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1.
Int J Biol Macromol ; 269(Pt 1): 132033, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38702000

RESUMEN

The role of mannanases is diverse and they are used in many industrial applications, in animal feed, in the food industry and in healthcare. They are also applied in biomass processing, because they play an important role in the breakdown of hemicellulose. Among the mannanase inhibitors, heavy metal ions and general enzyme inhibitors are mainly mentioned. Unfortunately, almost no data are available on carbohydrate-based natural inhibitors of mannanases. According to the literature, carbohydrates do not play an important role in the inhibition of mannanases, so neither do oligosaccharides. This is in contrast to the action and inhibition of other O-glycosyl hydrolases. My hypothesis is that mannanases, like other polysaccharide-degrading enzymes, work in the same way and can be inhibited by oligosaccharides. Evidence from docking and modeling results supports and makes probable the hypothesis that oligosaccharides can inhibit the activity of mannanases, similar to the inhibition of other O-glycosyl hydrolases. Among natural carbohydrate oligomers, several potential mannanase inhibitors have been identified and characterized. In addition to expensive research, it is very important to use research based on cheaper modeling to explore the processes. The results obtained are novel and forward-looking, enabling in-depth and targeted research to be carried out.


Asunto(s)
Inhibidores Enzimáticos , Manosidasas , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Manosidasas/antagonistas & inhibidores , Manosidasas/metabolismo , Manosidasas/química , Simulación del Acoplamiento Molecular , Oligosacáridos/química , Oligosacáridos/farmacología , Hidrólisis
2.
J Biomol Struct Dyn ; 42(5): 2714-2725, 2024 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-37158092

RESUMEN

The search for Golgi α-mannosidase II (GMII) potent and specific inhibitors has been a focus of many studies for the past three decades since this enzyme is a key target for cancer treatment. α-Mannosidases, such as those from Drosophila melanogaster or Jack bean, have been used as functional models of the human Golgi α-mannosidase II (hGMII) because mammalian mannosidases are difficult to purify and characterize experimentally. Meanwhile, computational studies have been seen as privileged tools able to explore assertive solutions to specific enzymes, providing molecular details of these macromolecules, their protonation states and their interactions. Thus, modelling techniques can successfully predict hGMII 3D structure with high confidence, speeding up the development of new hits. In this study, Drosophila melanogaster Golgi mannosidase II (dGMII) and a novel human model, developed in silico and equilibrated via molecular dynamics simulations, were both opposed for docking. Our findings highlight that the design of novel inhibitors should be carried out considering the human model's characteristics and the enzyme operating pH. A reliable model is evidenced, showing a good correlation between Ki/IC50 experimental data and theoretical ΔGbinding estimations in GMII, opening the possibility of optimizing the rational drug design of new derivatives.Communicated by Ramaswamy H. Sarma.


Asunto(s)
Drosophila melanogaster , Simulación de Dinámica Molecular , Animales , Humanos , alfa-Manosidasa/química , Drosophila melanogaster/metabolismo , Manosidasas/química , Manosidasas/metabolismo , Aparato de Golgi/metabolismo , Mamíferos/metabolismo
3.
Chemphyschem ; 24(24): e202300628, 2023 Dec 14.
Artículo en Inglés | MEDLINE | ID: mdl-37782219

RESUMEN

The catalytic mechanism of a C a + 2 ${C{a}^{+2}}$ -dependent family 92 α ${{\rm \alpha }}$ -mannosidase, which is abundantly present in human gut flora and malfunctions leading to the lysosomal storage disease α-mannosidosis, has been investigated using quantum mechanics/molecular mechanics and metadynamics methods. Computational efforts show that the enzyme follows a conformational itinerary of and the C a + 2 ${C{a}^{+2}}$ ion serves a dual purpose, as it not only distorts the sugar ring but also plays a crucial role in orchestrating the arrangement of catalytic residues. This orchestration, in turn, contributes to the facilitation of O S 2 ${{{\rm \ }}^{{\rm O}}{{\rm S}}_{2}}$ conformers for the ensuing reaction. This mechanistic insight is well-aligned with the experimental predictions of the catalytic pathway, and the computed energies are of the same order of magnitude as the experimental estimations. Hence, our results extend the mechanistic understanding of glycosidases.


