Structure and substrate recognition by the Ruminococcus bromii amylosome pullulanases

Highlights

• The gut bacterium Ruminococcus bromii secretes two pullulanases, Amy10 and Amy12.

• Amy10 and Amy12 have different activities on starch and maltoligosaccharides.

• Amy12 structures demonstrate substrate accommodation within the −5 to + 4 subsites, including branch points.

• Bioinformatic analysis of Amy10 and Amy12 suggests their action on complex starches.

Abstract

Pullulanases are glycoside hydrolase family 13 (GH13) enzymes that target α1,6 glucosidic linkages within starch and aid in the degradation of the α1,4- and α1,6- linked glucans pullulan, glycogen and amylopectin. The human gut bacterium Ruminococcus bromii synthesizes two extracellular pullulanases, Amy10 and Amy12, that are incorporated into the multiprotein amylosome complex that enables the digestion of granular resistant starch from the diet. Here we provide a comparative biochemical analysis of these pullulanases and the x-ray crystal structures of the wild type and the nucleophile mutant D392A of Amy12 complexed with maltoheptaose and 6³-α-D glucosyl-maltotriose. While Amy10 displays higher catalytic efficiency on pullulan and cleaves only α1,6 linkages, Amy12 has some activity on α1,4 linkages suggesting that these enzymes are not redundant within the amylosome. Our structures of Amy12 include a mucin-binding protein (MucBP) domain that follows the C-domain of the GH13 fold, an atypical feature of these enzymes. The wild type Amy12 structure with maltoheptaose captured two oligosaccharides in the active site arranged as expected following catalysis of an α1,6 branch point in amylopectin. The nucleophile mutant D392A complexed with maltoheptaose or 6³-α-D glucosyl-maltotriose captured β-glucose at the reducing end in the −1 subsite, facilitated by the truncation of the active site aspartate and stabilized by stacking with Y279. The core interface between the co-crystallized ligands and Amy12 occurs within the −2 through + 1 subsites, which may allow for flexible recognition of α1,6 linkages within a variety of starch structures.

Introduction

The human gut microbiota is a metabolic bioreactor that plays a key role in the digestion of dietary carbohydrates that human enzymes cannot break down (Salyers et al., 1977, Flint and Bayer, 2008). Dietary polysaccharides that transit the distal gut are hydrolyzed and fermented by intestinal bacteria to short chain fatty acids such as butyrate, acetate and propionate that profoundly shape our physiology (Koh et al., 2016). The breakdown of complex polysaccharides is largely a team sport, with primary degraders initiating the breakdown of large or insoluble dietary fibers yielding partially digested fiber, oligosaccharides and smaller sugars that are then scavenged by secondary degrading species (Ze et al., 2012, Rakoff-Nahoum et al., 2014, Rogowski et al., 2015). Primary degraders typically possess enhanced hydrolytic potential for fiber degradation by packaging glycan-binding and hydrolysis functions in proximity. This is accomplished either by having a single polypeptide with discrete modules devoted to these functions or via the assembly into complexes of multiple proteins with these individual functions (Rogowski et al., 2015, Ze et al., 2015).

Starch is an important energy source and abundant polysaccharide in the Western diet (Cordain, et al., 2005). In plants, starch is produced as granules comprised of layers of amylose and amylopectin (Bertoft, 2017). Amylose is a predominantly α1,4-linked glucose polymer that forms helices by virtue of the glycosidic bond geometry, and these helices can pack tightly together (Buléon et al., 1998). Amylopectin is α1,4-linked glucose with infrequent α1,6-branch points; these branch points aid in solubilization of the polymer as they prevent packing of the α1,4 helical regions. Thus the α1,4 regions within amylopectin are more soluble than those in amylose and less resistant to enzymatic digestion by host enzymes (Buléon et al., 1998, Jane, 2006). However, the α1,6 linkages themselves are less well processed in the upper gastrointestinal tract. Though starch consists of a single monosaccharide and only two glycosidic linkages, there is an overwhelming variety in chain length and distribution of branch points within different starches that dictate its digestibility by the host as well as their gut bacteria (Ze et al., 2012, Englyst and Cummings, 1987, Muir et al., 1995).

