Dataset Information

Structural and kinetic analysis of substrate binding to the sialyltransferase Cst-II from Campylobacter jejuni.

ABSTRACT: Sialic acids play important roles in various biological processes and typically terminate the oligosaccharide chains on the cell surfaces of a wide range of organisms, including mammals and bacteria. Their attachment is catalyzed by a set of sialyltransferases with defined specificities both for their acceptor sugars and the position of attachment. However, little is known of how this specificity is encoded. The structure of the bifunctional sialyltransferase Cst-II of the human pathogen Campylobacter jejuni in complex with CMP and the terminal trisaccharide of its natural acceptor (Neu5Ac-?-2,3-Gal-?-1,3-GalNAc) has been solved at 1.95 Å resolution, and its kinetic mechanism was shown to be iso-ordered Bi Bi, consistent with its dual acceptor substrate specificity. The trisaccharide acceptor is seen to bind to the active site of Cst-II through interactions primarily mediated by Asn-51, Tyr-81, and Arg-129. Kinetic and structural analyses of mutants modified at these positions indicate that these residues are critical for acceptor binding and catalysis, thereby providing significant new insight into the kinetic and catalytic mechanism, and acceptor specificity of this pathogen-encoded bifunctional GT-42 sialyltransferase.

SUBMITTER: Lee HJ

PROVIDER: S-EPMC3195623 | biostudies-literature | 2011 Oct

REPOSITORIES: biostudies-literature

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Structural and kinetic analysis of substrate binding to the sialyltransferase Cst-II from Campylobacter jejuni.

Lee Ho Jun HJ Lairson Luke L LL Rich Jamie R JR Lameignere Emilie E Wakarchuk Warren W WW Withers Stephen G SG Strynadka Natalie C J NCJ

The Journal of biological chemistry 20110805 41

Sialic acids play important roles in various biological processes and typically terminate the oligosaccharide chains on the cell surfaces of a wide range of organisms, including mammals and bacteria. Their attachment is catalyzed by a set of sialyltransferases with defined specificities both for their acceptor sugars and the position of attachment. However, little is known of how this specificity is encoded. The structure of the bifunctional sialyltransferase Cst-II of the human pathogen Campylo ...[more]

PMID: 21832050

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Project description:Cerebroside sulfotransferase (CST) is emerging as an important therapeutic target to develop substrate reduction therapy (SRT) for metachromatic leukodystrophy (MLD), a rare neurodegenerative lysosomal storage disorder. MLD develops with progressive impairment and destruction of the myelin sheath as a result of accumulation of sulfatide around the nerve cells in the absence of its recycling mechanism with deficiency of arylsulfatase A (ARSA). Sulfatide is the product of the catalytic action of cerebroside sulfotransferase (CST), which needs to be regulated under pathophysiological conditions by inhibitor development. To carry out in silico-based preliminary drug screening or for designing new drug candidates, a high-quality three-dimensional (3D) structure is needed in the absence of an experimentally derived three-dimensional crystal structure. In this study, a 3D model of the protein was developed using a primary sequence with the SWISS-MODEL server by applying the top four GMEQ score-based templates belonging to the sulfotransferase family as a reference. The 3D model of CST highlights the features of the protein responsible for its catalytic action. The CST model comprises five β-strands, which are flanked by ten α-helices from both sides as well as form the upside cover of the catalytic pocket of CST. CST has two catalytic regions: PAPS (-sulfo donor) binding and galactosylceramide (-sulfo acceptor) binding. The catalytic action of CST was proposed via molecular docking and molecular dynamic (MD) simulation with PAPS, galactosylceramide (GC), PAPS-galactosylceramide, and PAP. The stability of the model and its catalytic action were confirmed using molecular dynamic simulation-based trajectory analysis. CST response against the inhibition potential of the experimentally reported competitive inhibitor of CST was confirmed via molecular docking and molecular dynamics simulation, which suggested the suitability of the CST model for future drug discovery to strengthen substrate reduction therapy for MLD.

Project description:BackgroundProtein glycosylation is of fundamental importance in many biological systems. The discovery of N-glycosylation in bacteria and the functional expression of the N-oligosaccharyltransferase PglB of Campylobacter jejuni in Escherichia coli enabled the production of engineered glycoproteins and the study of the underlying molecular mechanisms. A particularly promising application for protein glycosylation in recombinant bacteria is the production of potent conjugate vaccines where polysaccharide antigens of pathogenic bacteria are covalently bound to immunogenic carrier proteins.ResultsIn this study capsular polysaccharides of the clinically relevant pathogen Staphylococcus aureus serotype 5 (CP5) were expressed in Escherichia coli and linked in vivo to a detoxified version of Pseudomonas aeruginosa exotoxin (EPA). We investigated which amino acids of the periplasmic domain of PglB are crucial for the glycosylation reaction using a newly established 96-well screening system enabling the relative quantification of glycoproteins by enzyme-linked immunosorbent assay. A random mutant library was generated by error-prone PCR and screened for inactivating amino acid substitutions. In addition to 15 inactive variants with amino acid changes within the previously known, strictly conserved WWDYG motif of N-oligosaccharyltransferases, 8 inactivating mutations mapped to a flexible loop in close vicinity of the amide nitrogen atom of the acceptor asparagine as revealed in the crystal structure of the homologous enzyme C. lari PglB. The importance of the conserved loop residue H479 for glycosylation was confirmed by site directed mutagenesis, while a change to alanine of the adjacent, non-conserved L480 had no effect. In addition, we investigated functional requirements in the so-called MIV motif of bacterial N-oligosaccharyltransferases. Amino acid residues I571 and V575, which had been postulated to interact with the acceptor peptide, were subjected to cassette saturation mutagenesis. With the exception of I571C only hydrophobic residues were found in active variants. Variant I571V performed equally well as the wild type, cysteine at the same position reduced glycoprotein yield slightly, while a change to phenylalanine reduced activity by a factor of three.ConclusionsThis study provides novel structure-function relationships for the periplasmic domain of the Campylobacter jejuni N-oligosaccharyltransferase PglB and describes procedures for generating and screening oligosaccharyltransferase mutant libraries in an engineered E. coli system.

Dataset Information

Structural and kinetic analysis of substrate binding to the sialyltransferase Cst-II from Campylobacter jejuni.

Publications

Structural and kinetic analysis of substrate binding to the sialyltransferase Cst-II from Campylobacter jejuni.

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