Doctor of Philosophy in Chemistry (PhD)
Fundamental and applied carbohydrate enzymology
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Xyloglucan (XyG) is a ubiquitous plant heteropolysaccharide representing up to one-quarter of the total carbohydrate content of terrestrial plant cell walls. Given its structural complexity, XyG requires a consortium of backbone-cleaving endo-xyloglucanases and sidechain-cleaving exo-glycosidases for complete saccharification. The Gram-negative soil saprophyte Cellvibrio japonicus is a treasure trove for carbohydrate active enzymes (CAZymes) due to its robust capacity to degrade different plant polysaccharides. The XyG utilization machinery in C. japonicus is incompletely understood, despite recent characterization of associated sidechain-cleaving exo-glycosidases. I present here my attempts to identify and functionally characterize the endo-xyloglucanase(s) and the XyG-specific β-1,4 exo-glucosidase catalyzing the first and final steps, respectively, in the XyG saccharification pathway in C. japonicus. Bioinformatic analysis identified one Glycoside Hydrolase Family 74 (CjGH74), three GH5_4 (CjGH5D, CjGH5E and CjGH5F) and three GH9 (CjGH9A, CjGH9B and CjGH9C) candidates with putative endo-xyloglucanase activity. Biochemical and structural analyses that involved CjGH74 and the three CjGH5_4 enzymes clearly demonstrated the exquisite specificity of the four enzymes towards XyG. Scrutiny of the modular architecture of the CjGH5_4 enzyme, CjGH5F, identified the module of unknown function X181. Affinity gel electrophoresis and isothermal titration calorimetry identified the X181 module as a member of a new CBM family that exclusively binds galactose-containing polysaccharides including XyG and galactomannans, congruent with the displayed endo-xyloglucanase activity of the pendent catalytic domain. Surprisingly, reverse genetic analysis in C. japonicus displayed the lack of growth perturbation on XyG upon the deletion of the four specific endo-xyloglucanases, suggesting the presence of other enzyme(s) with potential endo-xyloglucanase activity. Biochemical characterization of the GH9 enzyme CjGH9B revealed its high catalytic efficiency towards mixed linkage β-glucan and its weak side-activity against xyloglucan, which might be sufficient to rescue the quadruple deletion mutant. Bioinformatic analysis identified the four CjGH3 enzymes Bgl3A, Bgl3B, Bgl3C, and Bgl3D as potential targets for the XyG-specific exo-β-glucosidase in C. japonicus. Comprehensive genetic and biochemical approaches interestingly revealed the fundamental contribution of Bgl3D in XyG utilization in C. japonicus. Together, these data shed light on the initial and final steps of xyloglucan saccharification in C. japonicus and identify useful enzymes for selective biomass deconstruction.
Plant biomass is both the most abundant organic carbon source and the most abundant organic carbon sink on our planet. This carbon is stored primarily in the cell wall, where carbohydrates, proteins and polyphenols are interwoven to form complex purpose-built composite materials. Within plants, diverse polysaccharides are built up and broken down as part of their natural life cycle. Within the guts of multicellular organisms, a diverse and adaptable collection of bacteria anaerobically ferments complex plant polysaccharides. In this thesis, the structure and function of enzymes involved in these two processes are described. The xyloglucan endo-transglycosylase/hydrolase (XTH) gene family encodes enzymes of central importance to plant cell wall remodelling. Investigations into the ancestry of the XTH family revealed a subfamily of endo-glucanases which share a common ancestor with the XTHs. Based on product analysis, kinetics, and X-ray crystallography these EG16s have been identified as a family of β(1,4)-specific endo-glucanases with an uncommon mode of substrate recognition. Although the biological role(s) of EG16 orthologues remains to be fully resolved, the presented biochemical and tertiary structural characterisation provide insight into plant glycoside hydrolase evolution, and will continue to inform studies of plant cell walls.Within the gut, Prevotella are an important genus of Gram-negative bacteria associated with carbohydrate-rich diets. Recent genome sequencing has shown that they possess many undescribed polysaccharide utilisation loci (PULs). A revisited broad-specificity cross-linking glycan-degrading endo-glucanase (PbGH5A) is associated with a PUL of unknown function within Prevotella bryantii, an obligate anaerobe originally isolated from the bovine gut. Based on X-ray crystallography, product identification, binding assays, and kinetic measurements, the structures and functions of a variety of proteins involved in the recognition and breakdown of complex β-mannans have been determined. These include a broad-specificity endo-β-glucanase, an endo-β-mannanase, two β-mannan-binding proteins, two β-mannan acetylesterases, a mannobiose-2-epimerase, and a mannosylglucose phosphorylase. Characterisation of the two β-mannan acetylesterases provides a basis for the expansion of CAZy family CE7 and the formation of a new CE family. Furthermore, the presented model of the Prevotella β-mannan utilisation locus provides a genetic template for identifying systems which degrade complex galactomannans and glucomannans across the Bacteroidetes phylum.