Phylogenetic analysis reveals key residues in substrate hydrolysis in the isomaltase domain of sucrase-isomaltase and its role in starch digestion.

BACKGROUND:Starch constitutes one of the main sources of nutrition in the human diet and is broken down through a number of stages of digestion. Small intestinal breakdown of starch-derived substrates occurs through the mechanisms of small intestinal brush border enzymes, maltase-glucoamylase and sucrase-isomaltase. These enzymes each contain two functional enzymatic domains, and though they share sequence and structural similarities due to their evolutionary conservation, they demonstrate distinct substrate preferences and catalytic efficiency. The N-terminal isomaltase domain of sucrase-isomaltase has a unique ability to actively hydrolyze isomaltose substrates in contrast to the sucrase, maltase and glucoamylase enzymes. METHODS:Through phylogenetic analysis, structural comparisons and mutagenesis, we were able to identify specific residues that play a role in the distinct substrate preference. Mutational analysis and comparison with wild-type activity provide evidence that this role is mediated in part by affecting interactions between the sucrase and isomaltase domains in the intact molecule. RESULTS:The sequence analysis revealed three residues proposed to play key roles in isomaltase specificity. Mutational analysis provided evidence that these residues in isomaltase can also affect activity in the partner sucrase domain, suggesting a close interaction between the domains. MAJOR CONCLUSIONS:The sucrase and isomaltase domains are closely interacting in the mature protein. The activity of each is affected by the presence of the other. GENERAL SIGNIFICANCE:There has been little experimental evidence previously of the effects on activity of interactions between the sucrase-isomaltase enzyme domains. By extension, similar interactions might be expected in the other intestinal α-glucosidase, maltase-glucoamylase.