RESUMEN
Class I inverting exo-acting α-1,2-mannosidases (CAZY family GH47) display an unusual catalytic itinerary featuring ring-flipped mannosides, 3S1 â 3H4 â 1C4. Conformationally locked 1C4 compounds, such as kifunensine, display nanomolar inhibition but large multigene GH47 mannosidase families render specific "isoform-dependent" inhibition impossible. Here we develop a bump-and-hole strategy in which a new mannose-configured 1,6-trans-cyclic sulfamidate inhibits α-d-mannosidases by virtue of its 1C4 conformation. This compound does not inhibit the wild-type GH47 model enzyme by virtue of a steric clash, a "bump", in the active site. An L310S (a conserved residue amongst human GH47 enzymes) mutant of the model Caulobacter GH47 awoke 574 nM inhibition of the previously dormant inhibitor, confirmed by structural analysis of a 0.97 Å structure. Considering that L310 is a conserved residue amongst human GH47 enzymes, this work provides a unique framework for future biotechnological studies on N-glycan maturation and ER associated degradation by isoform-specific GH47 α-d-mannosidase inhibition through a bump-and-hole approach.
RESUMEN
Human O-linked ß-N-acetylglucosaminidase (hOGA) is one of the two enzymes involved in nuclear and cytoplasmic protein O-GlcNAcylation, an essential post-translational modification. The enzyme catalyzes the hydrolysis of the GlcNAc-O-(Ser/Thr) glycosidic bonds via anchimeric assistance through the 2-acetamido group of the GlcNAc sugar. However, the conformational itinerary of the GlcNAc ring during catalysis remains unclear. Here we report the crystal structure of wild type hOGA in complex with a nonhydrolyzable glycopeptide substrate and elucidate the full enzyme catalytic mechanism using QM/MM metadynamics. We show that the enzyme can bind the substrate in either a chair- or a boat-like conformation, but only the latter is catalytically competent, leading to the reaction products via 1,4B/1S3 â [4E] â 4C1 and 4C1 â [4E] â 1,4B/1S3 conformational itineraries for the first and second catalytic reaction steps, respectively. Our results reconcile previous experimental observations for human and bacterial OGA and will aid the development of more effective OGA inhibitors for diseases associated with impaired O-GlcNAcylation.
RESUMEN
Owing to its roles in human health and disease, the modification of nuclear, cytoplasmic, and mitochondrial proteins with O-linked N-acetylglucosamine residues (O-GlcNAc) has emerged as a topic of great interest. Despite the presence of O-GlcNAc on hundreds of proteins within cells, only two enzymes regulate this modification. One of these enzymes is O-GlcNAcase (OGA), a dimeric glycoside hydrolase that has a deep active site cleft in which diverse substrates are accommodated. Chemical tools to control OGA are emerging as essential resources for helping to decode the biochemical and cellular functions of the O-GlcNAc pathway. Here we describe rationally designed bicyclic thiazolidine inhibitors that exhibit superb selectivity and picomolar inhibition of human OGA. Structures of these inhibitors in complex with human OGA reveal the basis for their exceptional potency and show that they extend out of the enzyme active site cleft. Leveraging this structure, we create a high affinity chemoproteomic probe that enables simple one-step purification of endogenous OGA from brain and targeted proteomic mapping of its post-translational modifications. These data uncover a range of new modifications, including some that are less-known, such as O-ubiquitination and N-formylation. We expect that these inhibitors and chemoproteomics probes will prove useful as fundamental tools to decipher the mechanisms by which OGA is regulated and directed to its diverse cellular substrates. Moreover, the inhibitors and structures described here lay out a blueprint that will enable the creation of chemical probes and tools to interrogate OGA and other carbohydrate active enzymes.