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Structural Insights into the Broad Substrate Specificity of a Novel Endoglycoceramidase I Belonging to a New Subfamily of GH5 Glycosidases.

ABSTRACT: Endoglycoceramidases (EGCases) specifically hydrolyze the glycosidic linkage between the oligosaccharide and the ceramide moieties of various glycosphingolipids, and they have received substantial attention in the emerging field of glycosphingolipidology. However, the mechanism regulating the strict substrate specificity of these GH5 glycosidases has not been identified. In this study, we report a novel EGCase I from Rhodococcus equi 103S (103S_EGCase I) with remarkably broad substrate specificity. Based on phylogenetic analyses, the enzyme may represent a new subfamily of GH5 glycosidases. The X-ray crystal structures of 103S_EGCase I alone and in complex with its substrates monosialodihexosylganglioside (GM3) and monosialotetrahexosylganglioside (GM1) enabled us to identify several structural features that may account for its broad specificity. Compared with EGCase II from Rhodococcus sp. M-777 (M777_EGCase II), which possesses strict substrate specificity, 103S_EGCase I possesses a longer ?7-helix and a shorter loop 4, which forms a larger substrate-binding pocket that could accommodate more extended oligosaccharides. In addition, loop 2 and loop 8 of the enzyme adopt a more open conformation, which also enlarges the oligosaccharide-binding cavity. Based on this knowledge, a rationally designed experiment was performed to examine the substrate specificity of EGCase II. The truncation of loop 4 in M777_EGCase II increased its activity toward GM1 (163%). Remarkably, the S63G mutant of M777_EGCase II showed a broader substrate spectra and significantly increased activity toward bulky substrates (up to >1370-fold for fucosyl-GM1). Collectively, the results presented here reveal the exquisite substrate recognition mechanism of EGCases and provide an opportunity for further engineering of these enzymes.

SUBMITTER: Han YB

PROVIDER: S-EPMC5377795 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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Structural Insights into the Broad Substrate Specificity of a Novel Endoglycoceramidase I Belonging to a New Subfamily of GH5 Glycosidases.

Han Yun-Bin YB Chen Liu-Qing LQ Li Zhuo Z Tan Yu-Meng YM Feng Yan Y Yang Guang-Yu GY

The Journal of biological chemistry 20170208 12

Endoglycoceramidases (EGCases) specifically hydrolyze the glycosidic linkage between the oligosaccharide and the ceramide moieties of various glycosphingolipids, and they have received substantial attention in the emerging field of glycosphingolipidology. However, the mechanism regulating the strict substrate specificity of these GH5 glycosidases has not been identified. In this study, we report a novel EGCase I from <i>Rhodococcus equi</i> 103S (103S_EGCase I) with remarkably broad substrate sp ...[more]

PMID: 28179425

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Project description:Aldosterone is a major mineralocorticoid hormone that plays a key role in the regulation of electrolyte balance and blood pressure. Excess aldosterone levels can arise from dysregulation of the renin-angiotensin-aldosterone system and are implicated in the pathogenesis of hypertension and heart failure. Aldosterone synthase (cytochrome P450 11B2, CYP11B2) is the sole enzyme responsible for the production of aldosterone in humans. Blocking of aldosterone synthesis by mediating aldosterone synthase activity is thus a recently emerging pharmacological therapy for hypertension, yet a lack of structural information has limited this approach. Here, we present the crystal structures of human aldosterone synthase in complex with a substrate deoxycorticosterone and an inhibitor fadrozole. The structures reveal a hydrophobic cavity with specific features associated with corticosteroid recognition. The substrate binding mode, along with biochemical data, explains the high 11β-hydroxylase activity of aldosterone synthase toward both gluco- and mineralocorticoid formation. The low processivity of aldosterone synthase with a high extent of intermediates release might be one of the mechanisms of controlled aldosterone production from deoxycorticosterone. Although the active site pocket is lined by identical residues between CYP11B isoforms, most of the divergent residues that confer additional 18-oxidase activity of aldosterone synthase are located in the I-helix (vicinity of the O(2) activation path) and loops around the H-helix (affecting an egress channel closure required for retaining intermediates in the active site). This intrinsic flexibility is also reflected in isoform-selective inhibitor binding. Fadrozole binds to aldosterone synthase in the R-configuration, using part of the active site cavity pointing toward the egress channel. The structural organization of aldosterone synthase provides critical insights into the molecular mechanism of catalysis and enables rational design of more specific antihypertensive agents.

Dataset Information

Structural Insights into the Broad Substrate Specificity of a Novel Endoglycoceramidase I Belonging to a New Subfamily of GH5 Glycosidases.

Publications

Structural Insights into the Broad Substrate Specificity of a Novel Endoglycoceramidase I Belonging to a New Subfamily of GH5 Glycosidases.

Similar Datasets

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