Abstract
The starch debranching enzymes isoamylase 1 and 2 (ISA1 and ISA2) are known to exist in a large complex and are involved in the biosynthesis and crystallization of starch. It is suggested that the function of the complex is to remove misplaced branches of growing amylopectin molecules, which would otherwise prevent the association and crystallization of adjacent linear chains. Here, we investigate the function of ISA1 and ISA2 from starch producing alga Chlamydomonas. Through complementation studies, we confirm that the STA8 locus encodes for ISA2 and sta8 mutants lack the ISA1·ISA2 heteromeric complex. However, mutants retain a functional dimeric ISA1 that is able to partly sustain starch synthesis in vivo. To better characterize ISA1, we have overexpressed and purified ISA1 from Chlamydomonas reinhardtii (CrISA1) and solved the crystal structure to 2.3 Å and in complex with maltoheptaose to 2.4 Å. Analysis of the homodimeric CrISA1 structure reveals a unique elongated structure with monomers connected end-to-end. The crystal complex reveals details about the mechanism of branch binding that explains the low activity of CrISA1 toward tightly spaced branches and reveals the presence of additional secondary surface carbohydrate binding sites.
Highlights
The ISA11⁄7ISA2 isoamylase complex is involved in starch synthesis
There was at least a 2-fold decrease in apparent size of the isoamylase complex possibly from a hexamer or tetramer to that of a dimer [17]. These observations have been previously observed in ISA2 knockdown and mutant studies in maize and rice [11, 14], the molecular nature of the STA8 locus and its relationship to the ISA2 gene was lacking from the initial studies in Chlamydomonas [17, 19]
We show that starch defects in the sta8 mutants can be almost fully reversed through functional complementation with wild-type ISA2 and ISA2 carrying a triple HA tag (ISA2-HA) (Table 1) suggesting the STA8 locus encodes for ISA2
Summary
The ISA11⁄7ISA2 isoamylase complex is involved in starch synthesis. Results: The ISA1 homodimer from the green algae Chlamydomonas is functional without ISA2 and its crystal structure is described. We confirm that the STA8 locus encodes for ISA2 and sta mutants lack the ISA11⁄7ISA2 heteromeric complex. Mutant studies in maize, rice, Arabidopsis, and Chlamydomonas revealed that debranching enzymes are involved in the crystallization of starch, its mechanism is not very well understood (6 – 8). The differences in the ISA1 and ISA11⁄7ISA2 protein complex distribution in the species correlate with the results observed upon inactivation of either ISA1 or ISA2 genes through mutagenesis in Arabidopsis and cereals or upon decreasing transcript abundance by antisense RNA approaches in potato. We show in Chlamydomonas that the STA8 locus encodes ISA2, that ISA2 interacts physically with ISA1, and confirm the presence of both homomeric ISA1 and heteromeric ISA11⁄7ISA2 complexes in vivo. We compare CrISA1 with other plant ISA1, propose the conservation of the dimeric ISA1 structure in plants, and suggest how it may serve as framework for the assembly of ISA11⁄7ISA2 heterocomplexes
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