scholarly journals Arabidopsis thaliana β1,2-xylosyltransferase: an unusual glycosyltransferase with the potential to act at multiple stages of the plant N-glycosylation pathway

2005 ◽  
Vol 388 (2) ◽  
pp. 515-525 ◽  
Author(s):  
Peter BENCÚR ◽  
Herta STEINKELLNER ◽  
Barbara SVOBODA ◽  
Jan MUCHA ◽  
Richard STRASSER ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Substrate Specificity ◽  
Metal Ion ◽  
Catalytic Domain ◽  
Protein Characterization ◽  
Reaction Products ◽  
Glycosylation Sites ◽  
Glycosylation Pathway ◽  
Multiple Stages

XylT (β1,2-xylosyltransferase) is a unique Golgi-bound glycosyltransferase that is involved in the biosynthesis of glycoprotein-bound N-glycans in plants. To delineate the catalytic domain of XylT, a series of N-terminal deletion mutants was heterologously expressed in insect cells. Whereas the first 54 residues could be deleted without affecting the catalytic activity of the enzyme, removal of an additional five amino acids led to the formation of an inactive protein. Characterization of the N-glycosylation status of recombinant XylT revealed that all three potential N-glycosylation sites of the protein are occupied by N-linked oligosaccharides. However, an unglycosylated version of the enzyme displayed substantial catalytic activity, demonstrating that N-glycosylation is not essential for proper folding of XylT. In contrast with most other glycosyltransferases, XylT is enzymatically active in the absence of added metal ions. This feature is not due to any metal ion directly associated with the enzyme. The precise acceptor substrate specificity of XylT was assessed with several physiologically relevant compounds and the xylosylated reaction products were subsequently tested as substrates of other Golgi-resident glycosyltransferases. These experiments revealed that the substrate specificity of XylT permits the enzyme to act at multiple stages of the plant N-glycosylation pathway.

scholarly journals Identification of three critical acidic residues of poly(ADP-ribose) glycohydrolase involved in catalysis: determining the PARG catalytic domain

2005 ◽  
Vol 388 (2) ◽  
pp. 493-500 ◽  
Author(s):  
Chandra N. PATEL ◽  
David W. KOH ◽  
Myron K. JACOBSON ◽  
Marcos A. OLIVEIRA
Keyword(s):  
Catalytic Activity ◽  
Catalytic Domain ◽  
Functional Characterization ◽  
Sequence Alignments ◽  
Inhibitor Binding ◽  
Conserved Sequence ◽  
Binding Residue ◽  
Acidic Residues ◽  
Hydrolysis Of

PARG [poly(ADP-ribose) glycohydrolase] catalyses the hydrolysis of α(1″→2′) or α(1‴→2″) O-glycosidic linkages of ADP-ribose polymers to produce free ADP-ribose. We investigated possible mechanistic similarities between PARG and glycosidases, which also cleave O-glycosidic linkages. Glycosidases typically utilize two acidic residues for catalysis, thus we targeted acidic residues within a conserved region of bovine PARG that has been shown to contain an inhibitor-binding site. The targeted glutamate and aspartate residues were changed to asparagine in order to minimize structural alterations. Mutants were purified and assayed for catalytic activity, as well as binding, to an immobilized PARG inhibitor to determine ability to recognize substrate. Our investigation revealed residues essential for PARG catalytic activity. Two adjacent glutamic acid residues are found in the conserved sequence Gln755-Glu-Glu757, and a third residue found in the conserved sequence Val737-Asp-Phe-Ala-Asn741. Our functional characterization of PARG residues, along with recent identification of an inhibitor-binding residue Tyr796 and a glycine-rich region Gly745-Gly-Gly747 important for PARG function, allowed us to define a PARG ‘signature sequence’ [vDFA-X3-GGg-X6–8-vQEEIRF-X3-PE-X14-E-X12-YTGYa], which we used to identify putative PARG sequences across a range of organisms. Sequence alignments, along with our mapping of PARG functional residues, suggest the presence of a conserved catalytic domain of approx. 185 residues which spans residues 610–795 in bovine PARG.

scholarly journals N-linked glycosylation enzymes in the diatom Thalassiosira oceanica exhibit a diel cycle in transcript abundance and favor for NXT-type sites

