Crystal Structure of the Chlamydomonas Starch Debranching Enzyme Isoamylase ISA1 Reveals Insights into the Mechanism of Branch Trimming and Complex Assembly

Abstract

Background:The ISA1⅐ISA2 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. Conclusion: The ISA1 structure reveals potential substrate recognition sites and explains its low selectivity toward tightly spaced branches. Significance: Structural conservation with plant ISA1 suggests it may be a useful model for studying branch trimming.

Biosynthesis of Reserve Starch

“…ISA3 likely plays a part in degrading branched glucans in the leaf (HennenBierwagen et al 2012), but not in the glucan synthesis in the endosperm (Yun et al 2011). It is interesting that both ISA1 homomer and ISA1-ISA2 heteromer were present in maize endosperm (Kubo et al 2010) and leaf , rice endosperm (Utsumi and Nakamura 2006;Utsumi et al 2011), and Chlamydomonas (Sim et al 2014). In contrast, only the ISA1-ISA2 heteromer existed in potato tuber (Hussain et al 2003) as in Arabidopsis leaves (Dellate et al 2005).…”

“…Information on the crystal structure of plant DBEs is available for Chlamydomonas ISA1 (CrISA1) (Sim et al 2014) and barley PUL (Vester-Christensen et al 2010). It is interesting that the homodimeric CrISA1 structure had a unique elongated structure with monomers connected end to end, and the distinct dimeric structure was commonly conserved in ISA1 homomer and ISA1-ISA2 heteromer at least in green plants (Dauvillée et al 2000;Sim et al 2014).…”

“…5.4a and 5.5) was reported by James et al (1995) using the gene-tagging method. Since then the essential roles of DBEs in amylopectin synthesis have been verified by extensive investigations of a variety of mutants and transformants from maize (James et al 1995;Beatty et al 1999;Dinges et al 2001;Dinges et al 2003;Kubo et al 2010;Lin et al 2012Lin et al , 2013, rice (Nakamura et al , 1997Kubo et al 1999Kubo et al , 2005Fujita et al 2003Fujita et al , 2009Utsumi et al 2011), potato (Hussain et al 2003;Bustos et al 2004), barley (Burton et al 2002), Arabidopsis (Zeeman et al 1998;Dellate et al 2005;Wattebled et al 2005Wattebled et al , 2008Streb et al 2008;Pfister et al 2014;Streb and Zeeman 2014), Chlamydomonas (Mouille et al 1996;Colleoni et al 1999aColleoni et al , 1999bDauvillée et al 2000Dauvillée et al , 2001Sim et al 2014), and Cyanobacterium sp. CLg1 (Cenci et al 2013) (also see review by Hennen-Bierwagen et al 2012), although in this chapter experimental results with maize and rice endosperm are mainly described.…”

“…The structural features of ISA (Katsuya et al 1998;Sim et al 2014) and PUL (Vester-Christensen et al 2010) were revealed.…”

Starch Biosynthesis in the Developing Endosperms of Grasses and Cereals

“…In this model, loss of ISA1 or ISA2 creates an abundance of branch points leading to unfavourable conditions for the formation of crystalline lamellae, resulting in the less ordered phytoglycogen. Recent structural studies with ISA1 from the green alga Chlamydomonas reinhardtii indicate that the enzyme has a relatively low affinity towards tightly spaced branches, supporting its proposed role in glucan trimming model [335]. Nevertheless, although ISA mutants accumulate large quantities of phytoglycogen (in maize, this is the basis of some "sweetcorn" varieties), many of these mutants are able to synthesize some starch [320,336], indicating that ISA activity is not a mandatory requirement for crystalline-competent amylopectin formation.…”