Akkermansia muciniphila is a human microbial symbiont residing in the mucosal layer of the large intestine. Its main carbon source is the highly heterogeneous mucin glycoprotein, and it uses an array of carbohydrate-active enzymes and sulfatases to access this complex energy source. Here we describe the biochemical characterization of 54 glycoside hydrolases, 11 sulfatases and 1 polysaccharide lyase from A.
View Article and Find Full Text PDFSeveral bacterial species initially colonise the infant gut, but are outcompeted. Human milk oligosaccharides (HMOs) in breast milk create an environment for Bifidobacterium to flourish. Laursen and Roager recently showed a clear link between breast milk and the dominance of Bifidobacterium longum subsp.
View Article and Find Full Text PDFThe modification of proteins by N-glycans is ubiquitous to most organisms and they have multiple biological functions, including protecting the adjoining protein from degradation and facilitating communication or adhesion between cells, for example. Microbes have evolved CAZymes to deconstruct different types of N-glycans and some of these have been characterised from microbes originating from different niches, both commensals and pathogens. The specificity of these CAZymes provides clues as to how different microbes breakdown these substrates and possibly cross-feed them.
View Article and Find Full Text PDFThe major nutrients available to the human colonic microbiota are complex glycans derived from the diet. To degrade this highly variable mix of sugar structures, gut microbes have acquired a huge array of different carbohydrate-active enzymes (CAZymes), predominantly glycoside hydrolases, many of which have specificities that can be exploited for a range of different applications. Plant -glycans are prevalent on proteins produced by plants and thus components of the diet, but the breakdown of these complex molecules by the gut microbiota has not been explored.
View Article and Find Full Text PDFDeciphering how enzymes interact, modify, and recognize carbohydrates has long been a topic of interest in academic, pharmaceutical, and industrial research. Carbohydrate-binding modules (CBMs) are noncatalytic globular protein domains attached to carbohydrate-active enzymes that strengthen enzyme affinity to substrates and increase enzymatic efficiency via targeting and proximity effects. CBMs are considered auspicious for various biotechnological purposes in textile, food, and feed industries, representing valuable tools in basic science research and biomedicine.
View Article and Find Full Text PDFThe bacterial heterodimeric ATP-binding cassette (ABC) multidrug exporter PatAB has a critical role in conferring antibiotic resistance in multidrug-resistant infections by Streptococcus pneumoniae. As with other heterodimeric ABC exporters, PatAB contains two transmembrane domains that form a drug translocation pathway for efflux and two nucleotide-binding domains that bind ATP, one of which is hydrolysed during transport. The structural and functional elements in heterodimeric ABC multidrug exporters that determine interactions with drugs and couple drug binding to nucleotide hydrolysis are not fully understood.
View Article and Find Full Text PDFChanges in human IgG galactosylation and sialylation have been associated with several inflammatory diseases which are a major burden on the health care system. A large body of work on well-established glycomic and glycopeptidomic assays has repeatedly demonstrated inflammation-induced changes in IgG glycosylation. However, these assays are usually based on specialized analytical instrumentation which could be considered a technical barrier for uptake by some laboratories.
View Article and Find Full Text PDFThe thick mucus layer of the gut provides a barrier to infiltration of the underlying epithelia by both the normal microbiota and enteric pathogens. Some members of the microbiota utilise mucin glycoproteins as a nutrient source, but a detailed understanding of the mechanisms used to breakdown these complex macromolecules is lacking. Here we describe the discovery and characterisation of endo-acting enzymes from prominent mucin-degrading bacteria that target the polyLacNAc structures within oligosaccharide side chains of both animal and human mucins.
View Article and Find Full Text PDFGlycans are the major carbon sources available to the human colonic microbiota. Numerous N-glycosylated proteins are found in the human gut, from both dietary and host sources, including immunoglobulins such as IgA that are secreted into the intestine at high levels. Here, we show that many mutualistic gut Bacteroides spp.
View Article and Find Full Text PDFA putative GH35 β-galactosidase gene from the mucin-degrading bacterium Akkermansia muciniphila was successfully cloned and further investigated. The recombinant enzyme with the molecular mass of 74 kDa was purified to homogeneity and biochemically characterised. The optimum temperature of the enzyme was 42 °C, and the optimum pH was determined to be pH 3.
View Article and Find Full Text PDFReaction centre/light harvesting proteins such as the RCLH1X complex from Rhodobacter sphaeroides carry out highly quantum-efficient conversion of solar energy through ultrafast energy transfer and charge separation, and these pigment-proteins have been incorporated into biohybrid photoelectrochemical cells for a variety of applications. In this work we demonstrate that, despite not being able to support normal photosynthetic growth of Rhodobacter sphaeroides, an engineered variant of this RCLH1X complex lacking the PufX protein and with an enlarged light harvesting antenna is unimpaired in its capacity for photocurrent generation in two types of bio-photoelectrochemical cells. Removal of PufX also did not impair the ability of the RCLH1 complex to act as an acceptor of energy from synthetic light harvesting quantum dots.