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Joerg Behnke ◽  
Alejandro M. Cohen ◽  
Julie LaRoche
Keyword(s):  
Cell Biology ◽  
Biochemical Characterization ◽  
Solid Phase ◽  
Transcript Abundance ◽  
Growth Conditions ◽  
Iron Starvation ◽  
Glycosylation Sites ◽  
Glycosylation Pathway ◽  
And Function

AbstractN-linked glycosylation is a posttranslational modification affecting protein folding and function. The N-linked glycosylation pathway in algae is poorly characterized, and further knowledge is needed to understand the cell biology of algae and the evolution of N-linked glycosylation. This study investigated the N-linked glycosylation pathway in Thalassiosira oceanica, an open ocean diatom adapted to survive at growth-limiting iron concentrations. Here we identified and annotated the genes coding for the essential enzymes involved in the N-linked glycosylation pathway of T. oceanica. Transcript levels for genes coding for calreticulin, oligosaccharyltransferase (OST), N-acetylglucosaminyltransferase (GnT1), and UDP-glucose glucosyltransferase (UGGT) under high- and low-iron growth conditions revealed diel transcription patterns with a significant decrease of calreticulin and OST transcripts under iron-limitation. Solid-phase extraction of N-linked glycosylated peptides (SPEG) revealed 118 N-linked glycosylated peptides from cells grown in high- and low-iron growth conditions. The identified peptides had 81% NXT-type motifs, with X being any amino acids except proline. The presence of N-linked glycosylation sites in the iron starvation-induced protein 1a (ISIP1a) confirmed its predicted topology, contributing to the biochemical characterization of ISIP1 proteins. Analysis of extensive oceanic gene databases showed a global distribution of calreticulin, OST, and UGGT, reinforcing the importance of glycosylation in microalgae.

A new subfamily of fungal subtilases: structural and functional analysis of a Pleurotus ostreatus member

Microbiology ◽  
2005 ◽  
Vol 151 (2) ◽  
pp. 457-466 ◽  
Author(s):  
Vincenza Faraco ◽  
Gianna Palmieri ◽  
Giovanna Festa ◽  
Maria Monti ◽  
Giovanni Sannia ◽  
...  
Keyword(s):  
Pleurotus Ostreatus ◽  
Catalytic Domain ◽  
Peptide Bond ◽  
Homology Search ◽  
Extracellular Proteases ◽  
Leading Role ◽  
Glycosylation Sites ◽  
Activation Mechanisms ◽  
Basidiomycete Fungi

Pleurotus ostreatus produces several extracellular proteases which are believed to be involved in the regulation of the ligninolytic activities of this fungus. Recently, purification and characterization of the most abundant P. ostreatus extracellular protease (PoSl) have been reported. The sequence of the posl gene and of the corresponding cDNA has been determined, allowing the identification of its pre- and pro-sequences. A mature protein sequence has been verified by mass spectrometry mapping, the N-glycosylation sites have been identified and the glycosidic moieties characterized. Mature PoSl shows a cleaved peptide bond in the C-terminal region, which remains associated with the catalytic domain in a non-covalent complex. Reported results indicate that this enzyme is involved in the activation of other P. ostreatus secreted proteases, thus suggesting its leading role in cascade activation mechanisms. Analyses of the PoSl sequence by homology search resulted in the identification of a DNA sequence encoding a new protease, homologous to PoSl, in the Phanerochaete chrysosporium genome. A new subgroup of subtilisin-like proteases, belonging to the pyrolysin family, has been defined, which includes proteases from ascomycete and basidiomycete fungi.

scholarly journals REASSESSMENT OF THE CATALYTIC ACTIVITY AND SUBSTRATE SPECIFICITY OF FKBP35 FROM Plasmodium knowlesi USING PROTEASE-FREE ASSAY

2020 ◽  
pp. 125
Author(s):  
Cahyo Budiman ◽  
Carlmond Goh Kah Wun ◽  
Lee Ping Chin ◽  
Rafida Razali ◽  
Thean Chor Leow
Keyword(s):  
Catalytic Activity ◽  
Substrate Specificity ◽  
Catalytic Domain ◽  
Plasmodium Knowlesi ◽  
Tetratricopeptide Repeat ◽  
Cleavage Sites ◽  
Repeat Domain ◽  
The Common ◽  
Tetratricopeptide Repeat Domain ◽  
Promising Avenue

