Structural basis for the exolytic activity of polysaccharide lyase family 6 alginate lyase BcAlyPL6 from human gut microbe Bacteroides clarus

Glycosaminoglycans (GAGs) and GAG-degrading enzymes have wide-ranging applications in the medical and biotechnological industries. The former are also an important nutrient source for select species of the human gut microbiota (HGM), a key player in host–microbial interactions. How GAGs are metabolized by the HGM is therefore of interest and has been extensively investigated in the model human gut microbe Bacteroides thetaiotaomicron. The presence of as-yet uncharacterized GAG-inducible genes in its genome and of related species, however, is testament to our incomplete understanding of this process. Nevertheless, it presents a potential opportunity for the discovery of additional GAG-degrading enzymes. Here, we investigated a gene of unknown function (BT_3328) from the chondroitin sulfate (CS) utilization locus of B. thetaiotaomicron. NMR and UV spectroscopic assays revealed that it encodes a novel polysaccharide lyase (PL), hereafter referred to as BtCDH, reflecting its source (B. thetaiotaomicron (Bt)) and its ability to degrade the GAGs CS, dermatan sulfate (DS), and hyaluronic acid (HA). When incubated with HA, BtCDH generated a series of unsaturated HA sugars, including Δ4,5UA-GlcNAc, Δ4,5UA-GlcNAc-GlcA-GlcNac, Δ4,5UA-[GlcNAc-GlcA]2-GlcNac, and Δ4,5UA-[GlcNAc-GlcA]3-GlcNac, as end products and hence was classed as endo-acting. A combination of genetic and biochemical assays revealed that BtCDH localizes to the cell surface of B. thetaiotaomicron where it enables extracellular GAG degradation. BtCDH homologs were also detected in several other HGM species, and we therefore propose that it represents the founding member of a new polysaccharide lyase family (PL29). The current discovery also contributes new insights into CS metabolism by the HGM.

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Structural and functional aspects of mannuronic acid–specific PL6 alginate lyase from the human gut microbe Bacteroides cellulosilyticus

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.010206 ◽

2019 ◽

Vol 294 (47) ◽

pp. 17915-17930 ◽

Cited By ~ 5

Author(s):

Emil G. P. Stender ◽

Christian Dybdahl Andersen ◽

Folmer Fredslund ◽

Jesper Holck ◽

Amalie Solberg ◽

...

Keyword(s):

Alginate Lyase ◽

Human Gut ◽

Mannuronic Acid ◽

Functional Aspects ◽

Gut Microbe

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Kin selection explains the evolution of cooperation in the gut microbiota

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2016046118 ◽

2021 ◽

Vol 118 (6) ◽

pp. e2016046118 ◽

Cited By ~ 1

Author(s):

Camille Simonet ◽

Luke McNally

Keyword(s):

Gut Microbiota ◽

Predictive Power ◽

Inclusive Fitness ◽

Evolution Of Cooperation ◽

Community Management ◽

Human Gut ◽

Full Diversity ◽

Gut Microbe ◽

Disease Understanding ◽

Key Parameter

Through the secretion of “public goods” molecules, microbes cooperatively exploit their habitat. This is known as a major driver of the functioning of microbial communities, including in human disease. Understanding why microbial species cooperate is therefore crucial to achieve successful microbial community management, such as microbiome manipulation. A leading explanation is that of Hamilton’s inclusive-fitness framework. A cooperator can indirectly transmit its genes by helping the reproduction of an individual carrying similar genes. Therefore, all else being equal, as relatedness among individuals increases, so should cooperation. However, the predictive power of relatedness, particularly in microbes, is surrounded by controversy. Using phylogenetic comparative analyses across the full diversity of the human gut microbiota and six forms of cooperation, we find that relatedness is predictive of the cooperative gene content evolution in gut-microbe genomes. Hence, relatedness is predictive of cooperation over broad microbial taxonomic levels that encompass variation in other life-history and ecology details. This supports the generality of Hamilton’s central insights and the relevance of relatedness as a key parameter of interest to advance microbial predictive and engineering science.

