Functional characterization and catalytic activity improvement of BAHD acyltransferase from Celastrus angulatus Maxim

Planta ◽  
2020 ◽  
Vol 252 (1) ◽  
Author(s):  
Xiaoguang Yan ◽  
Xiaoyu Qin ◽  
Weiguo Li ◽  
Dongmei Liang ◽  
Jianjun Qiao ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Functional Characterization ◽  
Bahd Acyltransferase

scholarly journals Human xylosyltransferase I and N-terminal truncated forms: functional characterization of the core enzyme

2006 ◽  
Vol 394 (1) ◽  
pp. 163-171 ◽  
Author(s):  
Sandra Müller ◽  
Jennifer Disse ◽  
Manuela Schöttler ◽  
Sylvia Schön ◽  
Christian Prante ◽  
...  
Keyword(s):  
Amino Acids ◽  
Catalytic Activity ◽  
Binding Capacity ◽  
Functional Characterization ◽  
Terminal Region ◽  
Binding Motif ◽  
Heparin Binding ◽  
Loss Of Function ◽  
The Impact

Human XT-I (xylosyltransferase I; EC 2.4.2.26) initiates the biosynthesis of the glycosaminoglycan linkage region and is a diagnostic marker of an enhanced proteoglycan biosynthesis. In the present study, we have investigated mutant enzymes of human XT-I and assessed the impact of the N-terminal region on the enzymatic activity. Soluble mutant enzymes of human XT-I with deletions at the N-terminal domain were expressed in insect cells and analysed for catalytic activity. As many as 260 amino acids could be truncated at the N-terminal region of the enzyme without affecting its catalytic activity. However, truncation of 266, 272 and 273 amino acids resulted in a 70, 90 and >98% loss in catalytic activity. Interestingly, deletion of the single 12 amino acid motif G261KEAISALSRAK272 leads to a loss-of-function XT-I mutant. This is in agreement with our findings analysing the importance of the Cys residues where we have shown that C276A mutation resulted in a nearly inactive XT-I enzyme. Moreover, we investigated the location of the heparin-binding site of human XT-I using the truncated mutants. Heparin binding was observed to be slightly altered in mutants lacking 289 or 568 amino acids, but deletion of the potential heparin-binding motif P721KKVFKI727 did not lead to a loss of heparin binding capacity. The effect of heparin or UDP on the XT-I activity of all mutants was not significantly different from that of the wild-type. Our study demonstrates that over 80% of the nucleotide sequence of the XT-I-cDNA is necessary for expressing a recombinant enzyme with full catalytic activity.

scholarly journals Functional characterization of the non-catalytic ectodomains of the nucleotide pyrophosphatase/phosphodiesterase NPP1

2003 ◽  
Vol 371 (2) ◽  
pp. 321-330 ◽  
Author(s):  
Rik GIJSBERS ◽  
Hugo CEULEMANS ◽  
Mathieu BOLLEN
Keyword(s):  
Catalytic Activity ◽  
Catalytic Domain ◽  
Transmembrane Domain ◽  
Functional Characterization ◽  
Structure Stability ◽  
Subunit Structure ◽  
Terminal Domain ◽  
Nucleotide Pyrophosphatase ◽  
Domain Truncation ◽  
And Function

The ubiquitous nucleotide pyrophosphatases/phosphodiesterases NPP1–3 consist of a short intracellular N-terminal domain, a single transmembrane domain and a large extracellular part, comprising two somatomedin-B-like domains, a catalytic domain and a poorly defined C-terminal domain. We show here that the C-terminal domain of NPP1–3 is structurally related to a family of DNA/RNA non-specific endonucleases. However, none of the residues that are essential for catalysis by the endonucleases are conserved in NPP1–NPP3, suggesting that the nuclease-like domain of NPP1–3 does not represent a second catalytic domain. Truncation analysis revealed that the nuclease-like domain of NPP1 is required for protein stability, for the targeting of NPP1 to the plasma membrane and for the expression of catalytic activity. We also demonstrate that 16 conserved cysteines in the somatomedin-B-like domains of NPP1, in concert with two flanking cysteines, mediate the dimerization of NPP1. The K173Q polymorphism of NPP1, which maps to the second somatomedin-B-like domain and has been associated with the aetiology of insulin resistance, did not affect the dimerization or catalytic activity of NPP1, and did not endow NPP1 with an affinity for the insulin receptor. Our data suggest that the non-catalytic ectodomains contribute to the subunit structure, stability and function of NPP1–3.

