Abstract
Starch is a complex branched glucose polymer whose branch molecular weight distribution (the chain-length distribution, CLD) influences nutritionally important properties such as digestion rate. Chain-stopping in starch biosynthesis is by starch branching enzyme (SBE). Site-directed mutagenesis was used to modify SBEIIa from Zea mays (mSBEIIa) to produce mutants, each differing in a single conserved amino-acid residue. Products at different times from in vitro branching were debranched and the time evolution of the CLD measured by size-exclusion chromatography. The results confirm that Tyr352, Glu513, and Ser349 are important for mSBEIIa activity while Arg456 is important for determining the position at which the linear glucan is cut. The mutant mSBEIIa enzymes have different activities and suggest the length of the transferred chain can be varied by mutation. The work shows analysis of the molecular weight distribution can yield information regarding the enzyme branching sites useful for development of plants yielding starch with improved functionality.
Highlights
Starch is a homopolymer of glucose with a complex hierarchical structure [1]
We examine what the results of changing a single amino-acid residue in starch branching enzyme (SBE), the objective being to change the chain-length distribution (CLD) by changing activity and/or X0 and Xmin, the latter being features of an SBE which is reasonable to suppose might be changed by slight alteration of the binding site
After examination of the homology model generated by SWISS-MODEL using the structure of rice (Oryza sativa L.) SBEI (PDB ID 3AML) as a template, four conserved residues that lay within the hydrophobic groove (Tyr352, Glu513, Ser349, and Arg456) together with another conserved residue (Arg363) located at the back of the groove were identified as potentially interacting with the glucan and selected for the mutation studies (Fig 2)
Summary
Starch is a homopolymer of glucose with a complex hierarchical structure [1]. It has two major components, amylose and amylopectin. The glucose units are connected by α-(1!4) glycosidic linkages in the linear glucan chains, from which there are α-(1!6) glycosidic branch linkages. The functional and nutritional properties of starch are related to its structure [2,3,4,5,6,7,8]. Starches with higher amylose content or with longer-branched amylopectin have a higher tendency to retrograde, slowing down enzymatic degradation in the digestive track [9].
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