Structure- and context-based analysis of the GxGYxYP family reveals a new putative class of glycoside hydrolase.

BackgroundGut microbiome metagenomics has revealed many protein families and domains found largely or exclusively in that environment. Proteins containing the GxGYxYP domain are over-represented in the gut microbiota, and are found in Polysaccharide Utilization Loci in the gut symbiont Bacteroides thetaiotaomicron, suggesting their involvement in polysaccharide metabolism, but little else is known of the function of this domain.ResultsGenomic context and domain architecture analyses support a role for the GxGYxYP domain in carbohydrate metabolism. Sparse occurrences in eukaryotes are the result of lateral gene transfer. The structure of the GxGYxYP domain-containing protein encoded by the BT2193 locus reveals two structural domains, the first composed of three divergent repeats with no recognisable homology to previously solved structures, the second a more familiar seven-stranded β/α barrel. Structure-based analyses including conservation mapping localise a presumed functional site to a cleft between the two domains of BT2193. Matching to a catalytic site template from a GH9 cellulase and other analyses point to a putative catalytic triad composed of Glu272, Asp331 and Asp333.ConclusionsWe suggest that GxGYxYP-containing proteins constitute a novel glycoside hydrolase family of as yet unknown specificity

Synergistic interaction of the cellulosome integrating protein (CipA) from Clostridium thermocellum with a cellulosomal endoglucanase

AbstractActivity of a cellulosomal endoglucanase (endoglucanase E; EGE) from Clostridium thermocellum against two crystalline forms of cellulose was enhanced by combination with the cellulosome integrating protein (CipA), but CipA did not enhance EGE activity against amorphous cellulose, even though it was able to bind to it. Similarly, CipA added in trans to genetically truncated EGE that was unable to combine with it nevertheless enhanced EGE activity against crystalline cellulose. These results indicate that the CipA cellulose binding domain does not mediate an increase in activity solely by bringing the catalytic subunits of the cellulosome complex into intimate contact with the substrate

Complexity of the Ruminococcus flavefaciens FD-1 cellulosome reflects an expansion of family-related protein-protein interactions

This work was supported in part by the European Union, Area NMP.2013.1.1–2: Self-assembly of naturally occurring nanosystems: CellulosomePlus Project number: 604530, and by the EU Seventh Framework Programme (FP7 2007–2013) under the WallTraC project (Grant Agreement no 263916), and BioStruct-X (grant agreement no 283570). This paper reflects the author’s views only. The European Community is not liable for any use that may be made of the information contained herein. CMGAF is also supported by Fundação para a Ciência e a Tecnologia (Lisbon, Portugal) through grants PTDC/BIA-PRO/103980/2008 and EXPL/BIA-MIC/1176/2012. EAB is also funded by a grant (No. 1349/13) from the Israel Science Foundation (ISF), Jerusalem, Israel and by a grant (No. 2013284) from the U.S.-Israel Binational Science Foundation (BSF). E.A.B. is the incumbent of The Maynard I. and Elaine Wishner Chair of Bio-organic Chemistry.Peer reviewedPublisher PD

Sulfation of Arabinogalactan Proteins Confers Privileged Nutrient Status to <i>Bacteroides plebeius</i>

The human gut microbiota (HGM) contributes to the physiology and health of its host. The health benefits provided by dietary manipulation of the HGM require knowledge of how glycans, the major nutrients available to this ecosystem, are metabolized. Arabinogalactan proteins (AGPs) are a ubiquitous feature of plant polysaccharides available to the HGM. Although the galactan backbone and galactooligosaccharide side chains of AGPs are conserved, the decorations of these structures are highly variable. Here, we tested the hypothesis that these variations in arabinogalactan decoration provide a selection mechanism for specific Bacteroides species within the HGM. The data showed that only a single bacterium, B. plebeius, grew on red wine AGP (Wi-AGP) and seaweed AGP (SW-AGP) in mono- or mixed culture. Wi-AGP thus acts as a privileged nutrient for a Bacteroides species within the HGM that utilizes marine and terrestrial plant glycans. The B. plebeius polysaccharide utilization loci (PULs) upregulated by AGPs encoded a polysaccharide lyase, located in the enzyme family GH145, which hydrolyzed Rha-Glc linkages in Wi-AGP. Further analysis of GH145 identified an enzyme with two active sites that displayed glycoside hydrolase and lyase activities, respectively, which conferred substrate flexibility for different AGPs. The AGP-degrading apparatus of B. plebeius also contained a sulfatase, BpS1_8, active on SW-AGP and Wi-AGP, which played a pivotal role in the utilization of these glycans by the bacterium. BpS1_8 enabled other Bacteroides species to access the sulfated AGPs, providing a route to introducing privileged nutrient utilization into probiotic and commensal organisms that could improve human health.Importance: Dietary manipulation of the HGM requires knowledge of how glycans available to this ecosystem are metabolized. The variable structures that decorate the core component of plant AGPs may influence their utilization by specific organisms within the HGM. Here, we evaluated the ability of Bacteroides species to utilize a marine and terrestrial AGP. The data showed that a single bacterium, B. plebeius, grew on Wi-AGP and SW-AGP in mono- or mixed culture. Wi-AGP is thus a privileged nutrient for a Bacteroides species that utilizes marine and terrestrial plant glycans. A key component of the AGP-degrading apparatus of B. plebeius is a sulfatase that conferred the ability of the bacterium to utilize these glycans. The enzyme enabled other Bacteroides species to access the sulfated AGPs, providing a route to introducing privileged nutrient utilization into probiotic and commensal organisms that could improve human health

