scholarly journals Crystal structure of the N-terminal anticodon-binding domain of the nondiscriminating aspartyl-tRNA synthetase fromHelicobacter pylori

2017 ◽  
Vol 73 (2) ◽  
pp. 62-69 ◽  
Author(s):  
Chomphunuch Songsiriritthigul ◽  
Suwimon Suebka ◽  
Chun-Jung Chen ◽  
Pitchayada Fuengfuloy ◽  
Pitak Chuawong
Keyword(s):  
Crystal Structure ◽  
Crucial Role ◽  
Structural Similarity ◽  
Trna Synthetase ◽  
Binding Domain ◽  
Trna Recognition ◽  
Binding Domains ◽  
Human Pathogenic Bacterium ◽  
High Structural Similarity ◽  
H Pylori

The N-terminal anticodon-binding domain of the nondiscriminating aspartyl-tRNA synthetase (ND-AspRS) plays a crucial role in the recognition of both tRNAAspand tRNAAsn. Here, the first X-ray crystal structure of the N-terminal domain of this enzyme (ND-AspRS1–104) from the human-pathogenic bacteriumHelicobacter pyloriis reported at 2.0 Å resolution. The apo form ofH. pyloriND-AspRS1–104shares high structural similarity with the N-terminal anticodon-binding domains of the discriminating aspartyl-tRNA synthetase (D-AspRS) fromEscherichia coliand ND-AspRS fromPseudomonas aeruginosa, allowing recognition elements to be proposed for tRNAAspand tRNAAsn. It is proposed that a long loop (Arg77–Lys90) in thisH. pyloridomain influences its relaxed tRNA specificity, such that it is classified as nondiscriminating. A structural comparison between D-AspRS fromE. coliand ND-AspRS fromP. aeruginosasuggests that turns E and F (78GAGL81and83NPKL86) inH. pyloriND-AspRS play a crucial role in anticodon recognition. Accordingly, the conserved Pro84 in turn F facilitates the recognition of the anticodons of tRNAAsp(34GUC36) and tRNAAsn(34GUU36). The absence of the amide H atom allows both C and U bases to be accommodated in the tRNA-recognition site.

scholarly journals Crystal structure of JHP933 from Helicobacter pylori J99

2014 ◽  
Vol 70 (a1) ◽  
pp. C823-C823
Author(s):  
Sang Jae Lee ◽  
Ji Young Yoon ◽  
Bong-Jin Lee ◽  
Se Won Suh
Keyword(s):  
Crystal Structure ◽  
Helicobacter Pylori ◽  
Peptic Ulcer ◽  
Functional Characterization ◽  
Domain Architecture ◽  
Structural Similarity ◽  
Terminal Domain ◽  
Plasticity Region ◽  
H Pylori ◽  
And Function

Helicobacter pylori infection is the main cause of chronic gastritis, gastric mucosal atrophy, peptic ulcer, and some forms of gastric cancer. There has been considerable interest in strain-specific genes found outside of the cag pathogenicity island, especially genes in the plasticity regions of H. pylori. In H. pylori strain J99, the plasticity region contains 48 genes ranging from jhp0914 to jhp0961. Because little is known about many of these genes in the plasticity region, further studies are necessary to elucidate their roles in H. pylori-associated pathogenesis. The JHP933 protein, encoded by the jhp0933 gene in the plasticity region of H. pylori J99, is one of the prevalently expressed proteins in some gastritis and peptic ulcer patients. However, its structure and function remain unknown. Here, we have determined the crystal structure of JHP933, revealing the first two-domain architecture of DUF1814 family. The N-terminal domain has the nucleotidyltransferase fold and the C-terminal domain is a helix bundle. Structural similarity of JHP933 to known nucleotidyltransferases is very remote, suggesting that it may function as a novel nucleotidyltransferase. It is expected that this study will facilitate functional characterization of JHP933 to obtain an insight into its role in pathogenesis by the H. pylori plasticity region.

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 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 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.

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.

Transcriptional expression of aminoacyl tRNA synthetase genes of Xanthomonas oryzae pv. oryzae (Xoo) on rice-leaf extract treatment and crystal structure of Xoo glutamyl-tRNA synthetase

2017 ◽  
Vol 68 (5) ◽  
pp. 434
Author(s):  
Thien-Hoang Ho ◽  
Myoung-Ki Hong ◽  
Seunghwan Kim ◽  
Jeong-Gu Kim ◽  
Jongha Lee ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Bacterial Blight ◽  
Leaf Extract ◽  
Catalytic Domain ◽  
Trna Synthetase ◽  
Binding Domain ◽  
Xanthomonas Oryzae ◽  
Transcriptional Expression ◽  
Aminoacyl Trna Synthetase ◽  
Rice Leaf

Xanthomonas oryzae pv. oryzae (Xoo) is the causal agent of bacterial blight of rice, one of the most devastating rice diseases. We analysed the time-resolved transcriptional expression of aminoacyl-tRNA synthetase (aaRS) genes in Xoo cells treated with rice-leaf extract. Most aaRS genes showed decreased expression in the initial 30 min and recovered or increased expression in the later 30 min. The protein-synthetic machinery of bacterial cells is an important target for developing antibiotic agents; aaRSs play an essential role in peptide synthesis by attaching amino acids onto the corresponding tRNA. In bacteria, glutaminyl-tRNA (Gln-tRNAGln) is synthesised in two steps by glutamyl-tRNA synthetase (GluRS) and tRNA-dependent aminotransferase, the indirect biosynthetic mechanism of which is not present in eukaryotes. We determined the crystal structure of GluRS from Xoo (XoGluRS) at resolution of 3.0 Å, this being the first GluRS structure from a plant pathogen such as Xoo. The XoGluRS structure consists of five domains, which are conserved in other bacterial GluRS structures. In the bacterial GluRS structures, the Rossmann-fold catalytic domain and the stem-contact domain are most conserved in both sequence and structure. The anticodon-binding domain 1 is less conserved in sequence but overall structure is conserved. The connective-polypeptide domain and the anticodon-binding domain 2 show various conformations in structure. The XoGluRS structure could provide useful information to develop a new pesticide against Xoo and bacterial blight.