FK506-binding protein35 of Plasmodium knowlesi (Pk-FKBP35) is a member of peptidyl prolyl cis-trans isomerase (PPIase) and is considered as a promising avenue of antimalarial drug target development. This protein is organized into the N-terminal domain responsible for PPIase catalytic activity followed and the tetratricopeptide repeat domain for its dimerization. The protease-coupling and protease-free assays are known to be the common methods for investigating the catalytic properties of PPIase. Earlier, the protease-coupling assay was used to confirm the catalytic activity of Pk-FKBP35 in accelerating cis-trans isomerization of the peptide substrate. This report is aimed to re-assess the catalytic and substrate specificity of Pk-FKBP35 using an alternative method of a protease-free assay. The result indicated that while Pk-FKBP35 theoretically contained many possible cleavage sites of chymotrypsin, experimentally, the catalytic domain was relatively stable from chymotrypsin. Furthermore, under protease-free assay, Pk-FKBP35 also demonstrated remarkable PPIase catalytic activity with kcat/KM of 4.5 + 0.13 × 105 M−1 s−1, while the kcat/KM of active site mutant of D55A is 0.81 + 0.05 × 105 M−1 s−1. These values were considered comparable to kcat/KM obtained from the protease-coupling assay. Interestingly, the substrate specificities of Pk-FKBP35 obtained from both methods are also similar, with the preference of Pk-FKBP35 towards Xaa at P1 position was Leu>Phe>Lys>Trp>Val>Ile>His>Asp>Ala>Gln>Glu. Altogether, we proposed that protease-free and protease-coupling assays arereliable for Pk-FKBP35.

Characterization of pH3DZ1 — An RNA-cleaving deoxyribozyme with optimal activity at pH 3

2007 ◽  
Vol 85 (4) ◽  
pp. 261-273 ◽  
Author(s):  
Md. Monsur Ali ◽  
Srinivas A Kandadai ◽  
Yingfu Li
Keyword(s):  
Catalytic Activity ◽  
In Vitro Selection ◽  
Catalytic Domain ◽  
Ph Profile ◽  
Cis Acting ◽  
Optimal Activity ◽  
Catalytic Function ◽  
Covalently Linked

We previously described a cis-acting RNA-cleaving deoxyribozyme known as pH3DZ1 that exhibits optimal catalytic activity at pH 3.0 (Zhongjie Liu, Shirley H. Mei, John D. Brennan, and Yingfu Li. J. Am. Chem. Soc. 125, 7539 (2003)). This DNA catalyst was made of a 99-nucleotide (nt) catalytic domain covalently linked to a 23-nt DNA–RNA chimeric substrate containing a single ribonucleotide as the cleavage site. In the present work, we conducted an extensive sequence examination of this deoxyribozyme via nucleotide truncation and reselection experiments, with a goal to minimize its size and identify the nucleotides that are crucial to its catalytic function. A trans-acting deoxyribozyme that can process an external substrate was also successfully designed. Stretches of 30 and 17 nucleotides from the 5′ and 3′ ends of the trans catalyst, respectively, were found to be completely dispensable; in contrast, few nucleotides could be deleted internally without producing a detrimental effect. The reselection experiment led to the discovery of 7 and 5 absolutely conserved nucleotides located at the 5′ and 3′ ends of the minimized catalyst, respectively, separated by a 31-nt element in which 14 highly conserved nucleotides were scattered among 17 variable nucleotides. The shortened deoxyribozyme and the original catalyst showed a similar pH profile with the optimal activity at pH 3; however, the minimized deoxyribozyme still exhibited strong catalytic activity at pH 2.5, while the full-length catalyst was barely active at this pH. Finally, it was found that this deoxyribozyme generated two cleavage fragments, one with 2′,3′-cyclic phosphate and the other with 5′-OH.Key words: DNA, deoxyribozyme, RNA cleavage, in vitro selection, catalysis.

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