Arnaud Baslé

Glycan complexity dictates microbial resource allocation in the large intestine

The structure of the human gut microbiota is controlled primarily through the degradation of complex dietary carbohydrates, but the extent to which carbohydrate breakdown products are shared between members of the microbiota is unclear. We show here, using xylan as a model, that sharing the breakdown products of complex carbohydrates by key members of the microbiota, such as Bacteroides ovatus, is dependent on the complexity of the target glycan. Characterization of the extensive xylan degrading apparatus expressed by B. ovatus reveals that the breakdown of the polysaccharide by the human gut microbiota is significantly more complex than previous models suggested, which were based on the deconstruction of xylans containing limited monosaccharide side chains. Our report presents a highly complex and dynamic xylan degrading apparatus that is fine-tuned to recognize the different forms of the polysaccharide presented to the human gut microbiota.

Complex pectin metabolism by gut bacteria reveals novel catalytic functions

The metabolism of carbohydrate polymers drives microbial diversity in the human gut microbiota. It is unclear, however, whether bacterial consortia or single organisms are required to depolymerize highly complex glycans. Here we show that the gut bacterium Bacteroides thetaiotaomicron uses the most structurally complex glycan known: the plant pectic polysaccharide rhamnogalacturonan-II, cleaving all but 1 of its 21 distinct glycosidic linkages. The deconstruction of rhamnogalacturonan-II side chains and backbone are coordinated to overcome steric constraints, and the degradation involves previously undiscovered enzyme families and catalytic activities. The degradation system informs revision of the current structural model of rhamnogalacturonan-II and highlights how individual gut bacteria orchestrate manifold enzymes to metabolize the most challenging glycan in the human diet.

How Nature Can Exploit Nonspecific Catalytic and Carbohydrate Binding Modules to Create Enzymatic Specificity

Noncatalytic carbohydrate binding modules (CBMs) are components of glycoside hydrolases that attack generally inaccessible substrates. CBMs mediate a two- to fivefold elevation in the activity of endo-acting enzymes, likely through increasing the concentration of the appended enzymes in the vicinity of the substrate. The function of CBMs appended to exo-acting glycoside hydrolases is unclear because their typical endo-binding mode would not fulfill a targeting role. Here we show that the Bacillus subtilis exo-acting β-fructosidase SacC, which specifically hydrolyses levan, contains the founding member of CBM family 66 (CBM66). The SacC-derived CBM66 (BsCBM66) targets the terminal fructosides of the major fructans found in nature. The crystal structure of BsCBM66 in complex with ligands reveals extensive interactions with the terminal fructose moiety (Fru-3) of levantriose but only limited hydrophobic contacts with Fru-2, explaining why the CBM displays broad specificity. Removal of BsCBM66 from SacC results in a ∼100-fold reduction in activity against levan. The truncated enzyme functions as a nonspecific β-fructosidase displaying similar activity against β-2,1– and β-2,6–linked fructans and their respective fructooligosaccharides. Conversely, appending BsCBM66 to BT3082, a nonspecific β-fructosidase from Bacteroides thetaiotaomicron, confers exolevanase activity on the enzyme. We propose that BsCBM66 confers specificity for levan, a branched fructan, through an “avidity” mechanism in which the CBM and the catalytic module target the termini of different branches of the same polysaccharide molecule. This report identifies a unique mechanism by which CBMs modulate enzyme function, and shows how specificity can be tailored by integrating nonspecific catalytic and binding modules into a single enzyme.

A scissor blade-like closing mechanism implicated in transmembrane signaling in a Bacteroides hybrid two-component system

Signaling across the membrane in response to extracellular stimuli is essential for survival of all cells. In bacteria, responses to environmental changes are predominantly mediated by two-component systems, which are typically composed of a membrane-spanning sensor histidine kinase and a cytoplasmic response regulator. In the human gut symbiont Bacteroides thetaiotaomicron, hybrid two-component systems are a key part of the bacterium’s ability to sense and degrade complex carbohydrates in the gut. Here, we identify the activating ligand of the hybrid two-component system, BT4663, which controls heparin and heparan sulfate acquisition and degradation in this prominent gut microbe, and report the crystal structure of the extracellular sensor domain in both apo and ligand-bound forms. Current models for signal transduction across the membrane involve either a piston-like or rotational displacement of the transmembrane helices to modulate activity of the linked cytoplasmic kinases. The structures of the BT4663 sensor domain reveal a significant conformational change in the homodimer on ligand binding, which results in a scissor-like closing of the C-termini of each protomer. We propose this movement activates the attached intracellular kinase domains and represents an allosteric mechanism for bacterial transmembrane signaling distinct from previously described models, thus expanding our understanding of signal transduction across the membrane, a fundamental requirement in many important biological processes.