A chimaeric glutamyl:glutaminyl-tRNA synthetase: implications for evolution

2008 ◽  
Vol 417 (2) ◽  
pp. 449-455 ◽  
Author(s):  
Rajesh Saha ◽  
Saumya Dasgupta ◽  
Gautam Basu ◽  
Siddhartha Roy
Keyword(s):  
Catalytic Domain ◽  
Trna Synthetase ◽  
Binding Domain ◽  
Temperature Sensitive ◽  
Trna Synthetases ◽  
E Coli ◽  
Discrimination Capability ◽  
Binding Domains ◽  
History Of ◽  
Substrate Binding Domain

aaRSs (aminoacyl-tRNA synthetases) are multi-domain proteins that have evolved by domain acquisition. The anti-codon binding domain was added to the more ancient catalytic domain during aaRS evolution. Unlike in eukaryotes, the anti-codon binding domains of GluRS (glutamyl-tRNA synthetase) and GlnRS (glutaminyl-tRNA synthetase) in bacteria are structurally distinct. This originates from the unique evolutionary history of GlnRSs. Starting from the catalytic domain, eukaryotic GluRS evolved by acquiring the archaea/eukaryote-specific anti-codon binding domain after branching away from the eubacteria family. Subsequently, eukaryotic GlnRS evolved from GluRS by gene duplication and horizontally transferred to bacteria. In order to study the properties of the putative ancestral GluRS in eukaryotes, formed immediately after acquiring the anti-codon binding domain, we have designed and constructed a chimaeric protein, cGluGlnRS, consisting of the catalytic domain, Ec GluRS (Escherichia coli GluRS), and the anti-codon binding domain of EcGlnRS (E. coli GlnRS). In contrast to the isolated EcN-GluRS, cGluGlnRS showed detectable activity of glutamylation of E. coli tRNAglu and was capable of complementing an E. coli ts (temperature-sensitive)-GluRS strain at non-permissive temperatures. Both cGluGlnRS and EcN-GluRS were found to bind E. coli tRNAglu with native EcGluRS-like affinity, suggesting that the anticodon-binding domain in cGluGlnRS enhances kcat for glutamylation. This was further confirmed from similar experiments with a chimaera between EcN-GluRS and the substrate-binding domain of EcDnaK (E. coli DnaK). We also show that an extended loop, present in the anticodon-binding domains of GlnRSs, is absent in archaeal GluRS, suggesting that the loop was a later addition, generating additional anti-codon discrimination capability in GlnRS as it evolved from GluRS in eukaryotes.

scholarly journals Crystal Structure of the Simian Virus 40 Large T-Antigen Origin-Binding Domain

2006 ◽  
Vol 80 (9) ◽  
pp. 4304-4312 ◽  
Author(s):  
Gretchen Meinke ◽  
Peter A. Bullock ◽  
Andrew Bohm
Keyword(s):  
Crystal Structure ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Binding Domain ◽  
Large T Antigen ◽  
Binding Domains ◽  
Multiple Binding Sites ◽  
Tumor Viruses ◽  
Resolution Model

ABSTRACT The origins of replication of DNA tumor viruses have a highly conserved feature, namely, multiple binding sites for their respective initiator proteins arranged as inverted repeats. In the 1.45-Å crystal structure of the simian virus 40 large T-antigen (T-ag) origin-binding domain (obd) reported herein, T-ag obd monomers form a left-handed spiral with an inner channel of 30 Å having six monomers per turn. The inner surface of the spiral is positively charged and includes residues known to bind DNA. Residues implicated in hexamerization of full-length T-ag are located at the interface between adjacent T-ag obd monomers. These data provide a high-resolution model of the hexamer of origin-binding domains observed in electron microscopy studies and allow the obd's to be oriented relative to the hexamer of T-ag helicase domains to which they are connected.

scholarly journals A highly conserved cryptic epitope in the receptor-binding domains of SARS-CoV-2 and SARS-CoV

2020 ◽  
Author(s):  
Meng Yuan ◽  
Nicholas C. Wu ◽  
Xueyong Zhu ◽  
Chang-Chun D. Lee ◽  
Ray T. Y. So ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Receptor Binding ◽  
Neutralizing Antibody ◽  
Binding Domain ◽  
Receptor Binding Domain ◽  
S Protein ◽  
Binding Domains ◽  
Cryptic Epitope ◽  
Insight Into ◽  
Conserved Epitope

ABSTRACTThe outbreak of COVID-19, which is caused by SARS-CoV-2 virus, continues to spread globally, but there is currently very little understanding of the epitopes on the virus. In this study, we have determined the crystal structure of the receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein in complex with CR3022, a neutralizing antibody previously isolated from a convalescent SARS patient. CR3022 targets a highly conserved epitope that enables cross-reactive binding between SARS-CoV-2 and SARS-CoV. Structural modeling further demonstrates that the binding site can only be accessed when at least two RBDs on the trimeric S protein are in the “up” conformation. Overall, this study provides structural and molecular insight into the antigenicity of SARS-CoV-2.ONE SENTENCE SUMMARYStructural study of a cross-reactive SARS antibody reveals a conserved epitope on the SARS-CoV-2 receptor-binding domain.

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