Biology subjects

Pilgaard, B.

Publications and source records attributed to Pilgaard, B..

4 recordsLinked to original sources

bioRxiv2024

Unraveling the Molecular mechanism of Polysaccharide Lyases for Efficient Alginate Degradation

Alginate lyases (ALs) are essential for breaking down brown macroalgae alginates, widely used naturally-occurring polysaccharides. Their molecular mechanisms remain challenging due to the lack of catalytically competent Michaelis-Menten complex structures. We here provide structural snap-shots and dissect the mechanism of mannuronan-specific ALs from family 7 polysaccharide lyases (PL7), employing time-resolved NMR, X-ray, neutron crystallography, and QM/MM simulations. We reveal the protonation state of critical active site residues, enabling atomic-level analysis of the reaction coordinate. Our approach reveals an endolytic and asynchronous syn {beta}-elimination reaction, with Tyr serving as both Bronsted base and acid, involving a carbanion-type of transition state. This study not only reconciles previous structural and kinetic discrepancies, but also establishes a comprehensive PL reaction mechanism applicable across lyase families, which can guide the engineering of ALs for tailored alginate oligosaccharide production.

molecular biology↗

bioRxiv2024

Carbohydrate-active enzymes from Akkermansia muciniphila breakdown mucin O-glycans to completion

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 A. muciniphila uses an array of Carbohydrate-active enzymes and sulfatases to access this complex energy source. Here we describe the biochemical characterisation of fifty-four glycoside hydrolases, eleven sulfatases, and one polysaccharide lyase from A. muciniphila to provide a holistic understanding of the carbohydrate-degrading activities. The results provide an extensive insight into the sequence of O-glycan degradation and how A. muciniphila can access this structurally variable substrate. One of the most outstanding elements of this work was the demonstration that these enzymes can act synergistically to degrade the O-glycans on the mucin polypeptide to completion, down to the core GalNAc. Additionally, human breast milk oligosaccharide, ganglioside, and globoside glycan structures were included in the study to understand the full degradative capability of A. muciniphila.

biochemistry↗

bioRxiv2020

First draft genome of the Labyrinthula genus, an opportunistic seagrass pathogen, reveals novel insight into marine protist phylogeny, ecology and CAZyme cell-wall degradation

Labyrinthula spp. are saprobic, marine protists that also act as opportunistic pathogens and are the causative agents of seagrass wasting disease (SWD). Despite the threat of local- and large-scale SWD outbreaks, there are currently gaps in our understanding of the drivers of SWD, particularly surrounding Labyrinthula virulence and ecology. Given these uncertainties, we investigated Labyrinthula from a novel genomic perspective by presenting the first draft genome and predicted proteome of a pathogenic isolate of Labyrinthula SR_Ha_C, generated from a hybrid assembly of Nanopore and Illumina sequences. Phylogenetic and cross-phyla comparisons revealed insights into the evolutionary history of Stramenopiles. Genome annotation showed evidence of glideosome-type machinery and an apicoplast protein typically found in protist pathogens and parasites. Proteins involved in Labyrinthulas actin-myosin mode of transport, as well as carbohydrate degradation were also prevalent. Further, CAZyme functional predictions revealed a repertoire of enzymes involved in breakdown of cell-wall and carbohydrate storage compounds common to seagrasses. The relatively low number of CAZymes annotated from the genome of Labyrinthula SR_Ha_C compared to other Labyrinthulea species may reflect the conservative annotation parameters, a specialised substrate affinity and the scarcity of characterised protist enzymes. Inherently, there is high probability for finding both unique and novel enzymes from Labyrinthula spp. This study provides resources for further exploration of Labyrinthula ecology and evolution, and will hopefully be the catalyst for new hypothesis-driven SWD research revealing more details of molecular interactions between Labyrinthula species and its host substrate.

genomics↗

bioRxiv2018

The origin of fungi-culture in termites was associated with a shift to a mycolytic gut bacteria community

Termites forage on a range of substrates, and it has been suggested that diet shapes the composition and function of termite gut bacterial communities. Through comparative analyses of gut metagenomes in nine termite species with distinct diets, we characterise bacterial community compositions and identify biomass-degrading enzymes and the bacterial taxa that encode them. We find that fungus-growing termite guts are enriched in fungal cell wall-degrading and proteolytic enzymes, while wood-feeding termite gut communities are enriched for plant cell wall-degrading enzymes. Interestingly, wood-feeding termite gut bacteria code for abundant chitinolytic enzymes, suggesting that fungal biomass within the decaying wood likely contributes to gut bacteria or termite host nutrition. Across diets, the dominant biomass-degrading enzymes are predominantly coded for by the most abundant bacterial taxa, suggesting tight links between diet and gut community composition, with the most marked shift being the communities coding for the mycolytic capacity of the fungus-growing termite gut.

ecology↗