Publications by authors named "Gideon J Davies"

263 Publications

Molecular Basis of Sulfosugar Selectivity in Sulfoglycolysis.

ACS Cent Sci 2021 Mar 23;7(3):476-487. Epub 2021 Feb 23.

School of Chemistry and Bio21 Molecular Science and Biotechnology Institute and University of Melbourne, Parkville, Victoria 3010, Australia.

The sulfosugar sulfoquinovose (SQ) is produced by essentially all photosynthetic organisms on Earth and is metabolized by bacteria through the process of sulfoglycolysis. The sulfoglycolytic Embden-Meyerhof-Parnas pathway metabolizes SQ to produce dihydroxyacetone phosphate and sulfolactaldehyde and is analogous to the classical Embden-Meyerhof-Parnas glycolysis pathway for the metabolism of glucose-6-phosphate, though the former only provides one C3 fragment to central metabolism, with excretion of the other C3 fragment as dihydroxypropanesulfonate. Here, we report a comprehensive structural and biochemical analysis of the three core steps of sulfoglycolysis catalyzed by SQ isomerase, sulfofructose (SF) kinase, and sulfofructose-1-phosphate (SFP) aldolase. Our data show that despite the superficial similarity of this pathway to glycolysis, the sulfoglycolytic enzymes are specific for SQ metabolites and are not catalytically active on related metabolites from glycolytic pathways. This observation is rationalized by three-dimensional structures of each enzyme, which reveal the presence of conserved sulfonate binding pockets. We show that SQ isomerase acts preferentially on the β-anomer of SQ and reversibly produces both SF and sulforhamnose (SR), a previously unknown sugar that acts as a derepressor for the transcriptional repressor CsqR that regulates SQ-utilization. We also demonstrate that SF kinase is a key regulatory enzyme for the pathway that experiences complex modulation by the metabolites SQ, SLA, AMP, ADP, ATP, F6P, FBP, PEP, DHAP, and citrate, and we show that SFP aldolase reversibly synthesizes SFP. This body of work provides fresh insights into the mechanism, specificity, and regulation of sulfoglycolysis and has important implications for understanding how this biochemistry interfaces with central metabolism in prokaryotes to process this major repository of biogeochemical sulfur.
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http://dx.doi.org/10.1021/acscentsci.0c01285DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8006165PMC
March 2021

Fungal GH25 muramidases: New family members with applications in animal nutrition and a crystal structure at 0.78Å resolution.

PLoS One 2021 12;16(3):e0248190. Epub 2021 Mar 12.

Department of Chemistry, Structural Biology Laboratory, The University of York, York, United Kingdom.

Muramidases/lysozymes hydrolyse the peptidoglycan component of the bacterial cell wall. They are found in many of the glycoside hydrolase (GH) families. Family GH25 contains muramidases/lysozymes, known as CH type lysozymes, as they were initially discovered in the Chalaropsis species of fungus. The characterized enzymes from GH25 exhibit both β-1,4-N-acetyl- and β-1,4-N,6-O-diacetylmuramidase activities, cleaving the β-1,4-glycosidic bond between N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) moieties in the carbohydrate backbone of bacterial peptidoglycan. Here, a set of fungal GH25 muramidases were identified from a sequence search, cloned and expressed and screened for their ability to digest bacterial peptidoglycan, to be used in a commercial application in chicken feed. The screen identified the enzyme from Acremonium alcalophilum JCM 736 as a suitable candidate for this purpose and its relevant biochemical and biophysical and properties are described. We report the crystal structure of the A. alcalophilum enzyme at atomic, 0.78 Å resolution, together with that of its homologue from Trichobolus zukalii at 1.4 Å, and compare these with the structures of homologues. GH25 enzymes offer a new solution in animal feed applications such as for processing bacterial debris in the animal gut.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0248190PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7954357PMC
March 2021

Cysteine Nucleophiles in Glycosidase Catalysis: Application of a Covalent β-l-Arabinofuranosidase Inhibitor.

Angew Chem Int Ed Engl 2021 03 2;60(11):5754-5758. Epub 2021 Feb 2.

York Structural Biology Laboratory, Department of Chemistry, The University of York, Heslington, York, YO10 5DD, UK.

The recent discovery of zinc-dependent retaining glycoside hydrolases (GHs), with active sites built around a Zn(Cys) (Glu) coordination complex, has presented unresolved mechanistic questions. In particular, the proposed mechanism, depending on a Zn-coordinated cysteine nucleophile and passing through a thioglycosyl enzyme intermediate, remains controversial. This is primarily due to the expected stability of the intermediate C-S bond. To facilitate the study of this atypical mechanism, we report the synthesis of a cyclophellitol-derived β-l-arabinofuranosidase inhibitor, hypothesised to react with the catalytic nucleophile to form a non-hydrolysable adduct analogous to the mechanistic covalent intermediate. This β-l-arabinofuranosidase inhibitor reacts exclusively with the proposed cysteine thiol catalytic nucleophiles of representatives of GH families 127 and 146. X-ray crystal structures determined for the resulting adducts enable MD and QM/MM simulations, which provide insight into the mechanism of thioglycosyl enzyme intermediate breakdown. Leveraging the unique chemistry of cyclophellitol derivatives, the structures and simulations presented here support the assignment of a zinc-coordinated cysteine as the catalytic nucleophile and illuminate the finely tuned energetics of this remarkable metalloenzyme clan.
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http://dx.doi.org/10.1002/anie.202013920DOI Listing
March 2021

Activity-Based Protein Profiling of Retaining α-Amylases in Complex Biological Samples.

J Am Chem Soc 2021 Feb 26;143(5):2423-2432. Epub 2021 Jan 26.

Department of Chemistry, York Structural Biology Laboratory, University of York, Heslington, York YO10 5DD, United Kingdom.

