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 Different modes of internalization of apoptotic alkyl-lysophospholipid and cell-rescuing lysophosphatidylcholine

2003 ◽  
Vol 374 (3) ◽  
pp. 747-753 ◽  
Author(s):  
Arnold H. van der LUIT ◽  
Marianne BUDDE ◽  
Marcel VERHEIJ ◽  
Wim J. van BLITTERSWIJK
Keyword(s):  
Lipid Rafts ◽  
De Novo ◽  
Structural Similarity ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Chemotherapeutic Drugs ◽  
Alternative Substrate ◽  
High Structural Similarity ◽  
Additional Cell ◽  
Cell Membrane Level

The synthetic alkyl-lysophospholipid (ALP), Et-18-OCH3 (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine), can induce apoptosis in tumour cells. Unlike conventional chemotherapeutic drugs, ALP acts at the cell-membrane level. We have reported previously that ALP is internalized, and interferes with phosphatidylcholine (PC) biosynthesis de novo, which appeared to be essential for survival in lymphoma cells [Van der Luit, Budde, Ruurs, Verheij and Van Blitterswijk (2002) J. Biol. Chem. 277, 39541–39547]. Here, we report that, in HeLa cells, ALP accumulates in lipid rafts, and that internalization is inhibited by low temperature, monensin, disruption of lipid rafts and expression of a dominant-negative mutant of dynamin bearing a replacement of Lys44 with alanine (K44A). Thus ALP is internalized via raft- and dynamin-mediated endocytosis. Dynamin-K44A alleviated the ALP-induced inhibition of PC synthesis and rescued the cells from apoptosis induction. Additional cell rescue was attained by exogenous lysoPC, which after internalization serves as an alternative substrate for PC synthesis (through acylation). Unlike ALP, and despite the high structural similarity to ALP, lysoPC uptake did not occur via lipid rafts and did not depend on functional dynamin, indicating no involvement of endocytosis. Albumin back-extraction experiments suggested that (radiolabelled) lysoPC undergoes transbilayer movement (flipping). We conclude that ALP is internalized by endocytosis via lipid rafts to cause apoptosis, while exogenous cell-rescuing lysoPC traverses the plasma membrane outside rafts by flipping. Additionally, our data imply the importance of ether bonds in lyso-phospholipids, such as in ALP, for partitioning in lipid rafts.

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.

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.

Insights into the biochemical and functional characterization of sortase E transpeptidase of Corynebacterium glutamicum

2019 ◽  
Vol 476 (24) ◽  
pp. 3835-3847 ◽  
Author(s):  
Aliyath Susmitha ◽  
Kesavan Madhavan Nampoothiri ◽  
Harsha Bajaj
Keyword(s):  
Protein Engineering ◽  
Cell Attachment ◽  
Functional Characterization ◽  
Protein Structures ◽  
Structural Similarity ◽  
Surface Proteins ◽  
Sortase A ◽  
Peptide Cleavage ◽  
New Findings

Most Gram-positive bacteria contain a membrane-bound transpeptidase known as sortase which covalently incorporates the surface proteins on to the cell wall. The sortase-displayed protein structures are involved in cell attachment, nutrient uptake and aerial hyphae formation. Among the six classes of sortase (A–F), sortase A of S. aureus is the well-characterized housekeeping enzyme considered as an ideal drug target and a valuable biochemical reagent for protein engineering. Similar to SrtA, class E sortase in GC rich bacteria plays a housekeeping role which is not studied extensively. However, C. glutamicum ATCC 13032, an industrially important organism known for amino acid production, carries a single putative sortase (NCgl2838) gene but neither in vitro peptide cleavage activity nor biochemical characterizations have been investigated. Here, we identified that the gene is having a sortase activity and analyzed its structural similarity with Cd-SrtF. The purified enzyme showed a greater affinity toward LAXTG substrate with a calculated KM of 12 ± 1 µM, one of the highest affinities reported for this class of enzyme. Moreover, site-directed mutation studies were carried to ascertain the structure functional relationship of Cg-SrtE and all these are new findings which will enable us to perceive exciting protein engineering applications with this class of enzyme from a non-pathogenic microbe.

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