Functional characterization of arabidopsis α-amylase3 (AMY3): amylose preference and structural insights into its duplex carbohydrate-binding module

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, the function of AMY3 remains largely unknown, although dimerization is thought to regulate its activity. We observed that purified AMY3 decreased the amylose content of corn starch relative to amylopectin in vitro, suggesting a role for this enzyme in amylose metabolism. Enzyme assays using individual starch components revealed that AMY3 has a higher affinity for amylose compared to amylopectin. Furthermore, we found that the starch in leaves from a mutant lacking AMY3 had twice as much amylose as WT leaves at the end of the day. To gain a deeper understanding of this preference, we purified and characterized the structure and function of the N-terminal carbohydrate-binding modules. Through bioinformatic analysis, we found that the organization and overall structure of the AMY3 CBMs was distinct from that of the CBMs in GWD1, another CBM45 member; however, the carbohydrate-binding site is predicted to be structurally conserved. Further, we found that the AMY3 CBMs form a unique duplex in solution, which is unusual for carbohydrate-binding modules, and that the CBMs can bind both amylose and amylopectin. Interestingly, AMY3 without the CBMs still prefers amylose, indicating that the duplex CBM is not essential for amylose preference in vitro; but the CBM is important for AMY3 dimerization and regulation. Additionally, our data also show the importance of using structural comparisons rather than sequence alignments when assigning CBM family designations.