Marie Sofie Møller

Enzymology and Structure of the GH13_31 Glucan 1,6-α-Glucosidase That Confers Isomaltooligosaccharide Utilization in the Probiotic Lactobacillus acidophilus NCFM

Isomaltooligosaccharides (IMO) have been suggested as promising prebiotics that stimulate the growth of probiotic bacteria. Genomes of probiotic lactobacilli from the acidophilus group, as represented by Lactobacillus acidophilus NCFM, encode α-1,6 glucosidases of the family GH13_31 (glycoside hydrolase family 13 subfamily 31) that confer degradation of IMO. These genes reside frequently within maltooligosaccharide utilization operons, which include an ATP-binding cassette transporter and α-glucan active enzymes, e.g., maltogenic amylases and maltose phosphorylases, and they also occur separated from any carbohydrate transport or catabolism genes on the genomes of some acidophilus complex members, as in L. acidophilus NCFM. Besides the isolated locus encoding a GH13_31 enzyme, the ABC transporter and another GH13 in the maltooligosaccharide operon were induced in response to IMO or maltotetraose, as determined by reverse transcription-PCR (RT-PCR) transcriptional analysis, suggesting coregulation of α-1,6- and α-1,4-glucooligosaccharide utilization loci in L. acidophilus NCFM. The L. acidophilus NCFM GH13_31 (LaGH13_31) was produced recombinantly and shown to be a glucan 1,6-α-glucosidase active on IMO and dextran and product-inhibited by glucose. The catalytic efficiency of LaGH13_31 on dextran and the dextran/panose (trisaccharide) efficiency ratio were the highest reported for this class of enzymes, suggesting higher affinity at distal substrate binding sites. The crystal structure of LaGH13_31 was determined to a resolution of 2.05 Å and revealed additional substrate contacts at the +2 subsite in LaGH13_31 compared to the GH13_31 from Streptococcus mutans (SmGH13_31), providing a possible structural rationale to the relatively high affinity for dextran. A comprehensive phylogenetic and activity motif analysis mapped IMO utilization enzymes from gut microbiota to rationalize preferential utilization of IMO by gut residents.

Structure and function of α-glucan debranching enzymes

Abstractα-Glucan debranching enzymes hydrolyse α-1,6-linkages in starch/glycogen, thereby, playing a central role in energy metabolism in all living organisms. They belong to glycoside hydrolase families GH13 and GH57 and several of these enzymes are industrially important. Nine GH13 subfamilies include α-glucan debranching enzymes; isoamylase and glycogen debranching enzymes (GH13_11); pullulanase type I/limit dextrinase (GH13_12–14); pullulan hydrolase (GH13_20); bifunctional glycogen debranching enzyme (GH13_25); oligo-1 and glucan-1,6-α-glucosidases (GH13_31); pullulanase type II (GH13_39); and α-amylase domains (GH13_41) in two-domain amylase–pullulanases. GH57 harbours type II pullulanases. Specificity differences, domain organisation, carbohydrate binding modules, sequence motifs, three-dimensional structures and specificity determinants are discussed. The phylogenetic analysis indicated that GH13_39 enzymes could represent a “missing link” between the strictly α-1,6-specific debranching enzymes and the enzymes with dual specificity and α-1,4-linkage preference.

Recent insight into oligosaccharide uptake and metabolism in probiotic bacteria

Abstract In recent years, a plethora of studies have demonstrated the paramount physiological importance of the gut microbiota on various aspects of human health and development. Particular focus has been set on probiotic members of this community, the best studied of which are assigned into the Lactobacillus and Bifidobacterium genera. Effects such as pathogen exclusion, alleviation of inflammation and allergies, colon cancer, and other bowel disorders are attributed to the activity of probiotic bacteria, which selectively ferment prebiotics comprising mainly non-digestible oligosaccharides. Thus, glycan metabolism is an important attribute of probiotic action and a factor influencing the composition of the gut microbiota. In the quest to understand the molecular mechanism of this selectivity for certain glycans, we have explored the routes of uptake and utilization of a variety of oligosaccharides differing in size, composition, and glycosidic linkages. A combination of “omics” technologies bioinformatics, enzymology and protein characterization proved fruitful in elucidating the protein transport and catabolic machinery conferring the utilization of glucosides, galactosides, and xylosides in the two clinically validated probiotic strains Lactobacillus acidophilus NCFM and Bifidobacterium animalis subsp. lactis Bl-04. Importantly, we have been able to identify and in some cases validate the specificity of several transport systems, which are otherwise poorly annotated. Further, we have demonstrated for the first time that non-naturally occurring tri- and tetra-saccharides are internalized and efficiently utilized by probiotic bacteria in some cases better than well-established natural prebiotics. Selected highlights of these data are presented, emphasising the importance and the diversity of oligosaccharide transport in probiotic bacteria.

