Structure of the gas vesicle protein GvpF from the cyanobacteriumMicrocystis aeruginosa

2014 ◽  
Vol 70 (11) ◽  
pp. 3013-3022 ◽  
Author(s):  
Bo-Ying Xu ◽  
Ya-Nan Dai ◽  
Kang Zhou ◽  
Yun-Tao Liu ◽  
Qianqian Sun ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Gene Cluster ◽  
Microcystis Aeruginosa ◽  
Immunogold Labelling ◽  
Gas Vesicles ◽  
Structural Protein ◽  
Apparent Variation ◽  
Vesicle Protein

Gas vesicles are gas-filled proteinaceous organelles that provide buoyancy for bacteria and archaea. A gene cluster that is highly conserved in various species encodes about 8–14 proteins (Gvp proteins) that are involved in the formation of gas vesicles. Here, the first crystal structure of the gas vesicle protein GvpF fromMicrocystis aeruginosaPCC 7806 is reported at 2.7 Å resolution. GvpF is composed of two structurally distinct domains (the N-domain and C-domain), both of which display an α+β class overall structure. The N-domain adopts a novel fold, whereas the C-domain has a modified ferredoxin fold with an apparent variation owing to an extension region consisting of three sequential helices. The two domains pack against each otherviainteractions with a C-terminal tail that is conserved among cyanobacteria. Taken together, it is concluded that the overall architecture of GvpF presents a novel fold. Moreover, it is shown that GvpF is most likely to be a structural protein that is localized at the gas-facing surface of the gas vesicle by immunoblotting and immunogold labelling-based tomography.

scholarly journals The Gas Vesicle Gene Cluster from Microcystis aeruginosa and DNA Rearrangements That Lead to Loss of Cell Buoyancy

2004 ◽  
Vol 186 (8) ◽  
pp. 2355-2365 ◽  
Author(s):  
Alyssa Mlouka ◽  
Katia Comte ◽  
Anne-Marie Castets ◽  
Christiane Bouchier ◽  
Nicole Tandeau de Marsac
Keyword(s):  
Electron Microscopy ◽  
Gene Cluster ◽  
Microcystis Aeruginosa ◽  
Gas Vesicles ◽  
Structural Protein ◽  
Dna Rearrangements ◽  
Unicellular Cyanobacterium ◽  
Laboratory Cultures ◽  
Insertion Elements ◽  
Freshwater Environments

ABSTRACT Microcystis aeruginosa is a planktonic unicellular cyanobacterium often responsible for seasonal mass occurrences at the surface of freshwater environments. An abundant production of intracellular structures, the gas vesicles, provides cells with buoyancy. A 8.7-kb gene cluster that comprises twelve genes involved in gas vesicle synthesis was identified. Ten of these are organized in two operons, gvpAI AII AIII CNJX and gvpKFG, and two, gvpV and gvpW, are individually expressed. In an attempt to elucidate the basis for the frequent occurrence of nonbuoyant mutants in laboratory cultures, four gas vesicle-deficient mutants from two strains of M. aeruginosa, PCC 7806 and PCC 9354, were isolated and characterized. Their molecular analysis unveiled DNA rearrangements due to four different insertion elements that interrupted gvpN, gvpV, or gvpW or led to the deletion of the gvpAI -AIII region. While gvpA, encoding the major gas vesicle structural protein, was expressed in the gvpN, gvpV, and gvpW mutants, immunodetection revealed no corresponding GvpA protein. Moreover, the absence of a gas vesicle structure was confirmed by electron microscopy. This study brings out clues concerning the process driving loss of buoyancy in M. aeruginosa and reveals the requirement for gas vesicle synthesis of two newly described genes, gvpV and gvpW.

scholarly journals The SARS-CoV-2 conserved macrodomain is a mono-ADP-ribosylhydrolase

2020 ◽  
Author(s):  
Yousef M.O. Alhammad ◽  
Maithri M. Kashipathy ◽  
Anuradha Roy ◽  
Jean-Philippe Gagné ◽  
Peter McDonald ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Therapeutic Target ◽  
Viral Infections ◽  
Protein Domain ◽  
Structural Protein ◽  
Potential Therapeutic Target ◽  
Novel Therapeutic Strategies ◽  
Design And Testing ◽  
Translational Process ◽  
Novel Therapeutic

ABSTRACTSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other SARS-like-CoVs encode 3 tandem macrodomains within non-structural protein 3 (nsp3). The first macrodomain, Mac1, is conserved throughout CoVs, and binds to and hydrolyzes mono-ADP-ribose (MAR) from target proteins. Mac1 likely counters host-mediated anti-viral ADP-ribosylation, a posttranslational modification that is part of the host response to viral infections. Mac1 is essential for pathogenesis in multiple animal models of CoV infection, implicating it as a virulence factor and potential therapeutic target. Here we report the crystal structure of SARS-CoV-2 Mac1 in complex with ADP-ribose. SARS-CoV-2, SARS-CoV and MERS-CoV Mac1 exhibit similar structural folds and all 3 proteins bound to ADP-ribose with low μM affinities. Importantly, using ADP-ribose detecting binding reagents in both a gel-based assay and novel ELISA assays, we demonstrated de-MARylating activity for all 3 CoV Mac1 proteins, with the SARS-CoV-2 Mac1 protein leading to a more rapid loss of substrate compared to the others. In addition, none of these enzymes could hydrolyze poly-ADP-ribose. We conclude that the SARS-CoV-2 and other CoV Mac1 proteins are MAR-hydrolases with similar functions, indicating that compounds targeting CoV Mac1 proteins may have broad anti-CoV activity.IMPORTANCESARS-CoV-2 has recently emerged into the human population and has led to a worldwide pandemic of COVID-19 that has caused greater than 900 thousand deaths worldwide. With, no currently approved treatments, novel therapeutic strategies are desperately needed. All coronaviruses encode for a highly conserved macrodomain (Mac1) that binds to and removes ADP-ribose adducts from proteins in a dynamic post-translational process increasingly recognized as an important factor that regulates viral infection. The macrodomain is essential for CoV pathogenesis and may be a novel therapeutic target. Thus, understanding its biochemistry and enzyme activity are critical first steps for these efforts. Here we report the crystal structure of SARS-CoV-2 Mac1 in complex with ADP-ribose, and describe its ADP-ribose binding and hydrolysis activities in direct comparison to SARS-CoV and MERS-CoV Mac1 proteins. These results are an important first step for the design and testing of potential therapies targeting this unique protein domain.

scholarly journals Biochemical and Structural Insights into Xylan Utilization by the Thermophilic Bacterium Caldanaerobius polysaccharolyticus

2012 ◽  
Vol 287 (42) ◽  
pp. 34946-34960 ◽  
Author(s):  
Yejun Han ◽  
Vinayak Agarwal ◽  
Dylan Dodd ◽  
Jason Kim ◽  
Brian Bae ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Gene Cluster ◽  
Plant Cell Wall ◽  
Hydrolytic Enzymes ◽  
Thermophilic Bacterium ◽  
Pentose Phosphate ◽  
Structural Basis ◽  
Resolution Analysis ◽  
Calorimetric Studies ◽  
Hydrolysis Of

Hemicellulose is the next most abundant plant cell wall component after cellulose. The abundance of hemicellulose such as xylan suggests that their hydrolysis and conversion to biofuels can improve the economics of bioenergy production. In an effort to understand xylan hydrolysis at high temperatures, we sequenced the genome of the thermophilic bacterium Caldanaerobius polysaccharolyticus. Analysis of the partial genome sequence revealed a gene cluster that contained both hydrolytic enzymes and also enzymes key to the pentose-phosphate pathway. The hydrolytic enzymes in the gene cluster were demonstrated to convert products from a large endoxylanase (Xyn10A) predicted to anchor to the surface of the bacterium. We further use structural and calorimetric studies to demonstrate that the end products of Xyn10A hydrolysis of xylan are recognized and bound by XBP1, a putative solute-binding protein, likely for transport into the cell. The XBP1 protein showed preference for xylo-oligosaccharides as follows: xylotriose > xylobiose > xylotetraose. To elucidate the structural basis for the oligosaccharide preference, we solved the co-crystal structure of XBP1 complexed with xylotriose to a 1.8-Å resolution. Analysis of the biochemical data in the context of the co-crystal structure reveals the molecular underpinnings of oligosaccharide length specificity.

