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

Abstract

Starch synthase (SS) (ADP-glucose:1,4-α-D-glucan 4-α-D-glucosyltransferase) elongates α-(1→4)-linked linear glucans within plastids to generate the storage polymers that constitute starch granules. Multiple SS classes are conserved throughout the plant kingdom, indicating that each provides a unique function responsible for evolutionary selection. Evidence has been presented arguing for addition of glucosyl units from the ADPglucose donor to either the reducing end or the non-reducing end of the acceptor substrate, although until recently direct evidence addressing this question was not available. Characterization of newly incorporated glucosyl units determined that recombinant maize (Zea mays L.) SSIIa elongates its substrates at the non-reducing end. However, the possibility remained that other SSs might utilize distinct mechanisms, and that one or more of the conserved enzyme classes could elongate acceptors at the reducing end. This study characterized the reaction mechanism of recombinant maize SSI regarding its addition site. Newly incorporated residues were labeled with ¹³C, and reducing ends of the elongation products were labeled by chemical derivitization. Electrospray ionization-tandem mass spectroscopy traced the two parameters, i.e., the newly added residue and the reducing end. The results determined that SSI elongates glucans at the non-reducing end. The study also confirmed previous findings showing recombinant SSI can generate glucans of at least 25 units, that it is active using acceptors as short as maltotriose, that recombinant forms of the enzyme absolutely require an acceptor for activity, and that it is not saturable with maltooligosaccharide acceptor substrates.

Keywords: amylopectin metabolism; enzymology; maize; starch; starch synthase.

FIGURE 1

NS-SSI purification. NS-SSI was purified from total soluble E. coli extract by binding to S-protein agarose. Proteins in each fraction were visualized by SDS-PAGE and Coomassie blue staining. The purified fraction was analyzed after boiling of the affinity matrix in SDS-PAGE loading buffer.

FIGURE 2

Reaction velocity with variable concentration of acceptor substrate. Reactions were saturated for ADPGlc and limited for NS-SSI. Reaction rates were determined by measuring molecules of ADP released at each time point.

FIGURE 3

Thin layer chromatography (TLC) analysis of NS-SSI reaction products. Reactions were in enzyme excess conditions. The maltooligosaccharide acceptor used in each reaction, or lack therof, is indicated. Numbers indicate reaction times in hours. “St” indicates maltooligosaccharide standard for each acceptor.

FIGURE 4

HPAEC-PAD analysis of NS-SSI reaction products. The initial acceptor substrate was DP4, and reactions were in conditions of enzyme excess. Reaction times are indicated. Products are identified based on standards. Asterisk indicates an unidentified peak in the reaction mixture.

FIGURE 5

MS/MS analysis of NS-SSI reaction products. The initial acceptor substrate used in each column is indicated. Symbols indicate the molecule generating each peak, including glucosyl residues and N-EDA reducing end label. Open or closed hexagons represent [¹²C]- or [¹³C]-glucosyl units, respectively. (A) MS1 spectra of control reaction lacking enzyme. (B) MS1 spectra NS-SSI-catalyzed reaction products. (C) MS2 spectra of products elongated by one residue. (D) MS2 spectra of products elongated by two residues.