Abstract
Flowering plants contain a plastid-localized ɑ-amylase (AMY3) with several recognized domains: two N-terminal carbohydrate-binding modules, an alpha-alpha hairpin that binds the catalytically inactive activator β-amylase9, and a C-terminal catalytic domain. Despite considerable research on leaf starch metabolism in Arabidopsis, little is known about the function of AMY3. Starch is composed of two glucose polymers: highly branched amylopectin (70-90%) and largely unbranched amylose (10-30%). Because ɑ-amylases are endo-amylases, we predicted that AMY3 might prefer to hydrolyze amylose, in contrast to the exo-amylase β-amylase1 (BAM1), which we predicted would prefer amylopectin. Using iodine spectroscopy, we observed that AMY3 caused the percent amylose of corn starch to decrease, whereas BAM1 caused the percent amylose to increase before a gradual decrease. Enzyme assays revealed that AMY3 has a higher affinity for amylose compared to amylopectin, whereas BAM1 has a higher affinity for amylopectin. Furthermore, we found that the starch in amy3 leaves had twice as much amylose as WT leaves at the end of the day which suggests potential health and industrial applications. We purified the AMY3 CBM region and studied its structure with small-angle X-ray scattering, seeking to understand its role in substrate specificity. The AMY3 CBMs form a unique duplex where two interlocking β-strands connect CBM1 and CBM2. The duplex CBM forms a dimer in solution and can bind amylose and amylopectin. However, AMY3 without the CBMs still prefers amylose, indicating the duplex CBM is not essential for amylose specificity in vitro. However, the duplex CBM is important for AMY3 dimerization.