Abstract
Chloroplast-containing species possess 2 α-(1→6)-glucosidases that share a common ancestor but were independently acquired by horizontal gene transfer from separate eubacterial donors. The pullulanase-type enzyme (CAZy subfamily GH13_13) and the isoamylase-type enzyme (CAZy subfamily GH13_11) both hydrolyze branch linkages in α-polyglucans. Thus, both enzyme types function as debranching enzymes (DBE) in starch metabolism. As both enzyme types are conserved, distinct selectable functions are expected. This study describes the functional interactions between maize (Zea mays L.) pullulanase1 (ZPU1) and the isoamylase-type enzyme complex comprising the paralogous proteins isoamylase1 (ISA1) and isoamylase2 (ISA2). Mutation of ISA1 or ISA2 caused reduced ZPU1 activity in developing endosperm extracts, and the addition of ISA1 to ZPU1-expressing yeast (Saccharomyces cerevisiae) cells caused increased ZPU1 activity. Specific amino acid substitutions in ISA1 resulted in altered ZPU1 mobility in SDS-PAGE. In vivo protein-protein interaction tests and co-immunoprecipitation revealed that ZPU1 and ISA1 interact in multi-subunit complexes. Maize lines harboring ISA1 mutations, exhibiting a classical low-starch, high-phytoglycogen-accumulation phenotype, were altered by recurrent selection so that kernel appearance reverted to near normal. Extragenic suppression indicated the requirement for ISA1/ISA2 activity had been bypassed. These results are consistent with a functional overlap between the GH13_11 and GH13_13 DBE types and raise the possibility that multiple GH13 proteins, namely ZPU1, ISA1 and ISA2, act together to physically coordinate their hydrolytic activities on precursor α-polyglucans.