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Starch accumulates in the plastids of green plant tissue during the day to provide carbon for metabolism at night. Starch hydrolysis is catalyzed by members of the β-amylase (BAM) family, which in Arabidopsis thaliana (At), includes nine structurally and functionally diverse members. One of these enzymes, AtBAM2, is a plastid-localized enzyme that is unique among characterized β-amylases since it is tetrameric and exhibits sigmoidal kinetics. Sequence alignments show that the BAM domains of AtBAM7, a catalytically inactive, nuclear-localized transcription factor with an N-terminal DNA binding domain, and AtBAM2 are more closely related to each other than they are to any other AtBAM. Since BAM2 is found in more ancient lineages, it was hypothesized that BAM7 evolved from BAM2. However, analysis of the genomes of 48 flowering plants revealed 12 species that appear to have a BAM7 gene but lack a BAM2 gene. Upon closer inspection, these BAM7 proteins have a greater percent identity to AtBAM2 than to AtBAM7, and they share all of the AtBAM2 functional residues that BAM7 proteins normally lack. We hypothesize that these genes may encode a BAM2-like protein although they are currently annotated as BAM7-like genes. To test this hypothesis, we designed a cDNA of the short form of corn BAM7 (ZmBAM7-S) for expression in E. coli. Small Angle X-Ray Scattering data indicate that ZmBAM7-S has a tetrameric solution structure more similar to that of AtBAM2 than AtBAM1. In addition, partially purified ZmBAM7-S is catalytically active and exhibits sigmoidal kinetics. Together these data suggest that some BAM7 genes may encode a functional BAM2. Exploring and understanding β-amylase gene structure could have impacts on the current annotation of genes. |