Because of the massive size of starch polymers, bacteria initiate the breakdown of this nutrient at the cell surface, then import the liberated glucose and maltooligosaccharides. Primarily, enzymes of the glycoside hydrolase family 13 (GH13) endow various species of gut bacteria with the ability to degrade dietary starch (Kaoutari et al., 2013). The GH13 family includes both amylase and pullulanase enzymes that target α1,4- and α1,6- glucosidic linkages within starch polymers, respectively (Møller and Svensson, 2016). The repertoire of GH13 enzymes possessed by individual bacteria and the organization of these enzymes greatly influences bacterial starch digestion (Cerqueira et al., 2020).

While host glucoamylases can digest more soluble or processed types of starch, certain retrograded or granular starches, and starch with extensive α1,6 branching, can escape digestion in the upper gastrointestinal tract (Brownlee et al., 2018). Resistant starch (RS) describes starch that is inaccessible to host digestion or is minimally processed and becomes food for gut bacteria in the colon (Birt, et al., 2013). While many gut bacteria possess the genomic potential to degrade starch, few can directly metabolize RS (Ze et al., 2012, Jung et al., 2019, Baxter et al., 2019).

Ruminococcus bromii is a primary degrader of RS and thus considered a keystone species in the gut (Ze et al., 2012). Its RS metabolism supports the growth of secondary degraders that produce butyrate, a short-chain fatty acid with anti-inflammatory and anti-tumorigenic properties (Ze et al., 2012, Baxter et al., 2019). The ability of R. bromii to degrade RS is attributed to its expression of an amylosome, a multiprotein complex comprised of starch-binding and -hydrolyzing components (Ze et al., 2015, Mukhopadhya et al., 2018). This assembly is akin to the cellulosome, a multiprotein complex for cellulose degradation that is found in some soil bacteria and ruminant bacterial species, as well as fungi and the human gut isolate Ruminococcus champenellensis (Artzi et al., 2017, Morais, 2016, Haitjema, 2017). The R. bromii amylosome is predicted to be comprised of scaffolding proteins, called scaffoldins (Bayer et al., 1994) that nucleate the assembly of additional proteins into the complex (Ze et al., 2015, Mukhopadhya et al., 2018). Cohesin modules on scaffoldins mediate high-affinity protein–protein interactions with matching dockerin modules on starch-binding and starch-hydrolyzing enzymes. Like the observed synergistic action of the cellulosomal enzymes, it is believed that on the amylosome the proximity of multiple GH13s and/or carbohydrate-binding modules (CBMs) on the scaffoldin proteins facilitate the enhanced efficiency of RS degradation.

There are only five dockerin-containing GH13 enzymes within the predicted R. bromii amylosome architecture including two pullulanases, Amy10 and Amy12, that preferentially target α1,6 branch points (Mukhopadhya et al., 2018). The α1,6-glycosidic branch points of amylopectin prevent complete starch digestion in the upper gastrointestinal tract, in part because the linkage itself is not hydrolyzed by extracellular host enzymes (Brownlee et al., 2018, Lee and Hamaker, 2017, Lee, 2013). Also, branching of the glucan chain prevents enzymes from accessing α1,4 linked regions of the polysaccharide up- and downstream of this junction (Møller and Svensson, 2016, Bijttebier et al., 2008). Dietary starch processed by host enzymes generates limit dextrins enriched in α1,6 linkages that then traverse the large bowel (Lee and Hamaker, 2017, Lee et al., 2014). Therefore, gut bacterial pullulanases may have essential roles in providing nutrient access to limit dextrins and in the efficient breakdown of starch granules. Indeed, pullulanases from some Bifidobacterial species are hypothesized to be key for degradation of RS, and Lactobacillus pullulanases are important for hydrolyzing branched α-glucans (Centanni, 2018, Moller, 2017). Beyond utility in RS degradation, pullulanases have important starch-debranching activity in the food and beverage industry (van der Maarel, 2002, Domań-Pytka and Bardowski, 2004). Additionally, some pathogenic bacteria such as Klebsiella and Streptococcus pneumoniae rely on debranching GH13 enzymes to access host glycogen (Lammerts van Bueren et al., 2011, Paczosa and Mecsas, 2016). To understand the features of the R. bromii amylosome pullulanases Amy10 and Amy12, we performed a comparative biochemical characterization of these enzymes. We also determined four crystal structures of Amy12 with maltooligosaccharides to discern the molecular features that afford recognition of α1,6 glycosidic linkages by the R. bromii amylosome.