Sulfation of Arabinogalactan Proteins Confers Privileged Nutrient Status to <i>Bacteroides plebeius</i>

The human gut microbiota (HGM) contributes to the physiology and health of its host. The health benefits provided by dietary manipulation of the HGM require knowledge of how glycans, the major nutrients available to this ecosystem, are metabolized. Arabinogalactan proteins (AGPs) are a ubiquitous feature of plant polysaccharides available to the HGM. Although the galactan backbone and galactooligosaccharide side chains of AGPs are conserved, the decorations of these structures are highly variable. Here, we tested the hypothesis that these variations in arabinogalactan decoration provide a selection mechanism for specific Bacteroides species within the HGM. The data showed that only a single bacterium, B. plebeius, grew on red wine AGP (Wi-AGP) and seaweed AGP (SW-AGP) in mono- or mixed culture. Wi-AGP thus acts as a privileged nutrient for a Bacteroides species within the HGM that utilizes marine and terrestrial plant glycans. The B. plebeius polysaccharide utilization loci (PULs) upregulated by AGPs encoded a polysaccharide lyase, located in the enzyme family GH145, which hydrolyzed Rha-Glc linkages in Wi-AGP. Further analysis of GH145 identified an enzyme with two active sites that displayed glycoside hydrolase and lyase activities, respectively, which conferred substrate flexibility for different AGPs. The AGP-degrading apparatus of B. plebeius also contained a sulfatase, BpS1_8, active on SW-AGP and Wi-AGP, which played a pivotal role in the utilization of these glycans by the bacterium. BpS1_8 enabled other Bacteroides species to access the sulfated AGPs, providing a route to introducing privileged nutrient utilization into probiotic and commensal organisms that could improve human health.Importance: Dietary manipulation of the HGM requires knowledge of how glycans available to this ecosystem are metabolized. The variable structures that decorate the core component of plant AGPs may influence their utilization by specific organisms within the HGM. Here, we evaluated the ability of Bacteroides species to utilize a marine and terrestrial AGP. The data showed that a single bacterium, B. plebeius, grew on Wi-AGP and SW-AGP in mono- or mixed culture. Wi-AGP is thus a privileged nutrient for a Bacteroides species that utilizes marine and terrestrial plant glycans. A key component of the AGP-degrading apparatus of B. plebeius is a sulfatase that conferred the ability of the bacterium to utilize these glycans. The enzyme enabled other Bacteroides species to access the sulfated AGPs, providing a route to introducing privileged nutrient utilization into probiotic and commensal organisms that could improve human health

Structure and Kinetic Investigation of Streptococcus pyogenes Family GH38 α-Mannosidase

