Structural and Mechanistic Analyses of endo-Glycoceramidase II, a Membrane-associated Family 5 Glycosidase in the Apo and GM3 Ganglioside-bound Forms

Caines, M.E.C.; Vaughan, Mark D.; Tarling, Chris A.; Hancock, Susan M.; Warren, R. A. J.; Withers, Stephen G.; Strynadka, N.C.J.

doi:10.1074/jbc.m611455200

Cited by 45 publications

(49 citation statements)

References 65 publications

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“…That is, a fl exible loop hanging over the catalytic cleft is present in EGCase II but not EGCase I. The complex structure of EGCase II with GM3 (NeuAc ␣ 2-3 Gal ␤ 1-4Glc ␤ -Cer) revealed that the 4-hydroxyl group of the internal ␤ -galactose in GM3 is located close to the loop ( 17 ). On the other hand, the 4-hydroxyl group is substituted with ␣ -galactose in globo-series GSLs such as Gb3Cer (Gal ␣ 1-4Gal ␤ 1-4Glc ␤ -Cer), suggesting that the presence of a loop could affect the turnover of substrates and products in the catalytic region.…”

Section: Mechanical Insights Into the Difference In Reaction Effi Ciementioning

confidence: 99%

“…The model showed a ( ␤ / ␣ ) 8 barrel structure typical for GH family 5 glycosidase. The crystal structure of EGCase II shows a highly disordered and fl exible loop region (Pro 145 to Gly 154 ) hanging over the active site ( 17 ). This region was absent in EGCase I ( Fig.…”

Section: Substrate Specifi City Of the Recombinant Egcase Imentioning

confidence: 99%

“…The molecular cloning of animal EGCase has been performed in the last two animals. Molecular cloning ( 11 ) and X-ray crystal analysis ( 17 ) of EGCase II revealed that the enzyme, belonging to glycoside hydrolase (GH) family 5, was composed of an N-terminal ( ␤ / ␣ ) 8 domain and a C-terminal ␤ -sandwich domain.…”

Section: Expression and Purifi Cation Of The Recombinant Egcase Imentioning

confidence: 99%

“…However, the expression level of the protein was insuffi cient to characterize this enzyme. Thus, we used a novel rhodococcal expression system with R. erythropolis as the host cell ( 30 ) to express the recombinant catalyst and nucleophile, respectively ( 12,17 ) ( Fig. 1 ) ( Fig.…”

Section: Downloaded Frommentioning

confidence: 99%

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Preparation and characterization of EGCase I, applicable to the comprehensive analysis of GSLs, using a rhodococcal expression system

Ishibashi

Kobayashi

Hijikata

et al. 2012

Journal of Lipid Research

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This article is available online at http://www.jlr.org Glycosphingolipids (GSLs), amphipathic compounds consisting of oligosaccharides and ceramide (Cer) moieties, are ubiquitous in the outer leafl et of the plasma membrane. More than 500 species of GSLs differing in oligosaccharides and Cer moieties have been characterized, some of which represent potential biomarkers for diseases and cell development. For instance, Forssman antigen is specifi cally detected in some gastric, colonic, and lung cancers ( 1 ), and stage-specifi c embryonic antigen (SSEA)-3 and SSEA-4, highly expressed at a stage of embryonic development, are utilized as a marker of embryonic stem cells ( 2 ) and induced pluripotent stem cells ( 3 ).The classifi cation of GSLs, into ganglio-series, globoseries and lacto-series GSLs, is entirely based on the structure of the saccharide moiety. However, the Cer moiety, composed of a sphingoid base and fatty acyl chain linked by an N -acyl linkage, shows heterogeneity in carbon chain length, and saturation and hydroxylation status, etc. The complexity of the Cer moiety prevents the comprehensive analysis of cellular GSLs by MS and/or LC-MS. Removal of the Cer moiety makes the analysis of GSLs much simpler and easier. For this purpose, chemical methods such as ozonolysis ( 4 ) and osmium-catalyzed periodate oxidation ( 5 ) have been developed. These chemical methods, however, require complicated and time-consuming procedures with relatively low yield ( 6 ). Alternatively, enzymes capable of detaching intact oligosaccharides from various GSLs Abstract Endoglycoceramidase (EGCase) is a glycosidase capable of hydrolyzing the ␤ -glycosidic linkage between the oligosaccharides and ceramides of glycosphingolipids (GSLs). Three molecular species of EGCase differing in specifi city were found in the culture fl uid of Rhodococcus equi (formerly Rhodococcus sp. M-750) and designated EGCase I, II, and III. This study describes the molecular cloning of EGCase I and characterization of the recombinant enzyme, which was highly expressed in a rhodococcal expression system using Rhodococcus erythropolis. Kinetic analysis revealed the turnover number (k cat ) ( k cat ) of the recombinant EGCase I to be 22-and 1,200-fold higher than that of EGCase II toward GM1a and Gb3Cer, respectively, although the K m of both enzymes was almost the same for these substrates.

