scholarly journals THAP9 Transposase Cleaves DNA via Conserved Acidic Residues in an RNaseH-Like Domain

Cells ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1351
Author(s):  
Vasudha Sharma ◽  
Prachi Thakore ◽  
Sharmistha Majumdar
Keyword(s):  
Catalytic Activity ◽  
Catalytic Domain ◽  
Catalytic Triad ◽  
Beta Sheets ◽  
Rnase H ◽  
Site Directed Mutagenesis ◽  
P Element ◽  
Alpha Helices ◽  
Acidic Residues ◽  
Subsequent Integration

The catalytic domain of most ‘cut and paste’ DNA transposases have the canonical RNase-H fold, which is also shared by other polynucleotidyl transferases such as the retroviral integrases and the RAG1 subunit of V(D)J recombinase. The RNase-H fold is a mixture of beta sheets and alpha helices with three acidic residues (Asp, Asp, Glu/Asp—DDE/D) that are involved in the metal-mediated cleavage and subsequent integration of DNA. Human THAP9 (hTHAP9), homologous to the well-studied Drosophila P-element transposase (DmTNP), is an active DNA transposase that, although domesticated, still retains the catalytic activity to mobilize transposons. In this study we have modeled the structure of hTHAP9 using the recently available cryo-EM structure of DmTNP as a template to identify an RNase-H like fold along with important acidic residues in its catalytic domain. Site-directed mutagenesis of the predicted catalytic residues followed by screening for DNA excision and integration activity has led to the identification of candidate Ds and Es in the RNaseH fold that may be a part of the catalytic triad in hTHAP9. This study has helped widen our knowledge about the catalytic activity of a functionally uncharacterized transposon-derived gene in the human genome.

scholarly journals THAP9 transposase cleaves DNA via conserved acidic residues in an RNaseH-like domain

2021 ◽  
Author(s):  
Vasudha Sharma ◽  
Prachi Thakore ◽  
Sharmistha MAJUMDAR
Keyword(s):  
Catalytic Activity ◽  
Genome Engineering ◽  
Catalytic Domain ◽  
Catalytic Triad ◽  
Beta Sheets ◽  
Rnase H ◽  
Site Directed Mutagenesis ◽  
P Element ◽  
Alpha Helices ◽  
Acidic Residues

Abstract Background: The catalytic domain of most ‘cut and paste’ DNA transposases have the canonical RNase-H fold which is also shared by other polynucleotidyl transferases like the retroviral integrases and the RAG1 subunit of V(D)J recombinase. The RNase-H fold is a mixture of beta sheets and alpha helices with three acidic residues (Asp, Asp, Glu/Asp - DDE/D) that are involved in the metal-mediated cleavage and subsequent integration of DNA. Human THAP9 (hTHAP9), homologous to the well-studied Drosophila P-element transposase (DmTNP), is an active DNA transposase that, although domesticated, still retains the catalytic activity to mobilize transposons.Results: In this study we have modelled the structure of hTHAP9 using the recently available cryo-EM structure of DmTNP as a template to identify an RNase-H like fold along with important acidic residues in its catalytic domain. Site-directed mutagenesis of the predicted catalytic residues followed by screening for DNA excision and integration activity, has led to the identification of candidate Ds and Es in the RNaseH fold that can be a part of the catalytic triad in hTHAP9.Conclusions: Many DNA transposases execute DNA excision via a catalytic domain, which has a canonical RNase-H fold. Despite the similar nature of the catalytic domain, these transposases exhibit mechanistically different strategies of transposition. We identify a potential RNase-H fold in hTHAP9 with conserved DDE motif required for cutting DNA. Additionally, we have found a residue, which when mutated, leads to an increase in hTHAP9’s transposition activity. Such hyperactive transposase mutants can be exploited as tools in genome engineering and gene therapy. This study has helped widen our knowledge about the catalytic activity of a functionally uncharacterised transposon-derived gene in the human genome.