Copper-Binding Properties and Structures of Methanobactins from Methylosinus trichosporium OB3b

Methanobactins (mbs) are a class of copper-binding peptides produced by aerobic methane oxidizing bacteria (methanotrophs) that have been linked to the substantial copper needs of these environmentally important microorganisms. The only characterized mbs are those from Methylosinus trichosporium OB3b and Methylocystis strain SB2. M. trichosporium OB3b produces a second mb (mb-Met), which is missing the C-terminal Met residue from the full-length form (FL-mb). The as-isolated copper-loaded mbs bind Cu(I). The absence of the Met has little influence on the structure of the Cu(I) site, and both molecules mediate switchover from the soluble iron methane mono-oxygenase to the particulate copper-containing enzyme in M. trichosporium OB3b cells. Cu(II) is reduced in the presence of the mbs under our experimental conditions, and the disulfide plays no role in this process. The Cu(I) affinities of these molecules are extremely high with values of (6-7) × 10(20) M(-1) determined at pH ≥ 8.0. The affinity for Cu(I) is 1 order of magnitude lower at pH 6.0. The reduction potentials of copper-loaded FL-mb and mb-Met are 640 and 590 mV respectively, highlighting the strong preference for Cu(I) and indicating different Cu(II) affinities for the two forms. Cleavage of the disulfide bridge results in a decrease in the Cu(I) affinity to ∼9 × 10(18) M(-1) at pH 7.5. The two thiolates can also bind Cu(I), albeit with much lower affinity (∼ 3 × 10(15) M(-1) at pH 7.5). The high affinity of mbs for Cu(I) is consistent with a physiological role in copper uptake and protection.

Variations in methanobactin structure influences copper utilization by methane-oxidizing bacteria

Methane-oxidizing bacteria are nature’s primary biological mechanism for suppressing atmospheric levels of the second-most important greenhouse gas via methane monooxygenases (MMOs). The copper-containing particulate enzyme is the most widespread and efficient MMO. Under low-copper conditions methane-oxidizing bacteria secrete the small copper-binding peptide methanobactin (mbtin) to acquire copper, but how variations in the structures of mbtins influence copper metabolism and species selection are unknown. Methanobactins have been isolated from Methylocystis strains M and hirsuta CSC1, organisms that can switch to using an iron-containing soluble MMO when copper is limiting, and the nonswitchover Methylocystis rosea. These mbtins are shorter, and have different amino acid compositions, than the characterized mbtin from Methylosinus trichosporium OB3b. A coordinating pyrazinedione ring in the Methylocystis mbtins has little influence on the Cu(I) site structure. The Methylocystis mbtins have a sulfate group that helps stabilize the Cu(I) forms, resulting in affinities of approximately 1021 M-1. The Cu(II) affinities vary over three orders of magnitude with reduction potentials covering approximately 250 mV, which may dictate the mechanism of intracellular copper release. Copper uptake and the switchover from using the iron-containing soluble MMO to the copper-containing particulate enzyme is faster when mediated by the native mbtin, suggesting that the amino acid sequence is important for the interaction of mbtins with receptors. The differences in structures and properties of mbtins, and their influence on copper utilization by methane-oxidizing bacteria, have important implications for the ecology and global function of these environmentally vital organisms.

Evidence that GH115 α-glucuronidase activity, which is required to degrade plant biomass, is dependent on conformational flexibility.

Background: The structure of GH115 glucuronidases that remove glucuronic acid from xylan chains is unknown. Results: Bacteroides ovatus GH115 glucuronidase is a dimeric enzyme that contains a flexible active site pocket. Conclusion: The assembly of the catalytic apparatus of the glucuronidase requires substantial conformational changes. Significance: Conformational changes are highly unusual in glycoside hydrolases. The microbial degradation of the plant cell wall is an important biological process that is highly relevant to environmentally significant industries such as the bioenergy and biorefining sectors. A major component of the wall is glucuronoxylan, a β1,4-linked xylose polysaccharide that is decorated with α-linked glucuronic and/or methylglucuronic acid (GlcA/MeGlcA). Recently three members of a glycoside hydrolase family, GH115, were shown to hydrolyze MeGlcA side chains from the internal regions of xylan, an activity that has not previously been described. Here we show that a dominant member of the human microbiota, Bacteroides ovatus, contains a GH115 enzyme, BoAgu115A, which displays glucuronoxylan α-(4-O-methyl)-glucuronidase activity. The enzyme is significantly more active against substrates in which the xylose decorated with GlcA/MeGlcA is flanked by one or more xylose residues. The crystal structure of BoAgu115A revealed a four-domain protein in which the active site, comprising a pocket that abuts a cleft-like structure, is housed in the second domain that adopts a TIM barrel-fold. The third domain, a five-helical bundle, and the C-terminal β-sandwich domain make inter-chain contacts leading to protein dimerization. Informed by the structure of the enzyme in complex with GlcA in its open ring form, in conjunction with mutagenesis studies, the potential substrate binding and catalytically significant amino acids were identified. Based on the catalytic importance of residues located on a highly flexible loop, the enzyme is required to undergo a substantial conformational change to form a productive Michaelis complex with glucuronoxylan.