Amylases are key enzymes in the processing of starch in many kingdoms of life. They are important catalysts in industrial biotechnology where they are applied in, among others, food processing and the production of detergents. In man amylases are the first enzymes in the digestion of starch to glucose and arguably also the preferred target in therapeutic strategies aimed at the treatment of type 2 diabetes patients through down-tuning glucose assimilation. Efficient and sensitive assays that report selectively on retaining amylase activities irrespective of the nature and complexity of the biomaterial studied are of great value both in finding new and effective human amylase inhibitors and in the discovery of new microbial amylases with potentially advantageous features for biotechnological application. Activity-based protein profiling (ABPP) of retaining glycosidases is inherently suited for the development of such an assay format. We here report on the design and synthesis of 1,6--cyclophellitol-based pseudodisaccharides equipped with a suite of reporter entities and their use in ABPP of retaining amylases from human saliva, murine tissue as well as secretomes from fungi grown on starch. The activity and efficiency of the inhibitors and probes are substantiated by extensive biochemical analysis, and the selectivity for amylases over related retaining endoglycosidases is validated by structural studies.
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http://dx.doi.org/10.1021/jacs.0c13059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7883350PMC
February 2021

Structure of human endo-α-1,2-mannosidase (MANEA), an antiviral host-glycosylation target.

Proc Natl Acad Sci U S A 2020 11 5;117(47):29595-29601. Epub 2020 Nov 5.

Department of Chemistry, University of York, York YO10 5DD, United Kingdom;

Mammalian protein N-linked glycosylation is critical for glycoprotein folding, quality control, trafficking, recognition, and function. N-linked glycans are synthesized from GlcManGlcNAc precursors that are trimmed and modified in the endoplasmic reticulum (ER) and Golgi apparatus by glycoside hydrolases and glycosyltransferases. Endo-α-1,2-mannosidase (MANEA) is the sole -acting glycoside hydrolase involved in N-glycan trimming and is located within the Golgi, where it allows ER-escaped glycoproteins to bypass the classical N-glycosylation trimming pathway involving ER glucosidases I and II. There is considerable interest in the use of small molecules that disrupt N-linked glycosylation as therapeutic agents for diseases such as cancer and viral infection. Here we report the structure of the catalytic domain of human MANEA and complexes with substrate-derived inhibitors, which provide insight into dynamic loop movements that occur on substrate binding. We reveal structural features of the human enzyme that explain its substrate preference and the mechanistic basis for catalysis. These structures have inspired the development of new inhibitors that disrupt host protein N-glycan processing of viral glycans and reduce the infectivity of bovine viral diarrhea and dengue viruses in cellular models. These results may contribute to efforts aimed at developing broad-spectrum antiviral agents and help provide a more in-depth understanding of the biology of mammalian glycosylation.
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http://dx.doi.org/10.1073/pnas.2013620117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7703563PMC
November 2020

Structure of a GH51 α-L-arabinofuranosidase from Meripilus giganteus: conserved substrate recognition from bacteria to fungi.

Acta Crystallogr D Struct Biol 2020 Nov 16;76(Pt 11):1124-1133. Epub 2020 Oct 16.

York Structural Biology Laboratory, University of York, Heslington, York YO10 5DD, United Kingdom.

α-L-Arabinofuranosidases from glycoside hydrolase family 51 use a stereochemically retaining hydrolytic mechanism to liberate nonreducing terminal α-L-arabinofuranose residues from plant polysaccharides such as arabinoxylan and arabinan. To date, more than ten fungal GH51 α-L-arabinofuranosidases have been functionally characterized, yet no structure of a fungal GH51 enzyme has been solved. In contrast, seven bacterial GH51 enzyme structures, with low sequence similarity to the fungal GH51 enzymes, have been determined. Here, the crystallization and structural characterization of MgGH51, an industrially relevant GH51 α-L-arabinofuranosidase cloned from Meripilus giganteus, are reported. Three crystal forms were grown in different crystallization conditions. The unliganded structure was solved using sulfur SAD data collected from a single crystal using the I23 in vacuo diffraction beamline at Diamond Light Source. Crystal soaks with arabinose, 1,4-dideoxy-1,4-imino-L-arabinitol and two cyclophellitol-derived arabinose mimics reveal a conserved catalytic site and conformational itinerary between fungal and bacterial GH51 α-L-arabinofuranosidases.
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http://dx.doi.org/10.1107/S205979832001253XDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7604909PMC
November 2020

Structural insights into heparanase activity using a fluorogenic heparan sulfate disaccharide.

Chem Commun (Camb) 2020 Nov;56(89):13780-13783

School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, QLD, Australia.

A synthetic heparan sulfate disaccharide has been assessed as a fluorogenic heparanase substrate, enabling enzyme turnover and inhibition kinetics measurements despite slow turnover. Crystal structures with human heparanase also provide the first ever observation of a substrate in an activated 1S3 conformation, highlighting previously unknown interactions involved in enzymatic processing. Our data provide insights into the heparanase catalytic mechanism, and will inform the design of improved heparanase substrates and inhibitors.
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http://dx.doi.org/10.1039/d0cc05932cDOI Listing
November 2020

Mechanistic Insights into the Chaperoning of Human Lysosomal-Galactosidase Activity: Highly Functionalized Aminocyclopentanes and -5a-Substituted Derivatives of 4--Isofagomine.

Molecules 2020 Sep 3;25(17). Epub 2020 Sep 3.

Glycogroup, Institute of Chemistry and Technology of Biobased Systems, Graz University of Technology, Stremayrgasse 9, A-8010 Graz, Austria.

