scholarly journals The Exopolyphosphatase Gene from Sulfolobus solfataricus: Characterization of the First Gene Found To Be Involved in Polyphosphate Metabolism in Archaea

2002 ◽  
Vol 68 (10) ◽  
pp. 4812-4819 ◽  
Author(s):  
Silvia T. Cardona ◽  
Francisco P. Chávez ◽  
Carlos A. Jerez
Keyword(s):  
Sulfolobus Solfataricus ◽  
Open Reading Frame ◽  
Inorganic Polyphosphate ◽  
Reading Frame ◽  
Highly Active ◽  
Calculated Mass ◽  
Polyphosphate Metabolism ◽  
Long Chain

ABSTRACT Inorganic polyphosphate (polyP) polymers are widely distributed in all kinds of organisms. Although the presence of polyP in members of the domain Archaea has been described, at present nothing is known about the enzymology of polyP metabolism or the genes involved in this domain. We have cloned, sequenced, and overexpressed an exopolyphosphatase (PPX) gene (ppx) from thermophilic Sulfolobus solfataricus. The gene codes for a functional PPX and possesses an open reading frame for 417 amino acids (calculated mass, 47.9 kDa). The purified recombinant PPX was highly active, degrading long-chain polyP (700 to 800 residues) in vitro at 50 to 60°C. The putative PPXs present in known archaeal genomes showed the highest similarity to yeast PPXs. In contrast, informatic analysis revealed that the deduced amino acid sequence of S. solfataricus PPX showed the highest similarity (25 to 45%) to sequences of members of the bacterial PPXs, possessing all of their conserved motifs. To our knowledge, this is the first report of an enzyme characterized to be involved in polyP metabolism in members of the Archaea.

Characterization of the Chromosomalaac(6′)-Iz Gene of Stenotrophomonas maltophilia

1999 ◽  
Vol 43 (10) ◽  
pp. 2366-2371 ◽  
Author(s):  
Thierry Lambert ◽  
Marie-Cécile Ploy ◽  
François Denis ◽  
Patrice Courvalin
Keyword(s):  
Stenotrophomonas Maltophilia ◽  
Type I ◽  
Aminoglycoside Resistance ◽  
Reading Frame ◽  
A Value ◽  
Calculated Mass ◽  
Total Dna ◽  
Good Agreement

ABSTRACT The aac(6′)-Iz gene of Stenotrophomonas maltophilia BM2690 encoding an aminoglycoside 6′-N-acetyltransferase was characterized. The gene was identified as a coding sequence of 462 bp corresponding to a protein with a calculated mass of 16,506 Da, a value in good agreement with that of ca. 16,000 found by in vitro coupled transcription-translation. Analysis of the deduced amino acid sequence indicated that the protein was a member of the major subfamily of aminoglycoside 6′-N-acetyltransferases. The enzyme conferred resistance to amikacin but not to gentamicin, indicating that it was an AAC(6′) of type I. The open reading frame upstream from the aac(6′)-Izgene was homologous to the fprA gene of Myxococcus xanthus (61% identity), which encodes a putative pyridoxine (pyridoxamine) 5′-phosphate oxidase. Pulsed-field gel electrophoresis of total DNA from BM2690 and S. maltophilia ATTC 13637 digested with XbaI, DraI, and SpeI followed by hybridization with rRNA and aac(6′)-Iz-specific probes indicated that the gene was located in the chromosome. Theaac(6′)-Iz gene was detected by DNA-DNA hybridization in all 80 strains of S. maltophilia tested. The MICs of gentamicin against these strains of S. maltophilia were lower than those of amikacin, netilmicin, and tobramycin, indicating that production of AAC(6′)-Iz contributes to aminoglycoside resistance in S. maltophilia.

scholarly journals A Phosphohexomutase from the Archaeon Sulfolobus solfataricus Is Covalently Modified by Phosphorylation on Serine

2005 ◽  
Vol 187 (12) ◽  
pp. 4270-4275 ◽  
Author(s):  
W. Keith Ray ◽  
Sabrina M. Keith ◽  
Andrea M. DeSantis ◽  
Jeremy P. Hunt ◽  
Timothy J. Larson ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Sulfolobus Solfataricus ◽  
Catalytic Efficiency ◽  
Protein Product ◽  
Open Reading Frame ◽  
Site Directed Mutagenesis ◽  
Phosphoryl Group ◽  
Reading Frame

