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

Lyann Sim, Sophie R. Beeren, Justin Findinier, David Dauvillee, Steven G. Ball, Anette Henriksen, Monica M. Palcic

Research output: Contribution to journalJournal articleResearchpeer-review

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 angstrom and in complex with maltoheptaose to 2.4 angstrom 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.
Original languageEnglish
JournalJournal of Biological Chemistry
Volume289
Issue number33
Pages (from-to)22991-23003
Number of pages14
ISSN0021-9258
DOIs
Publication statusPublished - 2014
Externally publishedYes

Keywords

  • Biochemistry
  • Enzymes
  • Amylopectin molecules
  • Carbohydrate binding
  • Chlamydomonas reinhardtii
  • Complex assembly
  • Elongated structures
  • Heteromeric complexes
  • Starch debranching enzymes
  • Starch synthesis
  • Starch
  • Cell Biology
  • Molecular Biology
  • Isoamylase
  • Isoamylase 1
  • Isoamylase 2
  • Unclassified drug
  • Binding affinity
  • Binding site
  • Carbohydrate synthesis
  • Complex formation
  • Controlled study
  • Crystal structure
  • Enzyme active site
  • Enzyme activity
  • Enzyme mechanism
  • Enzyme substrate
  • Molecular recognition
  • Nonhuman
  • Priority journal
  • Protein protein interaction
  • sequence alignment
  • Structure analysis
  • Branch trimming
  • Algae Plantae (Algae, Microorganisms, Nonvascular Plants, Plants) - Chlorophyta [13300] Chlamydomonas genus Chlamydomonas reinhardtii species strain-BafV13
  • ISA1-ISA2 heterocomplex
  • Isoamylase ISA1 starch debranching enzyme, crystal complex, carbohydrate binding site
  • 10802, Enzymes - General and comparative studies: coenzymes
  • 51518, Plant physiology - Enzymes
  • Biochemistry and Molecular Biophysics
  • Enzymology
  • Rice endosperm
  • Amylopectin biosynthesis
  • Homo-oligomer
  • Data quality
  • REINHARDTII
  • MODEL
  • Glycogen
  • Mutants
  • Granule
  • Plants
  • Plant Biology

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