scholarly journals The crystal structure of mycobacterial epoxide hydrolase A

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Eike C. Schulz ◽  
Sara R. Henderson ◽  
Boris Illarionov ◽  
Thomas Crosskey ◽  
Stacey M. Southall ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Substrate Specificity ◽  
Drug Targets ◽  
Epoxide Hydrolase ◽  
Sequence Similarity ◽  
Structural Similarity ◽  
High Sequence Similarity ◽  
High Structural Similarity ◽  
Potential Drug Targets ◽  
Insight Into

Abstract The human pathogen Mycobacterium tuberculosis is the causative agent of tuberculosis resulting in over 1 million fatalities every year, despite decades of research into the development of new anti-TB compounds. Unlike most other organisms M. tuberculosis has six putative genes for epoxide hydrolases (EH) of the α/β-hydrolase family with little known about their individual substrates, suggesting functional significance for these genes to the organism. Due to their role in detoxification, M. tuberculosis EH’s have been identified as potential drug targets. Here, we demonstrate epoxide hydrolase activity of M. thermoresistibile epoxide hydrolase A (Mth-EphA) and report its crystal structure in complex with the inhibitor 1,3-diphenylurea at 2.0 Å resolution. Mth-EphA displays high sequence similarity to its orthologue from M. tuberculosis and generally high structural similarity to α/β-hydrolase EHs. The structure of the inhibitor bound complex reveals the geometry of the catalytic residues and the conformation of the inhibitor. Comparison to other EHs from mycobacteria allows insight into the active site plasticity with respect to substrate specificity. We speculate that mycobacterial EHs may have a narrow substrate specificity providing a potential explanation for the genetic repertoire of epoxide hydrolase genes in M. tuberculosis.

scholarly journals Probing the promiscuity of ent-kaurene oxidases via combinatorial biosynthesis

2016 ◽  
Vol 113 (9) ◽  
pp. 2526-2531 ◽  
Author(s):  
Sibongile Mafu ◽  
Meirong Jia ◽  
Jiachen Zi ◽  
Dana Morrone ◽  
Yisheng Wu ◽  
...  
Keyword(s):  
Natural Products ◽  
Metabolic Engineering ◽  
Substrate Specificity ◽  
Plant Hormone ◽  
Structural Similarity ◽  
Combinatorial Biosynthesis ◽  
Underlying Structure ◽  
Engineering System ◽  
Insight Into

The substrate specificity of enzymes from natural products’ metabolism is a topic of considerable interest, with potential biotechnological use implicit in the discovery of promiscuous enzymes. However, such studies are often limited by the availability of substrates and authentic standards for identification of the resulting products. Here, a modular metabolic engineering system is used in a combinatorial biosynthetic approach toward alleviating this restriction. In particular, for studies of the multiply reactive cytochrome P450, ent-kaurene oxidase (KO), which is involved in production of the diterpenoid plant hormone gibberellin. Many, but not all, plants make a variety of related diterpenes, whose structural similarity to ent-kaurene makes them potential substrates for KO. Use of combinatorial biosynthesis enabled analysis of more than 20 such potential substrates, as well as structural characterization of 12 resulting unknown products, providing some insight into the underlying structure–function relationships. These results highlight the utility of this approach for investigating the substrate specificity of enzymes from complex natural products’ biosynthesis.

scholarly journals Distribution and Characterization of AKT Homologs in the Tangerine Pathotype of Alternaria alternata

2000 ◽  
Vol 90 (7) ◽  
pp. 762-768 ◽  
Author(s):  
A. Masunaka ◽  
A. Tanaka ◽  
T. Tsuge ◽  
T. L. Peever ◽  
L. W. Timmer ◽  
...  
Keyword(s):  
Alternaria Alternata ◽  
Sequence Similarity ◽  
Structural Similarity ◽  
Toxin Production ◽  
Brown Spot ◽  
Japanese Pear ◽  
Black Rot ◽  
Acid Moiety ◽  
High Sequence Similarity ◽  
Rough Lemon

