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Tertiary structure of the covalent fluoroglycosyl-enzyme intermediate formed by cocrystallization of CjGH5D(E255A) with XXXG(2F)-β-DNP. a. Divergent (wall-eyed) stereo cartoon representation of the secondary structure, colour ramped from the N-terminus (blue) to the C-terminus (red), with the inhibitor represented as sticks with C atoms in green, and the catalytic residues with C atoms in tan. b: Maximum-likelihood/σ A weighted 2F obs − F calc electron density map contoured at an r.m.s.d. level of 1 σ for the ligand XXXG-2F. c: Hydrogen bonding interactions with the ligand. In panels b and c, side chains of interacting residues are shown in ice blue and hydrogen bonds are shown as dashed lines.
Source publication
Xyloglucan (XyG) is a complex polysaccharide that is ubiquitous and often abundant in the cell walls of terrestrial plants. XyG metabolism is therefore a key component of the global carbon cycle, and hence XyG enzymology is of significant fundamental and applied importance in biomass conversion. To facilitate structure-function analyses of XyG-spec...
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... Selectfluor™ by-product and the filtrate was concentrated by rotary evaporation. Elution of the crude product through a silica column (ethyl acetate/hexanes, 3.5 : 1) to remove polar impurities and remaining by-products yielded a mixture of anomers (yield 0.24 g, 37%), which was used directly in the next step without further separation. 19 F-NMR (Fig. S3, † 376.5 MHz, CDCl 3 ) gluco β-anomer: −197.91 (ddd, J H2-F2 = 51.1 Hz, J H3-F2 = 13.7 Hz, J H1-F2 = 1.6 Hz, F2), gluco α-anomer: −199.30 (dd, J H2-F2 = 49.5 Hz, J H3-F2 = 12.0 Hz, J H1-F2 = 0 Hz, F2), consistent with values for the corresponding cellobioside. 21 The synthesis of the per-O-acetylated 2′4′-dinitrophenyl β-glycoside of ...
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... e.g. cellobiose, the amount of the 2F-gluco epimer reached 80% in polar solvents. 44,45 In our case, we were pleased to discover that only the desired gluco configured product was obtained in dry nitromethane, 43 albeit as a mixture of α/β anomers, as indicated by 19 F NMR (trace amounts of manno epimers were perhaps also present, 45 Fig. S3 †). As this represents the only example, to our knowledge, of the application of Selectfluor™ to such a large oligosaccharide glucal, the origins of this high stereoselectivity are not fully clear, but may be based in increased local steric congestion or altered access to boat-conformer addition products. 45 The reaction of the anomeric ...
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... turnover of the fluoroglycosyl-enzyme (Fig. 3b) precluded the observation of a covalent complex by protein crystallography, despite MS evidence of its accumulation (Fig. S10 †). To overcome this problem, 30 we produced a site-directed mutation of the general acid/base residue Glu255 to alanine in order to further reduce the rate of hydrolysis of the fluoroglycosyl-enzyme ...
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... was by soaking of E255A crystals with the inhibitor, which surprisingly led to an unreacted complex; with a −3 to +1 binding mode of the glycone with the aryl glycosidic bond still intact (Fig. S11 †). For this reason, co-crystallisation was instead used to access the reacted tertiary structure of XXXG(2F)-CjGH5D(E255A) at 1.7 Å resolution (Fig. 3). The overall structures corresponded well to that of the wild-type enzyme in free, or "apo", form (PBD ID 5OYC), which has a classic (β/α) 8 -barrel fold. 46 Crystallographic data collection and refinement statistics are given in Table S1. † Privateer results showing validation for Glc (BGC) and Xyl (XYS) residues in ...
Citations
... One promising technique relies on devising and synthesizing surrogate ligand mimetics that act as mechanism-based inhibitors. 76, 77 We conjecture that this mechanism may also play a role in other carbohydrate-active enzymes, such as lytic transglycosylases, expansins, loosenins, and swollenins, which are evolutionarily close to PcCe-l45A. 22,23,49 We expect our findings motivate further experimental and theoretical studies aimed at probing the inverting endocyclic mechanism in PcCel45A and correlated enzymes. ...
Glycoside hydrolases (GH) cleave carbohydrate glycosidic bonds and play pivotal roles in living organisms and in many industrial processes. Unlike acid-catalyzed hydrolysis of carbohydrates in solution, which can occur either via cyclic or acyclic oxocarbenium-like transition states, it is widely accepted that GH-catalyzed hydrolysis proceeds via a general acid mechanism involving a cyclic oxocarbenium-like transition state with protonation of the glycosidic oxygen. The GH45 subfamily C inverting endoglucanase from Phanerochaete chrysosporium (PcCel45A) defies the classical inverting mechanism as its crystal structure conspicuously lacks a general Asp or Glu base residue. Instead, PcCel45A has an Asn residue, a notoriously weak base in solution, as one of its catalytic residues at position 92. Moreover, unlike other inverting GHs, the relative position of the catalytic residues in PcCel45A impairs the proton abstraction from the nucleophilic water that attacks the anomeric carbon, a key step in the classical mechanism. Here, we investigate the viability of an endocyclic mechanism for PcCel45A using hybrid quantum mechanics/molecular mechanics (QM/MM) simulations, with the QM region treated with the self-consistent-charge density-functional tight-binding level of theory. In this mechanism, an acyclic oxocarbenium-like transition state is stabilized leading to the opening of the glucopyranose ring and formation of an unstable acyclic hemiacetal that can be readily decomposed into hydrolysis product. In silico characterization of the Michaelis complex shows that PcCel45A significantly restrains the sugar ring to the ⁴C1 chair conformation at the −1 subsite of the substrate binding cleft, in contrast to the classical exocyclic mechanism in which ring puckering is critical. We also show that PcCel45A provides an environment where the catalytic Asn92 residue in its standard amide form participates in a cooperative hydrogen bond network resulting in its increased nucleophilicity due to an increased negative charge on the oxygen atom. Our results for PcCel45A suggest that carbohydrate hydrolysis catalyzed by GHs may take an alternative route from the classical mechanism.