Asunto(s)
Manosidasas , Simulación de Dinámica Molecular , alfa-Manosidosis , Catálisis , Manosidasas/química , Conformación Molecular , Microbioma Gastrointestinal/fisiología , alfa-Manosidosis/metabolismo , alfa-Manosidosis/microbiología , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo
4.
Acta Crystallogr D Struct Biol ; 79(Pt 5): 387-400, 2023 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-37071393

RESUMEN

Many secreted eukaryotic proteins are N-glycosylated with oligosaccharides composed of a high-mannose N-glycan core and, in the specific case of yeast cell-wall proteins, an extended α-1,6-mannan backbone carrying a number of α-1,2- and α-1,3-mannose substituents of varying lengths. α-Mannosidases from CAZy family GH92 release terminal mannose residues from these N-glycans, providing access for the α-endomannanases, which then degrade the α-mannan backbone. Most characterized GH92 α-mannosidases consist of a single catalytic domain, while a few have extra domains including putative carbohydrate-binding modules (CBMs). To date, neither the function nor the structure of a multi-domain GH92 α-mannosidase CBM has been characterized. Here, the biochemical investigation and crystal structure of the full-length five-domain GH92 α-1,2-mannosidase from Neobacillus novalis (NnGH92) with mannoimidazole bound in the active site and an additional mannoimidazole bound to the N-terminal CBM32 are reported. The structure of the catalytic domain is very similar to that reported for the GH92 α-mannosidase Bt3990 from Bacteroides thetaiotaomicron, with the substrate-binding site being highly conserved. The function of the CBM32s and other NnGH92 domains was investigated by their sequential deletion and suggested that whilst their binding to the catalytic domain was crucial for the overall structural integrity of the enzyme, they appear to have little impact on the binding affinity to the yeast α-mannan substrate. These new findings provide a better understanding of how to select and optimize other multi-domain bacterial GH92 α-mannosidases for the degradation of yeast α-mannan or mannose-rich glycans.


Asunto(s)
Mananos , Manosidasas , Manosidasas/química , Manosidasas/metabolismo , alfa-Manosidasa/metabolismo , Mananos/química , Mananos/metabolismo , Manosa/química , Manosa/metabolismo , Saccharomyces cerevisiae/metabolismo , Modelos Moleculares , Polisacáridos/química , Especificidad por Sustrato
5.
Org Biomol Chem ; 20(45): 8932-8943, 2022 11 23.
Artículo en Inglés | MEDLINE | ID: mdl-36322142

RESUMEN

The development of effective inhibitors of Golgi α-mannosidase II (GMII, E.C.3.2.1.114) with minimal off-target effects on phylogenetically-related lysosomal α-mannosidase (LMan, E.C.3.2.1.24) is a complex task due to the complicated structural and chemical properties of their active sites. The pKa values (and also protonation forms in some cases) of several ionizable amino acids, such as Asp, Glu, His or Arg of enzymes, can be changed upon the binding of the inhibitor. Moreover, GMII and LMan work under different pH conditions. The pKa calculations on large enzyme-inhibitor complexes and FMO-PIEDA energy decomposition analysis were performed on the structures of selected inhibitors obtained from docking and hybrid QM/MM calculations. Based on the calculations, the roles of the amino group incorporated in the ring of the imino-D-lyxitol inhibitors and some ionizable amino acids of Golgi-type (Asp270-Asp340-Asp341 of Drosophila melanogaster α-mannosidase dGMII) and lysosomal-type enzymes (His209-Asp267-Asp268 of Canavalia ensiformis α-mannosidase, JBMan) were explained in connection with the observed inhibitory properties. The pyrrolidine ring of the imino-D-lyxitols prefers at the active site of dGMII the neutral form while in JBMan the protonated form, whereas that of imino-L-lyxitols prefers the protonation form in both enzymes. The calculations indicate that the binding mechanism of inhibitors to the active-site of α-mannosidases is dependent on the inhibitor structure and could be used to design new selective inhibitors of GMII. A series of novel synthetic N-substituted imino-D-lyxitols were evaluated with four enzymes from the glycoside hydrolase GH38 family (two of Golgi-type, Drosophila melanogaster GMIIb and Caenorhabditis elegans AMAN-2, and two of lysosomal-type, Drosophila melanogaster LManII and Canavalia ensiformis JBMan, enzymes). The most potent structures [N-9-amidinononyl and N-2-(1-naphthyl)ethyl derivatives] inhibited GMIIb (Ki = 40 nM) and AMAN-2 (Ki = 150 nM) with a weak selectivity index (SI) toward Golgi-type enzymes of IC50(LManII)/IC50(GMIIb) = 35 or IC50(JBMan)/IC50(AMAN-2) = 86. On the other hand, weaker micromolar inhibitors, such as N-2-naphthylmethyl or 4-iodobenzyl derivatives [IC50(GMIIb) = 2.4 µM and IC50 (AMAN-2) = 7.6 µM], showed a significant SI in the range from 111 to 812.