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Compuestos Bicíclicos con Puentes/química , Inhibidores Enzimáticos/química , Histona Acetiltransferasas/metabolismo , Hialuronoglucosaminidasa/metabolismo , Secuencia de Aminoácidos , Encéfalo/metabolismo , Compuestos Bicíclicos con Puentes/metabolismo , Dominio Catalítico , Cromatografía Líquida de Alta Presión , Inhibidores Enzimáticos/metabolismo , Histona Acetiltransferasas/antagonistas & inhibidores , Humanos , Hialuronoglucosaminidasa/antagonistas & inhibidores , Espectrometría de Masas , Péptidos/análisis , Péptidos/química , Procesamiento Proteico-Postraduccional , Proteómica/métodos , Relación Estructura-Actividad , Tiazolidinas/química , Tiazolidinas/metabolismo , Cadena alfa de beta-Hexosaminidasa/antagonistas & inhibidores , Cadena alfa de beta-Hexosaminidasa/metabolismoRESUMEN
There is a vast genomic resource for enzymes active on carbohydrates. Lagging far behind, however, are functional chemical tools for the rapid characterization of carbohydrate-active enzymes. Activity-based probes (ABPs) offer one chemical solution to these issues with ABPs based upon cyclophellitol epoxide and aziridine covalent and irreversible inhibitors representing a potent and widespread approach. Such inhibitors for enzymes active on polysaccharides are potentially limited by the requirement for several glycosidic bonds, themselves substrates for the enzyme targets. Here, it is shown that non-hydrolysable trisaccharide can be synthesized and applied even to enzymes with challenging subsite requirements. It was found that incorporation of carbasugar moieties, which was accomplished by cuprate-assisted regioselective trans-diaxial epoxide opening of carba-mannal synthesised for this purpose, yields inactivators that act as powerful activity-based inhibitors for α-1,6 endo-mannanases. 3-D structures at 1.35-1.47â Å resolutions confirm the design rationale and binding to the enzymatic nucleophile. Carbasugar oligosaccharide cyclophellitols offer a powerful new approach for the design of robust endoglycosidase inhibitors, while the synthesis procedures presented here should allow adaptation towards activity-based endoglycosidase probes as well as configurational isosteres targeting other endoglycosidase families.
Asunto(s)
Carba-azúcares , Glicósido Hidrolasas , Oligosacáridos , Compuestos EpoxiRESUMEN
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 B2,5 (or enantiomeric 2,5B) boat and a 3H4 half-chair. This extends to a new family (GT108) of GDPMan-dependent ß-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.
Asunto(s)
Manosidasas/química , Biocatálisis , Manosa/metabolismo , Conformación MolecularRESUMEN
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.
Asunto(s)
Acetilglucosamina/metabolismo , Hidrolasas/metabolismo , N-Acetilglucosaminiltransferasas/metabolismo , Humanos , Hidrolasas/química , Hidrolasas/genética , N-Acetilglucosaminiltransferasas/química , N-Acetilglucosaminiltransferasas/genética , Procesamiento Proteico-Postraduccional , Especificidad por Sustrato , Activación TranscripcionalRESUMEN
Parasitic protists belonging to the genus Leishmania synthesize the non-canonical carbohydrate reserve, mannogen, which is composed of ß-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.
Asunto(s)
Glicosiltransferasas/clasificación , Glicosiltransferasas/metabolismo , Leishmania/enzimología , Manosiltransferasas/metabolismo , Fosforilasas/clasificación , Fosforilasas/metabolismo , Cristalografía por Rayos X , Transferencia de Gen Horizontal , Glicosiltransferasas/química , Glicosiltransferasas/genética , Mananos , Manosiltransferasas/química , Manosiltransferasas/genética , Modelos Moleculares , Oligosacáridos , Fosforilasas/química , Fosforilasas/genética , Conformación Proteica , Termotolerancia , VirulenciaRESUMEN
Enzyme transition-state mimics can act as powerful inhibitors and allow structural studies that report on the conformation of the transition-state. Here, mannoimidazole, a mimic of the transition state of mannosidase catalyzed hydrolysis of mannosides, is shown to bind in a B2,5 conformation on the Clostridium perfringens GH125 α-1,6-mannosidase, providing additional evidence of a OS2-B2,5-1S5 conformational itinerary for enzymes of this family.
