Harry Brumer

Professor

Research Classification

Research Interests

Biological and Biochemical Mechanisms
biomass
carbohydrates
cellulose
Chemical Synthesis and Catalysis
Enzymes
microbiota
plant cell walls
polysaccharides

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Doctoral students
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I am open to hosting Visiting International Research Students (non-degree, up to 12 months).

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Postdoctoral Fellows

Graduate Student Supervision

Doctoral Student Supervision (Jan 2008 - Nov 2020)
Covalent probes for functional and structural characterization of glycoside hydrolases (2020)

Carbohydrates are ubiquitous in Nature and fundamental to the sustenance of organisms across all domains of life. Carbohydrates serve as sources and reserves of metabolic energy, participate in various cellular communication events, and provide structural support to plant and animal cells. Highly specific enzymes have evolved over several millennia to bind and manipulate carbohydrate substrates. Glycoside hydrolases (GHs) are a class of carbohydrate-active enzymes that cleave glycosidic linkages in complex carbohydrates. Organisms across all domains of life dedicate part of their genome to the production of GHs. New GHs are continually discovered through genome sequencing, while their structural and functional characterization, particularly in complex native environments, poses a persistent challenge to the dynamic field of GH characterization. One of the fundamental ways of ascribing protein function is the exploration of protein active sites, which can be used to deduce important details regarding substrate-enzyme interactions.The work presented in this thesis describes the development of six new probes targeting a variety of GHs by irreversible covalent inhibition. These probes, developed on oligosaccharide scaffolds, feature either an N-bromoacetylglycosylamine electrophilic warhead or 2', 4' dinitrophenyl 2-deoxy-2-fluoro substitutions, facilitating irreversible inhibition of the target GH. The analysis presented in this work reveals key information about enzyme-inhibitor interactions through enzyme kinetic analyses, intact-protein mass spectrometry, and inhibitor-bound protein X-ray crystallography. Enzymes of diverse GH families and substrate preferences including endo-xyloglucanases, mixed-linkage glucanases, and β-(1,3) glucanases are featured to demonstrate the potency of this library of inhibitors. This small-molecule inhibitor toolkit targeting specific GH enzymes has the potential to enhance our knowledge of the structural and functional characteristics of GHs and to provide a platform for activity-based enzyme profiling.

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Enzymology of the xyloglucan utilization system in the soil saprophyte Cellvibrio japonicus (2018)

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.

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Structure-function analyses of plant glycan-degrading enzymes (2018)

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.

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