Dataset Information

CUPRA-ZYME: An Assay for Measuring Carbohydrate-Active Enzyme Activities, Pathways, and Substrate Specificities.

ABSTRACT: Carbohydrate-Active enZymes (CAZymes) are involved in the synthesis, degradation, and modification of carbohydrates. They play critical roles in diverse physiological and pathophysiological processes, have important industrial and biotechnological applications, are important drug targets, and represent promising biomarkers for the diagnosis of a variety of diseases. Measurements of their activities, catalytic pathway, and substrate specificities are essential to a comprehensive understanding of the biological functions of CAZymes and exploiting these enzymes for industrial and biomedical applications. For glycosyl hydrolases a variety of sensitive and quantitative spectrophotometric techniques are available. However, measuring the activity of glycosyltransferases is considerably more challenging. Here, we introduce CUPRA-ZYME, a versatile and quantitative electrospray ionization mass spectrometry (ESI-MS) assay for measuring the kinetic parameters of CAZymes, monitoring reaction pathways, and profiling substrate specificities. The method employs the recently developed competitive universal proxy receptor assay (CUPRA), implemented in a time-resolved manner. Measurements of the hydrolysis kinetics of CUPRA substrates containing ganglioside oligosaccharides by the glycosyl hydrolase human neuraminidase 3 served to validate the reliability of kinetic parameters measured by CUPRA-ZYME and highlight its use in establishing catalytic pathways. Applications to libraries of substrates demonstrate the potential of the assay for quantitative profiling of the substrate specificities glycosidases and glycosyltransferases. Finally, we show how the comparison of the reactivity of CUPRA substrates and glycan substrates present on glycoproteins, measured simultaneously, affords a unique opportunity to quantitatively study how the structure and protein environment of natural glycoconjugate substrates influences CAZyme activity.

SUBMITTER: Li Z

PROVIDER: S-EPMC7586304 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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CUPRA-ZYME: An Assay for Measuring Carbohydrate-Active Enzyme Activities, Pathways, and Substrate Specificities.

Li Zhixiong Z Kitov Pavel I PI Kitova Elena N EN Mozenah Fahima F Rodrigues Emily E Chapla Digantkumar G DG Moremen Kelley W KW Macauley Matthew S MS Klassen John S JS

Analytical chemistry 20200207 4

Carbohydrate-Active enZymes (CAZymes) are involved in the synthesis, degradation, and modification of carbohydrates. They play critical roles in diverse physiological and pathophysiological processes, have important industrial and biotechnological applications, are important drug targets, and represent promising biomarkers for the diagnosis of a variety of diseases. Measurements of their activities, catalytic pathway, and substrate specificities are essential to a comprehensive understanding of ...[more]

PMID: 31961140

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Project description:Background:Deposition of new genetic sequences in online databases is expanding at an unprecedented rate. As a result, sequence identification continues to outpace functional characterization of carbohydrate active enzymes (CAZymes). In this paradigm, the discovery of enzymes with novel functions is often hindered by high volumes of uncharacterized sequences particularly when the enzyme sequence belongs to a family that exhibits diverse functional specificities (i.e., polyspecificity). Therefore, to direct sequence-based discovery and characterization of new enzyme activities we have developed an automated in silico pipeline entitled: Sequence Analysis and Clustering of CarboHydrate Active enzymes for Rapid Informed prediction of Specificity (SACCHARIS). This pipeline streamlines the selection of uncharacterized sequences for discovery of new CAZyme or CBM specificity from families currently maintained on the CAZy website or within user-defined datasets. Results:SACCHARIS was used to generate a phylogenetic tree of a GH43, a CAZyme family with defined subfamily designations. This analysis confirmed that large datasets can be organized into sequence clusters of manageable sizes that possess related functions. Seeding this tree with a GH43 sequence from Bacteroides dorei DSM 17855 (BdGH43b, revealed it partitioned as a single sequence within the tree. This pattern was consistent with it possessing a unique enzyme activity for GH43 as BdGH43b is the first described ?-glucanase described for this family. The capacity of SACCHARIS to extract and cluster characterized carbohydrate binding module sequences was demonstrated using family 6 CBMs (i.e., CBM6s). This CBM family displays a polyspecific ligand binding profile and contains many structurally determined members. Using SACCHARIS to identify a cluster of divergent sequences, a CBM6 sequence from a unique clade was demonstrated to bind yeast mannan, which represents the first description of an ?-mannan binding CBM. Additionally, we have performed a CAZome analysis of an in-house sequenced bacterial genome and a comparative analysis of B. thetaiotaomicron VPI-5482 and B. thetaiotaomicron 7330, to demonstrate that SACCHARIS can generate "CAZome fingerprints", which differentiate between the saccharolytic potential of two related strains in silico. Conclusions:Establishing sequence-function and sequence-structure relationships in polyspecific CAZyme families are promising approaches for streamlining enzyme discovery. SACCHARIS facilitates this process by embedding CAZyme and CBM family trees generated from biochemically to structurally characterized sequences, with protein sequences that have unknown functions. In addition, these trees can be integrated with user-defined datasets (e.g., genomics, metagenomics, and transcriptomics) to inform experimental characterization of new CAZymes or CBMs not currently curated, and for researchers to compare differential sequence patterns between entire CAZomes. In this light, SACCHARIS provides an in silico tool that can be tailored for enzyme bioprospecting in datasets of increasing complexity and for diverse applications in glycobiotechnology.

