Davies GJ

References (9)

Title : Detecting and identifying glycoside hydrolases using cyclophellitol-derived activity-based probes - McGregor_2022_Methods.Enzymol_664_103
Author(s) : McGregor NGS , Overkleeft HS , Davies GJ
Ref : Methods Enzymol , 664 :103 , 2022
Abstract : 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.
ESTHER : McGregor_2022_Methods.Enzymol_664_103
PubMedSearch : McGregor_2022_Methods.Enzymol_664_103
PubMedID: 35331370

Title : An Epoxide Intermediate in Glycosidase Catalysis - Sobala_2020_ACS.Cent.Sci_6_760
Author(s) : Sobala LF , Speciale G , Zhu S , Raich L , Sannikova N , Thompson AJ , Hakki Z , Lu D , Shamsi Kazem Abadi S , Lewis AR , Rojas-Cervellera V , Bernardo-Seisdedos G , Zhang Y , Millet O , Jimenez-Barbero J , Bennet AJ , Sollogoub M , Rovira C , Davies GJ , Williams SJ
Ref : ACS Cent Sci , 6 :760 , 2020
Abstract : 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 endo-alpha-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 beta-1,2-aziridine and beta-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 (E 3) conformation. Kinetic isotope effects (k cat/K M) for anomeric-(2)H and anomeric-(13)C support an oxocarbenium ion-like transition state, and that for C2-(18)O (1.052 +/- 0.006) directly implicates nucleophilic participation by the C2-hydroxyl. Collectively, these data substantiate this unprecedented and long-imagined enzymatic mechanism.
ESTHER : Sobala_2020_ACS.Cent.Sci_6_760
PubMedSearch : Sobala_2020_ACS.Cent.Sci_6_760
PubMedID: 32490192

Title : Complex pectin metabolism by gut bacteria reveals novel catalytic functions - Ndeh_2017_Nature_544_65
Author(s) : Ndeh D , Rogowski A , Cartmell A , Luis AS , Basle A , Gray J , Venditto I , Briggs J , Zhang X , Labourel A , Terrapon N , Buffetto F , Nepogodiev S , Xiao Y , Field RA , Zhu Y , O'Neil MA , Urbanowicz BR , York WS , Davies GJ , Abbott DW , Ralet MC , Martens EC , Henrissat B , Gilbert HJ
Ref : Nature , 544 :65 , 2017
Abstract : The metabolism of carbohydrate polymers drives microbial diversity in the human gut microbiota. It is unclear, however, whether bacterial consortia or single organisms are required to depolymerize highly complex glycans. Here we show that the gut bacterium Bacteroides thetaiotaomicron uses the most structurally complex glycan known: the plant pectic polysaccharide rhamnogalacturonan-II, cleaving all but 1 of its 21 distinct glycosidic linkages. The deconstruction of rhamnogalacturonan-II side chains and backbone are coordinated to overcome steric constraints, and the degradation involves previously undiscovered enzyme families and catalytic activities. The degradation system informs revision of the current structural model of rhamnogalacturonan-II and highlights how individual gut bacteria orchestrate manifold enzymes to metabolize the most challenging glycan in the human diet.
ESTHER : Ndeh_2017_Nature_544_65
PubMedSearch : Ndeh_2017_Nature_544_65
PubMedID: 28329766