Asunto(s)
Drosophila melanogaster , Manosidasas , Animales , alfa-Manosidasa/química , Drosophila melanogaster/metabolismo , Manosidasas/química , Manosidasas/metabolismo , Inhibidores Enzimáticos/química , Aminoácidos , Amantadina
6.
Chemistry ; 28(14): e202200148, 2022 Mar 07.
Artículo en Inglés | MEDLINE | ID: mdl-35049087

RESUMEN

The conformational changes in a sugar moiety along the hydrolytic pathway are key to understand the mechanism of glycoside hydrolases (GHs) and to design new inhibitors. The two predominant itineraries for mannosidases go via O S2 →B2,5 →1 S5 and 3 S1 →3 H4 →1 C4 . For the CAZy family 92, the conformational itinerary was unknown. Published complexes of Bacteroides thetaiotaomicron GH92 catalyst with a S-glycoside and mannoimidazole indicate a 4 C1 →4 H5 /1 S5 →1 S5 mechanism. However, as observed with the GH125 family, S-glycosides may not act always as good mimics of GH's natural substrate. Here we present a cooperative study between computations and experiments where our results predict the E5 →B2,5 /1 S5 →1 S5 pathway for GH92 enzymes. Furthermore, we demonstrate the Michaelis complex mimicry of a new kind of C-disaccharides, whose biochemical applicability was still a chimera.


Asunto(s)
Glicósidos , Manosidasas , Glicósido Hidrolasas/metabolismo , Glicósidos/química , Manosidasas/química , Conformación Molecular
7.
Cells ; 10(11)2021 11 10.
Artículo en Inglés | MEDLINE | ID: mdl-34831340

RESUMEN

Congenital disorders of glycosylation (CDG), inherited metabolic diseases caused by defects in glycosylation, are characterized by a high frequency of intellectual disability (ID) and various clinical manifestations. Two siblings with ID, dysmorphic features, and epilepsy were examined using mass spectrometry of serum transferrin, which revealed a CDG type 2 pattern. Whole-exome sequencing showed that both patients were homozygous for a novel pathogenic variant of MAN1B1 (NM_016219.4:c.1837del) inherited from their healthy parents. We conducted a HPLC analysis of sialylated N-linked glycans released from total plasma proteins and characterized the α1,2-mannosidase I activity of the lymphocyte microsome fraction. The accumulation of monosialoglycans was observed in MAN1B1-deficient patients, indicating N-glycan-processing defects. The enzymatic activity of MAN1B1 was compromised in patient-derived lymphocytes. The present patients exhibited unique manifestations including early-onset epileptic encephalopathy and cerebral infarction. They also showed coagulation abnormalities and hypertransaminasemia. Neither sibling had truncal obesity, which is one of the characteristic features of MAN1B1-CDG.


Asunto(s)
Trastornos Congénitos de Glicosilación/genética , Manosidasas/genética , Hermanos , Adolescente , Secuencia de Aminoácidos , Secuencia de Bases , Proteínas Sanguíneas/metabolismo , Niño , Preescolar , Femenino , Humanos , Lactante , Recién Nacido , Linfocitos/metabolismo , Masculino , Manosidasas/química , Manosidasas/metabolismo , Microsomas/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Linaje , Polisacáridos/química , Espectrometría de Masa por Ionización de Electrospray , Secuenciación del Exoma
8.
Org Lett ; 23(8): 3053-3057, 2021 04 16.
Artículo en Inglés | MEDLINE | ID: mdl-33793242

RESUMEN

Oligomannose glycans are of interest as HIV vaccine components, but they are subject to mannosidase degradation in vivo. Herein, we report the synthesis of oligosaccharides containing a thio linkage at the nonreducing end. A thio-linked dimannose donor participates in highly stereoselective glycosylations to afford trimannose and tetramannose fragments. Saturation transfer difference nuclear magnetic resonance (STD NMR) studies show that these glycans are recognized by HIV antibody 2G12, and we confirm that the reducing terminal S-linkage confers complete stability against x. manihotis mannosidase.