Glycosidase inhibitors have shown great potential as pharmacological chaperones for lysosomal storage diseases. In light of this, a series of new cyclopentanoid β-galactosidase inhibitors were prepared and their inhibitory and pharmacological chaperoning activities determined and compared with those of lipophilic analogs of the potent β-d-galactosidase inhibitor 4--isofagomine. Structure-activity relationships were investigated by X-ray crystallography as well as by alterations in the cyclopentane moiety such as deoxygenation and replacement by fluorine of a "strategic" hydroxyl group. New compounds have revealed highly promising activities with a range of β-galactosidase-compromised human cell lines and may serve as leads towards new pharmacological chaperones for G-gangliosidosis and Morquio B disease.
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http://dx.doi.org/10.3390/molecules25174025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7504770PMC
September 2020

Mechanistic basis of substrate-O coupling within a chitin-active lytic polysaccharide monooxygenase: An integrated NMR/EPR study.

Proc Natl Acad Sci U S A 2020 08 28;117(32):19178-19189. Epub 2020 Jul 28.

Norwegian Biopolymer Laboratory (NOBIPOL), Department of Biotechnology and Food Science, Norwegian University of Science and Technology, N-7491 Trondheim, Norway;

Lytic polysaccharide monooxygenases (LPMOs) have a unique ability to activate molecular oxygen for subsequent oxidative cleavage of glycosidic bonds. To provide insight into the mode of action of these industrially important enzymes, we have performed an integrated NMR/electron paramagnetic resonance (EPR) study into the detailed aspects of an AA10 LPMO-substrate interaction. Using NMR spectroscopy, we have elucidated the solution-phase structure of -LPMO10A from , along with solution-phase structural characterization of the Cu(I)-LPMO, showing that the presence of the metal has minimal effects on the overall protein structure. We have, moreover, used paramagnetic relaxation enhancement (PRE) to characterize Cu(II)-LPMO by NMR spectroscopy. In addition, a multifrequency continuous-wave (CW)-EPR and N-HYSCORE spectroscopy study on the uniformly isotope-labeled Cu(II)-bound N-LPMO10A along with its natural abundance isotopologue determined copper spin-Hamiltonian parameters for LPMOs to markedly improved accuracy. The data demonstrate that large changes in the Cu(II) spin-Hamiltonian parameters are induced upon binding of the substrate. These changes arise from a rearrangement of the copper coordination sphere from a five-coordinate distorted square pyramid to one which is four-coordinate near-square planar. There is also a small reduction in metal-ligand covalency and an attendant increase in the d(x-y) character/energy of the singly occupied molecular orbital (SOMO), which we propose from density functional theory (DFT) calculations predisposes the copper active site for the formation of a stable Cu-O intermediate. This switch in orbital character upon addition of chitin provides a basis for understanding the coupling of substrate binding with O activation in chitin-active AA10 LPMOs.
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http://dx.doi.org/10.1073/pnas.2004277117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7431007PMC
August 2020

Profiling Substrate Promiscuity of Wild-Type Sugar Kinases for Multi-fluorinated Monosaccharides.

Cell Chem Biol 2020 09 2;27(9):1199-1206.e5. Epub 2020 Jul 2.

Department of Chemistry, University of York, Heslington, York YO10 5DD, UK. Electronic address:

Fluorinated sugar-1-phosphates are of emerging importance as intermediates in the chemical and biocatalytic synthesis of modified oligosaccharides, as well as probes for chemical biology. Here we present a systematic study of the activity of a wide range of anomeric sugar kinases (galacto- and N-acetylhexosamine kinases) against a panel of fluorinated monosaccharides, leading to the first examples of polyfluorinated substrates accepted by this class of enzymes. We have discovered four new N-acetylhexosamine kinases with a different substrate scope, thus expanding the number of homologs available in this subclass of kinases. Lastly, we have solved the crystal structure of a galactokinase in complex with 2-deoxy-2-fluorogalactose, giving insight into changes in the active site that may account for the specificity of the enzyme toward certain substrate analogs.
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http://dx.doi.org/10.1016/j.chembiol.2020.06.005DOI Listing
September 2020

Manno--cyclophellitols Enable Activity-Based Protein Profiling of Human α-Mannosidases and Discovery of New Golgi Mannosidase II Inhibitors.

J Am Chem Soc 2020 07 16;142(30):13021-13029. Epub 2020 Jul 16.

Structural Biology Laboratory, Department of Chemistry, The University of York, York YO10 5DD, United Kingdom.

Golgi mannosidase II (GMII) catalyzes the sequential hydrolysis of two mannosyl residues from GlcNAcManGlcNAc to produce GlcNAcManGlcNAc, the precursor for all complex -glycans, including the branched -glycans associated with cancer. Inhibitors of GMII are potential cancer therapeutics, but their usefulness is limited by off-target effects, which produce α-mannosidosis-like symptoms. Despite many structural and mechanistic studies of GMII, we still lack a potent and selective inhibitor of this enzyme. Here, we synthesized manno--cyclophellitol epoxide and aziridines and demonstrate their covalent modification and time-dependent inhibition of GMII. Application of fluorescent manno--cyclophellitol aziridine derivatives enabled activity-based protein profiling of α-mannosidases from both human cell lysate and mouse tissue extracts. Synthesized probes also facilitated a fluorescence polarization-based screen for dGMII inhibitors. We identified seven previously unknown inhibitors of GMII from a library of over 350 iminosugars and investigated their binding modalities through X-ray crystallography. Our results reveal previously unobserved inhibitor binding modes and promising scaffolds for the generation of selective GMII inhibitors.
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http://dx.doi.org/10.1021/jacs.0c03880DOI Listing
July 2020

A baculoviral system for the production of human β-glucocerebrosidase enables atomic resolution analysis.

Acta Crystallogr D Struct Biol 2020 Jun 29;76(Pt 6):565-580. Epub 2020 May 29.

Department of Chemistry, York Structural Biology Laboratory, University of York, Heslington, York YO10 5DD, United Kingdom.