BACKGROUND: The enzymatic hydrolysis of alpha-mannosides is catalyzed by glycoside hydrolases (GH), termed alpha-mannosidases. These enzymes are found in different GH sequence-based families. Considerable research has probed the role of higher eukaryotic "GH38" alpha-mannosides that play a key role in the modification and diversification of hybrid N-glycans; processes with strong cellular links to cancer and autoimmune disease. The most extensively studied of these enzymes is the Drosophila GH38 alpha-mannosidase II, which has been shown to be a retaining alpha-mannosidase that targets both alpha-1,3 and alpha-1,6 mannosyl linkages, an activity that enables the enzyme to process GlcNAc(Man)(5)(GlcNAc)(2) hybrid N-glycans to GlcNAc(Man)(3)(GlcNAc)(2). Far less well understood is the observation that many bacterial species, predominantly but not exclusively pathogens and symbionts, also possess putative GH38 alpha-mannosidases whose activity and specificity is unknown. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that the Streptococcus pyogenes (M1 GAS SF370) GH38 enzyme (Spy1604; hereafter SpGH38) is an alpha-mannosidase with specificity for alpha-1,3 mannosidic linkages. The 3D X-ray structure of SpGH38, obtained in native form at 1.9 A resolution and in complex with the inhibitor swainsonine (K(i) 18 microM) at 2.6 A, reveals a canonical GH38 five-domain structure in which the catalytic "-1" subsite shows high similarity with the Drosophila enzyme, including the catalytic Zn(2+) ion. In contrast, the "leaving group" subsites of SpGH38 display considerable differences to the higher eukaryotic GH38s; features that contribute to their apparent specificity. CONCLUSIONS/SIGNIFICANCE: Although the in vivo function of this streptococcal GH38 alpha-mannosidase remains unknown, it is shown to be an alpha-mannosidase active on N-glycans. SpGH38 lies on an operon that also contains the GH84 hexosaminidase (Spy1600) and an additional putative glycosidase. The activity of SpGH38, together with its genomic context, strongly hints at a function in the degradation of host N- or possibly O-glycans. The absence of any classical signal peptide further suggests that SpGH38 may be intracellular, perhaps functioning in the subsequent degradation of extracellular host glycans following their initial digestion by secreted glycosidases

An invariant distribution in static granular media

We have discovered an invariant distribution for local packing configurations in static granular media. This distribution holds in experiments for packing fractions covering most of the range from random loose packed to random close packed, for beads packed both in air and in water. Assuming only that there exist elementary cells in which the system volume is subdivided, we derive from statistical mechanics a distribution that is in accord with the observations. This universal distribution function for granular media is analogous to the Maxwell-Boltzmann distribution for molecular gasses.Comment: 4 pages 3 figure

J-Band Infrared Spectroscopy of a Sample of Brown Dwarfs Using Nirspec on Keck II

Near-infrared spectroscopic observations of a sample of very cool, low-mass objects are presented with higher spectral resolution than in any previous studies. Six of the objects are L-dwarfs, ranging in spectral class from L2 to L8/9, and the seventh is a methane or T-dwarf. These new observations were obtained during commissioning of NIRSPEC, the first high-resolution near-infrared cryogenic spectrograph for the Keck II 10-meter telescope on Mauna Kea, Hawaii. Spectra with a resolving power of R=2500 from 1.135 to 1.360 microns (approximately J-band) are presented for each source. At this resolution, a rich spectral structure is revealed, much of which is due to blending of unresolved molecular transitions. Strong lines due to neutral potassium (K I), and bands due to iron hydride (FeH) and steam (H2O) change significantly throughout the L sequence. Iron hydride disappears between L5 and L8, the steam bands deepen and the K I lines gradually become weaker but wider due to pressure broadening. An unidentified feature occurs at 1.22 microns which has a temperature dependence like FeH but has no counterpart in the available FeH opacity data. Because these objects are 3-6 magnitudes brighter in the near-infrared compared to the I-band, spectral classification is efficient. One of the objects studied (2MASSW J1523+3014) is the coolest L-dwarf discovered so far by the 2-Micron All-Sky Survey (2MASS), but its spectrum is still significantly different from the methane-dominated objects such as Gl229B or SDSS 1624+0029.Comment: New paper, Latex format, 2 figures, accepted to ApJ Letter

Measurement of [OIII] Emission in Lyman Break Galaxies

Measurements of [OIII] emission in Lyman Break galaxies (LBGs) at z>3 are presented. Four galaxies were observed with narrow-band filters using the Near-IR Camera on the Keck I 10-m telescope. A fifth galaxy was observed spectroscopically during the commissioning of NIRSPEC, the new infrared spectrometer on Keck II. The emission-line spectrum is used to place limits on the metallicity. Comparing these new measurements with others available from the literature, we find that strong oxygen emission in LBGs may suggest sub-solar metallicity for these objects. The [OIII]5007 line is also used to estimate the star formation rate (SFR) of the LBGs. The inferred SFRs are higher than those estimated from the UV continuum, and may be evidence for dust extinction.Comment: 25 pages, including 6 figures. Accepted for publication in Ap