The tangerine pathotype of Alternaria alternata produces a host-selective toxin (HST), known as ACT-toxin, and causes Alternaria brown spot disease of citrus. The structure of ACT-toxin is closely related to AK- and AF-toxins, which are HSTs produced by the Japanese pear and strawberry pathotypes of A. alternata, respectively. AC-, AK-, and AF-toxins are chemically similar and share a 9,10-epoxy-8-hydroxy-9-methyl-decatrienoic acid moiety. Two genes controlling AK-toxin biosynthesis (AKT1 and AKT2) were recently cloned from the Japanese pear pathotype of A. alternata. Portions of these genes were used as heterologous probes in Southern blots, that detected homologs in 13 isolates of A. alternata tangerine pathotype from Minneola tangelo in Florida. Partial sequencing of the homologs in one of these isolates demonstrated high sequence similarity to AKT1 (89.8%) and to AKT2 (90.7%). AKT homologs were not detected in nine isolates of A. alternata from rough lemon, six isolates of nonpathogenic A. alternata, and one isolate of A. citri that causes citrus black rot. The presence of homologs in the Minneola isolates and not in the rough lemon isolates, nonpathogens or black rot isolates, correlates perfectly to pathogenicity on Iyo tangerine and ACT-toxin production. Functionality of the homologs was demonstrated by detection of transcripts using reverse transcription-polymerase chain reaction (RT-PCR) in total RNA of the tangerine pathotype of A. alternata. The high sequence similarity of AKT and AKT homologs in the tangerine patho-type, combined with the structural similarity of AK-toxin and ACT-toxin, may indicate that these homologs are involved in the biosynthesis of the decatrienoic acid moiety of ACT-toxin.

scholarly journals Crystal structure of gluconate 5-dehydrogenase from Lentibacter algarum

2020 ◽  
Vol 76 (5) ◽  
pp. 228-234
Author(s):  
Dengke Tian ◽  
Xueqi Fu ◽  
Wenqiang Cao ◽  
Hong Yuan
Keyword(s):  
Crystal Structure ◽  
Escherichia Coli ◽  
Structural Similarity ◽  
Unit Cell ◽  
Unit Cell Parameters ◽  
Monoclinic System ◽  
Space Group ◽  
Cell Parameters ◽  
High Structural Similarity

Gluconate 5-dehydrogenase (Ga5DH; EC 1.1.1.69) from Lentibacter algarum (LaGa5DH) was recombinantly expressed in Escherichia coli and purified to homogeneity. The protein was crystallized and the crystal structure was solved at 2.1 Å resolution. The crystal belonged to the monoclinic system, with space group P1 and unit-cell parameters a = 55.42, b = 55.48, c = 79.16 Å, α = 100.51, β = 105.66, γ = 97.99°. The structure revealed LaGaDH to be a tetramer, with each subunit consisting of six α-helices and three antiparallel β-hairpins. LaGa5DH has high structural similarity to other Ga5DH proteins, demonstrating that this enzyme is highly conserved.

scholarly journals Correction: Crystal Structure of Human Myotubularin-Related Protein 1 Provides Insight into the Structural Basis of Substrate Specificity

PLoS ONE ◽  
2018 ◽  
Vol 13 (1) ◽  
pp. e0190844
Author(s):  
Seoung Min Bong ◽  
Kka-bi Son ◽  
Seung-Won Yang ◽  
Jae-Won Park ◽  
Jea-Won Cho ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Substrate Specificity ◽  
Structural Basis ◽  
Related Protein ◽  
Insight Into

scholarly journals Considerations on activity determinants of fungal polyphenol oxidases based on mutational and structural studies

2021 ◽  
Author(s):  
Efstratios Nikolaivits ◽  
Alexandros Valmas ◽  
Grigorios Dedes ◽  
Evangelos Topakas ◽  
Maria Dimarogona
Keyword(s):  
Crystal Structure ◽  
Fruits And Vegetables ◽  
Structural Data ◽  
Structural Similarity ◽  
Site Directed Mutagenesis ◽  
Special Focus ◽  
Structural Determinants ◽  
Polyphenol Oxidases ◽  
High Structural Similarity ◽  
Tree Branch

