We are interested in understanding the function of various members of the β-amylase (BAM) gene family in plants by investigating their structure and biochemical properties, and the phenotypes of mutant plants in which one or more of the BAM genes is defective. For a review of the gene family in Arabidopsis, see: Monroe and Storm, 2018. The model system we use is Arabidopsis thaliana which contains nine BAM genes. Using the many sequenced plant genomes, we can also trace the evolutionary history of the gene family, noting when new forms evolved and how their functions might have changed over time. We are very fortunate to have joined forces on this project with Dr. Chris Berndsen in the Chemistry and Biochemistry Department at JMU, who adds his expertise in biochemistry and structural biology to our emphasis on plant physiology and molecular biology.
Our work has been generously funded by the National Science Foundation.
Highlights
A quintuple mutant lacking all five of the catalytically active BAMs (BAM1, -2, -3, -5, and -6) that we call B-Null, grows slowly, accumulates starch, and importantly, has no detectable β-amylase activity (Monroe 2020).
BAM2 is unique among plant BAM proteins in requiring K+ for activity, being a tetramer, and having sigmoidal kinetics (Monroe et al., 2017; 2018; Chandrasekharan et al., 2020). Catalytically active dimers containing a secondary starch binding site that is required for activity (Monroe et al., 2017; 2018). Our analysis of BAM orthologs and intron positions among BAM genes indicates that BAM2 and BAM1/3 existed in the first land plants and probably gave rise to all of the others by duplication events (Monroe et al., 2017).
A conserved serine residue in BAM2, adjacent to one of the catalytic residues, is a conserved glycine in all of the other catalytically active BAMs. BAM2 containing a S464G mutation has the same Vmax as the WT BAM2 enzyme but it has hyperbolic kinetics suggesting that the mutation eliminates the allosteric activation by starch binding in the groove (Monroe et al., 2018). The physiological function of BAM2 is not yet known and mutant plants lacking BAM2 appear to be normal.
After 4 days of 4°C cold stress, BAM3 transcript in leaves is elevated, but the BAM3 activity is much lower than in control plants (Monroe et al, 2014; Monroe 2020). Purified BAM3 is highly sensitive to glutathionylation at C433 (Storm et al., 2018), but it is not yet known if this mechanism operates in vivo to lower the activity in cold-stressed plants.
BAM3 and BAM1 are catalytically active BAMs that are thought to function during the night (BAM3) and day (BAM1). Transcription of BAM3 is induced by cold stress, and BAM1 is induced by heat and osmotic stress. We discovered that the optimal temperature for BAM3 activity is about 10°C lower than that of BAM1, and that differences in the amino acid composition of the two enzymes resembles the differences seen in thermophilic and psychrophilic enzymes generally (Monroe et al, 2014).
BAM7 and BAM8 have in addition to their BAM domains, an N-terminal BZR1-like domain that binds DNA and contains a nuclear localization signal directing the proteins to the nucleus (Reinhold et al. 2011). These catalytically inactive proteins act as transcription factors regulating the expression of other genes, probably dependent on levels of unknown sugars that bind to the “active” site.