Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 291, Issue 48, Pages 24951-24960Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M116.754705
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Funding
- NIGMS, National Institutes of Health [R01-GM115489]
- Roy J. Carver Department of Biochemistry, Biophysics AMP
- Molecular Biology at Iowa State University
- Div Of Molecular and Cellular Bioscience
- Direct For Biological Sciences [1517256] Funding Source: National Science Foundation
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A comprehensive description of starch biosynthesis and granule assembly remains undefined despite the central nature of starch as an energy storage molecule in plants and as a fundamental calorie source for many animals. Multiple theories regarding the starch synthase (SS)-catalyzed assembly of (alpha 1-4)-linked D-glucose molecules into maltodextrins generally agree that elongation occurs at the non-reducing terminus based on the degradation of radiolabeled maltodextrins, although recent reports challenge this hypothesis. Surprisingly, a direct analysis of the SS catalytic product has not been reported, to our knowledge. We expressed and characterized recombinant Zea mays SSIIa and prepared pure ADP-[C-13(U)] glucose in a one-pot enzymatic synthesis to address the polarity of maltodextrin chain elongation. We synthesized maltoheptaose (degree of polymerization 7) using ADP-[C-13(U)] glucose, maltohexaose (degree of polymerization 6), and SSIIa. Product analysis by ESI-MS revealed that the [C-13(U)] glucose unit was added to the non-reducing end of the growing chain, and SSIIa demonstrated a > 7,850-fold preference for addition to the non-reducing end versus the reducing end. Independent analysis of [C-13(U)] glucose added to maltohexaose by SSIIa using solution NMR spectroscopy confirmed the polarity of maltodextrin chain elongation.
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