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Ruminococcus gnavus, a member of the human gut microbiome associated with Crohn’s disease, produces an inflammatory polysaccharide

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1904099116 ◽

2019 ◽

Vol 116 (26) ◽

pp. 12672-12677 ◽

Cited By ~ 54

Author(s):

Matthew T. Henke ◽

Douglas J. Kenny ◽

Chelsi D. Cassilly ◽

Hera Vlamakis ◽

Ramnik J. Xavier ◽

...

Keyword(s):

Crohn’S Disease ◽

Crohn's Disease ◽

Biosynthetic Gene Cluster ◽

Spectroscopic Studies ◽

Major Type ◽

Human Gut ◽

Toll Like Receptor 4 ◽

Gut Microbe ◽

Research Goal ◽

Inflammatory Bowel

A substantial and increasing number of human diseases are associated with changes in the gut microbiota, and discovering the molecules and mechanisms underlying these associations represents a major research goal. Multiple studies associateRuminococcus gnavus, a prevalent gut microbe, with Crohn’s disease, a major type of inflammatory bowel disease. We have found thatR. gnavussynthesizes and secretes a complex glucorhamnan polysaccharide with a rhamnose backbone and glucose sidechains. Chemical and spectroscopic studies indicated that the glucorhamnan was largely a repeating unit of five sugars with a linear backbone formed from three rhamnose units and a short sidechain composed of two glucose units. The rhamnose backbone is made from 1,2- and 1,3-linked rhamnose units, and the sidechain has a terminal glucose linked to a 1,6-glucose. This glucorhamnan potently induces inflammatory cytokine (TNFα) secretion by dendritic cells, and TNFα secretion is dependent on toll-like receptor 4 (TLR4). We also identify a putative biosynthetic gene cluster for this molecule, which has the four biosynthetic genes needed to convert glucose to rhamnose and the five glycosyl transferases needed to build the repeating pentasaccharide unit of the inflammatory glucorhamnan.

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Infection with Bacteroides Phage BV01 Alters the Host Transcriptome and Bile Acid Metabolism in a Common Human Gut Microbe

Cell Reports ◽

10.1016/j.celrep.2020.108142 ◽

2020 ◽

Vol 32 (11) ◽

pp. 108142

Author(s):

Danielle E. Campbell ◽

Lindsey K. Ly ◽

Jason M. Ridlon ◽

Ansel Hsiao ◽

Rachel J. Whitaker ◽

...

Keyword(s):

Bile Acid ◽

Acid Metabolism ◽

Bile Acid Metabolism ◽

Human Gut ◽

Gut Microbe

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Gut Microbiota Metabolism and Interaction with Food Components

International Journal of Molecular Sciences ◽

10.3390/ijms21103688 ◽

2020 ◽

Vol 21 (10) ◽

pp. 3688 ◽

Cited By ~ 2

Author(s):

Pamela Vernocchi ◽

Federica Del Chierico ◽

Lorenza Putignani

Keyword(s):

Gut Microbiota ◽

Disease Onset ◽

Human Gut ◽

Food Components ◽

Host Health ◽

Wide Variability ◽

Gut Microbe ◽

A Cell ◽

Health And Disease ◽

Biochemical Mechanisms

The human gut contains trillions of microbes that play a central role in host biology, including the provision of key nutrients from the diet. Food is a major source of precursors for metabolite production; in fact, diet modulates the gut microbiota (GM) as the nutrients, derived from dietary intake, reach the GM, affecting both the ecosystem and microbial metabolic profile. GM metabolic ability has an impact on human nutritional status from childhood. However, there is a wide variability of dietary patterns that exist among individuals. The study of interactions with the host via GM metabolic pathways is an interesting field of research in medicine, as microbiota members produce myriads of molecules with many bioactive properties. Indeed, much evidence has demonstrated the importance of metabolites produced by the bacterial metabolism from foods at the gut level that dynamically participate in various biochemical mechanisms of a cell as a reaction to environmental stimuli. Hence, the GM modulate homeostasis at the gut level, and the alteration in their composition can concur in disease onset or progression, including immunological, inflammatory, and metabolic disorders, as well as cancer. Understanding the gut microbe–nutrient interactions will increase our knowledge of how diet affects host health and disease, thus enabling personalized therapeutics and nutrition.

