Analysis of Keystone Enzyme in Agar Hydrolysis Provides Insight into the Degradation (of a Polysaccharide from) Red Seaweeds

Hehemann, Jan‐Hendrik; Smyth, L.; Yadav, Anuj; Vocadlo, David J.; Boraston, A.B.

doi:10.1074/jbc.m112.345645

Cited by 82 publications

(79 citation statements)

References 50 publications

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“…The catalytic domains of the porphyranases and agarase were cloned from B. plebeius DSM 17135 genomic DNA (28). Crystallization screenings were carried out in sitting-drop experiments by using commercial screens and optimizations in hanging-drop setups by grid screen expansions around initially successful hits.…”

Section: Methodsmentioning

confidence: 99%

Bacteria of the human gut microbiome catabolize red seaweed glycans with carbohydrate-active enzyme updates from extrinsic microbes

Hehemann

Kelly

Pudlo

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Humans host an intestinal population of microbes—collectively referred to as the gut microbiome—which encode the carbohydrate active enzymes, or CAZymes, that are absent from the human genome. These CAZymes help to extract energy from recalcitrant polysaccharides. The question then arises as to if and how the microbiome adapts to new carbohydrate sources when modern humans change eating habits. Recent metagenome analysis of microbiomes from healthy American, Japanese, and Spanish populations identified putative CAZymes obtained by horizontal gene transfer from marine bacteria, which suggested that human gut bacteria evolved to degrade algal carbohydrates—for example, consumed in form of sushi. We approached this hypothesis by studying such a polysaccharide utilization locus (PUL) obtained by horizontal gene transfer by the gut bacterium Bacteroides plebeius . Transcriptomic and growth experiments revealed that the PUL responds to the polysaccharide porphyran from red algae, enabling growth on this carbohydrate but not related substrates like agarose and carrageenan. The X-ray crystallographic and biochemical analysis of two proteins encoded by this PUL, BACPLE_01689 and BACPLE_01693 , showed that they are β-porphyranases belonging to glycoside hydrolase families 16 and 86, respectively. The product complex of the GH86 at 1.3 Å resolution highlights the molecular details of porphyran hydrolysis by this new porphyranase. Combined, these data establish experimental support for the argument that CAZymes and associated genes obtained from extrinsic microbes add new catabolic functions to the human gut microbiome.

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Section: Methodsmentioning

confidence: 99%

Bacteria of the human gut microbiome catabolize red seaweed glycans with carbohydrate-active enzyme updates from extrinsic microbes

Hehemann

Kelly

Pudlo

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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278

250

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“…Whereas endo-acting ␤-agarases, such as GH16 and GH86, cleave randomly along the agarose polymer chains (9,21), Aga50D appears as a key component to degrade those neoagarooligosaccharides into neoagarobiose units. The disaccharide units produced are then further degraded by ␣-agarases, such as GH117 (17), into monosaccharides useable as carbon and energy sources by the bacterium.…”

Section: Discussionmentioning

confidence: 99%

“…500 mg of the sample was redissolved in 1 ml of water prior to filtration through a 0.2-m membrane (Millipore) and size exclusion chromatography using Bio-gel P2 resin equilibrated with 50 mM ammonium carbonate buffer (pH 7.5). Samples from the 2-ml fractions were analyzed by TLC as described previously, and oligomers were identified by comparison with standards of the neoagarobiose series (17). Fractions with purified oligosaccharides were pooled and lyophilized.…”

Section: Methodsmentioning

confidence: 99%

“…In accordance with their role of generating longer oligosaccharides, the crystal structures of GH16 endo-␤-agarases (AgaA, AgaB, and AgaD) from Zobellia galactanivorans and the GH86 endo-␤-porphyranase from Bacteroides plebeius, revealed open and extended substrate binding clefts (11,21,25,26). In contrast, the structures of GH117 exo-␣-neoagarobiose hydrolases, from S. degradans (27), Z. galactanivorans (28), and B. plebeius (17), displayed small substrate binding pockets sufficient to bind and cleave a disaccharide. Structural analysis of a neoagarobiohydrolase, such as those from GH50, which have been found critical for agarose metabolism in S. degradans (9), has not been reported, limiting our understanding of the agarolytic system of this and other marine microbes.…”

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confidence: 98%

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Substrate Recognition and Hydrolysis by a Family 50 exo-β-Agarase, Aga50D, from the Marine Bacterium Saccharophagus degradans

Pluvinage

Hehemann

Boraston

2013

Journal of Biological Chemistry

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Background:The catalytic mechanism and substrate recognition required for exo-␤-agarase activity are unclear. Results: Structural analysis of a family 50 glycoside hydrolase details substrate recognition and supports a retaining mechanism. Conclusion: The active site architecture dictates exo-activity, whereas substrate distortion aids glycosidic bond hydrolysis. Significance: This structural work offers the first snapshots of Michaelis complexes formed during hydrolysis of the ␤-linkages in agarose.

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“…While no cation was found in the structure of SdNABH ), BpGH117 features a magnesium ion at the position conserved with ZgAhgA (Hehemann et al 2012c), suggesting a degree of plasticity for this cation-binding site. The structure of an inactive mutant of BpGH117 has also been determined, in a complex with neoagarobiose, identifying key residues for substrate recognition and catalysis.…”

Section: α-13-(36-anhydro)-l-galactosidasesmentioning

confidence: 95%

Microorganisms living on macroalgae: diversity, interactions, and biotechnological applications

Martin

Portetelle

Michel

et al. 2014

Appl Microbiol Biotechnol

144

107

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Marine microorganisms play key roles in every marine ecological process, hence the growing interest in studying their populations and functions. Microbial communities on algae remain underexplored, however, despite their huge biodiversity and the fact that they differ markedly from those living freely in seawater. The study of this microbiota and of its relationships with algal hosts should provide crucial information for ecological investigations on algae and aquatic ecosystems. Furthermore, because these microorganisms interact with algae in multiple, complex ways, they constitute an interesting source of novel bioactive compounds with biotechnological potential, such as dehalogenases, antimicrobials and alga-specific polysaccharidases (e.g. agarases, carrageenases, alginate lyases). Here, to demonstrate the huge potential of alga-associated organisms and their metabolites in developing future biotechnological applications, we first describe the immense diversity and density of these microbial biofilms. We further describe their complex interactions with algae, leading to the production of specific bioactive compounds and hydrolytic enzymes of biotechnological interest.We end with a glance at their potential use in medical and industrial applications.

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Analysis of Keystone Enzyme in Agar Hydrolysis Provides Insight into the Degradation (of a Polysaccharide from) Red Seaweeds

Cited by 82 publications

References 50 publications

Bacteria of the human gut microbiome catabolize red seaweed glycans with carbohydrate-active enzyme updates from extrinsic microbes

Bacteria of the human gut microbiome catabolize red seaweed glycans with carbohydrate-active enzyme updates from extrinsic microbes

Substrate Recognition and Hydrolysis by a Family 50 exo-β-Agarase, Aga50D, from the Marine Bacterium Saccharophagus degradans

Microorganisms living on macroalgae: diversity, interactions, and biotechnological applications

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