Asunto(s)
Manosidasas/química , Oligosacáridos/química , Polisacáridos/química , Glicosilación , Humanos , Manosidasas/metabolismo , Estructura Molecular
9.
Cells ; 11(1)2021 12 29.
Artículo en Inglés | MEDLINE | ID: mdl-35011665

RESUMEN

Mutations in the apically located kidney Na-K-2Cl cotransporter NKCC2 cause type I Bartter syndrome, a life-threatening kidney disorder. We previously showed that transport from the ER represents the limiting phase in NKCC2 journey to the cell surface. Yet very little is known about the ER quality control components specific to NKCC2 and its disease-causing mutants. Here, we report the identification of Golgi alpha1, 2-mannosidase IA (ManIA) as a novel binding partner of the immature form of NKCC2. ManIA interaction with NKCC2 takes place mainly at the cis-Golgi network. ManIA coexpression decreased total NKCC2 protein abundance whereas ManIA knock-down produced the opposite effect. Importantly, ManIA coexpression had a more profound effect on NKCC2 folding mutants. Cycloheximide chase assay showed that in cells overexpressing ManIA, NKCC2 stability and maturation are heavily hampered. Deleting the cytoplasmic region of ManIA attenuated its interaction with NKCC2 and inhibited its effect on the maturation of the cotransporter. ManIA-induced reductions in NKCC2 expression were offset by the proteasome inhibitor MG132. Likewise, kifunensine treatment greatly reduced ManIA effect, strongly suggesting that mannose trimming is involved in the enhanced ERAD of the cotransporter. Moreover, depriving ManIA of its catalytic domain fully abolished its effect on NKCC2. In summary, our data demonstrate the presence of a ManIA-mediated ERAD pathway in renal cells promoting retention and degradation of misfolded NKCC2 proteins. They suggest a model whereby Golgi ManIA contributes to ERAD of NKCC2, by promoting the retention, recycling, and ERAD of misfolded proteins that initially escape protein quality control surveillance within the ER.


Asunto(s)
Degradación Asociada con el Retículo Endoplásmico , Aparato de Golgi/enzimología , Manosidasas/metabolismo , Miembro 1 de la Familia de Transportadores de Soluto 12/metabolismo , Animales , Línea Celular , Humanos , Manosa/metabolismo , Manosidasas/química , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Zarigüeyas , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Dominios Proteicos , Pliegue de Proteína , Estabilidad Proteica
10.
Int J Biol Macromol ; 166: 778-788, 2021 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-33144255

RESUMEN

A novel GH5 endo-1,4-ß-mannanase (BaMan5A) was identified from Bacillus sp. KW1, it shares the highest sequence identity (86%) with another characterized Bacillus endo-1,4-ß-mannanase. The recombinant BaMan5A displayed maximum activity at pH 7.0 and 70 °C, it was stable at a broad pH range (pH 3.5-11.0) after 12-h incubation at 25 °C, and exhibited good thermostability, retaining about 100% and 85% activity after incubating at 60 °C for 12 h and 65 °C for 8 h, respectively. The results of polysaccharide hydrolysis revealed that the enzyme can only hydrolyze mannan substrates, including carob galactomannan, konjac glucomannan, 1,4-ß-D-mannan, locust bean gum, and guar gum, yielding mannose, mannobiose, mannotriose, and some other oligosaccharides. The best substrate was carob galactomannan, the corresponding specific activity and Km value were 10,886 µmol/min/µmol and 3.31 mg/mL, respectively. Interestingly, BaMan5A was capable to hydrolyze both manno-oligosaccharides and cello-oligosaccharides, including mannotetraose, mannopentaose, mannohexaose, cellopentaose and cellohexaose. Furthermore, BaMan5A acted synergistically with a commercial α-galactosidase (CbAgal) on galactomannan depolymerization, a best synergy degree of 1.58 was achieved after optimizing enzyme ratios. This study not only expands the diversity of Bacillus GH5 ß-mannanase, but also discloses the potential of BaMan5A in industrial application.