View Article and Find Full Text PDFThe enzymatic degradation of plant cell walls is an important biological process of increasing environmental and industrial significance. Xylan, a major component of the plant cell wall, consists of a backbone of β-1,4-xylose (Xylp) units that are often decorated with arabinofuranose (Araf) side chains. A large penta-modular enzyme, CtXyl5A, was shown previously to specifically target arabinoxylans.
View Article and Find Full Text PDFCellvibrio japonicusis a Gram-negative soil bacterium that is primarily known for its ability to degrade plant cell wall polysaccharides through utilization of an extensive repertoire of carbohydrate-active enzymes. Several putative chitin-degrading enzymes are also found among these carbohydrate-active enzymes, such as chitinases, chitobiases, and lytic polysaccharide monooxygenases (LPMOs). In this study, we have characterized the chitin-active LPMO,CjLPMO10A, a tri-modular enzyme containing a catalytic family AA10 LPMO module, a family 5 chitin-binding module, and a C-terminal unclassified module of unknown function.
View Article and Find Full Text PDFType A non-catalytic carbohydrate-binding modules (CBMs), exemplified by CtCBM3acipA, are widely believed to specifically target crystalline cellulose through entropic forces. Here we have tested the hypothesis that type A CBMs can also bind to xyloglucan (XG), a soluble β-1,4-glucan containing α-1,6-xylose side chains. CtCBM3acipA bound to xyloglucan in cell walls and arrayed on solid surfaces.
View Article and Find Full Text PDFMicrobial depolymerization of plant cell walls contributes to global carbon balance and is a critical component of renewable energy. The genomes of lignocellulose degrading microorganisms encode diverse classes of carbohydrate modifying enzymes, although currently there is a paucity of knowledge on the role of these proteins in vivo. We report the comprehensive analysis of the cellulose degradation system in the saprophytic bacterium Cellvibrio japonicus.
View Article and Find Full Text PDFPhotosynthesis in purple bacteria is performed by pigment-protein complexes that are closely packed within specialized intracytoplasmic membranes. Here we report on the influence of carotenoid composition on the organization of RC-LH1 pigment-protein complexes in intact membranes and cells of Rhodobacter sphaeroides. Mostly dimeric RC-LH1 complexes could be isolated from strains expressing native brown carotenoids when grown under illuminated/anaerobic conditions, or from strains expressing green carotenoids when grown under either illuminated/anaerobic or dark/semiaerobic conditions.
View Article and Find Full Text PDFIn purple bacteria of the genus Rhodobacter (Rba.), an LH1 antenna complex surrounds the photochemical reaction centre (RC) with a PufX protein preventing the LH1 complex from completely encircling the RC. In membranes of Rba.
View Article and Find Full Text PDFThe innately highly efficient light-powered separation of charge that underpins natural photosynthesis can be exploited for applications in photoelectrochemistry by coupling nanoscale protein photoreaction centers to man-made electrodes. Planar photoelectrochemical cells employing purple bacterial reaction centers have been constructed that produce a direct current under continuous illumination and an alternating current in response to discontinuous illumination. The present work explored the basis of the open-circuit voltage (V(OC)) produced by such cells with reaction center/antenna (RC-LH1) proteins as the photovoltaic component.
View Article and Find Full Text PDFPhotochemical charge separation in isolated reaction center-light harvesting 1 (RC-LH1) complexes from Rhodobacter sphaeroides was examined using time-resolved mid-infrared pump-probe spectroscopy. Absorption difference spectra were recorded between 1760 and 1610 cm(-1) with subpicosecond time resolution to characterize excited-state and radical pair dynamics in these complexes, via the induced absorption changes in the keto carbonyl modes of the bacteriochlorophylls and bacteriopheophytins. Experiments on RC-LH1 complexes with and without the polypeptide PufX show that its presence is required to achieve generation of the radical pair P(+)Q(A)(-) under mildly reducing conditions.
View Article and Find Full Text PDFIn well-characterised species of the Rhodobacter (Rba.) genus of purple photosynthetic bacteria it is known that the photochemical reaction centre (RC) is intimately-associated with an encircling LH1 antenna pigment protein, and this LH1 antenna is prevented from completely surrounding the RC by a single copy of the PufX protein. In Rba.
View Article and Find Full Text PDFThe relationship between membrane protein structure and thermal stability has been examined in the reaction centre from the bacterium Rhodobacter sphaeroides, a complex membrane protein comprising three polypeptide chains and 10 cofactors. The core of this protein exhibits an approximate twofold symmetry, the cofactors being held in two membrane-spanning branches by two polypeptides, termed L and M, that have very similar folds. In assays of the thermal stability of wild-type and mutant reaction centres embedded in the native bilayer membrane, replacement of a Phe at position 197 of the M polypeptide by His produced an increase in stability, whereas an opposing replacement of His by Phe at the symmetrical position 168 of the L-polypeptide produced a decrease in stability.
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