Comparative in vitro analyses of recombinant maize starch synthases SSI, SSIIa, and SSIII reveal direct regulatory interactions and thermosensitivity

• Published in: Archives of biochemistry and biophysics Vol. 596; pp. 63 - 72
• Main Authors: Huang, Binquan, Keeling, Peter L., Hennen-Bierwagen, Tracie A., Myers, Alan M.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15.04.2016
• Subjects: Amylopectin - chemistry; Amylopectin - metabolism; Catalysis; Endosperm; Enzyme Stability - physiology; Hot Temperature; Maize; Plant Proteins - chemistry; Plant Proteins - genetics; Protein Domains; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Starch structure; Starch synthase; Starch Synthase - chemistry; Starch Synthase - genetics; Thermosensitivity; Zea mays - enzymology; Zea mays - genetics; SS; SSIII-HDCAT; SSIII-NHD; SSIII-N; Starch structure; Maize; DP; Thermosensitivity; SSIII-CAT; Starch synthase; Endosperm; ADPGlc; SSIII-HD
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1016/j.abb.2016.02.032

Starch synthases SSI, SSII, and SSIII function in assembling the amylopectin component of starch, but their specific roles and means of coordination are not fully understood. Genetic analyses indicate regulatory interactions among SS classes, and physical interactions among them are known. The N terminal extension of cereal SSIII, comprising up to 1200 residues beyond the catalytic domain, is responsible at least in part for these interactions. Recombinant maize SSI, SSIIa, and full-length or truncated SSIII, were tested for functional interactions regarding enzymatic activity. Amino-terminal truncated SSIII exhibited reduced activity compared to full-length enzyme, and addition of the N terminus to the truncated protein stimulated catalytic activity. SSIII and SSI displayed a negative interaction that reduced total activity in a reconstituted system. These data demonstrate that SSIII is both a catalytic and regulatory factor. SSIII activity was reduced by approximately 50% after brief incubation at 45 °C, suggesting a role in reduced starch accumulation during growth in high temperatures. Buffer effects were tested to address a current debate regarding the SS mechanism. Glucan stimulated the SSIIa and SSIII reaction rate regardless of the buffer system, supporting the accepted mechanism in which glucosyl units are added to exogenous primer substrates. [Display omitted] •SSI and SSIII mixed in a reconstituted system exhibited a negative regulatory interaction.•The reconstituted system recapitulated the in vivo effects of SSIII mutation.•SS activities were stimulated by glucans in various buffers, upholding the accepted mechanism.•Purified SSIII lost activity after brief incubation at relatively low temperatures.