Asunto(s)
Bacillus/enzimología , Proteínas Bacterianas/metabolismo , Mananos/metabolismo , Manosidasas/metabolismo , beta-Glucosidasa/metabolismo , Bacillus/genética , Proteínas Bacterianas/química , Dominio Catalítico , Clonación Molecular , Estabilidad de Enzimas , Galactosa/análogos & derivados , Calor , Hidrólisis , Microbiología Industrial/métodos , Manosidasas/química , Especificidad por Sustrato , alfa-Glucosidasas/metabolismo , beta-Glucosidasa/química
11.
Proc Natl Acad Sci U S A ; 117(47): 29595-29601, 2020 11 24.
Artículo en Inglés | MEDLINE | ID: mdl-33154157

RESUMEN

Mammalian protein N-linked glycosylation is critical for glycoprotein folding, quality control, trafficking, recognition, and function. N-linked glycans are synthesized from Glc3Man9GlcNAc2 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 endo-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.


Asunto(s)
Antivirales/química , Antivirales/farmacología , Glicosilación/efectos de los fármacos , Manosidasas/química , Manosidasas/farmacología , Animales , Diarrea Mucosa Bovina Viral/tratamiento farmacológico , Bovinos , Línea Celular , Virus del Dengue/efectos de los fármacos , Perros , Glucosidasas/metabolismo , Humanos , Células de Riñón Canino Madin Darby , Polisacáridos/metabolismo , Vías Secretoras/efectos de los fármacos
12.
Proc Natl Acad Sci U S A ; 117(40): 24825-24836, 2020 10 06.
Artículo en Inglés | MEDLINE | ID: mdl-32958677

RESUMEN

The failure of polypeptides to achieve conformational maturation following biosynthesis can result in the formation of protein aggregates capable of disrupting essential cellular functions. In the secretory pathway, misfolded asparagine (N)-linked glycoproteins are selectively sorted for endoplasmic reticulum-associated degradation (ERAD) in response to the catalytic removal of terminal alpha-linked mannose units. Remarkably, ER mannosidase I/Man1b1, the first alpha-mannosidase implicated in this conventional N-glycan-mediated process, can also contribute to ERAD in an unconventional, catalysis-independent manner. To interrogate this functional dichotomy, the intracellular fates of two naturally occurring misfolded N-glycosylated variants of human alpha1-antitrypsin (AAT), Null Hong Kong (NHK), and Z (ATZ), in Man1b1 knockout HEK293T cells were monitored in response to mutated or truncated forms of transfected Man1b1. As expected, the conventional catalytic system requires an intact active site in the Man1b1 luminal domain. In contrast, the unconventional system is under the control of an evolutionarily extended N-terminal cytoplasmic tail. Also, N-glycans attached to misfolded AAT are not required for accelerated degradation mediated by the unconventional system, further demonstrating its catalysis-independent nature. We also established that both systems accelerate the proteasomal degradation of NHK in metabolic pulse-chase labeling studies. Taken together, these results have identified the previously unrecognized regulatory capacity of the Man1b1 cytoplasmic tail and provided insight into the functional dichotomy of Man1b1 as a component in the mammalian proteostasis network.


Asunto(s)
Manosidasas/metabolismo , alfa 1-Antitripsina/química , Biocatálisis , Degradación Asociada con el Retículo Endoplásmico , Células HEK293 , Humanos , Manosidasas/química , Manosidasas/genética , Unión Proteica , Dominios Proteicos , Pliegue de Proteína , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/metabolismo
13.
Biochim Biophys Acta Proteins Proteom ; 1868(8): 140437, 2020 08.
Artículo en Inglés | MEDLINE | ID: mdl-32325255