The lysosomal glycoside hydrolase β-glucocerebrosidase (GBA; sometimes called GBA1 or GC) catalyses the hydrolysis of glycosphingolipids. Inherited deficiencies in GBA cause the lysosomal storage disorder Gaucher disease (GD). Consequently, GBA is of considerable medical interest, with continuous advances in the development of inhibitors, chaperones and activity-based probes. The development of new GBA inhibitors requires a source of active protein; however, the majority of structural and mechanistic studies of GBA today rely on clinical enzyme-replacement therapy (ERT) formulations, which are incredibly costly and are often difficult to obtain in adequate supply. Here, the production of active crystallizable GBA in insect cells using a baculovirus expression system is reported, providing a nonclinical source of recombinant GBA with comparable activity and biophysical properties to ERT preparations. Furthermore, a novel crystal form of GBA is described which diffracts to give a 0.98 Å resolution unliganded structure. A structure in complex with the inactivator 2,4-dinitrophenyl-2-deoxy-2-fluoro-β-D-glucopyranoside was also obtained, demonstrating the ability of this GBA formulation to be used in ligand-binding studies. In light of its purity, stability and activity, the GBA production protocol described here should circumvent the need for ERT formulations for structural and biochemical studies and serve to support GD research.
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http://dx.doi.org/10.1107/S205979832000501XDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7271948PMC
June 2020

An Epoxide Intermediate in Glycosidase Catalysis.

ACS Cent Sci 2020 May 16;6(5):760-770. Epub 2020 Apr 16.

School of Chemistry and Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria 3010, Australia.

Retaining glycoside hydrolases cleave their substrates through stereochemical retention at the anomeric position. Typically, this involves two-step mechanisms using either an enzymatic nucleophile via a covalent glycosyl enzyme intermediate or neighboring-group participation by a substrate-borne 2-acetamido neighboring group via an oxazoline intermediate; no enzymatic mechanism with participation of the sugar 2-hydroxyl has been reported. Here, we detail structural, computational, and kinetic evidence for neighboring-group participation by a mannose 2-hydroxyl in glycoside hydrolase family 99 -α-1,2-mannanases. We present a series of crystallographic snapshots of key species along the reaction coordinate: a Michaelis complex with a tetrasaccharide substrate; complexes with intermediate mimics, a sugar-shaped cyclitol β-1,2-aziridine and β-1,2-epoxide; and a product complex. The 1,2-epoxide intermediate mimic displayed hydrolytic and transfer reactivity analogous to that expected for the 1,2-anhydro sugar intermediate supporting its catalytic equivalence. Quantum mechanics/molecular mechanics modeling of the reaction coordinate predicted a reaction pathway through a 1,2-anhydro sugar via a transition state in an unusual flattened, envelope () conformation. Kinetic isotope effects (/) for anomeric-H and anomeric-C support an oxocarbenium ion-like transition state, and that for C2-O (1.052 ± 0.006) directly implicates nucleophilic participation by the C2-hydroxyl. Collectively, these data substantiate this unprecedented and long-imagined enzymatic mechanism.
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http://dx.doi.org/10.1021/acscentsci.0c00111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7256955PMC
May 2020

A Sulfoglycolytic Entner-Doudoroff Pathway in Rhizobium leguminosarum bv. trifolii SRDI565.

Appl Environ Microbiol 2020 07 20;86(15). Epub 2020 Jul 20.

School of Chemistry, University of Melbourne, Parkville, Victoria, Australia

Rhizobia are nitrogen-fixing bacteria that engage in symbiotic relationships with plant hosts but can also persist as free-living bacteria in the soil and rhizosphere. Here, we show that free-living SRDI565 can grow on the sulfosugar sulfoquinovose (SQ) or the related glycoside SQ-glycerol using a sulfoglycolytic Entner-Doudoroff (sulfo-ED) pathway, resulting in production of sulfolactate (SL) as the major metabolic end product. Comparative proteomics supports the involvement of a sulfo-ED operon encoding an ABC transporter, sulfo-ED enzymes, and an SL exporter. Consistent with an oligotrophic lifestyle, proteomics data revealed little change in expression of the sulfo-ED proteins during growth on SQ versus mannitol, a result confirmed through biochemical assay of sulfoquinovosidase activity in cell lysates. Metabolomics analysis showed that growth on SQ involves gluconeogenesis to satisfy metabolic requirements for glucose-6-phosphate and fructose-6-phosphate. Metabolomics analysis also revealed the unexpected production of small amounts of sulfofructose and 2,3-dihydroxypropanesulfonate, which are proposed to arise from promiscuous activities of the glycolytic enzyme phosphoglucose isomerase and a nonspecific aldehyde reductase, respectively. The discovery of a rhizobium isolate with the ability to degrade SQ builds our knowledge of how these important symbiotic bacteria persist within soil. Sulfonate sulfur is a major form of organic sulfur in soils but requires biomineralization before it can be utilized by plants. Very little is known about the biochemical processes used to mobilize sulfonate sulfur. We show that a rhizobial isolate from soil, SRDI565, possesses the ability to degrade the abundant phototroph-derived carbohydrate sulfonate SQ through a sulfoglycolytic Entner-Doudoroff pathway. Proteomics and metabolomics demonstrated the utilization of this pathway during growth on SQ and provided evidence for gluconeogenesis. Unexpectedly, off-cycle sulfoglycolytic species were also detected, pointing to the complexity of metabolic processes within cells under conditions of sulfoglycolysis. Thus, rhizobial metabolism of the abundant sulfosugar SQ may contribute to persistence of the bacteria in the soil and to mobilization of sulfur in the pedosphere.
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http://dx.doi.org/10.1128/AEM.00750-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7376563PMC
July 2020

An Overview of the Structure, Mechanism and Specificity of Human Heparanase.