... Whilst 'monosaccharide' ABPs that target retaining exo-glycosidases have been relatively well explored, profiling of retaining endo-glycosidases will require more complex ABPs structures to mimic the length and/or branching of their natural substrates. Given past and recent successes in the glycosylation of fluoroglycoside [20,60], cyclophellitol-derived [58 ,61], and bromoketone [37] warheads, the general profiling of retaining endo-glycosidases appears to be within reach. ...
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.
Glycans are the most abundant and diverse group of biomolecules with a crucial role in all the biological processes. Their structural and functional diversity is not genetically encoded, but depends on Carbohydrate Active Enzymes (CAZymes) which carry out all catalytic activities in terms of synthesis, modification, and degradation. CAZymes comprise large families of enzymes with specific functions and are widely used for various commercial applications ranging from biofuel production to textile and food industries with impact on biorefineries. To understand the structure and functional mechanism of these CAZymes for their modification for industrial use, together with knowledge of therapeutic aspects of their dysfunction associated with various diseases, CAZyme inhibitors can be used as a valuable tool. In search for new inhibitors, the screening of various secondary metabolites using high-throughput techniques and rational design techniques have been explored. The inhibitors can thus help tune CAZymes and are emerging as a potential research interest.
The ability to detect active enzymes in a complex mixture of folded proteins (e.g., secretome, cell lysate) generally relies on observations of catalytic ability, necessitating the development of an activity assay that is compatible with the sample and selective for the enzyme(s) of interest. Deconvolution of the contributions of different enzymes to an observed catalytic ability further necessitates an often-challenging protein separation. The advent of broadly reactive activity-based probes (ABPs) for retaining glycoside hydrolases (GHs) has enabled an alternative, often complementary, assay for active GHs. Using activity-based protein profiling (ABPP) techniques, many retaining glycoside hydrolases can be separated, detected, and identified with high sensitivity and selectivity. This chapter outlines ABPP methods for the detection and identification of retaining glycoside hydrolases from microbial sources, including protein sample preparation from bacterial lysates and fungal secretomes, enzyme labeling and detection via fluorescence, and enzyme identification using affinity-based enrichment coupled to peptide sequencing following isobaric labeling.
This review is the tenth update of the original article published in 1999 on the application of matrix‐assisted laser desorption/ionization mass spectrometry (MALDI) mass spectrometry to the analysis of carbohydrates and glycoconjugates and brings coverage of the literature to the end of 2018. Also included are papers that describe methods appropriate to glycan and glycoprotein analysis by MALDI, such as sample preparation techniques, even though the ionization method is not MALDI. Topics covered in the first part of the review include general aspects such as theory of the MALDI process, new methods, matrices, derivatization, MALDI imaging, fragmentation and the use of arrays. The second part of the review is devoted to applications to various structural types such as oligo‐ and poly‐saccharides, glycoproteins, glycolipids, glycosides, and biopharmaceuticals. Most of the applications are presented in tabular form. The third part of the review covers medical and industrial applications of the technique, studies of enzyme reactions, and applications to chemical synthesis. The reported work shows increasing use of combined new techniques such as ion mobility and highlights the impact that MALDI imaging is having across a range of diciplines. MALDI is still an ideal technique for carbohydrate analysis and advancements in the technique and the range of applications continue steady progress.
A urea with a pendant acetal has been reacted with nucleophilic aromatics to give a range of heterocyclic products. The acid‐catalyzed reaction has a complex mechanistic manifold, with the nature of the product dependent on the nucleophilicity of the arene and the strength of the acid. Catalytic glycosylations, with a particular focus on synthesis of oligosaccharides, are the subject of a comprehensive review. β‐Glucosyl fluoride has been used to identify the acid‐base catalytic residue of a glycosyltransferase enzyme. A computational study of fast pyrolysis of glucose has focused on four mechanisms of water loss; Maccoll elimination, pinacol ring contraction, cyclic Grob fragmentation, and alcohol condensation. Iridium‐catalyzed asymmetric hydrogenation of prochiral imines is very useful for preparing chiral amines. Aldehydes and ketones have been reacted with α‐nucleophiles such as hydrazines and alkoxy‐amines to give oximes and hydrazones using simple non‐toxic bifunctional buffers to both control pH and accelerate the reactions.
This chapter reviews the reactivity of fluorographene towards nucleophilic substitution, reduction and radical substitution pathways. Imperfections in the fluorographene sheets induce the observed reactivity patterns in the intrinsically unreactive perfluorinated structure. A review of substitutions of arenediazonium salts includes metal catalysis and single‐electron transfer processes, and emphasises the application of weak inorganic bases to promote substitution pathways. The electrochemical reduction of trichlorobiphenyls was studied by cyclic voltammetry and bulk electrolysis. A DFT study of the amine exchange reactions of the naphthalene derivative, with ammonia and primary and secondary amines showed that the amine reactivity order depends on the stability of the Meisenheimer intermediates, which may be stabilised by hydrogen bonding between the amine and the carbonyl group. There have been reviews of experimental and computational studies of carbon‐hydrogen bond activations and functionalisations by ruthenium catalysis, and of carbon‐hydrogen substitutions in N‐heteroaromatic compounds catalysed by Group 3 and 4 organometallic complexes.