RESUMEN

The endo-ß-1,4-mannanase from the hyperthermostable bacterium Thermotoga petrophila (TpMan) is an enzyme that catalyzes the hydrolysis of mannan and heteromannan polysaccharides. Of the three domains that comprise TpMan, the N-terminal GH5 catalytic domain and the C-terminal carbohydrate-binding domain are connected through a central ancillary domain of unknown structure and function. In this study, we report the partial crystal structure of the TpMan at 1.45 Å resolution, so far, the first modular hyperthermostable endo-ß-1,4-mannanase structure determined. The structure exhibits two domains, a (ß/α)8-barrel GH5 catalytic domain connected via a linker to the central domain with an immunoglobulin-like ß-sandwich fold formed of seven ß-strands. Functional analysis showed that whereas the immunoglobulin-like domain does not have the carbohydrate-binding function, it stacks on the GH5 catalytic domain acting as a thermostabilizing domain and allowing operation at hyperthermophilic conditions. The carbohydrate-binding domain is absent in the crystal structure most likely due to its high flexibility around the immunoglobulin-like domain which may act also as a pivot. These results represent new structural and functional information useful on biotechnological applications for biofuel and food industries.


Asunto(s)
Bacterias/química , Proteínas Bacterianas/química , Dominios de Inmunoglobulinas , Mananos/química , Manosidasas/química , Bacterias/enzimología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Dominio Catalítico , Clonación Molecular , Cristalografía por Rayos X , Estabilidad de Enzimas , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Hidrólisis , Interacciones Hidrofóbicas e Hidrofílicas , Mananos/metabolismo , Manosidasas/genética , Manosidasas/metabolismo , Modelos Moleculares , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Thermotoga
14.
J Biol Chem ; 295(15): 5012-5021, 2020 04 10.
Artículo en Inglés | MEDLINE | ID: mdl-32139511

RESUMEN

ß-Mannanases from the glycoside hydrolase 26 (GH26) family are retaining hydrolases that are active on complex heteromannans and whose genes are abundant in rumen metagenomes and metatranscriptomes. These enzymes can exhibit distinct modes of substrate recognition and are often fused to carbohydrate-binding modules (CBMs), resulting in a molecular puzzle of mechanisms governing substrate preference and mode of action that has not yet been pieced together. In this study, we recovered a novel GH26 enzyme with a CBM35 module linked to its N terminus (CrMan26) from a cattle rumen metatranscriptome. CrMan26 exhibited a preference for galactomannan as substrate and the crystal structure of the full-length protein at 1.85 Å resolution revealed a unique orientation of the ancillary domain relative to the catalytic interface, strategically positioning a surface aromatic cluster of the ancillary domain as an extension of the substrate-binding cleft, contributing to galactomannan preference. Moreover, systematic investigation of nonconserved residues in the catalytic interface unveiled that residues Tyr195 (-3 subsite) and Trp234 (-5 subsite) from distal negative subsites have a key role in galactomannan preference. These results indicate a novel and complex mechanism for substrate recognition involving spatially remote motifs, distal negative subsites from the catalytic domain, and a surface-associated aromatic cluster from the ancillary domain. These findings expand our molecular understanding of the mechanisms of substrate binding and recognition in the GH26 family and shed light on how some CBMs and their respective orientation can contribute to substrate preference.


Asunto(s)
Mananos/metabolismo , Manosidasas/química , Manosidasas/metabolismo , Metagenoma , Mutación , Rumen/metabolismo , Secuencia de Aminoácidos , Animales , Catálisis , Dominio Catalítico , Bovinos , Cristalografía por Rayos X , Galactosa/análogos & derivados , Hidrólisis , Manosidasas/genética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Filogenia , Unión Proteica , Homología de Secuencia , Especificidad por Sustrato
15.
Curr Opin Struct Biol ; 62: 79-92, 2020 06.
Artículo en Inglés | MEDLINE | ID: mdl-31891872

RESUMEN

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 Molecular
16.
FEBS Lett ; 594(3): 439-451, 2020 02.
Artículo en Inglés | MEDLINE | ID: mdl-31552675

RESUMEN

While multiple α 1-2-mannosidases are necessary for glycoprotein N-glycan maturation in vertebrates, a single bacterial α1-2-mannosidase can be sufficient to cleave all α1-2-linked mannose residues in host glycoprotein N-glycans. We report here the characterization and crystal structure of a new α1-2-mannosidase (EfMan-I) from Enterococcus faecalis, a Gram-positive opportunistic human pathogen. EfMan-I catalyzes the cleavage of α1-2-mannose from not only oligomannoses but also high-mannose-type N-glycans on glycoproteins. Its 2.15 Å resolution crystal structure reveals a two-domain enzyme fold similar to other CAZy GH92 mannosidases. An unexpected potassium ion was observed bridging two domains near the active site. These findings support EfMan-I as an effective catalyst for in vitro N-glycan modification of glycoproteins with high-mannose-type N-glycans.


