title: Direct Characterization of the Maize Starch Synthase IIa Product Shows Maltodextrin Elongation Occurs at the Non-reducing End.
abstract: 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 (α1-4)-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-[13CU]glucose in a one-pot enzymatic synthesis to address the polarity of maltodextrin chain elongation. We synthesized maltoheptaose (degree of polymerization 7) using ADP-[13CU]glucose, maltohexaose (degree of polymerization 6), and SSIIa. Product analysis by ESI-MS revealed that the [13CU]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 [13CU]glucose added to maltohexaose by SSIIa using solution NMR spectroscopy confirmed the polarity of maltodextrin chain elongation.
authors.affiliation: From the Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, Iowa 50011.
authors.forename: Mark E
authors.initials: ME
authors.lastname: Larson
authors.affiliation: From the Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, Iowa 50011.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Falconer
authors.affiliation: From the Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, Iowa 50011.
authors.forename: Alan M
authors.initials: AM
authors.lastname: Myers
authors.affiliation: From the Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, Iowa 50011 abarb@iastate.edu.
authors.forename: Adam W
authors.initials: AW
authors.lastname: Barb
mesh_terms: D005947:Glucose / Q000737:chemistry / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry* / Q000378:metabolism; D011134:Polysaccharides / Q000096:biosynthesis / Q000737:chemistry*; D013214:Starch Synthase / Q000737:chemistry* / Q000378:metabolism; D003313:Zea mays / Q000201:enzymology*
chemical_list: D009844:Oligosaccharides; D010940:Plant Proteins; D011134:Polysaccharides; C016549:maltohexaose; C008315:maltodextrin; C111377:starch synthase II; D013214:Starch Synthase; D005947:Glucose
keywords: SS2a; SSIIa; adenosine; enzyme mechanism; maltohexaose; mass spectrometry (MS); nuclear magnetic resonance (NMR); oligosaccharide