Project description:The 3C-like protease (3CLpro) of nidovirus plays an important role in viral replication and manipulation of host antiviral innate immunity, which makes it an ideal antiviral target. Here, we characterized that porcine torovirus (PToV; family Tobaniviridae, order Nidovirales) 3CLpro autocatalytically releases itself from the viral precursor protein by self-cleavage. Site-directed mutagenesis suggested that PToV 3CLpro, as a serine protease, employed His53 and Ser160 as the active-site residues. Interestingly, unlike most nidovirus 3CLpro, the P1 residue plays a less essential role in N-terminal self-cleavage of PToV 3CLpro Substituting either P1 or P4 residue of substrate alone has little discernible effect on N-terminal cleavage. Notably, replacement of the two residues together completely blocks N-terminal cleavage, suggesting that N-terminal self-cleavage of PToV 3CLpro is synergistically affected by both P1 and P4 residues. Using a cyclized luciferase-based biosensor, we systematically scanned the polyproteins for cleavage sites and identified (FXXQ↓A/S) as the main consensus sequences. Subsequent homology modeling and biochemical experiments suggested that the protease formed putative pockets S1 and S4 between the substrate. Indeed, mutants of both predicted S1 (D159A, H174A) and S4 (P62G/L185G) pockets completely lost the ability of cleavage activity of PToV 3CLpro In conclusion, the characterization of self-processing activities and substrate specificities of PToV 3CLpro will offer helpful information for the mechanism of nidovirus 3C-like proteinase's substrate specificities and the rational development of the antinidovirus drugs.IMPORTANCE Currently, the active-site residues and substrate specificities of 3C-like protease (3CLpro) differ among nidoviruses, and the detailed catalytic mechanism remains largely unknown. Here, porcine torovirus (PToV) 3CLpro cleaves 12 sites in the polyproteins, including its N- and C-terminal self-processing sites. Unlike coronaviruses and arteriviruses, PToV 3CLpro employed His53 and Ser160 as the active-site residues that recognize a glutamine (Gln) at the P1 position. Surprisingly, mutations of P1-Gln impaired the C-terminal self-processing but did not affect N-terminal self-processing. The "noncanonical" substrate specificity for its N-terminal self-processing was attributed to the phenylalanine (Phe) residue at the P4 position in the N-terminal site. Furthermore, a double glycine (neutral) substitution at the putative P4-Phe-binding residues (P62G/L185G) abolished the cleavage activity of PToV 3CLpro suggested the potential hydrophobic force between the PToV 3CLpro and P4-Phe side chains.

Dataset Information

CUPRA-ZYME: An Assay for Measuring Carbohydrate-Active Enzyme Activities, Pathways, and Substrate Specificities.

Publications

CUPRA-ZYME: An Assay for Measuring Carbohydrate-Active Enzyme Activities, Pathways, and Substrate Specificities.

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