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-[C]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]glucose, maltohexaose (degree of polymerization 6), and SSIIa. Product analysis by ESI-MS revealed that the [C]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]glucose added to maltohexaose by SSIIa using solution NMR spectroscopy confirmed the polarity of maltodextrin chain elongation.

A Review of Starch Biosynthesis in Relation to the Building Block-Backbone Model

“…Three of the six known SS isoforms are directly involved in amylopectin biosynthesis, producing α-(1,4)-linked glucan chains of varying chain lengths, which can be further elongated, branched (by SBEs) or debranched (DBEs). These SS isoforms are SSI, SSII and SSIII, and are involved in the elongation of successively longer (higher DP) α-(1,4)-linked glucan chains by the addition of glucosyl residues onto the non-reducing end of a pre-existing glucan chain, ADP-Glc serving as the glucosyl donor [ 189 , 190 , 191 ]. Unlike GBSS, all the SS isoforms involved in amylopectin biosynthesis have a distributive mode of action whereby the α-(1,4)-linked chain is extended by a single Glc per enzyme-substrate interaction.…”

Direct Determination of the Site of Addition of Glucosyl Units to Maltooligosaccharide Acceptors Catalyzed by Maize Starch Synthase I

“…That conclusion was confirmed by 13 C-NMR analyses that distinguished between free anomeric carbons and those bound in α-(1→4) glycoside bonds. Anomeric carbons in newly incorporated residues were found to be glycoside-bound, not free, thus confirming non-reducing end addition ( Larson et al, 2016 ).…”

“…Beads were removed by brief centrifugation and ADP product in 5 μL of the supernatant was quantified using the ADP Glo Kinase Assay Kit (Promega No. V6930) as described previously ( Huang et al, 2016 ; Larson et al, 2016 ).…”

“…Other reactions were carried out in conditions of enzyme excess, using [ 13 C U ]-ADPGlc prepared as previously described ( Larson et al, 2016 ). These “enzyme excess” reactions varied from the kinetic analyses in that they contained 50 μL of SSI bead slurry, 4 mM [ 13 C U ]-ADPGlc, and 2 mM maltooligosaccharide, either maltotriose (DP3), maltotetraose (DP4), or maltopentaose (DP5).…”

“…Electrospray ionization-tandem mass spectroscopy (ESI-MS/MS) detected addition of [ 13 C U ]-Glc units, then determined the molecular mass of the derivatized reducing end residue that was covalently labeled with the N-EDA tag. In these experiments, N-EDA mass label was attached uniquely to 12 C U -glucose, meaning only pre-existing residues within maltohexaose, not newly added glucosyl units from the enzymatic reaction, were located at the reducing end ( Larson et al, 2016 ). This provides direct evidence for non-reducing end addition by maize SSIIa.…”