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 The differential catalytic activity of ribosome-inactivating proteins saporin 5 and 6 is due to a single substitution at position 162

2006 ◽  
Vol 400 (1) ◽  
pp. 99-104 ◽  
Author(s):  
Paroma Ghosh ◽  
Janendra K. Batra
Keyword(s):  
Catalytic Activity ◽  
Amino Acid ◽  
Active Sites ◽  
Functional Characterization ◽  
Cytotoxic Activities ◽  
Type I ◽  
Ribosome Inactivating Protein ◽  
Aspartic Acid Residue ◽  
Ribosome Inactivating Proteins ◽  
Saponaria Officinalis

Saporin, a type I ribosome-inactivating protein produced by the soapwort plant Saponaria officinalis belongs to a multigene family that encodes its several isoforms. The saporin seed isoform 6 has significantly higher N-glycosidase and cytotoxic activities compared with the seed isoform 5, although the two have identical active sites. In the present study, we have investigated the contribution of non-conservative amino acid changes outside the active sites of these isoforms towards their differential catalytic activity. The saporin 6 residues Lys134, Leu147, Phe149, Asn162, Thr188 and Asp196 were replaced by the corresponding saporin 5 residues, Gln134, Ser147, Ser149, Asp162, Ile188 and Asn196, to generate six variants of saporin 6, K134Q, L147S, F149S, N162D, T188I and D196N. By functional characterization, we show that the change in amino acid Asn162 in saporin 6 to aspartic acid residue of saporin 5 contributes mainly to the lower catalytic activity of saporin 5 compared with saporin 6. The non-involvement of other non-conservative amino acids in the differential catalytic activity of these isoforms was confirmed with the help of the double mutations N162D/K134Q, N162D/L147S, N162D/F149S, N162D/T188I and N162D/D196N.

Identification and functional characterization of novel CYP2J2 variants: G312R variant causes loss of enzyme catalytic activity

2005 ◽  
Vol 15 (2) ◽  
pp. 105-113 ◽  
Author(s):  
Sang Seop Lee ◽  
Hye-Eun Jeong ◽  
Kwang-Hyeon Liu ◽  
Ji-Young Ryu ◽  
Taesung Moon ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Functional Characterization

Production, purification and functional characterization of phospholipase C from Bacillus thuringiensis with high catalytic activity

2019 ◽  
Vol 83 ◽  
pp. 122-130 ◽  
Author(s):  
Ahlem Eddehech ◽  
Nabil Smichi ◽  
Yani Arhab ◽  
Alexandre Noiriel ◽  
Abdelkarim Abousalham ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Bacillus Thuringiensis ◽  
Phospholipase C ◽  
Functional Characterization ◽  
High Catalytic Activity

Functional Analysis Of TTP-Associated ADAMTS13 Mutants Under Shear Flow

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1094-1094
Author(s):  
Maria A. Brehm ◽  
Emma Kraus ◽  
Tobias Obser ◽  
Reinhard Schneppenheim
Keyword(s):  
Endothelial Cells ◽  
Catalytic Activity ◽  
Shear Flow ◽  
Proteolytic Activity ◽  
Binding Sites ◽  
Conflicts Of Interest ◽  
Functional Characterization ◽  
Residual Activity ◽  
Single Point Mutation ◽  
Physiological Conditions