scholarly journals X-ray crystal structure of ornithine acetyltransferase from the clavulanic acid biosynthesis gene cluster

2005 ◽  
Vol 385 (2) ◽  
pp. 565-573 ◽  
Author(s):  
Jonathan M. ELKINS ◽  
Nadia J. KERSHAW ◽  
Christopher J. SCHOFIELD
Keyword(s):  
Crystal Structure ◽  
Clavulanic Acid ◽  
Gene Cluster ◽  
Acid Biosynthesis ◽  
Biosynthesis Gene Cluster ◽  
Reading Frame ◽  
X Ray ◽  
Orf6 Gene ◽  
Biosynthesis Gene ◽  
Acetyl Transfer

The orf6 gene from the clavulanic acid biosynthesis gene cluster encodes an OAT (ornithine acetyltransferase). Similar to other OATs the enzyme has been shown to catalyse the reversible transfer of an acetyl group from N-acetylornithine to glutamate. OATs are Ntn (N-terminal nucleophile) enzymes, but are distinct from the better-characterized Ntn hydrolase enzymes as they catalyse acetyl transfer rather than a hydrolysis reaction. In the present study, we describe the X-ray crystal structure of the OAT, corresponding to the orf6 gene product, to 2.8 Å (1 Å=0.1 nm) resolution. The larger domain of the structure consists of an αββα sandwich as in the structures of Ntn hydrolase enzymes. However, differences in the connectivity reveal that OATs belong to a structural family different from that of other structurally characterized Ntn enzymes, with one exception: unexpectedly, the αββα sandwich of ORF6 (where ORF stands for open reading frame) displays the same fold as an DmpA (L-aminopeptidase D-ala-esterase/amidase from Ochrobactrum anthropi), and so the OATs and DmpA form a new structural subfamily of Ntn enzymes. The structure reveals an α2β2-heterotetrameric oligomerization state in which the intermolecular interface partly defines the active site. Models of the enzyme–substrate complexes suggest a probable oxyanion stabilization mechanism as well as providing insight into how the enzyme binds its two differently charged substrates.

scholarly journals Crystal structure of the SARS-CoV-2 non-structural protein 9, Nsp9

2020 ◽  
Author(s):  
D. R. Littler ◽  
B. S. Gully ◽  
R. N. Colson ◽  
J Rossjohn
Keyword(s):  
Crystal Structure ◽  
Binding Site ◽  
Peptide Binding ◽  
Structural Protein ◽  
Dimer Interface ◽  
X Ray ◽  
3C Protease ◽  
Peptide Binding Site ◽  
High Level ◽  
Viral Genomic Rna

AbstractMany of the proteins produced by SARS-CoV-2 have related counterparts across the Severe Acute Respiratory Syndrome (SARS-CoV) family. One such protein is non-structural protein 9 (Nsp9), which is thought to mediate both viral replication and virulence. Current understanding suggests that Nsp9 is involved in viral genomic RNA reproduction. Nsp9 is thought to bind RNA via a fold that is unique to this class of betacoronoaviruses although the molecular basis for this remains ill-defined. We sought to better characterise the SARS-CoV-2 Nsp9 protein and subsequently solved its X-ray crystal structure, in an apo-form and, unexpectedly, in a peptide-bound form with a sequence originating from a rhinoviral 3C protease sequence (LEVL). The structure of the SARS-CoV-2 Nsp9 revealed the high level of structural conservation within the Nsp9 family. The exogenous peptide binding site is close to the dimer interface and impacted on the relative juxtaposition of the monomers within the homodimer. Together we have established a protocol for the production of SARS-CoV-2 Nsp9, determined its structure and identified a peptide-binding site that may warrant further study from the perspective of understanding Nsp9 function.

scholarly journals Crystal structure of the African swine fever virus structural protein p35 reveals its role for core shell assembly

2020 ◽  
Vol 11 (8) ◽  
pp. 600-605
Author(s):  
Guobang Li ◽  
Dan Fu ◽  
Guangshun Zhang ◽  
Dongming Zhao ◽  
Mingyu Li ◽  
...  
Keyword(s):  
Crystal Structure ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Core Shell ◽  
Structural Protein

scholarly journals Characterization of the 2-Hydroxy-acid Dehydrogenase McyI, Encoded within the Microcystin Biosynthesis Gene Cluster of Microcystis aeruginosa PCC7806