Title : Evidence that family 35 carbohydrate binding modules display conserved specificity but divergent function - Montanier_2009_Proc.Natl.Acad.Sci.U.S.A_106_3065
Author(s) : Montanier C , van Bueren AL , Dumon C , Flint JE , Correia MA , Prates JA , Firbank SJ , Lewis RJ , Grondin GG , Ghinet MG , Gloster TM , Herve C , Knox JP , Talbot BG , Turkenburg JP , Kerovuo J , Brzezinski R , Fontes CM , Davies GJ , Boraston AB , Gilbert HJ
Ref : Proc Natl Acad Sci U S A , 106 :3065 , 2009
Abstract : Enzymes that hydrolyze complex carbohydrates play important roles in numerous biological processes that result in the maintenance of marine and terrestrial life. These enzymes often contain noncatalytic carbohydrate binding modules (CBMs) that have important substrate-targeting functions. In general, there is a tight correlation between the ligands recognized by bacterial CBMs and the substrate specificity of the appended catalytic modules. Through high-resolution structural studies, we demonstrate that the architecture of the ligand binding sites of 4 distinct family 35 CBMs (CBM35s), appended to 3 plant cell wall hydrolases and the exo-beta-D-glucosaminidase CsxA, which contributes to the detoxification and metabolism of an antibacterial fungal polysaccharide, is highly conserved and imparts specificity for glucuronic acid and/or Delta4,5-anhydrogalaturonic acid (Delta4,5-GalA). Delta4,5-GalA is released from pectin by the action of pectate lyases and as such acts as a signature molecule for plant cell wall degradation. Thus, the CBM35s appended to the 3 plant cell wall hydrolases, rather than targeting the substrates of the cognate catalytic modules, direct their appended enzymes to regions of the plant that are being actively degraded. Significantly, the CBM35 component of CsxA anchors the enzyme to the bacterial cell wall via its capacity to bind uronic acid sugars. This latter observation reveals an unusual mechanism for bacterial cell wall enzyme attachment. This report shows that the biological role of CBM35s is not dictated solely by their carbohydrate specificities but also by the context of their target ligands.
ESTHER : Montanier_2009_Proc.Natl.Acad.Sci.U.S.A_106_3065
PubMedSearch : Montanier_2009_Proc.Natl.Acad.Sci.U.S.A_106_3065
PubMedID: 19218457
Gene_locus related to this paper: celju-b3pei5

Title : The active site of a carbohydrate esterase displays divergent catalytic and noncatalytic binding functions - Montanier_2009_PLoS.Biol_7_e71
Author(s) : Montanier C , Money VA , Pires VM , Flint JE , Pinheiro BA , Goyal A , Prates JA , Izumi A , Stalbrand H , Morland C , Cartmell A , Kolenova K , Topakas E , Dodson EJ , Bolam DN , Davies GJ , Fontes CM , Gilbert HJ
Ref : PLoS Biol , 7 :e71 , 2009
Abstract : Multifunctional proteins, which play a critical role in many biological processes, have typically evolved through the recruitment of different domains that have the required functional diversity. Thus the different activities displayed by these proteins are mediated by spatially distinct domains, consistent with the specific chemical requirements of each activity. Indeed, current evolutionary theory argues that the colocalization of diverse activities within an enzyme is likely to be a rare event, because it would compromise the existing activity of the protein. In contrast to this view, a potential example of multifunctional recruitment into a single protein domain is provided by CtCel5C-CE2, which contains an N-terminal module that displays cellulase activity and a C-terminal module, CtCE2, which exhibits a noncatalytic cellulose-binding function but also shares sequence identity with the CE2 family of esterases. Here we show that, unlike other CE2 members, the CtCE2 domain displays divergent catalytic esterase and noncatalytic carbohydrate binding functions. Intriguingly, these diverse activities are housed within the same site on the protein. Thus, a critical component of the active site of CtCE2, the catalytic Ser-His dyad, in harness with inserted aromatic residues, confers noncatalytic binding to cellulose whilst the active site of the domain retains its esterase activity. CtCE2 catalyses deacetylation of noncellulosic plant structural polysaccharides to deprotect these substrates for attack by other enzymes. Yet it also acts as a cellulose-binding domain, which promotes the activity of the appended cellulase on recalcitrant substrates. The CE2 family encapsulates the requirement for multiple activities by biocatalysts that attack challenging macromolecular substrates, including the grafting of a second, powerful and discrete noncatalytic binding functionality into the active site of an enzyme. This article provides a rare example of "gene sharing," where the introduction of a second functionality into the active site of an enzyme does not compromise the original activity of the biocatalyst.
ESTHER : Montanier_2009_PLoS.Biol_7_e71
PubMedSearch : Montanier_2009_PLoS.Biol_7_e71
PubMedID: 19338387