Abstract Upon agonist stimulation endothelial cells secrete ultralarge von Willebrand Factor (VWF) multimers. These multimers form prothrombotic VWF strings on the cell's surface which are size-regulated by ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13). Under physiological conditions, this metalloprotease cleaves VWF between Tyr1605 and Met1606 in its A2 domain only when this domain is unfolded by shear stress and the respective binding sites of both, VWF and ADAMTS13, adjoin each other (Gao W, et al., Blood. 2008 Sep 1;112(5):1713-9). This sophisticated substrate recognition involves distinct steps during which the TSP5-CUB domains of ADAMTS13 initially bind to VWF shear force independent, then, upon shear induced elongation of VWF, additional exosite binding sites become exposed which subsequently can be bound by the complementary spacer domain of ADAMTS13 (Crawley JT, et al., Blood. 2011 Sep 22;118(12):3212-21). Mutations in ADAMTS13 cause Upshaw-Schulman Syndrome, the inherited form of Thrombotic Thrombocytopenic Purpura (TTP), a microangiopathy characterized by platelet clumping, hemolytic anemia, and subsequent organ failure (Coppo P & Veyradier A, Cardiovasc Hematol Disord Drug Targets. 2009 Mar;9(1):36-50). State-of-the-art ADAMTS13 tests of patients' plasma use static approaches based on either proteolysis of VWF minimal fragments or full length VWF under denaturing conditions. In some cases the results of these static assays could be without pathological finding, e.g. when a mutation in the shear dependent spacer region of ADAMTS13 would prevent binding to VWF strings only under flow conditions without affecting its proteolytic activity towards denatured VWF substrates in static assays. We developed an assay based on gain of function GPIba mutants immobilized on latex particles that facilitates the visualization of VWF strings on the surface of stimulated endothelial cells without the need for whole blood or isolated platelets. We then used this assay to observe the proteolytic activity of wildtype (wt) ADAMTS13. Under shear flow imitating the physiological conditions of the venous blood stream, VWF string formation was induced by histamine stimulation and visualized by binding of the GPIba coated particles. wtADAMTS13 was then floated over the cells at the same shear rate (500 s-1) thereby offering the VWF strings as substrate. VWF string proteolysis was observed live and the time was determined that was needed for complete degradation. wtADAMTS13 degraded the up to 1mm long VWF strings within seconds. 5-10 minutes after ADAMTS13 addition only a few string fragments with a maximal length of 10 µm remained intact. To determine the accuracy of our assay, we then determined the catalytic activity of two ADAMTS13 mutants with mutations in the C-terminal TPS1 domains. Since these domains are responsible for shear flow independent substrate binding we expected the same results as in the static assays. Mutant ADAMTS13-R1060W possesses a single point-mutation in its TSP1-7 domain which leads to a secretion defect and a catalytic activity that is reduced by 65% in a static assay. We indeed also determined a decrease in proteolytic activity by 65% and VWF multimeric fragments with a size of 10-30 µm remained intact. The truncated mutant ADAMTS13-R910*, lacking the TSP1-7, TSP1-8 and the CUP domains was incapable of producing VWF-fragments smaller than 80 µm within one hour and therefore only showed a minimal residual activity in accordance to the previously published catalytic activity of 2%. After these prove-of-principle experiments our assay could now be adapted to be used with blood samples of TTP patients who exhibit reduced proteolytic band patterns in VWF multimer analysis but possess normal ADAMTS13 activity in static assays. Summarizing we developed an assay that allows functional characterization of TTP-associated ADAMTS13 mutants with shear flow dependent dis-functionalities. The assay may serve as a valuable tool for research but could also broaden the range of TTP diagnostics. Disclosures: No relevant conflicts of interest to declare.

High resolution electron microscopy of lanthanum phosphate crystals

1978 ◽  
Vol 36 (1) ◽  
pp. 274-275
Author(s):  
J. C. Wheatley ◽  
J. M. Cowley
Keyword(s):  
Electron Microscopy ◽  
Catalytic Activity ◽  
High Resolution ◽  
Petroleum Industry ◽  
High Resolution Electron Microscopy ◽  
Lanthanum Phosphate ◽  
Good Catalyst ◽  
Resolution Electron ◽  
Good Catalytic Activity ◽  
Physical And Chemical

Rare-earth phosphates are of particular interest because of their catalytic properties associated with the hydrolysis of many aromatic chlorides in the petroleum industry. Lanthanum phosphates (LaPO4) which have been doped with small amounts of copper have shown increased catalytic activity (1). However the physical and chemical characteristics of the samples leading to good catalytic activity are not known.Many catalysts are amorphous and thus do not easily lend themselves to methods of investigation which would include electron microscopy. However, the LaPO4, crystals are quite suitable samples for high resolution techniques.The samples used were obtained from William L. Kehl of Gulf Research and Development Company. The electron microscopy was carried out on a JEOL JEM-100B which had been modified for high resolution microscopy (2). Standard high resolution techniques were employed. Three different sample types were observed: 669A-1-5-7 (poor catalyst), H-L-2 (good catalyst) and 27-011 (good catalyst).

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