No 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.

The β‐amylase family in Arabidopsis thaliana has nine members, four of which; BAM1, BAM2, BAM3 and BAM6, are both plastid‐localized and, based on active‐site sequence conservation, potentially capable of hydrolyzing starch to maltose. BAM1 and BAM3 are known to play roles in leaf starch degradation. BAM6 is poorly understood but may also contribute to leaf starch degradation. In contrast, BAM2 was previously thought to be catalytically inactive. We recently reported that BAM2 is catalytically activity in the presence of KCl and, unlike all other characterized BAMs, exhibits sigmoidal kinetics with a Hill coefficient of over 3. Mapping of conserved residues onto a homology model of BAM2 revealed the presence of a putative secondary binding site (SBS) for starch on one side of the protein. Two point mutations in this putative SBS caused activity to decline by up to 95%. In addition, Multi‐Angle Light Scattering revealed that BAM2 is a tetramer whereas other active BAMs are all monomeric. A tetrameric model of Arabidopsis BAM2 was then generated comprised of a pair of dimers in which the putative SBSs of two subunits form a deep groove between the subunits of each dimer set. To test this model, 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 that prevented pairs of dimers from forming was active but with slightly altered kinetics. 2) The model revealed that N‐terminal acidic domains, not found in other BAMs, appear to form part of the interface containing the putative starch‐binding groove. Several mutations altering or removing the N‐terminal acidic domain did not disrupt the tetramer but did affect the enzyme's requirement for KCl. 3) 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 towards the putative starch‐binding groove. Mutating the serine in BAM2 to a glycine resulted in an enzyme with a Vmax that was similar to that of wild type enzyme but with a 10‐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. Molecular dynamics analysis is underway to attempt to understand these observations. In order to investigate the physiological function of BAM2 in Arabidopsis plants we are characterizing plant mutants with multiple BAM KOs by measuring the changes in BAM activity and starch accumulation associated with the presence/loss of each BAM gene. So far we have observed starch accumulating in older bam2 plants.Support or Funding InformationThis work was supported by an NSF RUI grant to JDMThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.