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.

Structural Studies of the Intestinal α-Glucosidases, Maltase-glucoamylase and Sucrase-isomaltase.

OBJECTIVES: Maltase-glucoamylase and sucrase-isomaltase are enzymes in the brush-border membrane of the small intestinal lumen responsible for the breakdown of postamylase starch polysaccharides to release monomeric glucose. As such, they are critical players in healthy nutrition and their malfunction can lead to severe disorders. METHODS: This review covers investigations of the structures and functions of these enzymes. RESULTS: Each consists of 2 enzyme domains of the glycoside hydrolase family GH31 classification, yet with somewhat differing enzymatic properties. CONCLUSIONS: Crystallographic structures of 3 of the domains have been published. Insights into substrate binding and specificity will be discussed, along with future lines of inquiry related to the enzymes' roles in disease and potential avenues for therapeutics.

Suggested alternative starch utilization system from the human gut bacterium Bacteroides thetaiotaomicron.

The human digestive system is host to a highly populated ecosystem of bacterial species that significantly contributes to our assimilation of dietary carbohydrates. Bacteroides thetaiotaomicron is a member of this ecosystem, and participates largely in the role of the gut microbiome by breaking down dietary complex carbohydrates. This process of acquiring glycans from the colon lumen is predicted to rely on the mechanisms of proteins that are part of a classified system known as polysaccharide utilization loci (PUL). These loci are responsible for binding substrates at the cell outer membrane, internalizing them, and then hydrolyzing them within the periplasm into simple sugars. Here we report our investigation into specific components of a PUL, and suggest an alternative starch utilization system in B. thetaiotaomicron. Our analysis of an outer membrane binding protein, a SusD homolog, highlights its contribution to this PUL by acquiring starch-based sugars from the colon lumen. Through our structural characterization of two Family GH31 α-glucosidases, we reveal the flexibility of this bacterium with respect to utilizing a range of starch-derived glycans with an emphasis on branched substrates. With these results we demonstrate the predicted function of a gene locus that is capable of contributing to starch hydrolysis in the human colon.

Crystallographic structure of ChitA, a glycoside hydrolase family 19, plant class IV chitinase from Zea mays.

Maize ChitA chitinase is composed of a small, hevein-like domain attached to a carboxy-terminal chitinase domain. During fungal ear rot, the hevein-like domain is cleaved by secreted fungal proteases to produce truncated forms of ChitA. Here, we report a structural and biochemical characterization of truncated ChitA (ChitA ΔN), which lacks the hevein-like domain. ChitA ΔN and a mutant form (ChitA ΔN-EQ) were expressed and purified; enzyme assays showed that ChitA ΔN activity was comparable to the full-length enzyme. Mutation of Glu62 to Gln (ChitA ΔN-EQ) abolished chitinase activity without disrupting substrate binding, demonstrating that Glu62 is directly involved in catalysis. A crystal structure of ChitA ΔN-EQ provided strong support for key roles for Glu62, Arg177, and Glu165 in hydrolysis, and for Ser103 and Tyr106 in substrate binding. These findings demonstrate that the hevein-like domain is not needed for enzyme activity. Moreover, comparison of the crystal structure of this plant class IV chitinase with structures from larger class I and II enzymes suggest that class IV chitinases have evolved to accommodate shorter substrates.

Expression and purification of two Family GH31 α-glucosidases from Bacteroides thetaiotaomicron.

Microorganisms in the human gut outnumber human cells by a factor of 10. These microbes have been shown to have relevance to the human immune, nutrition and metabolic systems. A dominant symbiont of this environment is Bacteroides thetaiotaomicron which is characterized as being involved in degrading non-digestible plant polysaccharides. This organism's genome is highly enriched in genes predicted to be involved in the hydrolysis of various glycans. Presented here is a comparative functional analysis of two α-glucosidases (designated BT_0339 and BT_3299), Family 31 Glycoside Hydrolases from B. thetaiotaomicron. The purpose of this research is to explore the contributions these enzymes may have to human nutrition and specifically starch digestion. Expression of both α-glucosidases in pET-29a expression vector resulted in high levels of expressed protein in the soluble fraction. Two-step purification allowed for the isolation of the enzymes of interest in significant yield and fractions were observed to be homogenous. Both enzymes demonstrated activity on maltose, isomaltose and malto-oligosaccharide substrates and low level of activity on lactose and sucrose. Enzymatic kinetics revealed these enzymes both preferentially cleave the α1-6 linkage in comparison to the expected α1-4 and specifically favor maltose-derived substrates of longer length. The flexible hydrolytic capabilities of BT_0339 and BT_3299 reveal the ability of this bacterium to maintain its dominant position in its environment by utilizing an array of substrates. Specifically, these enzymes demonstrate an important aspect of this organism's contribution to starch digestion in the distal gut and the overall energy intake of humans.