Abstract
Starch branching enzyme IIb (SBEIIb) plays a crucial role in amylopectin biosynthesis in maize endosperm by defining the structural and functional properties of storage starch and is regulated by protein phosphorylation. Native and recombinant maize SBEIIb were used as substrates for amyloplast protein kinases to identify phosphorylation sites on the protein. A multidisciplinary approach involving bioinformatics, site-directed mutagenesis, and mass spectrometry identified three phosphorylation sites at Ser residues: Ser(649), Ser(286), and Ser(297). Two Ca(2+)-dependent protein kinase activities were partially purified from amyloplasts, termed K1, responsible for Ser(649) and Ser(286) phosphorylation, and K2, responsible for Ser(649) and Ser(297) phosphorylation. The Ser(286) and Ser(297) phosphorylation sites are conserved in all plant branching enzymes and are located at opposite openings of the 8-stranded parallel β-barrel of the active site, which is involved with substrate binding and catalysis. Molecular dynamics simulation analysis indicates that phospho-Ser(297) forms a stable salt bridge with Arg(665), part of a conserved Cys-containing domain in plant branching enzymes. Ser(649) conservation appears confined to the enzyme in cereals and is not universal, and is presumably associated with functions specific to seed storage. The implications of SBEIIb phosphorylation are considered in terms of the role of the enzyme and the importance of starch biosynthesis for yield and biotechnological application.
Highlights
Starch is the major component of cereal yield, yet the biochemical regulation of its synthesis is poorly understood
Of the current work was to identify the phosphorylation sites on maize Starch branching enzyme IIb (SBEIIb), the major branching enzyme involved in amylopectin synthesis, to understand how the activities of the enzyme are coordinated with other components of the starch biosynthetic pathway through protein phosphorylation, and to begin to characterize the protein kinases and phosphatases that regulate starch branching enzymes (SBE)
Truncated forms of recombinant SBEIIb were helpful in determining the location of the most phosphorylated region of the protein, and bioinformatics was employed to guide site-directed mutagenesis and biochemical analysis of likely phosphorylation sites
Summary
Starch is the major component of cereal yield, yet the biochemical regulation of its synthesis is poorly understood. Starch branching enzyme IIb (SBEIIb) plays a crucial role in amylopectin biosynthesis in maize endosperm by defining the structural and functional properties of storage starch and is regulated by protein phosphorylation. Different SS isoforms have distinct biochemical properties and produce ␣-glucan chains of different length, or degree of polymerization; which are substrates for starch branching enzymes (SBE) (1,4-␣-glucan: 1,4-␣-glucan 6-glucosyltransferase; E.C. 2.4.1.18). An emerging aspect of our understanding of the regulation of storage starch biosynthesis is the physical association of different classes of amylopectin-synthesizing enzymes in protein complexes (19 –21) within starch-synthesizing plastids, and the important regulatory role played by protein phosphorylation. This article reports on the identification of multiple Ser phosphorylation sites on SBEIIb, a key enzyme in amylopectin biosynthesis, and component of a number of heteromeric protein complexes whose activity influences the functional properties of starch. Two distinct Ca2ϩ-dependent protein kinase activities have been partially purified that show differential specificity for Ser residues on SBEIIb
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