2006 ◽  
Vol 282 (7) ◽  
pp. 4681-4692 ◽  
Author(s):  
Leanne A. Pearson ◽  
Kevin D. Barrow ◽  
Brett A. Neilan
Keyword(s):  
Gene Cluster ◽  
Microcystis Aeruginosa ◽  
Hydroxy Acid ◽  
Biosynthesis Gene Cluster ◽  
Glutamate Mutase ◽  
Acid Dehydrogenase ◽  
Methyl Aspartate ◽  
Catalyzed Reactions

The cyanobacterium Microcystis aeruginosa is widely known for its production of the potent hepatotoxin microcystin. This cyclic heptapeptide is synthesized non-ribosomally by the thio-template function of a large modular enzyme complex encoded within the 55-kb microcystin synthetase gene (mcy) cluster. The mcy gene cluster also encodes several stand-alone enzymes, putatively involved in the tailoring and export of microcystin. This study describes the characterization of the 2-hydroxy-acid dehydrogenase McyI, putatively involved in the production of d-methyl aspartate at position 3 within the microcystin cyclic structure. A combination of bioinformatics, molecular, and biochemical techniques was used to elucidate the structure, function, regulation, and evolution of this unique enzyme. The recombinant McyI enzyme was overexpressed in Escherichia coli and enzymatically characterized. The hypothesized native activity of McyI, the interconversion of 3-methyl malate to 3-methyl oxalacetate, was demonstrated using an in vitro spectrophotometric assay. The enzyme was also able to reduce α-ketoglutarate to 2-hydroxyglutarate and to catalyze the interconversion of malate and oxalacetate. Although NADP(H) was the preferred cofactor of the McyI-catalyzed reactions, NAD(H) could also be utilized, although rates of catalysis were significantly lower. The combined results of this study suggest that hepatotoxic cyanobacteria such as M. aeruginosa PCC7806 are capable of producing methyl aspartate via a novel glutamate mutase-independent pathway, in which McyI plays a pivotal role.

scholarly journals The movement protein encoded by gene 3 of rice transitory yellowing virus is associated with virus particles

2012 ◽  
Vol 93 (10) ◽  
pp. 2290-2298 ◽  
Author(s):  
Akihiro Hiraguri ◽  
Hiroyuki Hibino ◽  
Takaharu Hayashi ◽  
Osamu Netsu ◽  
Takumi Shimizu ◽  
...  
Keyword(s):  
Movement Protein ◽  
Cell Movement ◽  
Immunogold Labelling ◽  
Tomato Mosaic Virus ◽  
Structural Protein ◽  
Western Blots ◽  
Thin Sections ◽  
Virus Particles ◽  
E Coli ◽  
Yellowing Virus

Gene 3 in the genomes of several plant-infecting rhabdoviruses, including rice transitory yellowing virus (RTYV), has been postulated to encode a cell-to-cell movement protein (MP). Trans-complementation experiments using a movement-defective tomato mosaic virus and the P3 protein of RTYV, encoded by gene 3, facilitated intercellular transport of the mutant virus. In transient-expression experiments with the GFP-fused P3 protein in epidermal leaf cells of Nicotiana benthamiana, the P3 protein was associated with the nucleus and plasmodesmata. Immunogold-labelling studies of thin sections of RTYV-infected rice plants using an antiserum against Escherichia coli-expressed His6-tagged P3 protein indicated that the P3 protein was located in cell walls and on virus particles. In Western blots using antisera against E. coli-expressed P3 protein and purified RTYV, the P3 protein was detected in purified RTYV, whilst antiserum against purified RTYV reacted with the E. coli-expressed P3 protein. After immunogold labelling of crude sap from RTYV-infected rice leaves, the P3 protein, as well as the N protein, was detected on the ribonucleocapsid core that emerged from partially disrupted virus particles. These results provide evidence that the P3 protein of RTYV, which functions as a viral MP, is a viral structural protein and seems to be associated with the ribonucleocapsid core of virus particles.

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