ABSTRACTPolyphenol oxidases (PPOs) are an industrially relevant family of enzymes, being involved in the post-harvest browning of fruits and vegetables, as well as in human melanogenesis. Their involvement lies in their ability to oxidize phenolic or polyphenolic compounds, that subsequently form pigments. PPO family includes tyrosinases and catechol oxidases, which in spite of their high structural similarity, exhibit different catalytic activities. Long-standing research efforts have not yet managed to decipher the structural determinants responsible for this differentiation, as every new theory is disproved by a more recent study. In the present work, we combined biochemical along with structural data, in order to rationalize the function of a previously characterized PPO from Thermothelomyces thermophila (TtPPO). The crystal structure of a TtPPO variant, determined at 1.55 Å resolution, represents the second known structure of an ascomycete PPO. Kinetic data of structure-guided mutants prove the implication of “gate” residue L306, residue HB1+1 (G292) and HB2+1 (Y296) in TtPPO function against various substrates. Our findings demonstrate the role of L306 in the accommodation of bulky substrates and that residue HB1+1 is unlikely to determine monophenolase activity as suggested from previous studies.IMPORTANCEPPOs are enzymes of biotechnological interest. They have been extensively studied both biochemically and structurally, with a special focus on the plant-derived counterparts. Even so, explicit description of the molecular determinants of their substrate specificity is still pending. Especially for ascomycete PPOs, only one crystal structure has been determined so far, thus limiting our knowledge on this tree branch of the family. In the present study, we report the second crystal structure of an ascomycete PPO. Combined with site-directed mutagenesis and biochemical studies, we depict the amino acids in the vicinity of the active site that affect enzyme activity, and perform a detailed analysis on a variety of substrates. Our findings improve current understanding of structure-function relations of microbial PPOs, which is a prerequisite for the engineering of biocatalysts of desired properties.

scholarly journals Considerations on activity determinants of fungal polyphenol oxidases based on mutational and structural studies

2021 ◽  
Author(s):  
Efstratios Nikolaivits ◽  
Alexandros Valmas ◽  
Grigorios Dedes ◽  
Evangelos Topakas ◽  
Maria Dimarogona
Keyword(s):  
Crystal Structure ◽  
Fruits And Vegetables ◽  
Structural Data ◽  
Structural Similarity ◽  
Site Directed Mutagenesis ◽  
Special Focus ◽  
Structural Determinants ◽  
Polyphenol Oxidases ◽  
High Structural Similarity ◽  
Tree Branch

Polyphenol oxidases (PPOs) are an industrially relevant family of enzymes, being involved in the post-harvest browning of fruits and vegetables, as well as in human melanogenesis. Their involvement lies in their ability to oxidize phenolic or polyphenolic compounds, that subsequently form pigments. PPO family includes tyrosinases and catechol oxidases, which in spite of their high structural similarity, exhibit different catalytic activities. Long-standing research efforts have not yet managed to decipher the structural determinants responsible for this differentiation, as every new theory is disproved by a more recent study. In the present work, we combined biochemical along with structural data, in order to rationalize the function of a previously characterized PPO from Thermothelomyces thermophila (TtPPO). The crystal structure of a TtPPO variant, determined at 1.55 Å resolution, represents the second known structure of an ascomycete PPO. Kinetic data of structure-guided mutants prove the implication of “gate” residue L306, residue HB1+1 (G292) and HB2+1 (Y296) in TtPPO function against various substrates. Our findings demonstrate the role of L306 in the accommodation of bulky substrates and that residue HB1+1 is unlikely to determine monophenolase activity as suggested from previous studies. IMPORTANCE PPOs are enzymes of biotechnological interest. They have been extensively studied both biochemically and structurally, with a special focus on the plant-derived counterparts. Even so, explicit description of the molecular determinants of their substrate specificity is still pending. Especially for ascomycete PPOs, only one crystal structure has been determined so far, thus limiting our knowledge on this tree branch of the family. In the present study, we report the second crystal structure of an ascomycete PPO. Combined with site-directed mutagenesis and biochemical studies, we depict the amino acids in the vicinity of the active site that affect enzyme activity, and perform a detailed analysis on a variety of substrates. Our findings improve current understanding of structure-function relations of microbial PPOs, which is a prerequisite for the engineering of biocatalysts of desired properties.

scholarly journals PANOPLY: A cloud-based platform for automated and reproducible proteogenomic data analysis

2020 ◽  
Author(s):  
D. R. Mani ◽  
Myranda Maynard ◽  
Ramani Kothadia ◽  
Karsten Krug ◽  
Karen E. Christianson ◽  
...  
Keyword(s):  
Data Analysis ◽  
Drug Targets ◽  
Complex Data ◽  
Potential Drug ◽  
Post Translational Modification ◽  
Simple Interface ◽  
The Impact ◽  
Potential Drug Targets ◽  
Insight Into ◽  
Generation Sequencing

ABSTRACTProteogenomics involves the integrative analysis of genomic, transcriptomic, proteomic and post-translational modification data produced by next-generation sequencing and mass spectrometry-based proteomics. Several publications by the Clinical Proteomic Tumor Analysis Consortium (CPTAC) and others have highlighted the impact of proteogenomics in enabling deeper insight into the biology of cancer and identification of potential drug targets. In order to encapsulate the complex data processing required for proteogenomics, and provide a simple interface to deploy a range of algorithms developed for data analysis, we have developed PANOPLY—a cloud-based platform for automated and reproducible proteogenomic data analysis. A wide array of algorithms have been implemented, and we highlight the application of PANOPLY to the analysis of cancer proteogenomic data.