Adv Exp Med Biol 2020 ;1221:139-167

York Structural Biology Laboratory, Department of Chemistry, The University of York, York, UK.

The retaining endo-β-D-glucuronidase Heparanase (HPSE) is the primary mammalian enzyme responsible for breakdown of the glycosaminoglycan heparan sulfate (HS). HPSE activity is essential for regulation and turnover of HS in the extracellular matrix, and its activity affects diverse processes such as inflammation, angiogenesis and cell migration. Aberrant heparanase activity is strongly linked to cancer metastasis, due to structural breakdown of extracellular HS networks and concomitant release of sequestered HS-binding growth factors. A full appreciation of HPSE activity in health and disease requires a structural understanding of the enzyme, and how it engages with its HS substrates. This chapter summarizes key findings from the recent crystal structures of human HPSE and its proenzyme. We present details regarding the 3-dimensional protein structure of HPSE and the molecular basis for its interaction with HS substrates of varying sulfation states. We also examine HPSE in a wider context against related β-D-glucuronidases from other species, highlighting the structural features that control exo/endo - glycosidase selectivity in this family of enzymes.
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http://dx.doi.org/10.1007/978-3-030-34521-1_5DOI Listing
July 2020

Insights from semi-oriented EPR spectroscopy studies into the interaction of lytic polysaccharide monooxygenases with cellulose.

Dalton Trans 2020 Mar 3;49(11):3413-3422. Epub 2020 Mar 3.

Department of Chemistry, University of York, Heslington, York, YO10 5DD, UK.

Probing the detailed interaction between lytic polysaccharide monooxygenases (LPMOs) and their polysaccharide substrates is key to revealing further insights into the mechanism of action of this class of enzymes on recalcitrant biomass. This investigation is somewhat hindered, however, by the insoluble nature of the substrates, which precludes the use of most optical spectroscopic techniques. Herein, we report a new semi-oriented EPR method which evaluates directly the binding of cellulose-active LPMOs to crystalline cellulose. We make use of the intrinsic order of cellulose fibres in Apium graveolens (celery) to orient the LPMO with respect to the magnetic field of an EPR spectrometer. The subsequent angle-dependent changes observed in the EPR spectra can then be related to the orientation of the g matrix principal directions with respect to the magnetic field of the spectrometer and, hence, to the binding of the enzyme onto the cellulose fibres. This method, which does not require specific modification of standard CW-EPR equipment, can be used as a general procedure to investigate LPMO-cellulose interactions.
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http://dx.doi.org/10.1039/c9dt04065jDOI Listing
March 2020

Selective Fluorogenic β-Glucocerebrosidase Substrates for Convenient Analysis of Enzyme Activity in Cell and Tissue Homogenates.

ACS Chem Biol 2020 04 16;15(4):824-829. Epub 2020 Mar 16.

Department of Chemistry, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada.

Within mammals, there are often several functionally related glycoside hydrolases, which makes monitoring their activities problematic. This problem is particularly acute for the enzyme β-glucocerebrosidase (GCase), the malfunction of which is a key driver of Gaucher's disease (GD) and a major risk factor for Parkinson's disease (PD). Humans harbor two other functionally related β-glucosidases known as GBA2 and GBA3, and the currently used fluorogenic substrates are not selective, which has driven the use of complicated subtractive assays involving the use of detergents and inhibitors. Here we describe the preparation of fluorogenic substrates based on the widely used nonselective substrate resorufin β-d-glucopyranoside. Using recombinant enzymes, we show that these substrates are highly selective for GCase. We also demonstrate their value through the analysis of GCase activity in brain tissue homogenates from transgenic mice expressing mutant human GCase and patient fibroblasts expressing mutant GCase. This approach simplifies the analysis of cell and tissue homogenates and should facilitate the analysis of clinical and laboratory tissues and samples.
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http://dx.doi.org/10.1021/acschembio.9b01044DOI Listing
April 2020

A Direct Fluorescent Activity Assay for Glycosyltransferases Enables Convenient High-Throughput Screening: Application to O-GlcNAc Transferase.

Angew Chem Int Ed Engl 2020 06 2;59(24):9601-9609. Epub 2020 Apr 2.

Department of Chemistry, Simon Fraser University, Burnaby, BC, V5A 1S6, Canada.

Glycosyltransferases carry out important cellular functions in species ranging from bacteria to humans. Despite their essential roles in biology, simple and robust activity assays that can be easily applied to high-throughput screening for inhibitors of these enzymes have been challenging to develop. Herein, we report a bead-based strategy to measure the group-transfer activity of glycosyltransferases sensitively using simple fluorescence measurements, without the need for coupled enzymes or secondary reactions. We validate the performance and accuracy of the assay using O-GlcNAc transferase (OGT) as a model system through detailed Michaelis-Menten kinetic analysis of various substrates and inhibitors. Optimization of this assay and application to high-throughput screening enabled screening for inhibitors of OGT, leading to a novel inhibitory scaffold. We believe this assay will prove valuable not only for the study of OGT, but also more widely as a general approach for the screening of glycosyltransferases and other group-transfer enzymes.
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http://dx.doi.org/10.1002/anie.202000621DOI Listing
June 2020

Rational Design of Mechanism-Based Inhibitors and Activity-Based Probes for the Identification of Retaining α-l-Arabinofuranosidases.

J Am Chem Soc 2020 03 26;142(10):4648-4662. Epub 2020 Feb 26.

York Structural Biology Laboratory, Department of Chemistry, The University of York, Heslington, York YO10 5DD, U.K.

