Origin of Chain Length Specificites of Starch Branching Enzyme

Abstract

Starch Branching enzyme (BE) is one of the three enzymes involved in starch biosynthesis. It is responsible for synthesizing the alpha-1,6-glucan branches, remodeling the linear alpha-1,4-glucan polymer to produce amylopectin. There are at least two isoforms of the enzyme in most plants, with each having distinct reactivities and product chain-length specificities. Though the chemistry of the active site of branching enzyme is relatively well studied, the origin of the chain length specificity is yet to be understood. Using protein crystallography and biochemical studies on rice branching enzyme, we aim to understand the factors controlling the chain-length specificity of branching enzymes. The mechanism of branching enzyme involves two steps: (1) An oligosaccharide (donor chain) binds to the enzyme and is cleaved by the action of a nucleophilic aspartate residue to form a covalently-linked enzyme-glucan intermediate. (2) A second oligosaccharide (acceptor chain) then reacts, by nucleophilic attack of one of its alpha-1,6-hydroxyl groups, to form a new alpha-1,6-branch. We have focused on discovering the surface glucan binding sites in branching enzyme because they are likely essential to understanding the specificity of the enzyme. To this end we have obtained a crystal structure of an oligosaccharide (M12)-bound rice branching enzyme, which reveals oligosaccharide binding from the outer surface of the enzyme almost to the active site. Mutations of the residues interacting with the M12 can substantially compromise the enzyme's activity, though none have affected the branch chain specificity. On the other hand, comparison of an isoamylase maltoheptaose (M7)-bound structure with rice branching enzyme suggested another potential glucan surface binding site. Interestingly, mutations in this new site did effect the chain-length specificity of the enzyme. These results suggest that the bound M12 is part of the acceptor chain and the newly identified binding site hosts the donor chain. Together, the data allow us to, for the first time propose a detailed mechanism for the enzyme, explaining how disparate surface glucan binding sites far from the active site create the enzyme's activity and specificity against its polymeric substrate.