Title : Molecular determinants of substrate specificity in the feruloyl esterase module of xylanase 10B from Clostridium thermocellum - Tarbouriech_2005_Acta.Crystallogr.D.Biol.Crystallogr_61_194
Author(s) : Tarbouriech N , Prates JA , Fontes CM , Davies GJ
Ref : Acta Crystallographica D Biol Crystallogr , 61 :194 , 2005
Abstract : Feruloyl esterases play a key role in the degradation of the intricate structure of the plant cell wall by hydrolysing the ferulate ester groups involved in the cross-linking between hemicelluloses and between hemicellulose and lignin. The structure of the feruloyl esterase module of Clostridium thermocellum cellulosomal xylanase 10B has been reported previously. It displays the alpha/beta hydrolase fold with a classical Ser-His-Asp catalytic triad. Here, the structures of a Ser-Ala mutant of this feruloyl esterase in complexes with methyl syringate, methyl sinapinate and methyl vanillate are described. Substrate binding is accompanied by subtle conformational changes at amino acids Trp982, Met955, Asn1023 and Ile1019 in the ligand-binding cavity. The structural determinants, particularly the m-methoxy substituent, governing the substrate specificity of Xyn10B feruloyl esterase are rationalized.
ESTHER : Tarbouriech_2005_Acta.Crystallogr.D.Biol.Crystallogr_61_194
PubMedSearch : Tarbouriech_2005_Acta.Crystallogr.D.Biol.Crystallogr_61_194
PubMedID: 15681871
Gene_locus related to this paper: clotm-xyny

Title : Multifunctional Xylooligosaccharide\/Cephalosporin C Deacetylase Revealed by the Hexameric Structure of the Bacillus subtilis Enzyme at 1.9A Resolution - Vincent_2003_J.Mol.Biol_330_593
Author(s) : Vincent F , Charnock SJ , Verschueren KH , Turkenburg JP , Scott DJ , Offen WA , Roberts S , Pell G , Gilbert HJ , Davies GJ , Brannigan JA
Ref : Journal of Molecular Biology , 330 :593 , 2003
Abstract : Esterases and deacetylases active on carbohydrate ligands have been classified into 14 families based upon amino acid sequence similarities. Enzymes from carbohydrate esterase family seven (CE-7) are unusual in that they display activity towards both acetylated xylooligosaccharides and the antibiotic, cephalosporin C. The 1.9A structure of the multifunctional CE-7 esterase (hereinafter CAH) from Bacillus subtilis 168 reveals a classical alpha/beta hydrolase fold encased within a 32 hexamer. This is the first example of a hexameric alpha/beta hydrolase and is further evidence of the versatility of this particular fold, which is used in a wide variety of biological contexts. A narrow entrance tunnel leads to the centre of the molecule, where the six active-centre catalytic triads point towards the tunnel interior and thus are sequestered away from cytoplasmic contents. By analogy to self-compartmentalising proteases, the tunnel entrance may function to hinder access of large substrates to the poly-specific active centre. This would explain the observation that the enzyme is active on a variety of small, acetylated molecules. The structure of an active site mutant in complex with the reaction product, acetate, reveals details of the putative oxyanion binding site, and suggests that substrates bind predominantly through non-specific contacts with protein hydrophobic residues. Protein residues involved in catalysis are tethered by interactions with protein excursions from the canonical alpha/beta hydrolase fold. These excursions also mediate quaternary structure maintenance, so it would appear that catalytic competence is only achieved on protein multimerisation. We suggest that the acetyl xylan esterase (EC and cephalosporin C deacetylase (EC enzymes of the CE-7 family represent a single class of proteins with a multifunctional deacetylase activity against a range of small substrates.
ESTHER : Vincent_2003_J.Mol.Biol_330_593
PubMedSearch : Vincent_2003_J.Mol.Biol_330_593
PubMedID: 12842474
Gene_locus related to this paper: bacsu-CAH