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Section: Mechanical Insights Into the Difference In Reaction Effi Ciementioning

confidence: 99%

Section: Substrate Specifi City Of the Recombinant Egcase Imentioning

confidence: 99%

Section: Expression and Purifi Cation Of The Recombinant Egcase Imentioning

confidence: 99%

Section: Downloaded Frommentioning

confidence: 99%

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Preparation and characterization of EGCase I, applicable to the comprehensive analysis of GSLs, using a rhodococcal expression system

Ishibashi

Kobayashi

Hijikata

et al. 2012

Journal of Lipid Research

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“…GH9 family enzymes have an (␣/␣) 6 -barrel fold, and this family of enzymes is known to contain members that exhibit different enzymatic activities, including endoglucanase (45), cellobiohydrolase (15), 1,4-␤-D-glucan glucohydrolase (43), ␤-glucosidase (40), and exo-␤-glucosaminidase (23). The GH5 family of enzymes have an (␤/␣) 8 -barrel fold, and this family also encodes diverse enzymatic activities, including endoglucanase (16), mannanase (20), mannosidase (20), xylanase (14), chitosanase (50), glucosidase (11), licheninase (25), fucosidase (61), galactanase (44), endoglycoceramidase (9), and mannan transglycosylase (6). The binding cleft in GH9 catalytic modules can hold six sugar molecules, among which four subsites (Ϫ4 to Ϫ1) are at the nonreducing end and the two remaining subsites are at the reducing end (ϩ1 to ϩ2) (45).…”

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confidence: 99%

Biochemical and Mutational Analyses of a Multidomain Cellulase/Mannanase from Caldicellulosiruptor bescii

Mackie

Cann

2012

Appl Environ Microbiol

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Thermophilic cellulases and hemicellulases are of significant interest to the biofuel industry due to their perceived advantages over their mesophilic counterparts. We describe here biochemical and mutational analyses of Caldicellulosiruptor bescii Cel9B/ Man5A (CbCel9B/Man5A), a highly thermophilic enzyme. As one of the highly secreted proteins of C. bescii, the enzyme is likely to be critical to nutrient acquisition by the bacterium. CbCel9B/Man5A is a modular protein composed of three carbohydratebinding modules flanked at the N terminus and the C terminus by a glycoside hydrolase family 9 (GH9) module and a GH5 module, respectively. Based on truncational analysis of the polypeptide, the cellulase and mannanase activities within CbCel9B/ Man5A were assigned to the N-and C-terminal modules, respectively. CbCel9B/Man5A and its truncational mutants, in general, exhibited a pH optimum of ϳ5.5 and a temperature optimum of 85°C. However, at this temperature, thermostability was very low. After 24 h of incubation at 75°C, the wild-type protein maintained 43% activity, whereas a truncated mutant, TM1, maintained 75% activity. The catalytic efficiency with phosphoric acid swollen cellulose as a substrate for the wild-type protein was 7.2 s ؊1 ml/mg, and deleting the GH5 module led to a mutant (TM1) with a 2-fold increase in this kinetic parameter. Deletion of the GH9 module also increased the apparent k cat of the truncated mutant TM5 on several mannan-based substrates; however, a concomitant increase in the K m led to a decrease in the catalytic efficiencies on all substrates. These observations lead us to postulate that the two catalytic activities are coupled in the polypeptide.

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The Structural Basis of Glycosidase Inhibition by Five‐Membered Iminocyclitols: The Clan A Glycoside Hydrolase Endoglycoceramidase as a Model System

Caines¹,

Hancock²,

Tarling³

et al. 2007

Angewandte Chemie

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Fünf passt auch: Die Kristallstruktur eines an Glycosidase gebundenen fünfgliedrigen Iminocyclitols wurde bestimmt (siehe Bild), und die Wechselwirkungen bei der Bindung dieses Inhibitors wurden mit denjenigen für die gängigen sechsgliedrigen Inhibitoren Isofagomin und Glucoimidazol und im Glycosyl‐Enzym‐Intermediat verglichen. Diese Daten können zu wirksameren und spezifischeren Glycosidase‐Inhibitoren für therapeutische Anwendungen führen.

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Structural and Mechanistic Analyses of endo-Glycoceramidase II, a Membrane-associated Family 5 Glycosidase in the Apo and GM3 Ganglioside-bound Forms

Cited by 45 publications

References 65 publications

Preparation and characterization of EGCase I, applicable to the comprehensive analysis of GSLs, using a rhodococcal expression system

Preparation and characterization of EGCase I, applicable to the comprehensive analysis of GSLs, using a rhodococcal expression system

Biochemical and Mutational Analyses of a Multidomain Cellulase/Mannanase from Caldicellulosiruptor bescii

The Structural Basis of Glycosidase Inhibition by Five‐Membered Iminocyclitols: The Clan A Glycoside Hydrolase Endoglycoceramidase as a Model System

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