingly, the mutant no longer exhibited sigmoidal kinetics, suggesting that allosteric communication between the putative SBS and the active site was disrupted. These results confirm the unusual structure and function of this widespread enzyme, and suggest that our understanding of starch degradation in plants is incomplete.

Protein Mediated Enzyme Immobilization

“…Because enzyme function and loading capacity during immobilization can be impacted by the oligomeric state of the protein, it is important to consider the quaternary structure of the enzyme of interest. [ 202 ] Many materials discussed in this work are based on the oligomeric state of the protein, such as protein scaffolds, phase‐separated proteins, and all‐enzyme hydrogels. Oligomeric structure is a dynamic thermodynamic state and is sensitive to the chemical environment, such as concentration of ions, temperature, pH, and presence of organic solvent.…”

Looking to Move Away from Expository General Chemistry Laboratories? We May Have a Cure for What “Ales” You

“…The project was proposed to the GCL curriculum committee by Dr. Berndsen, a biochemist who did not regularly teach in the GCL1 and 2 sequence. This project is based on Dr. Berndsen’s grant-funded research interests in enzymatic starch degradation. − The initial conversations focused on identifying general chemistry topics related to bioplastics, such as the role of intermolecular forces in plastic formation and spectroscopic characterization. From there, a general set of experiments was outlined.…”

Structure of maize BZR1-type β-amylase BAM8 provides new insights into its noncatalytic adaptation

“…Most BAMs are well characterized in Arabidopsis (At), which is reported to have nine members of BAMs (BAM1-9) (Monroe et al, 2017) with conserved catalytic domains and variable N-terminal regions involved in localization. Among nine distinctive members of AtBAMs, AtBAM1/2/3/5 encode catalytically active enzymes (Monroe et al, 2018;Monroe et al, 2017) whereas the other three AtBAM4/7/8 are catalytically inactive. The sequence conservation analysis of these nine BAMs indicates that none of them share more than 60 % homology, suggesting that they have not evolved recently through gene duplication events (Fulton et al, 2008).…”