ABSTRACT A phosphoserine-containing peptide was identified from tryptic digests from Sulfolobus solfataricus P1 by liquid chromatography-tandem mass spectrometry. Its amino acid sequence closely matched that bracketing Ser-309 in the predicted protein product of open reading frame sso0207, a putative phosphohexomutase, in the genome of S. solfataricus P2. Open reading frame sso0207 was cloned, and its protein product expressed in Escherichia coli. The recombinant protein proved capable of interconverting mannose 1-phosphate and mannose 6-phosphate, as well as glucose 1-phosphate and glucose 6-phosphate, in vitro. It displayed no catalytic activity toward glucosamine 6-phosphate or N-acetylglucosamine 6-phosphate. Models constructed using the X-ray crystal structure of a homologous phosphohexomutase from Pseudomonas aeruginosa predicted that Ser-309 of the archaeal protein lies within the substrate binding site. The presence of a phosphoryl group at this location would be expected to electrostatically interfere with the binding of negatively charged phosphohexose substrates, thus attenuating the catalytic efficiency of the enzyme. Using site-directed mutagenesis, Ser-309 was substituted by aspartic acid to mimic the presence of a phosphoryl group. The V max of the mutationally altered protein was only 4% that of the unmodified form. Substitution of Ser-309 with larger, but uncharged, amino acids, including threonine, also decreased catalytic efficiency, but to a lesser extent—three- to fivefold. We therefore predict that phosphorylation of the enzyme in vivo serves to regulate its catalytic activity.

scholarly journals Antigenic Characterization of ORF2 and ORF3 Proteins of Hepatitis E Virus (HEV)

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1385
Author(s):  
Giulia Pezzoni ◽  
Lidia Stercoli ◽  
Eleonora Pegoiani ◽  
Emiliana Brocchi
Keyword(s):  
Hepatitis E Virus ◽  
Hepatitis E ◽  
Recombinant Antigen ◽  
Open Reading Frame ◽  
Reading Frame ◽  
E Coli ◽  
Antigenic Characterization ◽  
Antigenic Properties ◽  
Open Reading Frame 2

To evaluate the antigenic properties of Hepatitis E Virus (HEV) Open Reading Frame 2 and 3 (ORF2 and ORF3) codified proteins, we expressed different portions of ORF2 and the entire ORF3 in E. coli, a truncated ORF2, was also expressed in baculovirus. A panel of 37 monoclonal antibodies (MAbs) was raised against ORF2 (1–660 amino acids) and MAbs were mapped and characterized using the ORF2 expressed portions. Selected HEV positive and negative swine sera were used to evaluate ORF2 and ORF3 antigens’ immunogenicity. The MAbs were clustered in six groups identifying six antigenic regions along the ORF2. Only MAbs binding to the sixth ORF2 antigenic region (394–608 aa) were found to compete with HEV positive sera and efficiently catch the recombinant antigen expressed in baculovirus. The ORF2 portion from 394–608 aa demonstrated to include most immunogenic epitopes with 85% of HEV positive swine sera reacting against the region from 461–544 aa. Only 5% of the selected HEV sera reacted against the ORF3 antigen.

scholarly journals Open Reading Frame sso2387 from the Archaeon Sulfolobus solfataricus Encodes a Polypeptide with Protein-Serine Kinase Activity

2003 ◽  
Vol 185 (11) ◽  
pp. 3436-3445 ◽  
Author(s):  
Brian H. Lower ◽  
Peter J. Kennelly
Keyword(s):  
Protein Kinases ◽  
Catalytic Domain ◽  
Sulfolobus Solfataricus ◽  
Open Reading Frame ◽  
Serine Kinase ◽  
Eukaryotic Protein Kinase ◽  
Reading Frame ◽  
Eukaryotic Protein ◽  
High Concentrations ◽  
Eukaryotic Protein Kinases

ABSTRACT The predicted polypeptide product of open reading frame sso2387 from the archaeon Sulfolobus solfataricus, SsoPK2, displayed several of the sequence features conserved among the members of the “eukaryotic” protein kinase superfamily. sso2387 was cloned, and its polypeptide product was expressed in Escherichia coli. The recombinant protein, rSsoPK2, was recovered in insoluble aggregates that could be dispersed by using high concentrations (5 M) of urea. The solubilized polypeptide displayed the ability to phosphorylate itself as well as several exogenous proteins, including mixed histones, casein, bovine serum albumin, and reduced carboxyamidomethylated and maleylated lysozyme, on serine residues. The source of this activity resided in that portion of the protein displaying homology to the catalytic domain of eukaryotic protein kinases. By use of mass spectrometry, the sites of autophosphorylation were found to be located in two areas, one immediately N terminal to the region corresponding to subdomain I of eukaryotic protein kinases, and the second N terminal to the presumed activation loop located between subdomains VII and VIII. Autophosphorylation of rSsoPK2 could be uncoupled from the phosphorylation of exogenous proteins by manipulation of the temperature or mutagenic alteration of the enzyme. Autophosphorylation was detected only at temperatures ≥60°C, whereas phosphorylation of exogenous proteins was detectable at 37°C. Similarly, replacement of one of the potential sites of autophosphorylation, Ser548, with alanine blocked autophosphorylation but not phosphorylation of an exogenous protein, casein.

scholarly journals Gene Cloning and Molecular Characterization of a Two-Enzyme System Catalyzing the Oxidative Detoxification of β-Endosulfan