scholarly journals THAP9 transposase cleaves DNA via conserved acidic residues in an RNaseH-like domain

2020 ◽  
Author(s):  
Vasudha Sharma ◽  
Prachi Thakore ◽  
Sharmistha Majumdar
Keyword(s):  
Catalytic Activity ◽  
Genome Engineering ◽  
Catalytic Domain ◽  
Catalytic Triad ◽  
Beta Sheets ◽  
Rnase H ◽  
Site Directed Mutagenesis ◽  
P Element ◽  
Alpha Helices ◽  
Acidic Residues

AbstractThe catalytic domain of most ‘cut and paste’ DNA transposases have the canonical RNase-H fold which is also shared by other polynucleotidyl transferases like retroviral integrases and the RAG1 subunit of V(D)J recombinase. The RNase-H fold is a mixture of beta sheets and alpha helices with three acidic residues (Asp, Asp, Glu/Asp - DDE/D) that are involved in metal-mediated cleavage and subsequent integration of DNA. Human THAP9 (hTHAP9), homologous to the well-studied Drosophila P-element transposase (DmTNP), is an active DNA transposase that, although domesticated, still retains the catalytic activity to mobilize transposons. In this study we have modelled the structure of hTHAP9 using the recently available cryo-EM structure of DmTNP as a template to identify an RNase-H like fold along with important acidic residues in its catalytic domain. Site-directed mutagenesis of the predicted catalytic residues followed by screening for DNA excision and integration activity, led to the identification of candidate Ds and Es in the RNaseH fold that appear to constitute the catalytic triad of hTHAP9.Significance statementMany DNA transposases execute DNA excision via a catalytic domain, which has a canonical RNase-H fold. Despite the similar nature of the catalytic domain, these transposases exhibit mechanistically different strategies of transposition. We identify a potential RNase-H fold in hTHAP9 with a conserved DDE motif required for cutting DNA. Additionally, we have found a residue, which when mutated, leads to an increase in hTHAP9’s transposition activity. Such hyperactive transposase mutants can be exploited as tools in genome engineering and gene therapy. This study has helped widen our knowledge about the catalytic activity of a functionally uncharacterized transposon-derived gene in the human genome.

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.

Implications of Stisa2 catalytic residue restoration through site directed mutagenesis

2016 ◽  
Vol 42 (2) ◽  
Author(s):  
Hasnain Hussain ◽  
Nikson Fatt Ming Chong
Keyword(s):  
Catalytic Activity ◽  
Catalytic Triad ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Qualitative And Quantitative Analysis ◽  
Conserved Residues ◽  
Overlap Extension Pcr ◽  
Overlap Extension ◽  
Catalytic Network ◽  
And Function

AbstractObjective:Restoration of catalytic activity of Isa2 fromMethods:The six conserved amino acid residues absent in the Stisa2 gene were restored by mutation using the overlap extension PCR and the asymmetrical overlap extension PCR methods. Next, mutant Stisa2 with restored catalytic residues was expressed inResults:Both qualitative and quantitative analysis showed that the restoration of the conserved residues in the catalytic site did not restore starch debranching activity. Molecular modeling showed greater than expected distances between the catalytic triad in mutant Stisa2. These additional distances are likely to prevent hydrogen bonding which stabilizes the reaction intermediate, and are critical for catalytic activity.Conclusions:These results suggest that during evolution, mutations in other highly conserved regions have caused significant changes to the structure and function of the catalytic network. Catalytically inactive Isa2, which is conserved in starch-producing plants, has evolved important non-catalytic roles such as in substrate binding and in regulating isoamylase activity.

scholarly journals Piv Site-Specific Invertase Requires a DEDD Motif Analogous to the Catalytic Center of the RuvC Holliday Junction Resolvases