A four-helix bundle stores copper for methane oxidation

Methane-oxidizing bacteria (methanotrophs) require large quantities of copper for the membrane-bound (particulate) methane monooxygenase. Certain methanotrophs are also able to switch to using the iron-containing soluble methane monooxygenase to catalyse methane oxidation, with this switchover regulated by copper. Methane monooxygenases are nature’s primary biological mechanism for suppressing atmospheric levels of methane, a potent greenhouse gas. Furthermore, methanotrophs and methane monooxygenases have enormous potential in bioremediation and for biotransformations producing bulk and fine chemicals, and in bioenergy, particularly considering increased methane availability from renewable sources and hydraulic fracturing of shale rock. Here we discover and characterize a novel copper storage protein (Csp1) from the methanotroph Methylosinus trichosporium OB3b that is exported from the cytosol, and stores copper for particulate methane monooxygenase. Csp1 is a tetramer of four-helix bundles with each monomer binding up to 13 Cu(I) ions in a previously unseen manner via mainly Cys residues that point into the core of the bundle. Csp1 is the first example of a protein that stores a metal within an established protein-folding motif. This work provides a detailed insight into how methanotrophs accumulate copper for the oxidation of methane. Understanding this process is essential if the wide-ranging biotechnological applications of methanotrophs are to be realized. Cytosolic homologues of Csp1 are present in diverse bacteria, thus challenging the dogma that such organisms do not use copper in this location.

A human mitochondrial poly(A) polymerase mutation reveals the complexities of post-transcriptional mitochondrial gene expression

The p.N478D missense mutation in human mitochondrial poly(A) polymerase (mtPAP) has previously been implicated in a form of spastic ataxia with optic atrophy. In this study, we have investigated fibroblast cell lines established from family members. The homozygous mutation resulted in the loss of polyadenylation of all mitochondrial transcripts assessed; however, oligoadenylation was retained. Interestingly, this had differential effects on transcript stability that were dependent on the particular species of transcript. These changes were accompanied by a severe loss of oxidative phosphorylation complexes I and IV, and perturbation of de novo mitochondrial protein synthesis. Decreases in transcript polyadenylation and in respiratory chain complexes were effectively rescued by overexpression of wild-type mtPAP. Both mutated and wild-type mtPAP localized to the mitochondrial RNA-processing granules thereby eliminating mislocalization as a cause of defective polyadenylation. In vitro polyadenylation assays revealed severely compromised activity by the mutated protein, which generated only short oligo(A) extensions on RNA substrates, irrespective of RNA secondary structure. The addition of LRPPRC/SLIRP, a mitochondrial RNA-binding complex, enhanced activity of the wild-type mtPAP resulting in increased overall tail length. The LRPPRC/SLIRP effect although present was less marked with mutated mtPAP, independent of RNA secondary structure. We conclude that (i) the polymerase activity of mtPAP can be modulated by the presence of LRPPRC/SLIRP, (ii) N478D mtPAP mutation decreases polymerase activity and (iii) the alteration in poly(A) length is sufficient to cause dysregulation of post-transcriptional expression and the pathogenic lack of respiratory chain complexes.

A Novel, Noncatalytic Carbohydrate-Binding Module Displays Specificity for Galactose-Containing Polysaccharides Through Calcium-Mediated Oligomerization.

The enzymic degradation of plant cell walls plays a central role in the carbon cycle and is of increasing environmental and industrial significance. The catalytic modules of enzymes that catalyze this process are generally appended to noncatalytic carbohydrate-binding modules (CBMs). CBMs potentiate the rate of catalysis by bringing their cognate enzymes into intimate contact with the target substrate. A powerful plant cell wall-degrading system is the Clostridium thermocellum multienzyme complex, termed the “cellulosome.” Here, we identify a novel CBM (CtCBM62) within the large C. thermocellum cellulosomal protein Cthe_2193 (defined as CtXyl5A), which establishes a new CBM family. Phylogenetic analysis of CBM62 members indicates that a circular permutation occurred within the family. CtCBM62 binds to d-galactose and l-arabinopyranose in either anomeric configuration. The crystal structures of CtCBM62, in complex with oligosaccharides containing α- and β-galactose residues, show that the ligand-binding site in the β-sandwich protein is located in the loops that connect the two β-sheets. Specificity is conferred through numerous interactions with the axial O4 of the target sugars, a feature that distinguishes galactose and arabinose from the other major sugars located in plant cell walls. CtCBM62 displays tighter affinity for multivalent ligands compared with molecules containing single galactose residues, which is associated with precipitation of these complex carbohydrates. These avidity effects, which confer the targeting of polysaccharides, are mediated by calcium-dependent oligomerization of the CBM.