Asunto(s)
Biocatálisis , Enterococcus faecalis/enzimología , Glicoproteínas/química , Manosidasas/metabolismo , Polisacáridos/metabolismo , Dominio Catalítico , Clonación Molecular , Cristalografía por Rayos X , Glicoproteínas/metabolismo , Concentración de Iones de Hidrógeno , Manosidasas/química , Manosidasas/genética , Especificidad por Sustrato
17.
World J Microbiol Biotechnol ; 35(2): 32, 2019 Jan 30.
Artículo en Inglés | MEDLINE | ID: mdl-30701316

RESUMEN

Extremophilic microorganisms are valuable sources of enzymes for various industrial applications. In fact, given their optimal catalytic activity and operational stability under harsh physical and chemical conditions, they represent a suitable alternative to their mesophilic counterparts. For instance, extremophilic enzymes are important to foster the switch from fossil-based to lignocellulose-based industrial processes. Indeed, more stable enzymes are needed, because the conversion of the lignocellulosic biomass to a wide palette of value-added products requires extreme chemo-physical pre-treatments. Galactomannans are part of the hemicellulose fraction in lignocellulosic biomass. They are heteropolymers constituted by a ß-1,4-linked mannan backbone substituted with side chains of α-1,6-linked galactose residues. Therefore, the joint action of different hydrolytic enzymes (i.e. ß-mannanase, ß-mannosidase and α-galactosidase) is needed to accomplish their complete hydrolysis. So far, numerous galactomannan-degrading enzymes have been isolated and characterized from extremophilic microorganisms. Besides applications in biorefinery, these biocatalysts are also useful to improve the quality (i.e. digestibility and prebiotic properties) of food and feed as well as in paper industries to aid the pulp bleaching process. In this review, an overview about the structure, function and applications of galactomannans is provided. Moreover, a survey of (hyper)-thermophilic galactomannans-degrading enzymes, mainly characterized in the last decade, has been carried out. These extremozymes are described in the light of their biotechnological application in industrial processes requiring harsh conditions.


Asunto(s)
Bacterias/enzimología , Mananos/metabolismo , Manosidasas/química , alfa-Galactosidasa/química , beta-Manosidasa/química , Bacterias/química , Bacterias/genética , Biotecnología , Estabilidad de Enzimas , Galactosa/análogos & derivados , Mananos/química , Manosidasas/genética , Manosidasas/metabolismo , Plantas/química , Plantas/enzimología , Plantas/genética , Plantas/metabolismo , alfa-Galactosidasa/genética , alfa-Galactosidasa/metabolismo , beta-Manosidasa/genética , beta-Manosidasa/metabolismo
18.
Biochem Biophys Res Commun ; 510(3): 358-363, 2019 03 12.
Artículo en Inglés | MEDLINE | ID: mdl-30717976

RESUMEN

Comprehensive knowledge on the role of substrate subsites is a prerequisite to understand the interaction between glycoside hydrolase and its substrate. The present study delineates the role of individual substrate subsites present in ManB-1601 (GH26 endo-mannanase from Bacillus sp.) towards interaction with mannans. Isothermal titration calorimetry of catalytic mutant (E167A/E266A) of ManB-1601 with mannobiose to mannohexose revealed presence of six substrate subsites in ManB-1601. The amino acids present in substrate subsites of ManB-1601 were found to be highly conserved among GH26 endo-mannanases from Bacillus spp. Qualitative substrate binding analysis of subsite mutants by native affinity gel electrophoresis suggested that -3, -2, -1, +1 and + 2 subsites have a major role while, -4 subsite had minor role towards mannan binding. Affinity gels also pointed out the pivotal role of -1 subsite towards glucomannan binding. Quantitative substrate binding analysis using fluorescence titration revealed that -1 and -2 subsite mutants had 27- and 30-fold higher binding affinity (KD) for carob galactomannan when compared with catalytic mutant. The -1 subsite mutant also had highest KD values for glucomannan (13.6-fold) and ivory nut mannan (5-fold) among all mutants. The positive subsites contributed more towards binding with glucomannan (up to 10-fold higher KD) and ivory nut mannan (up to 4.3-fold higher KD) rather than carob galactomannan (up to 4-fold higher KD). Between distal subsites, -3 mutant displayed 10-fold higher KD for both carob galactomannan and glucomannan while, -4 mutant did not show any noticeable change in KD values when compared to catalytic mutant.