Identifying and characterizing the enzymes responsible for an observed activity within a complex eukaryotic catabolic system remains one of the most significant challenges in the study of biomass-degrading systems. The debranching of both complex hemicellulosic and pectinaceous polysaccharides requires the production of α-l-arabinofuranosidases among a wide variety of coexpressed carbohydrate-active enzymes. To selectively detect and identify α-l-arabinofuranosidases produced by fungi grown on complex biomass, potential covalent inhibitors and probes which mimic α-l-arabinofuranosides were sought. The conformational free energy landscapes of free α-l-arabinofuranose and several rationally designed covalent α-l-arabinofuranosidase inhibitors were analyzed. A synthetic route to these inhibitors was subsequently developed based on a key Wittig-Still rearrangement. Through a combination of kinetic measurements, intact mass spectrometry, and structural experiments, the designed inhibitors were shown to efficiently label the catalytic nucleophiles of retaining GH51 and GH54 α-l-arabinofuranosidases. Activity-based probes elaborated from an inhibitor with an aziridine warhead were applied to the identification and characterization of α-l-arabinofuranosidases within the secretome of grown on arabinan. This method was extended to the detection and identification of α-l-arabinofuranosidases produced by eight biomass-degrading basidiomycete fungi grown on complex biomass. The broad applicability of the cyclophellitol-derived activity-based probes and inhibitors presented here make them a valuable new tool in the characterization of complex eukaryotic carbohydrate-degrading systems and in the high-throughput discovery of α-l-arabinofuranosidases.
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http://dx.doi.org/10.1021/jacs.9b11351DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7068720PMC
March 2020

Mannosidase mechanism: at the intersection of conformation and catalysis.

Curr Opin Struct Biol 2020 06 28;62:79-92. Epub 2019 Dec 28.

School of Chemistry and Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria 3010, Australia. Electronic address:

Mannosidases are a diverse group of enzymes that are important in the biological processing of mannose-containing polysaccharides and complex glycoconjugates. They are found in 12 of the >160 sequence-based glycosidase families. We discuss evidence that nature has evolved a small set of common mechanisms that unite almost all of these mannosidase families. Broadly, mannosidases (and the closely related rhamnosidases) perform catalysis through just two conformations of the oxocarbenium ion-like transition state: a B (or enantiomeric B) boat and a H half-chair. This extends to a new family (GT108) of GDPMan-dependent β-1,2-mannosyltransferases/phosphorylases that perform mannosyl transfer through a boat conformation as well as some mannosidases that are metalloenzymes and require divalent cations for catalysis. Yet, among this commonality lies diversity. New evidence shows that one unique family (GH99) of mannosidases use an unusual mechanism involving anchimeric assistance via a 1,2-anhydro sugar (epoxide) intermediate.
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http://dx.doi.org/10.1016/j.sbi.2019.11.008DOI Listing
June 2020

Structure and function of 164 β-mannosidase from the founding member of glycoside hydrolase family GH164.

J Biol Chem 2020 03 22;295(13):4316-4326. Epub 2019 Dec 22.

Department of Chemistry, Structural Biology Laboratory, The University of York, York YO10 5DD, United Kingdom. Electronic address:

Recent work exploring protein sequence space has revealed a new glycoside hydrolase (GH) family (GH164) of putative mannosidases. GH164 genes are present in several commensal bacteria, implicating these genes in the degradation of dietary glycans. However, little is known about the structure, mechanism of action, and substrate specificity of these enzymes. Herein we report the biochemical characterization and crystal structures of the founding member of this family (164) from the human gut symbiont Previous reports of this enzyme indicated that it has α-mannosidase activity, however, we conclusively show that it cleaves only β-mannose linkages. Using NMR spectroscopy, detailed enzyme kinetics of WT and mutant 164, and multiangle light scattering we found that it is a trimeric retaining β-mannosidase, that is susceptible to several known mannosidase inhibitors. X-ray crystallography revealed the structure of 164, the first known structure of a GH164, at 1.91 Å resolution. 164 is composed of three domains: a (β/α) barrel, a trimerization domain, and a β-sandwich domain, representing a previously unobserved structural-fold for β-mannosidases. Structures of 164 at 1.80-2.55 Å resolution in complex with the inhibitors noeuromycin, mannoimidazole, or 2,4-dinitrophenol 2-deoxy-2-fluoro-mannoside reveal the residues essential for specificity and catalysis including the catalytic nucleophile (Glu-297) and acid/base residue (Glu-160). These findings further our knowledge of the mechanisms commensal microbes use for nutrient acquisition.
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http://dx.doi.org/10.1074/jbc.RA119.011591DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7105311PMC
March 2020

Discovering the Microbial Enzymes Driving Drug Toxicity with Activity-Based Protein Profiling.

ACS Chem Biol 2020 01 12;15(1):217-225. Epub 2019 Dec 12.

Department of Bioorganic Synthesis, Leiden Institute of Chemistry , Leiden University , Leiden 2311 , The Netherlands.

It is increasingly clear that interindividual variability in human gut microbial composition contributes to differential drug responses. For example, gastrointestinal (GI) toxicity is not observed in all patients treated with the anticancer drug irinotecan, and it has been suggested that this variability is a result of differences in the types and levels of gut bacterial β-glucuronidases (GUSs). GUS enzymes promote drug toxicity by hydrolyzing the inactive drug-glucuronide conjugate back to the active drug, which damages the GI epithelium. Proteomics-based identification of the exact GUS enzymes responsible for drug reactivation from the complexity of the human microbiota has not been accomplished, however. Here, we discover the specific bacterial GUS enzymes that generate SN-38, the active and toxic metabolite of irinotecan, from human fecal samples using a unique activity-based protein profiling (ABPP) platform. We identify and quantify gut bacterial GUS enzymes from human feces with an ABPP-enabled proteomics pipeline and then integrate this information with kinetics to pinpoint the specific GUS enzymes responsible for SN-38 reactivation. Furthermore, the same approach also reveals the molecular basis for differential gut bacterial GUS inhibition observed between human fecal samples. Taken together, this work provides an unprecedented technical and bioinformatics pipeline to discover the microbial enzymes responsible for specific reactions from the complexity of human feces. Identifying such microbial enzymes may lead to precision biomarkers and novel drug targets to advance the promise of personalized medicine.
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http://dx.doi.org/10.1021/acschembio.9b00788DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7321802PMC
January 2020

The C-Type Lysozyme from the upper Gastrointestinal Tract of the Stinkbird.

