Dissecting conformational contributions to glycosidase catalysis and inhibition

Speciale, Gaetano; Thompson, Andrew J.; Davies, G.J.; Williams, Spencer J.

doi:10.1016/j.sbi.2014.06.003

Cited by 123 publications

(149 citation statements)

References 68 publications

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“…The unreactive nature of α -MTF supports the notion that Sco GlgEI-V279S likely proceeds though a late dissociate transition state, destabilized by the 2,2-fluoromethylene modification, in the first step of the enzymatic reaction. 18 The X-ray structure of the Sco GlgEI-V279S in complex with α -MTF was solved. These results also illustrate how Sco GlgEI-V279S can be used in lieu of Mtb GlgE to rapidly probing important protein-ligand interactions.…”

Section: Discussionmentioning

confidence: 99%

Synthesis of 2-deoxy-2,2-difluoro-α-maltosyl fluoride and its X-ray structure in complex with Streptomyces coelicolor GlgEI-V279S

et al. 2015

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Streptomyces coelicolor (Sco) GlgEI is a glycoside hydrolase involved in α-glucan biosynthesis and can be used as a model enzyme for structure-based inhibitor design targeting Mycobacterium tuberculosis (Mtb) GlgE. The latter is a genetically validated drug target for the development of anti-Tuberculosis (TB) treatments. Inhibition of Mtb GlgE results in a lethal buildup of the GlgE substrate maltose-1-phosphate (M1P). However, Mtb GlgE is difficult to crystallize and affords lower resolution X-ray structures. Sco GlgEI-V279S on the other hand crystallizes readily, produces high resolution X-ray data, and has active site topology identical to Mtb GlgE. We report the X-ray structure of Sco GlgEI-V279S in complex with 2-deoxy-2,2-difluoro-α-maltosyl fluoride (α-MTF, 5) at 2.3Å resolution. α-MTF was designed as a non-hydrolysable mimic of M1P to probe the active site of GlgE1 prior to covalent bond formation without disruption of catalytic residues. The α-MTF complex revealed hydrogen bonding between Glu423 and the C1′F which provides evidence that Glu423 functions as proton donor during catalysis. Further, hydrogen bonding between Arg392 and the axial C2′ difluoromethylene moiety of α-MTF was observed suggesting that the C2′ position tolerates substitution with hydrogen bond acceptors. The key step in the synthesis of α-MDF was transformation of peracetylated 2-fluoro-maltal 1 into peracetylated 2,2-difluoro-α-maltosyl fluoride 2 in a single step via the use of Selectfluor®

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Section: Discussionmentioning

confidence: 99%

Synthesis of 2-deoxy-2,2-difluoro-α-maltosyl fluoride and its X-ray structure in complex with Streptomyces coelicolor GlgEI-V279S

et al. 2015

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“…The enzymatic hydrolysis of rhamnosidic bonds is particularly challenging. In-line nucleophilic attack at C1 of rhamnose would involve steric clash with the axial O2 hydroxyl, analogous to the challenges in mannose chemistry (9). Distortion of the pyranoside ring, to place O2 pseudoequatorial, will be needed to minimize these clashes, as has been observed previously for an α-L-rhamnosidase from GH78 (10) and GH106 (11).…”

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confidence: 88%

Unusual active site location and catalytic apparatus in a glycoside hydrolase family

Muñoz‐Muñoz

Cartmell

Terrapon

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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The human gut microbiota use complex carbohydrates as major nutrients. The requirement for an efficient glycan degrading systems exerts a major selection pressure on this microbial community. Thus, we propose that these bacteria represent a substantial resource for discovering novel carbohydrate active enzymes. To test this hypothesis, we focused on enzymes that hydrolyze rhamnosidic bonds, as cleavage of these linkages is chemically challenging and there is a paucity of information on L-rhamnosidases. Here we screened the activity of enzymes derived from the human gut microbiota bacterium Bacteroides thetaiotaomicron, which are up-regulated in response to rhamnose-containing glycans. We identified an α-L-rhamnosidase, BT3686, which is the founding member of a glycoside hydrolase (GH) family, GH145. In contrast to other rhamnosidases, BT3686 cleaved L-Rha-α1,4-D-GlcA linkages through a retaining double-displacement mechanism. The crystal structure of BT3686 showed that the enzyme displayed a type A sevenbladed β-propeller fold. Mutagenesis and crystallographic studies, including the structure of BT3686 in complex with the reaction product GlcA, revealed a location for the active site among β-propeller enzymes cited on the posterior surface of the rhamnosidase. In contrast to the vast majority of GH, the catalytic apparatus of BT3686 does not comprise a pair of carboxylic acid residues but, uniquely, a single histidine functions as the only discernable catalytic amino acid. Intriguingly, the histidine, His48, is not invariant in GH145; however, when engineered into structural homologs lacking the imidazole residue, α-L-rhamnosidase activity was established. The potential contribution of His48 to the catalytic activity of BT3686 is discussed.rhamnosidase | human gut microbiota | gum arabic | Bacteroides thetaiotaomicron | catalytic histidine

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“…They are also important targets for the development of therapeutic agents against bacterial and viral infections, diabetes, and other diseases (4,5). Understanding their catalytic mechanisms and ligand binding characteristics requires knowledge of the protonation states and hydrogen bonding at the atomic level, which is of paramount importance for protein engineering and drug design (6).…”

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confidence: 99%

Direct determination of protonation states and visualization of hydrogen bonding in a glycoside hydrolase with neutron crystallography

Wan

Parks

Hanson

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Glycoside hydrolase (GH) enzymes apply acid/base chemistry to catalyze the decomposition of complex carbohydrates. These ubiquitous enzymes accept protons from solvent and donate them to substrates at close to neutral pH by modulating the pK a values of key side chains during catalysis. However, it is not known how the catalytic acid residue acquires a proton and transfers it efficiently to the substrate. To better understand GH chemistry, we used macromolecular neutron crystallography to directly determine protonation and ionization states of the active site residues of a family 11 GH at multiple pD (pD = pH + 0.4) values. The general acid glutamate (Glu) cycles between two conformations, upward and downward, but is protonated only in the downward orientation. We performed continuum electrostatics calculations to estimate the pK a values of the catalytic Glu residues in both the apo-and substrate-bound states of the enzyme. The calculated pK a of the Glu increases substantially when the side chain moves down. The energy barrier required to rotate the catalytic Glu residue back to the upward conformation, where it can protonate the glycosidic oxygen of the substrate, is 4.3 kcal/mol according to free energy simulations. These findings shed light on the initial stage of the glycoside hydrolysis reaction in which molecular motion enables the general acid catalyst to obtain a proton from the bulk solvent and deliver it to the glycosidic oxygen.glycoside hydrolase | protonation state | macromolecular neutron crystallography | xylanase | molecular simulations

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Dissecting conformational contributions to glycosidase catalysis and inhibition

Abstract: Graphical abstract

Cited by 123 publications

References 68 publications

Synthesis of 2-deoxy-2,2-difluoro-α-maltosyl fluoride and its X-ray structure in complex with Streptomyces coelicolor GlgEI-V279S

Synthesis of 2-deoxy-2,2-difluoro-α-maltosyl fluoride and its X-ray structure in complex with Streptomyces coelicolor GlgEI-V279S

Unusual active site location and catalytic apparatus in a glycoside hydrolase family

Direct determination of protonation states and visualization of hydrogen bonding in a glycoside hydrolase with neutron crystallography

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