Enzyme kinetics of deoxyuridine triphosphatase from Western corn rootworm

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Abstract

Objective

The Western corn rootworm (WCR), Diabrotica virgifera virgifera, is a highly adaptable insect pest that has evolved resistance to a variety of control strategies, including insecticides. Therefore, it is interesting to examine how housekeeping proteins in WCR have been changed under WCR-controlling strategies. In this study, we focused on one of such proteins in WCR, a ubiquitous enzyme 5'-triphosphate nucleotidohydrolase (dUTPase). In the thymidine synthetic pathway, dUTPase hydrolyzes deoxyuridine triphosphate (dUTP) and supplies the substrate, deoxyuridine monophosphate, for the thymidylate synthase (TS). It decreases the cellular content of uracil, reducing uracil misincorporation into DNA. Suppressing the dUTPase activity, therefore, contributes to thymineless death. In this study, we investigated the enzymatic properties of dUTPase.

Results

The WCR dUTPase gene ( DUT) was synthesized with the addition of His-tag corresponding DNA sequence and then cloned and expressed in Escherichia coli, and the protein product was purified. The product of WCR DUT hydrolyzed dUTP and was designated as dUTPase. WCR dUTPase did not hydrolyze dATP, dTTP, dCTP, or dGTP. WCR dUTPase was analyzed via size-exclusion chromatography and exhibited a molecular weight corresponding to that of trimer. The present format can be interpreted as nuclear trimer type. Possible isomers will be examined once transcriptome analyses are conducted.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13104-023-06618-2.

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Most cited references 52

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SWISS-MODEL: homology modelling of protein structures and complexes

Andrew Waterhouse, Martino Bertoni, Stefan Bienert … (2018)

Abstract Homology modelling has matured into an important technique in structural biology, significantly contributing to narrowing the gap between known protein sequences and experimentally determined structures. Fully automated workflows and servers simplify and streamline the homology modelling process, also allowing users without a specific computational expertise to generate reliable protein models and have easy access to modelling results, their visualization and interpretation. Here, we present an update to the SWISS-MODEL server, which pioneered the field of automated modelling 25 years ago and been continuously further developed. Recently, its functionality has been extended to the modelling of homo- and heteromeric complexes. Starting from the amino acid sequences of the interacting proteins, both the stoichiometry and the overall structure of the complex are inferred by homology modelling. Other major improvements include the implementation of a new modelling engine, ProMod3 and the introduction a new local model quality estimation method, QMEANDisCo. SWISS-MODEL is freely available at https://swissmodel.expasy.org.

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KEGG: integrating viruses and cellular organisms

Minoru Kanehisa, Miho Furumichi, Yoko Sato … (2020)

Abstract KEGG (https://www.kegg.jp/) is a manually curated resource integrating eighteen databases categorized into systems, genomic, chemical and health information. It also provides KEGG mapping tools, which enable understanding of cellular and organism-level functions from genome sequences and other molecular datasets. KEGG mapping is a predictive method of reconstructing molecular network systems from molecular building blocks based on the concept of functional orthologs. Since the introduction of the KEGG NETWORK database, various diseases have been associated with network variants, which are perturbed molecular networks caused by human gene variants, viruses, other pathogens and environmental factors. The network variation maps are created as aligned sets of related networks showing, for example, how different viruses inhibit or activate specific cellular signaling pathways. The KEGG pathway maps are now integrated with network variation maps in the NETWORK database, as well as with conserved functional units of KEGG modules and reaction modules in the MODULE database. The KO database for functional orthologs continues to be improved and virus KOs are being expanded for better understanding of virus-cell interactions and for enabling prediction of viral perturbations.

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Empirical Statistical Model To Estimate the Accuracy of Peptide Identifications Made by MS/MS and Database Search

Andrew Keller, Eugene Kolker, Ruedi Aebersold … (2003)

We present a statistical model to estimate the accuracy of peptide assignments to tandem mass (MS/MS) spectra made by database search applications such as SEQUEST. Employing the expectation maximization algorithm, the analysis learns to distinguish correct from incorrect database search results, computing probabilities that peptide assignments to spectra are correct based upon database search scores and the number of tryptic termini of peptides. Using SEQUEST search results for spectra generated from a sample of known protein components, we demonstrate that the computed probabilities are accurate and have high power to discriminate between correctly and incorrectly assigned peptides. This analysis makes it possible to filter large volumes of MS/MS database search results with predictable false identification error rates and can serve as a common standard by which the results of different research groups are compared.

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Author and article information

Contributors

Hideaki Moriyama: hideaki@unl.edu

Journal

Journal ID (nlm-ta): BMC Res Notes

Journal ID (iso-abbrev): BMC Res Notes

Title: BMC Research Notes

Publisher: BioMed Central (London )

ISSN (Electronic): 1756-0500

Publication date (Electronic): 16 November 2023

Publication date PMC-release: 16 November 2023

Publication date Collection: 2023

Volume: 16

Electronic Location Identifier: 336

Affiliations

[1 ]School of Biological Sciences, University of Nebraska–Lincoln, ( https://ror.org/043mer456) 243 Manter Hall, Lincoln, NE 68588-0118 USA

[2 ]Escuela Superior Politécnica del Litoral, Centro de Investigaciones Biotecnológicas del Ecuador, ESPOL Polytechnic University, ESPOL, ( https://ror.org/04qenc566) Campus Gustavo Galindo Km 30.5 Vía Perimetral, P.O Box 09-01-5863, Guayaquil, Ecuador

Article

Publisher ID: 6618

DOI: 10.1186/s13104-023-06618-2

PMC ID: 10652518

PubMed ID: 37974243

SO-VID: d76fdd9b-2868-4402-adf3-0a06025daae9

License:

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

History

Date received : 31 July 2023

Date accepted : 7 November 2023

Funding

Funded by: Research Council Interdisciplinary grant, Office of Research & Economic Development, UNL.

Custom metadata

ScienceOpen disciplines: Medicine

Keywords: western corn rootworm,diabrotica virgifera virgifera,5ʹ-triphosphate nucleotidohydrolase (dutpase),dutp,thymidylate synthase,dump

Data availability:

ScienceOpen disciplines: Medicine

Keywords: western corn rootworm, diabrotica virgifera virgifera, 5ʹ-triphosphate nucleotidohydrolase (dutpase), dutp, thymidylate synthase, dump

Enzyme kinetics of deoxyuridine triphosphatase from Western corn rootworm

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Abstract

Objective

Results

Supplementary Information

Related collections

Karger: Nephrology

Most cited references 52

SWISS-MODEL: homology modelling of protein structures and complexes

KEGG: integrating viruses and cellular organisms

Empirical Statistical Model To Estimate the Accuracy of Peptide Identifications Made by MS/MS and Database Search

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