Int J Mol Sci 2019 Nov 6;20(22). Epub 2019 Nov 6.

Structural Biology Laboratory, Department of Chemistry, The University of York, York YO10 5DD, UK.

Muramidases/lysozymes are important bio-molecules, which cleave the glycan backbone in the peptidoglycan polymer found in bacterial cell walls. The glycoside hydrolase (GH) family 22 C-type lysozyme, from the folivorous bird (stinkbird), was expressed in , and a set of variants was produced. All variants were enzymatically active, including those designed to probe key differences between the Hoatzin enzyme and Hen Egg White lysozyme. Four variants showed improved thermostability at pH 4.7, compared to the wild type. The X-ray structure of the enzyme was determined in the apo form and in complex with chitin oligomers. Bioinformatic analysis of avian GH22 amino acid sequences showed that they separate out into three distinct subgroups (chicken-like birds, sea birds and other birds). The Hoatzin is found in the "other birds" group and we propose that this represents a new cluster of avian upper-gut enzymes.
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http://dx.doi.org/10.3390/ijms20225531DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6887759PMC
November 2019

Formation of a Copper(II)-Tyrosyl Complex at the Active Site of Lytic Polysaccharide Monooxygenases Following Oxidation by HO.

J Am Chem Soc 2019 11 12;141(46):18585-18599. Epub 2019 Nov 12.

York Cross-Disciplinary Centre for Systems Analysis , University of York , Heslington, York YO10 5GE , United Kingdom.

Hydrogen peroxide is a cosubstrate for the oxidative cleavage of saccharidic substrates by copper-containing lytic polysaccharide monooxygenases (LPMOs). The rate of reaction of LPMOs with hydrogen peroxide is high, but it is accompanied by rapid inactivation of the enzymes, presumably through protein oxidation. Herein, we use UV-vis, CD, XAS, EPR, VT/VH-MCD, and resonance Raman spectroscopies, augmented with mass spectrometry and DFT calculations, to show that the product of reaction of an AA9 LPMO with HO at higher pHs is a singlet Cu(II)-tyrosyl radical species, which is inactive for the oxidation of saccharidic substrates. The Cu(II)-tyrosyl radical center entails the formation of significant Cu(II)-(OTyr) overlap, which in turn requires that the plane of the d(-) SOMO of the Cu(II) is orientated toward the tyrosyl radical. We propose from the Marcus cross-relation that the active site tyrosine is part of a "hole-hopping" charge-transfer mechanism formed of a pathway of conserved tyrosine and tryptophan residues, which can protect the protein active site from inactivation during uncoupled turnover.
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http://dx.doi.org/10.1021/jacs.9b09833DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7007232PMC
November 2019

Molecular mechanisms regulating O-linked N-acetylglucosamine (O-GlcNAc)-processing enzymes.

Curr Opin Chem Biol 2019 12 22;53:131-144. Epub 2019 Oct 22.

Department of Chemistry, Simon Fraser University, Burnaby, British Columbia, V5A 1S6, Canada; Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia, V5A 1S6, Canada. Electronic address:

The post-translational modification of proteins by O-linked N-acetylglucosamine (O-GlcNAc) dynamically programmes cellular physiology to maintain homoeostasis and tailor biochemical pathways to meet context-dependent cellular needs. Despite diverse roles of played by O-GlcNAc, only two enzymes act antagonistically to govern its cycling; O-GlcNAc transferase installs the monosaccharide on target proteins, and O-GlcNAc hydrolase removes it. The recent literature has exposed a network of mechanisms regulating these two enzymes to choreograph global, and target-specific, O-GlcNAc cycling in response to cellular stress and nutrient availability. Herein, we amalgamate these emerging mechanisms from a structural and molecular perspective to explore how the cell exerts fine control to regulate O-GlcNAcylation of diverse proteins in a selective fashion.
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http://dx.doi.org/10.1016/j.cbpa.2019.09.001DOI Listing
December 2019

Inverting family GH156 sialidases define an unusual catalytic motif for glycosidase action.

Nat Commun 2019 10 23;10(1):4816. Epub 2019 Oct 23.

Department of Chemistry, University of York, York, YO10 5DD, UK.

Sialic acids are a family of related sugars that play essential roles in many biological events intimately linked to cellular recognition in both health and disease. Sialidases are therefore orchestrators of cellular biology and important therapeutic targets for viral infection. Here, we sought to define if uncharacterized sialidases would provide distinct paradigms in sialic acid biochemistry. We show that a recently discovered sialidase family, whose first member EnvSia156 was isolated from hot spring metagenomes, defines an unusual structural fold and active centre constellation, not previously described in sialidases. Consistent with an inverting mechanism, EnvSia156 reveals a His/Asp active center in which the His acts as a Brønsted acid and Asp as a Brønsted base in a single-displacement mechanism. A predominantly hydrophobic aglycone site facilitates accommodation of a variety of 2-linked sialosides; a versatility that offers the potential for glycan hydrolysis across a range of biological and technological platforms.
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http://dx.doi.org/10.1038/s41467-019-12684-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6811678PMC
October 2019

Structural and Functional Characterization of Three Novel Fungal Amylases with Enhanced Stability and pH Tolerance.

Int J Mol Sci 2019 Oct 3;20(19). Epub 2019 Oct 3.