scholarly journals Crystal structure ofd-stereospecific amidohydrolase fromStreptomycessp. 82F2 - insight into the structural factors for substrate specificity

FEBS Journal ◽  
2015 ◽  
Vol 283 (2) ◽  
pp. 337-349 ◽  
Author(s):  
Jiro Arima ◽  
Kana Shimone ◽  
Kazusa Miyatani ◽  
Yuka Tsunehara ◽  
Yoshitaka Isoda ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Substrate Specificity ◽  
Structural Factors ◽  
Insight Into

Crystal structure and functional characterization of a glucosamine-6-phosphate N-acetyltransferase from Arabidopsis thaliana

2012 ◽  
Vol 443 (2) ◽  
pp. 427-437 ◽  
Author(s):  
Heike Riegler ◽  
Thomas Herter ◽  
Irina Grishkovskaya ◽  
Anja Lude ◽  
Malgorzata Ryngajllo ◽  
...  
Keyword(s):  
Crystal Structure ◽  
De Novo ◽  
Homo Sapiens ◽  
Functional Characterization ◽  
Catalytic Efficiency ◽  
Structural Similarity ◽  
Highly Active ◽  
Glycan Chain ◽  
High Structural Similarity ◽  
Protein Sequence Identity

GlcNAc (N-acetylglucosamine) is an essential part of the glycan chain in N-linked glycoproteins. It is a building block for polysaccharides such as chitin, and several glucosaminoglycans and proteins can be O-GlcNAcylated. The deacetylated form, glucosamine, is an integral part of GPI (glycosylphosphatidylinositol) anchors. Both are incorporated into polymers by glycosyltransferases that utilize UDP-GlcNAc. This UDP-sugar is synthesized in a short pathway comprising four steps starting from fructose 6-phosphate. GNA (glucosamine-6-phosphate N-acetyltransferase) catalyses the second of these four reactions in the de novo synthesis in eukaryotes. A phylogenetic analysis revealed that only one GNA isoform can be found in most of the species investigated and that the most likely Arabidopsis candidate is encoded by the gene At5g15770 (AtGNA). qPCR (quantitative PCR) revealed the ubiquitous expression of AtGNA in all organs of Arabidopsis plants. Heterologous expression of AtGNA showed that it is highly active between pH 7 and 8 and at temperatures of 30–40°C. It showed Km values of 231 μM for glucosamine 6-phosphate and 33 μM for acetyl-CoA respectively and a catalytic efficiency comparable with that of other GNAs characterized. The solved crystal structure of AtGNA at a resolution of 1.5 Å (1 Å=0.1 nm) revealed a very high structural similarity to crystallized GNA proteins from Homo sapiens and Saccharomyces cerevisiae despite less well conserved protein sequence identity.

scholarly journals Computational strategies to combat COVID-19: useful tools to accelerate SARS-CoV-2 and coronavirus research

2020 ◽  
Author(s):  
Franziska Hufsky ◽  
Kevin Lamkiewicz ◽  
Alexandre Almeida ◽  
Abdel Aouacheria ◽  
Cecilia Arighi ◽  
...  
Keyword(s):  
Drug Targets ◽  
Web Applications ◽  
Use Case ◽  
Sequencing Data ◽  
Fast Detection ◽  
The Family ◽  
Containment Measures ◽  
Novel Virus ◽  
Potential Drug Targets ◽  
Insight Into

Abstract SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) is a novel virus of the family Coronaviridae. The virus causes the infectious disease COVID-19. The biology of coronaviruses has been studied for many years. However, bioinformatics tools designed explicitly for SARS-CoV-2 have only recently been developed as a rapid reaction to the need for fast detection, understanding and treatment of COVID-19. To control the ongoing COVID-19 pandemic, it is of utmost importance to get insight into the evolution and pathogenesis of the virus. In this review, we cover bioinformatics workflows and tools for the routine detection of SARS-CoV-2 infection, the reliable analysis of sequencing data, the tracking of the COVID-19 pandemic and evaluation of containment measures, the study of coronavirus evolution, the discovery of potential drug targets and development of therapeutic strategies. For each tool, we briefly describe its use case and how it advances research specifically for SARS-CoV-2. All tools are free to use and available online, either through web applications or public code repositories. Contact:  [email protected]

Sign in / Sign up

Export Citation Format

Share Document