Asunto(s)
Clostridium perfringens/enzimología , Inhibidores Enzimáticos/metabolismo , Imidazoles/metabolismo , Manosa/metabolismo , alfa-Manosidasa/metabolismo , Biocatálisis , Dominio Catalítico , Inhibidores Enzimáticos/química , Imidazoles/química , Manosa/análogos & derivados , Conformación Molecular , Mutación , Unión Proteica , alfa-Manosidasa/química , alfa-Manosidasa/genéticaRESUMEN
The enzyme O-GlcNAcase catalyses the removal of the O-GlcNAc co/post-translational modification in multicellular eukaryotes. The enzyme has become of acute interest given the intimate role of O-GlcNAcylation in tau modification and stability; small-molecular inhibitors of human O-GlcNAcase are under clinical assessment for the treatment of tauopathies. Given the importance of structure-based and mechanism-based inhibitor design for O-GlcNAcase, it was sought to test whether different crystal forms of the human enzyme could be achieved by surface mutagenesis. Guided by surface-entropy reduction, a Glu602Ala/Glu605Ala variant [on the Gly11-Gln396/Lys535-Tyr715 construct; Roth et al. (2017), Nature Chem. Biol. 13, 610-612] was obtained which led to a new crystal form of the human enzyme. An increase in crystal contacts stabilized disordered regions of the protein, enabling 88% of the structure to be modelled; only 83% was possible for the wild-type construct. Although the binding of the C-terminus was consistent with the wild type, Lys713 in monomer A was bound in the -1 subsite of the symmetry-related monomer A and the active sites of the B monomers were vacant. The new crystal form presents an opportunity for enhanced soaking experiments that are essential to understanding the binding mechanism and substrate specificity of O-GlcNAcase.
Asunto(s)
Entropía , Proteínas Mutantes/química , beta-N-Acetilhexosaminidasas/química , Sitios de Unión , Humanos , Unión Proteica , Especificidad por Sustrato , beta-N-Acetilhexosaminidasas/genéticaRESUMEN
The varied yet family-specific conformational pathways used by individual glycoside hydrolases (GHs) offer a tantalising prospect for the design of tightly binding and specific enzyme inhibitors. A cardinal example of a GH-family-specific inhibitor, and one that finds widespread practical use, is the natural product kifunensine, which is a low-nanomolar inhibitor that is selective for GH family 47 inverting α-mannosidases. Here we show, through quantum-mechanical approaches, that kifunensine is restrained to a "ring-flipped" 1 C4 conformation with another accessible, but higher-energy, region around the 1,4 B conformation. The conformations of kifunensine in complex with a range of GH47 enzymes-including an atomic-level resolution (1â Å) structure of kifunensine with Caulobacter sp. CkGH47 reported herein and with GH family 38 and 92 α-mannosidases-were mapped onto the kifunensine free-energy landscape. These studies revealed that kifunensine has the ability to mimic the product state of GH47 enzymes but cannot mimic any conformational states relevant to the reaction coordinate of mannosidases from other families.
Asunto(s)
Alcaloides/química , Caulobacter/enzimología , alfa-Manosidasa/química , Calorimetría , Conformación Molecular , Teoría Cuántica , alfa-Manosidasa/antagonistas & inhibidoresRESUMEN
A set of mannuronic-acid-based iminosugars, consisting of the C-5-carboxylic acid, methyl ester and amide analogues of 1deoxymannorjirimicin (DMJ), was synthesised and their pH-dependent conformational behaviour was studied. Under acidic conditions the methyl ester and the carboxylic acid adopted an "inverted" 1 C4 chair conformation as opposed to the "normal" 4 C1 chair at basic pH. This conformational change is explained in terms of the stereoelectronic effects of the ring substituents and it parallels the behaviour of the mannuronic acid ester oxocarbenium ion. Because of this solution-phase behaviour, the mannuronic acid ester azasugar was examined as an inhibitor for a Caulobacter GH47 mannosidase that hydrolyses its substrates by way of a reaction itinerary that proceeds through a 3 H4 transition state. No binding was observed for the mannuronic acid ester azasugar, but sub-atomic resolution data were obtained for the DMJâ CkGH47 complex, showing two conformations-3 S1 and 1 C4 -for the DMJ inhibitor.