York Structural Biology Laboratory, Department of Chemistry, University of York, Heslington, York YO10 5DD, UK.

Amylases are probably the best studied glycoside hydrolases and have a huge biotechnological value for industrial processes on starch. Multiple amylases from fungi and microbes are currently in use. Whereas bacterial amylases are well suited for many industrial processes due to their high stability, fungal amylases are recognized as safe and are preferred in the food industry, although they lack the pH tolerance and stability of their bacterial counterparts. Here, we describe three amylases, two of which have a broad pH spectrum extending to pH 8 and higher stability well suited for a broad set of industrial applications. These enzymes have the characteristic GH13 α-amylase fold with a central (β/α)-domain, an insertion domain with the canonical calcium binding site and a C-terminal β-sandwich domain. The active site was identified based on the binding of the inhibitor acarbose in form of a transglycosylation product, in the amylases from and . The three amylases have shortened loops flanking the nonreducing end of the substrate binding cleft, creating a more open crevice. Moreover, a potential novel binding site in the C-terminal domain of the enzyme was identified, which might be part of a starch interaction site. In addition, amylase presented a successful example of using the microseed matrix screening technique to significantly speed-up crystallization.
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http://dx.doi.org/10.3390/ijms20194902DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6801514PMC
October 2019

Discovery, activity and characterisation of an AA10 lytic polysaccharide oxygenase from the shipworm symbiont .

Biotechnol Biofuels 2019 30;12:232. Epub 2019 Sep 30.

1Department of Chemistry, University of York, York, YO10 5DD UK.

Background: The quest for novel enzymes for cellulosic biomass-degradation has recently been focussed on lytic polysaccharide monooxygenases (LPMOs/PMOs), Cu-containing proteins that catalyse the oxidative degradation of otherwise recalcitrant polysaccharides using O or HO as a co-substrate.

Results: Although classical saprotrophic fungi and bacteria have been a rich source of lytic polysaccharide monooxygenases (LPMOs), we were interested to see if LPMOs from less evident bio-environments could be discovered and assessed for their cellulolytic activity in a biofuel context. In this regard, the marine shipworm represents an interesting source of new enzymes, since it must digest wood particles ingested during its natural tunnel boring behaviour and plays host to a symbiotic bacterium, , the genome of which has revealed a multitude of enzymes dedicated to biomass deconstruction. Here, we show that encodes a cellulose-active AA10 LPMO. The 3D structure, at 1.4 Å resolution, along with its EPR spectrum is distinct from other AA10 polysaccharide monooxygenases insofar as it displays a "histidine-brace" catalytic apparatus with changes to the surrounding coordination sphere of the copper. Furthermore, AA10A possesses a second, surface accessible, Cu site 14 Å from the classical catalytic centre. Activity measurements show that the LPMO oxidises cellulose and thereby significantly augments the rate of degradation of cellulosic biomass by classical glycoside hydrolases.

Conclusion: Shipworms are wood-boring marine molluscs that can live on a diet of lignocellulose. Bacterial symbionts of shipworms provide many of the enzymes needed for wood digestion. The shipworm symbiont produces one of the few LPMOs yet described from the marine environment, notably adding to the capability of shipworms to digest recalcitrant polysaccharides.
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http://dx.doi.org/10.1186/s13068-019-1573-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6767633PMC
September 2019

A Family of Dual-Activity Glycosyltransferase-Phosphorylases Mediates Mannogen Turnover and Virulence in Leishmania Parasites.

Cell Host Microbe 2019 Sep;26(3):385-399.e9

Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, VIC 3010, Australia. Electronic address:

Parasitic protists belonging to the genus Leishmania synthesize the non-canonical carbohydrate reserve, mannogen, which is composed of β-1,2-mannan oligosaccharides. Here, we identify a class of dual-activity mannosyltransferase/phosphorylases (MTPs) that catalyze both the sugar nucleotide-dependent biosynthesis and phosphorolytic turnover of mannogen. Structural and phylogenic analysis shows that while the MTPs are structurally related to bacterial mannan phosphorylases, they constitute a distinct family of glycosyltransferases (GT108) that have likely been acquired by horizontal gene transfer from gram-positive bacteria. The seven MTPs catalyze the constitutive synthesis and turnover of mannogen. This metabolic rheostat protects obligate intracellular parasite stages from nutrient excess, and is essential for thermotolerance and parasite infectivity in the mammalian host. Our results suggest that the acquisition and expansion of the MTP family in Leishmania increased the metabolic flexibility of these protists and contributed to their capacity to colonize new host niches.
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http://dx.doi.org/10.1016/j.chom.2019.08.009DOI Listing
September 2019

An overview of activity-based probes for glycosidases.

Curr Opin Chem Biol 2019 12 13;53:25-36. Epub 2019 Aug 13.

York Structural Biology Laboratory, Department of Chemistry, University of York, York YO10 5DD, United Kingdom. Electronic address:

As the scope of modern genomics technologies increases, so does the need for informative chemical tools to study functional biology. Activity-based probes (ABPs) provide a powerful suite of reagents to probe the biochemistry of living organisms. These probes, featuring a specificity motif, a reactive chemical group and a reporter tag, are opening-up large swathes of protein chemistry to investigation in vitro, as well as in cellular extracts, cells and living organisms in vivo. Glycoside hydrolases, by virtue of their prominent biological and applied roles, provide a broad canvas on which ABPs may illustrate their functions. Here we provide an overview of glycosidase ABP mechanisms, and review recent ABP work in the glycoside hydrolase field, encompassing their use in medical diagnosis, their application for generating chemical genetic disease models, their fine-tuning through conformational and reactivity insight, their use for high-throughput inhibitor discovery, and their deployment for enzyme discovery and dynamic characterization.
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http://dx.doi.org/10.1016/j.cbpa.2019.05.030DOI Listing
December 2019