2005 ◽  
Vol 187 (10) ◽  
pp. 3431-3437 ◽  
Author(s):  
John M. Buchner ◽  
Anne E. Robertson ◽  
David J. Poynter ◽  
Shelby S. Denniston ◽  
Anna C. Karls
Keyword(s):  
Catalytic Activity ◽  
Tertiary Structure ◽  
Rnase H ◽  
Holliday Junction ◽  
Site Specific ◽  
Acidic Residues ◽  
Insertion Elements ◽  
The Stability ◽  
Retroviral Integrase

ABSTRACT Piv, a unique prokaryotic site-specific DNA invertase, is related to transposases of the insertion elements from the IS110/IS492 family and shows no similarity to the site-specific recombinases of the tyrosine- or serine-recombinase families. Piv tertiary structure is predicted to include the RNase H-like fold that typically encompasses the catalytic site of the recombinases or nucleases of the retroviral integrase superfamily, including transposases and RuvC-like Holliday junction resolvases. Analogous to the DDE and DEDD catalytic motifs of transposases and RuvC, respectively, four Piv acidic residues D9, E59, D101, and D104 appear to be positioned appropriately within the RNase H fold to coordinate two divalent metal cations. This suggests mechanistic similarity between site-specific inversion mediated by Piv and transposition or endonucleolytic reactions catalyzed by enzymes of the retroviral integrase superfamily. The role of the DEDD motif in Piv catalytic activity was addressed using Piv variants that are substituted individually or multiply at these acidic residues and assaying for in vivo inversion, intermolecular recombination, and DNA binding activities. The results indicate that all four residues of the DEDD motif are required for Piv catalytic activity. The DEDD residues are not essential for inv recombination site recognition and binding, but this acidic tetrad does appear to contribute to the stability of Piv-inv interactions. On the basis of these results, a working model for Piv-mediated inversion that includes resolution of a Holliday junction is presented.

scholarly journals Enzymatic Properties of Dipeptidyl Aminopeptidase IV Produced by the Periodontal Pathogen Porphyromonas gingivalis and Its Participation in Virulence

2000 ◽  
Vol 68 (2) ◽  
pp. 716-724 ◽  
Author(s):  
Yumi Kumagai ◽  
Kiyoshi Konishi ◽  
Tomoharu Gomi ◽  
Hisao Yagishita ◽  
Ayako Yajima ◽  
...  
Keyword(s):  
Amino Acid ◽  
Porphyromonas Gingivalis ◽  
Catalytic Domain ◽  
Animal Experiments ◽  
Catalytic Triad ◽  
Amino Acid Sequences ◽  
Enzymatic Properties ◽  
Site Directed Mutagenesis ◽  
Periodontal Pathogen ◽  
Dipeptidyl Aminopeptidase

ABSTRACT Porphyromonas gingivalis is a major pathogen associated with adult periodontitis. We cloned and sequenced the gene (dpp) coding for dipeptidyl aminopeptidase IV (DPPIV) fromP. gingivalis W83, based on the amino acid sequences of peptide fragments derived from purified DPPIV. An Escherichia coli strain overproducing P. gingivalis DPPIV was constructed. The enzymatic properties of recombinant DPPIV purified from the overproducer were similar to those of DPPIV isolated fromP. gingivalis. The three amino acid residues Ser, Asp, and His, which are thought to form a catalytic triad in the C-terminal catalytic domain of eukaryotic DPPIV, are conserved in P. gingivalis DPPIV. When each of the corresponding residues of the enzyme was substituted with Ala by site-directed mutagenesis, DPPIV activity significantly decreased, suggesting that these three residues of P. gingivalis DPPIV are involved in the catalytic reaction. DPPIV-deficient mutants of P. gingivalis were constructed and subjected to animal experiments. Mice injected with the wild-type strain developed abscesses to a greater extent and died more frequently than those challenged with mutant strains. Mice injected with the mutants exhibited faster recovery from the infection, as assessed by weight gain and the rate of lesion healing. This decreased virulence of mutants compared with the parent strain suggests that DPPIV is a potential virulence factor of P. gingivalis and may play important roles in the pathogenesis of adult periodontitis induced by the organism.