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Biochemical Characterization and Elucidation of Action Pattern of a Novel Polysaccharide Lyase 6 Family Alginate Lyase from Marine Bacterium Flammeovirga sp. NJ-04

Marine Drugs ◽

10.3390/md17060323 ◽

2019 ◽

Vol 17 (6) ◽

pp. 323 ◽

Cited By ~ 7

Author(s):

Qian Li ◽

Fu Hu ◽

Benwei Zhu ◽

Yun Sun ◽

Zhong Yao

Keyword(s):

Biochemical Characterization ◽

Alginate Lyase ◽

Ionization Mass ◽

Action Pattern ◽

Polysaccharide Lyase ◽

Glycosidic Bonds ◽

Alginate Oligosaccharides ◽

Degradation Pattern ◽

Alginate Lyases ◽

Layer Chromatography

Alginate lyases have been widely used to prepare alginate oligosaccharides in food, agricultural, and medical industries. Therefore, discovering and characterizing novel alginate lyases with excellent properties has drawn increasing attention. Herein, a novel alginate lyase FsAlyPL6 of Polysaccharide Lyase (PL) 6 family is identified and biochemically characterized from Flammeovirga sp. NJ-04. It shows highest activity at 45 °C and could retain 50% of activity after being incubated at 45 °C for 1 h. The Thin-Layer Chromatography (TLC) and Electrospray Ionization Mass Spectrometry (ESI-MS) analysis indicates that FsAlyPL6 endolytically degrades alginate polysaccharide into oligosaccharides ranging from monosaccharides to pentasaccharides. In addition, the action pattern of the enzyme is also elucidated and the result suggests that FsAlyPL6 could recognize tetrasaccharide as the minimal substrate and cleave the glycosidic bonds between the subsites of −1 and +3. The research provides extended insights into the substrate recognition and degradation pattern of PL6 alginate lyases, which may further expand the application of alginate lyases.

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Ascertaining the biochemical function of an essential pectin methylesterase in the gut microbe Bacteroides thetaiotaomicron

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.014974 ◽

2020 ◽

Vol 295 (52) ◽

pp. 18625-18637

Author(s):

Cheng-Jie Duan ◽

Arnaud Baslé ◽

Marcelo Visona Liberato ◽

Joseph Gray ◽

Sergey A. Nepogodiev ◽

...

Keyword(s):

Gut Microbiota ◽

Critical Role ◽

Pectin Methylesterase ◽

Bacteroides Thetaiotaomicron ◽

Human Gut ◽

Human Gut Microbiota ◽

New Family ◽

Gut Microbe ◽

Dietary Nutrient ◽

Helical Proteins

Pectins are a major dietary nutrient source for the human gut microbiota. The prominent gut microbe Bacteroides thetaiotaomicron was recently shown to encode the founding member (BT1017) of a new family of pectin methylesterases essential for the metabolism of the complex pectin rhamnogalacturonan-II (RG-II). However, biochemical and structural knowledge of this family is lacking. Here, we showed that BT1017 is critical for the metabolism of an RG-II–derived oligosaccharide ΔBT1017oligoB generated by a BT1017 deletion mutant (ΔBT1017) during growth on carbohydrate extract from apple juice. Structural analyses of ΔBT1017oligoB using a combination of enzymatic, mass spectrometric, and NMR approaches revealed that it is a bimethylated nonaoligosaccharide (GlcA-β1,4-(2-O-Me-Xyl-α1,3)-Fuc-α1,4-(GalA-β1,3)-Rha-α1,3-Api-β1,2-(Araf-α1,3)-(GalA-α1,4)-GalA) containing components of the RG-II backbone and its side chains. We showed that the catalytic module of BT1017 adopts an α/β-hydrolase fold, consisting of a central twisted 10-stranded β-sheet sandwiched by several α-helices. This constitutes a new fold for pectin methylesterases, which are predominantly right-handed β-helical proteins. Bioinformatic analyses revealed that the family is dominated by sequences from prominent genera of the human gut microbiota, including Bacteroides and Prevotella. Our re-sults not only highlight the critical role played by this family of enzymes in pectin metabolism but also provide new insights into the molecular basis of the adaptation of B. thetaiotaomicron to the human gut.

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