Asunto(s)
Compuestos Aza/química , Ácidos Carboxílicos/química , Inhibidores Enzimáticos/química , Ácidos Hexurónicos/química , Iminoazúcares/química , Manosidasas/antagonistas & inhibidores , Compuestos Aza/síntesis química , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/química , Conformación de Carbohidratos , Secuencia de Carbohidratos , Caulobacter/química , Caulobacter/enzimología , Inhibidores Enzimáticos/síntesis química , Ésteres/química , Concentración de Iones de Hidrógeno , Iminoazúcares/síntesis química , Manosidasas/química , Modelos MolecularesRESUMEN
Conformational analysis of enzyme-catalyzed mannoside hydrolysis has revealed two predominant conformational itineraries through B2,5 or 3H4 transition-state (TS) conformations. A prominent unassigned catalytic itinerary is that of exo-1,6-α-mannosidases belonging to CAZy family 125. A published complex of Clostridium perfringens GH125 enzyme with a nonhydrolyzable 1,6-α-thiomannoside substrate mimic bound across the active site revealed an undistorted 4C1 conformation and provided no insight into the catalytic pathway of this enzyme. We show through a purely computational approach (QM/MM metadynamics) that sulfur-for-oxygen substitution in the glycosidic linkage fundamentally alters the energetically accessible conformational space of a thiomannoside when bound within the GH125 active site. Modeling of the conformational free energy landscape (FEL) of a thioglycoside strongly favors a mechanistically uninformative 4C1 conformation within the GH125 enzyme active site, but the FEL of corresponding O-glycoside substrate reveals a preference for a Michaelis complex in an OS2 conformation (consistent with catalysis through a B2,5 TS). This prediction was tested experimentally by determination of the 3D X-ray structure of the pseudo-Michaelis complex of an inactive (D220N) variant of C. perfringens GH125 enzyme in complex with 1,6-α-mannobiose. This complex revealed unambiguous distortion of the -1 subsite mannoside to an OS2 conformation, matching that predicted by theory and supporting an OS2 â B2,5 â 1S5 conformational itinerary for GH125 α-mannosidases. This work highlights the power of the QM/MM approach and identified shortcomings in the use of nonhydrolyzable substrate analogues for conformational analysis of enzyme-bound species.
Asunto(s)
Manosa/química , Simulación de Dinámica Molecular , Teoría Cuántica , alfa-Manosidasa/química , Clostridium perfringens/enzimología , Manosa/análogos & derivados , Manosa/metabolismo , Estructura Molecular , Conformación Proteica , alfa-Manosidasa/metabolismoRESUMEN
Penicillin-binding protein 3 (PBP3) from Pseudomonas aeruginosa is the molecular target of ß-lactam-based antibiotics. Structures of PBP3 in complexes with azlocillin and cefoperazone, which are in clinical use for the treatment of pseudomonad infections, have been determined to 2.0 Å resolution. Together with data from other complexes, these structures identify a common set of residues involved in the binding of ß-lactams to PBP3. Comparison of wild-type and an active site mutant (S294A) showed that increased thermal stability of PBP3 following azlocillin binding was entirely due to covalent binding to S294, whereas cefoperazone binding produces some increase in stability without the covalent link. Consistent with this, a third crystal structure was determined in which the hydrolysis product of cefoperazone was noncovalently bound in the active site of PBP3. This is the first structure of a complex between a penicillin-binding protein and cephalosporic acid and may be important in the design of new noncovalent PBP3 inhibitors.