scholarly journals Electrostatic interactions of domain III stabilize the inactive conformation of μ-calpain

2004 ◽  
Vol 382 (2) ◽  
pp. 607-617 ◽  
Author(s):  
Amaury FERNÁNDEZ-MONTALVÁN ◽  
Irmgard ASSFALG-MACHLEIDT ◽  
Dietmar PFEILER ◽  
Hans FRITZ ◽  
Marianne JOCHUM ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Electrostatic Interactions ◽  
Catalytic Domain ◽  
Structural Information ◽  
Cysteine Proteases ◽  
Enzymic Activity ◽  
Site Directed Mutagenesis ◽  
Activation Process ◽  
Acidic Residues ◽  
Domain Iii

The ubiquitous μ- and m-calpains are Ca2+-dependent cysteine proteases. They are activated via rearrangement of the catalytic domain II induced by cooperative binding of Ca2+ to several sites of the molecule. Based on the crystallographic structures, a cluster of acidic residues in domain III, the acidic loop, has been proposed to function as part of an electrostatic switch in the activation process. Experimental support for this hypothesis was obtained by site-directed mutagenesis of recombinant human μ-calpain expressed with the baculovirus system in insect cells. Replacing the acidic residues of the loop individually with alanine resulted in an up to 7-fold reduction of the half-maximal Ca2+ concentration required for conformational changes (probed with 2-p-toluidinylnapthalene-6-sulphonate fluorescence) and for enzymic activity. Along with structural information, the contribution of individual acidic residues to the Ca2+ requirement for activation revealed that interactions of the acidic loop with basic residues in the catalytic subdomain IIb and in the pre-transducer region of domain III stabilize the structure of inactive μ-calpain. Disruption of these electrostatic interactions makes the molecule more flexible and increases its Ca2+ sensitivity. It is proposed that the acidic loop and the opposing basic loop of domain III constitute a double-headed electrostatic switch controlling the assembly of the catalytic domain.

Characterization of Recombinant Human Coagulation Factor XFriuli

1996 ◽  
Vol 75 (02) ◽  
pp. 313-317 ◽  
Author(s):  
D J Kim ◽  
A Girolami ◽  
H L James
Keyword(s):  
Catalytic Domain ◽  
Coagulation Factor ◽  
Molecular Size ◽  
Binding Pocket ◽  
Site Directed Mutagenesis ◽  
Bleeding Tendency ◽  
Factor X ◽  
Amino Terminal ◽  
Normal Plasma ◽  
Plasma Factor

SummaryNaturally occurring plasma factor XFriuli (pFXFr) is marginally activated by both the extrinsic and intrinsic coagulation pathways and has impaired catalytic potential. These studies were initiated to obtain confirmation that this molecule is multi-functionally defective due to the substitution of Ser for Pro at position 343 in the catalytic domain. By the Nelson-Long site-directed mutagenesis procedure a construct of cDNA in pRc/CMV was derived for recombinant factor XFriuli (rFXFr) produced in human embryonic (293) kidney cells. The rFXFr was purified and shown to have a molecular size identical to that of normal plasma factor X (pFX) by gel electrophoretic, and amino-terminal sequencing revealed normal processing cleavages. Using recombinant normal plasma factor X (rFXN) as a reference, the post-translational y-carboxy-glutamic acid (Gla) and (β-hydroxy aspartic acid (β-OH-Asp) content of rFXFr was over 85% and close to 100%, respectively, of expected levels. The specific activities of rFXFr in activation and catalytic assays were the same as those of pFXFr. Molecular modeling suggested the involvement of a new H-bond between the side-chains of Ser-343 and Thr-318 as they occur in anti-parallel (3-pleated sheets near the substrate-binding pocket of pFXFr. These results support the conclusion that the observed mutation in pFXFr is responsible for its dysfunctional activation and catalytic potentials, and that it accounts for the moderate bleeding tendency in the homozygous individuals who possess this variant procoagulant.

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