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, 95 (2), 209-30

Molecular Details of a Starch Utilization Pathway in the Human Gut Symbiont Eubacterium Rectale

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Molecular Details of a Starch Utilization Pathway in the Human Gut Symbiont Eubacterium Rectale

Darrell W Cockburn et al. Mol Microbiol.

Abstract

Eubacterium rectale is a prominent human gut symbiont yet little is known about the molecular strategies this bacterium has developed to acquire nutrients within the competitive gut ecosystem. Starch is one of the most abundant glycans in the human diet, and E. rectale increases in vivo when the host consumes a diet rich in resistant starch, although it is not a primary degrader of this glycan. Here we present the results of a quantitative proteomics study in which we identify two glycoside hydrolase 13 family enzymes, and three ABC transporter solute-binding proteins that are abundant during growth on starch and, we hypothesize, work together at the cell surface to degrade starch and capture the released maltooligosaccharides. EUR_21100 is a multidomain cell wall anchored amylase that preferentially targets starch polysaccharides, liberating maltotetraose, whereas the membrane-associated maltogenic amylase EUR_01860 breaks down maltooligosaccharides longer than maltotriose. The three solute-binding proteins display a range of glycan-binding specificities that ensure the capture of glucose through maltoheptaose and some α1,6-branched glycans. Taken together, we describe a pathway for starch utilization by E. rectale DSM 17629 that may be conserved among other starch-degrading Clostridium cluster XIVa organisms in the human gut.

Figures

Figure 1
Figure 1. Growth of E. rectale DSM 17629 on starch and maltooligosaccharides
Bacteria were grown in 96 well plates in a 37°C anaerobic chamber with the OD600 automatically recorded every 20 min. YCFA media was supplemented with 2 mg mL−1 of the listed carbohydrates as the sole carbon source. A. Growth on starches (APM, maize amylopectin, APP, potato amylopectin, H7, Hylon VII); water is displayed as a negative control. Note that autoclaved H7 is a turbid suspension that was not blanked in this set of experiments. B. Growth on maltooligosaccharides (G3, maltotriose; G7, maltoheptaose; aCD, α-cyclodextrin; bCD, β-cyclodextrin; GM, glucosyl-α1,6-maltotriose; GMM, glucosyl-α1,6-maltotriose-α1,6-maltotriose).
Figure 2
Figure 2. Genomic neighborhood of the genes encoding the differentially expressed proteins detected during growth on starch
Each gene is drawn to scale as a rectangle with its orientation indicated by the closed triangle. Genes have been color coded according to annotated function (IMG: img.jgi.doe.gov), or labeled: efflux protein MATE, multi-antimicrobial efflux protein pump; DMT permease, drug metabolite transporter/permease; RNRII, ribonucleotide reductase class II activase subunit; TrxB, thioredoxin; ABC ATPase, ABC transporter ATP-binding protein.
Figure 3
Figure 3. Domain organization of EUR_21100 and EUR_01860
EUR_21100 is multimodular with several N-terminal domains that resemble CBMs (via BLAST search against isolated CBM sequences). Between the putative CBMs and the catalytic module (also GH13) there is a region of unknown function that does not resemble any previously characterized domains. At the C-terminal end there is a putative cell wall anchoring motif that likely attaches this protein to the peptidoglycan layer. In contrast, EUR_01860 consists of a single module, a GH13 catalytic domain, with an N-terminal transmembrane helix. Arrows indicate regions of the proteins that were cloned for biochemical characterization.
Figure 4
Figure 4. Thin layer chromatography of enzyme hydrolysis products
Each reaction was analyzed at four time points: 0, 10, 20, 30 min. Lanes denoted C are TLC controls consisting of 2 nmol each of glucose, maltose and maltotriose. A) EUR_01860 hydrolysis of maltooligosaccharides. B) EUR_01860 hydrolysis of GMM and BCD. Lane Pan is a 5 nmol panose standard, and lane GM is a 5 nmol glucose-α1,6-maltotriose (GM) standard. C) EUR_21100 hydrolysis of amylopectin. Lane G4 is a 5 nmol maltotetraose standard. D) EUR_21100 hydrolysis of maltooligosaccharides. Abbreviations are as follows: β-cyclodextrin (BCD), glucose (Glc), maltose (G2), maltotriose (G3), maltotetraose (G4), maltopentaose (G5), maltohexaose (G6), maltoheptaose (G7), glucose-α(1,6)-maltotriose (GM), glucose-α(1,6)-maltotriose-α(1,6)-maltotriose (GMM), panose (Pan).
Figure 5
Figure 5. Structure of the maltodextrin-binding protein EUR_01830
A. Omit maps (σ = 3.0) displaying representative electron density for maltotriose and acarbose. B. Overlay of the structures of EUR_01830 complexed with acarbose (grey ribbon) and with maltotriose (blue ribbon). Acarbose is displayed as black sticks and maltotriose as blue sticks. The aromatic residues that cradle the maltodextrins are shown as sticks. C. Close-up of the binding pocket of EUR_01830. Direct hydrogen-bonding interactions between the protein and maltrotriose/acarbose are displayed as dashed lines. K91 and D384 have been omitted for clarity. Waters involved in water-mediated hydrogen-bonds are shown as spheres. D. Close-up end view of the hydrogen-bonding network that confers recognition of the reducing end sugar. Acarbose and the relevant amino acids are shown as sticks, while waters involved in water-mediated hydrogen bonds are shown as red spheres. Dashed lines indicate interactions within hydrogen bonding distance, and distances are displayed in Å. Here E246, though unlikely to contribute to hydrogen-bonding, is shown for reference. E. Close-up side view of EUR_01830 binding pocket demonstrating the manner in which E246, S87 and D88 may occlude the front of the binding pocket. The loop of residues 242–248 is shown in black ribbon, and E246, S87 and D88 are shown in black sticks. Note that S87 was observed in two conformations in the crystal structure. F. Superposition of the coordinates of the acarbose-bound EUR_01830 (grey) and MalX of S. pneumoniae (blue, pdb 2XD3). The C-terminal strand-loop-helix comprising residues 236–259 of EUR_01830 is displayed in black, and the corresponding helix-loop-helix comprising residues 240–266 of MalX is displayed in dark blue. Acarbose is displayed as black sticks and maltoheptaose is displayed as blue sticks.
Figure 6
Figure 6. Model of starch digestion and maltooligosaccharide uptake by Eubacterium rectale
The multidomain EUR_21100 is tethered to the peptidoglycan layer, and may anchor the bacterium to starch via its five N-terminal CBMs (predicted CBM family number as indicated, U= unknown domain). Released oligosaccharides may be targeted by EUR_01830 and EUR_31480 for import into the cell. The location of EUR_01860 is unknown. This maltogenic amylase produces some glucose, as suggested by its breakdown of maltotriose, and its presence on the surface of the cell may generate glucose and maltose that can be captured by EUR_01240 and EUR_31480 respective

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