Asunto(s)
Mananos/metabolismo , Manosidasas/metabolismo , Bacillus/enzimología , Galactosa/análogos & derivados , Mananos/química , Manosa/metabolismo , Manosidasas/química , Unión Proteica
19.
PLoS One ; 13(9): e0204703, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30261037

RESUMEN

Commensal and pathogenic bacteria have evolved efficient enzymatic pathways to feed on host carbohydrates, including protein-linked glycans. Most proteins of the human innate and adaptive immune system are glycoproteins where the glycan is critical for structural and functional integrity. Besides enabling nutrition, the degradation of host N-glycans serves as a means for bacteria to modulate the host's immune system by for instance removing N-glycans on immunoglobulin G. The commensal bacterium Cutibacterium acnes is a gram-positive natural bacterial species of the human skin microbiota. Under certain circumstances, C. acnes can cause pathogenic conditions, acne vulgaris, which typically affects 80% of adolescents, and can become critical for immunosuppressed transplant patients. Others have shown that C. acnes can degrade certain host O-glycans, however, no degradation pathway for host N-glycans has been proposed. To investigate this, we scanned the C. acnes genome and were able to identify a set of gene candidates consistent with a cytoplasmic N-glycan-degradation pathway of the canonical eukaryotic N-glycan core. We also found additional gene sequences containing secretion signals that are possible candidates for initial trimming on the extracellular side. Furthermore, one of the identified gene products of the cytoplasmic pathway, AEE72695, was produced and characterized, and found to be a functional, dimeric exo-ß-1,4-mannosidase with activity on the ß-1,4 glycosidic bond between the second N-acetylglucosamine and the first mannose residue in the canonical eukaryotic N-glycan core. These findings corroborate our model of the cytoplasmic part of a C. acnes N-glycan degradation pathway.


Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Manosidasas/química , Manosidasas/metabolismo , Propionibacteriaceae/enzimología , Sustitución de Aminoácidos , Proteínas Bacterianas/genética , Dominio Catalítico , Cristalografía por Rayos X , Genes Bacterianos , Glicoproteínas/metabolismo , Interacciones Microbiota-Huesped , Humanos , Cinética , Manosidasas/genética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Polisacáridos/química , Polisacáridos/metabolismo , Propionibacteriaceae/genética , Propionibacteriaceae/patogenicidad , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato
20.
Int J Biol Macromol ; 119: 79-95, 2018 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-30048723

RESUMEN

This review aims to emphasize the occurrence and abundant presence of mannans in nature, their classification, structural differences and significance in food and feed industry. With rising demand from the consumers' end for novel natural foods, usage of galactomannan and glucomannan has also increased alternatively. Non toxicity of mannans permits their usage in the pharmaceutical, biomedical, cosmetics, and textile industries. In the food industry, mannans have various applications such as edible films/coating, gel formation, stiffeners, viscosity modifiers, stabilizers, texture improvers, water absorbants, as prebiotics in dairy products and bakery, seasonings, diet foods, coffee whiteners etc. Applications and functions of these commonly used commercially available mannans have therefore, been highlighted. Mannans improve the texture and appeal of food products and provide numerous health benefits like controlling obesity and body weight control, prebiotic benefits, constipation alleviaton, prevent occurrence of diarrhea, check inflammation due to gut related diseases, management of diverticular disease management, balance intestinal microbiota, immune system modulator, reduced risk of colorectal cancer etc. Mannan degrading enzymes are the key enzymes involved in degradation and are useful in various industrial processes such as fruit juice clarification, viscosity reduction of coffee extracts etc. besides facilitating the process steps and improving process quality.


Asunto(s)
Alimentos , Mananos/química , Alimentación Animal/análisis , Animales , Productos Biológicos/química , Análisis de los Alimentos , Humanos , Hidrólisis , Mananos/clasificación , Manosidasas/química , Polisacáridos/química
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