Oligosaccharide and substrate binding in the starch debranching enzyme barley limit dextrinase

Complete hydrolytic degradation of starch requires hydrolysis of both the α-1,4- and α-1,6-glucosidic bonds in amylopectin. Limit dextrinase (LD) is the only endogenous barley enzyme capable of hydrolyzing the α-1,6-glucosidic bond during seed germination, and impaired LD activity inevitably reduces the maltose and glucose yields from starch degradation. Crystal structures of barley LD and active-site mutants with natural substrates, products and substrate analogues were sought to better understand the facets of LD-substrate interactions that confine high activity of LD to branched maltooligosaccharides. For the first time, an intact α-1,6-glucosidically linked substrate spanning the active site of a LD or pullulanase has been trapped and characterized by crystallography. The crystal structure reveals both the branch and main-chain binding sites and is used to suggest a mechanism for nucleophilicity enhancement in the active site. The substrate, product and analogue complexes were further used to outline substrate binding subsites and substrate binding restraints and to suggest a mechanism for avoidance of dual α-1,6- and α-1,4-hydrolytic activity likely to be a biological necessity during starch synthesis.

Structure of the starch-debranching enzyme barley limit dextrinase reveals homology of the N-terminal domain to CBM21.

Barley limit dextrinase (HvLD) is a debranching enzyme from glycoside hydrolase family 13 subfamily 13 (GH13_13) that hydrolyses α-1,6-glucosidic linkages in limit dextrins derived from amylopectin. The structure of HvLD was solved and refined to 1.9 Å resolution. The structure has a glycerol molecule in the active site and is virtually identical to the structures of HvLD in complex with the competitive inhibitors α-cyclodextrin and β-cyclodextrin solved to 2.5 and 2.1 Å resolution, respectively. However, three loops in the N-terminal domain that are shown here to resemble carbohydrate-binding module family 21 were traceable and were included in the present HvLD structure but were too flexible to be traced and included in the structures of the two HvLD-inhibitor complexes.

Structural biology of starch-degrading enzymes and their regulation

Starch is a major energy source for all domains of life. Recent advances in structures of starch-degrading enzymes encompass the substrate complex of starch debranching enzyme, the function of surface binding sites in plant isoamylase, details on individual steps in the mechanism of plant disproportionating enzyme and a self-stabilised conformation of amylose accommodated in the active site of plant α-glucosidase. Important inhibitor complexes include a flavonol glycoside, montbretin A, binding at the active site of human pancreatic α-amylase and barley limit dextrinase inhibitor binding to the debranching enzyme, limit dextrinase using a new binding mode for cereal protein inhibitors.

Efficient secretory expression of functional barley limit dextrinase inhibitor by high cell-density fermentation of Pichia pastoris

The limit dextrinase inhibitor (LDI) from barley seeds acts specifically on limit dextrinase (LD), an endogenous starch debranching enzyme. LDI is a 14 kDa hydrophobic protein containing four disulfide bonds and one unpaired thiol group previously found to be either glutathionylated or cysteinylated. It is a member of the so-called CM-protein family that includes α-amylase and serine protease inhibitors, which have been extremely challenging to produce recombinantly in functional form and in good yields. Here, LDI is produced in very high yields by secretory expression by Pichia pastoris applying high cell-density fermentation in a 5L fed-batch bioreactor. Thus about 200mg of LDI, which showed twofold higher inhibitory activity towards LD than LDI from barley seeds, was purified from 1L of culture supernatant by His-tag affinity chromatography and gel filtration. Electrospray ionization mass spectrometry verified the identity of the produced glutathionylated LDI-His(6). At a 1:1M ratio the recombinant LDI completely inhibited hydrolysis of pullulan catalyzed by 5-10 nM LD. LDI retained stability in the pH 2-12 range and at pH 6.5 displayed a half-life of 53 and 33 min at 90 and 93°C, respectively. The efficient heterologous production of LDI suggests secretory expression by P. pastoris to be a promising strategy to obtain other recombinant CM-proteins.

Recent insight in α -glucan metabolism in probiotic bacteria

Abstractα-Glucans from bacterial exo-polysaccharides or diet, e.g., resistant starch, legumes and honey are abundant in the human gut and fermentation of resistant fractions of these α-glucans by probiotic lactobacilli and bifidobacteria impacts human health positively. The ability to degrade polymeric α-glucans is confined to few strains encoding extracellular amylolytic activities of glycoside hydrolase (GH) family 13. Debranching pullulanases of the subfamily GH13_14 are the most common extracellular GH13 enzymes in lactobacilli, whereas corresponding enzymes are mainly α-amylases and amylopullulanases in bifidobacteria. Extracellular GH13 enzymes from both genera are frequently modular and possess starch binding domains, which are important for efficient catalysis and possibly to mediate attachment of cells to starch granules. α-1,6-Linked glucans, e.g., isomalto-oligosaccharides are potential prebiotics. The enzymes targeting these glucans are the most abundant intracellular GHs in bifidobacteria and lactobacilli. A phosphoenolpyruvate-dependent phosphotransferase system and a GH4 phospho-α-glucosidase are likely involved in metabolism of isomaltose and isomaltulose in probiotic lactobacilli based on transcriptional analysis. This specificity within GH4 is unique for lactobacilli, whereas canonical GH13 31 α-1,6-glucosidases active on longer α-1,6-gluco-oligosaccharides are ubiquitous in bifidobacteria and lactobacilli. Malto-oligosaccharide utilization operons encode more complex, diverse, and less biochemically understood activities in bifidobacteria compared to lactobacilli, where important members have been recently described at the molecular level. This review presents some aspects of α-glucan metabolism in probiotic bacteria and highlights vague issues that merit experimental effort, especially oligosaccharide uptake and the functionally unassigned enzymes, featuring in this important facet of glycan turnover by members of the gut microbiota.