Title : The Structure of the Feruloyl Esterase Module of Xylanase 10B from Clostridium thermocellum Provides Insights into Substrate Recognition - Prates_2001_Structure_9_1183
Author(s) : Prates JA , Tarbouriech N , Charnock SJ , Fontes CM , Ferreira LM , Davies GJ
Ref : Structure , 9 :1183 , 2001
Abstract : BACKGROUND: Degradation of the plant cell wall requires the synergistic action of a consortium of predominantly modular enzymes. In Clostridiae, these biocatalysts are organized into a supramolecular assembly termed a "cellulosome." This multienzyme complex possesses, in addition to its well-described cellulolytic activity, an apparatus specific for xylan degradation. Cinnamic acid esterases hydrolyze the ferulate groups involved in the crosslinking of arabinoxylans to lignin and thus play a key role in the degradation of the plant cell wall in addition to having promising industrial and medical applications. RESULTS: We have cloned and overexpressed the feruloyl esterase module from a 5 domain xylanase, Xyn10B from Clostridium thermocellum. The native structure at 1.6 A resolution has been solved with selenomethionine multiple wavelength anomalous dispersion and refined to a final R(free) of 17.8%. The structure of a hydrolytically inactive mutant, S954A, in complex with the reaction product ferulic acid has been refined at a resolution of 1.4 A with an R(free) of 16.0%.
CONCLUSIONS: The C. thermocellum Xyn10B ferulic acid esterase displays the alpha/beta-hydrolase fold and possesses a classical Ser-His-Asp catalytic triad. Ferulate esterases are characterized by their specificity, and the active center reveals the binding site for ferulic acid and related compounds. Ferulate binds in a small surface depression that possesses specificity determinants for both the methoxy and hydroxyl ring substituents of the substrate. There appears to be a lack of specificity for the xylan backbone, which may reflect the intrinsic chemical heterogeneity of the natural substrate.
ESTHER : Prates_2001_Structure_9_1183
PubMedSearch : Prates_2001_Structure_9_1183
PubMedID: 11738044
Gene_locus related to this paper: clotm-xyny

Title : A new variant of the Ntn hydrolase fold revealed by the crystal structure of L-aminopeptidase D-Ala-esterase\/amidase from Ochrobactrum anthropi - Bompard-Gilles_2000_Structure.Fold.Des_8_153
Author(s) : Bompard-Gilles C , Villeret V , Davies GJ , Fanuel L , Joris B , Frere JM , Van Beeumen J
Ref : Structure Fold Des , 8 :153 , 2000
Abstract : Background The L-aminopeptidase D-Ala-esterase/amidase from Ochrobactrum anthropi (DmpA) releases the N-terminal L and/or D-Ala residues from peptide substrates. This is the only known enzyme to liberate N-terminal amino acids with both D and L stereospecificity. The DmpA active form is an alphabeta heterodimer, which results from a putative autocatalytic cleavage of an inactive precursor polypeptide. RESULTS: The crystal structure of the enzyme has been determined to 1.82 A resolution using the multiple isomorphous replacement method. The heterodimer folds into a single domain organised as an alphabetabetaalpha sandwich in which two mixed beta sheets are flanked on both sides by two alpha helices.
CONCLUSIONS: DmpA shows no similarity to other known aminopeptidases in either fold or catalytic mechanism, and thus represents the first example of a novel family of aminopeptidases. The protein fold of DmpA does, however, show structural homology to members of the N-terminal nucleophile (Ntn) hydrolase superfamily. DmpA presents functionally equivalent residues in the catalytic centre when compared with other Ntn hydrolases, and is therefore likely to use the same catalytic mechanism. In spite of this homology, the direction and connectivity of the secondary structure elements differ significantly from the consensus Ntn hydrolase topology. The DmpA structure thus characterises a new subfamily, but supports the common catalytic mechanism for these enzymes suggesting an evolutionary relationship.
ESTHER : Bompard-Gilles_2000_Structure.Fold.Des_8_153
PubMedSearch : Bompard-Gilles_2000_Structure.Fold.Des_8_153
PubMedID: 10673442