Crystal Structure of Barley Limit Dextrinase:Limit Dextrinase Inhibitor (Ld:Ldi) Complex Reveals Insights Into Mechanism and Diversity of Cereal-Type Inhibitors.

Background: Barley limit dextrinase (LD), the sole starch debranching enzyme active during seed germination, is regulated by an endogenous inhibitor (LDI). Results: The crystal structure of the LD-LDI complex reveals a new and unexpected binding mode among cereal type inhibitors. Conclusion: A hydrophobic cluster drives the picomolar affinity of LDI. Significance: The molecular understanding of regulation of starch mobilization during germination is augmented. Molecular details underlying regulation of starch mobilization in cereal seed endosperm remain unknown despite the paramount role of this process in plant growth. The structure of the complex between the starch debranching enzyme barley limit dextrinase (LD), hydrolyzing α-1,6-glucosidic linkages, and its endogenous inhibitor (LDI) was solved at 2.7 Å. The structure reveals an entirely new and unexpected binding mode of LDI as compared with previously solved complex structures of related cereal type family inhibitors (CTIs) bound to glycoside hydrolases but is structurally analogous to binding of dual specificity CTIs to proteases. Site-directed mutagenesis establishes that a hydrophobic cluster flanked by ionic interactions in the protein-protein interface is vital for the picomolar affinity of LDI to LD as assessed by analysis of binding by using surface plasmon resonance and also supported by LDI inhibition of the enzyme activity. A phylogenetic analysis identified four LDI-like proteins in cereals among the 45 sequences from monocot databases that could be classified as unique CTI sequences. The unprecedented binding mechanism shown here for LDI has likely evolved in cereals from a need for effective inhibition of debranching enzymes having characteristic open active site architecture. The findings give a mechanistic rationale for the potency of LD activity regulation and provide a molecular understanding of the debranching events associated with optimal starch mobilization and utilization during germination. This study unveils a hitherto not recognized structural basis for the features endowing diversity to CTIs.

An extracellular cell-attached pullulanase confers branched α-glucan utilization in human gut Lactobacillus acidophilus

ABSTRACT Of the few predicted extracellular glycan-active enzymes, glycoside hydrolase family 13 subfamily 14 (GH13_14) pullulanases are the most common in human gut lactobacilli. These enzymes share a unique modular organization, not observed in other bacteria, featuring a catalytic module, two starch binding modules, a domain of unknown function, and a C-terminal surface layer association protein (SLAP) domain. Here, we explore the specificity of a representative of this group of pullulanases, Lactobacillus acidophilus Pul13_14 (LaPul13_14), and its role in branched α-glucan metabolism in the well-characterized Lactobacillus acidophilus NCFM, which is widely used as a probiotic. Growth experiments with L. acidophilus NCFM on starch-derived branched substrates revealed a preference for α-glucans with short branches of about two to three glucosyl moieties over amylopectin with longer branches. Cell-attached debranching activity was measurable in the presence of α-glucans but was repressed by glucose. The debranching activity is conferred exclusively by LaPul13_14 and is abolished in a mutant strain lacking a functional LaPul13_14 gene. Hydrolysis kinetics of recombinant LaPul13_14 confirmed the preference for short-branched α-glucan oligomers consistent with the growth data. Curiously, this enzyme displayed the highest catalytic efficiency and the lowest Km reported for a pullulanase. Inhibition kinetics revealed mixed inhibition by β-cyclodextrin, suggesting the presence of additional glucan binding sites besides the active site of the enzyme, which may contribute to the unprecedented substrate affinity. The enzyme also displays high thermostability and higher activity in the acidic pH range, reflecting adaptation to the physiologically challenging conditions in the human gut. IMPORTANCE Starch is one of the most abundant glycans in the human diet. Branched α-1,6-glucans in dietary starch and glycogen are nondegradable by human enzymes and constitute a metabolic resource for the gut microbiota. The role of health-beneficial lactobacilli prevalent in the human small intestine in starch metabolism remains unexplored in contrast to colonic bacterial residents. This study highlights the pivotal role of debranching enzymes in the breakdown of starchy branched α-glucan oligomers (α-limit dextrins) by human gut lactobacilli exemplified by Lactobacillus acidophilus NCFM, which is one of the best-characterized strains used as probiotics. Our data bring novel insight into the metabolic preference of L. acidophilus for α-glucans with short α-1,6-branches. The unprecedented affinity of the debranching enzyme that confers growth on these substrates reflects its adaptation to the nutrient-competitive gut ecological niche and constitutes a potential advantage in cross-feeding from human and bacterial dietary starch metabolism.