2002 ◽  
Vol 68 (12) ◽  
pp. 6237-6245 ◽  
Author(s):  
Tara D. Sutherland ◽  
Irene Horne ◽  
Robyn J. Russell ◽  
John G. Oakeshott
Keyword(s):  
Shuttle Vector ◽  
Open Reading Frame ◽  
Cosmid Library ◽  
Flavin Mononucleotide ◽  
Flavin Reductase ◽  
Reading Frame ◽  
Reductase Gene ◽  
Degradation Activity ◽  
Layer Chromatography

ABSTRACT The gram-positive bacterium Mycobacterium sp. strain ESD is able to use the cyclodiene insecticide endosulfan as a source of sulfur for growth. This activity is dependent on the absence of sulfite or sulfate in the growth medium. A cosmid library of strain ESD DNA was constructed in a Mycobacterium-Escherichia coli shuttle vector and screened for endosulfan-degrading activity in Mycobacterium smegmatis, a species that does not degrade endosulfan. Using this method, we identified a single cosmid that conferred sulfur-dependent endosulfan-degrading activity on the host strain. An open reading frame (esd) was identified within this cosmid that, when expressed behind a constitutive promoter in a mycobacterial expression vector, conferred sulfite- and sulfate-independent β-endosulfan degradation activity on the recombinant strain. The translation product of this gene (Esd) had up to 50% sequence identity with an unusual family of monooxygenase enzymes that use reduced flavins, provided by a separate flavin reductase enzyme, as cosubstrates. An additional partial open reading frame was located upstream of the Esd gene that had sequence homology to the same monooxygenase family. A flavin reductase gene, identified in the M. smegmatis genome, was cloned, expressed, and used to provide reduced flavin mononucleotide for Esd in enzyme assays. Thin-layer chromatography and gas chromatography analyses of the enzyme assay mixtures revealed the disappearance of β-endosulfan and the appearance of the endosulfan metabolites, endosulfan monoaldehyde and endosulfan hydroxyether. This suggests that Esd catalyzes the oxygenation of β-endosulfan to endosulfan monoaldehyde and endosulfan hydroxyether. Esd did not degrade either α-endosulfan or the metabolite of endosulfan, endosulfan sulfate.

scholarly journals Cloning and characterization of α-pinene synthase from Chamaecyparis formosensis Matsum

Holzforschung ◽  
2009 ◽  
Vol 63 (1) ◽  
Author(s):  
Fang-Hua Chu ◽  
Pei-Min Kuo ◽  
Yu-Rong Chen ◽  
Sheng-Yang Wang
Keyword(s):  
Major Product ◽  
Open Reading Frame ◽  
Terpene Synthase ◽  
Geranyl Diphosphate ◽  
Reading Frame ◽  
E Coli ◽  
Phylogenetic Taxonomy ◽  
Chamaecyparis Formosensis ◽  
Pcr Method

AbstractAnalyzing the gene sequences of terpene synthase (TPS) may contribute to a better understanding of terpenes biosynthesis and evolution of phylogenetic taxonomy.Chamaecyparis formosensisis an endemic and precious conifer of Taiwan. To understand the biosynthesis mechanism of terpenes in this tree, a full length of putative mono-TPS, named asCf-Pin(GeneBank accession no. EU099434), was obtained by PCR method and RACE extension. TheCf-Pinhas an 1887-bp open reading frame and encodes 628 amino acids. To identify the function ofCf-Pin,the recombinant protein fromEscherichia coliwas incubated with geranyl diphosphate, produced one major product, the structure of which was elucidated. GC/MS analysis and matching of retention time and mass spectrum with authentic standards revealed that this product isα-pinene. This is the first report of cloning of a mono-TPS and functionally expressed inE. coliand which could be identified asα-pinene synthase from a Cupressaceae conifer.

Identification and characterization of a novel Neospora caninum immune mapped protein 1

Parasitology ◽  
2012 ◽  
Vol 139 (8) ◽  
pp. 998-1004 ◽  
Author(s):  
X. CUI ◽  
T. LEI ◽  
D. Y. YANG ◽  
P. HAO ◽  
Q. LIU
Keyword(s):  
Neospora Caninum ◽  
Polyclonal Antibodies ◽  
Functional Protein ◽  
Reading Frame ◽  
Protein Motifs ◽  
Apicomplexan Parasites ◽  
Potential Vaccine ◽  
Palmitoylation Site

SUMMARYImmune mapped protein 1 (IMP1) is a newly discovered protein in Eimeria maxima. It is recognized as a potential vaccine candidate against E. maxima and a highly conserved protein in apicomplexan parasites. Although the Neospora caninum IMP1 (NcIMP1) orthologue of E. maxima IMP1 was predicted in the N. caninum genome, it was still not identified and characterized. In this study, cDNA sequence encoding NcIMP1 was cloned by RT-PCR from RNA isolated from Nc1 tachyzoites. NcIMP1 was encoded by an open reading frame of 1182 bp, which encoded a protein of 393 amino acids with a predicted molecular weight of 42·9 kDa. Sequence analysis showed that there was neither a signal peptide nor a transmembrane region present in the NcIMP1 amino acid sequence. However, several kinds of functional protein motifs, including an N-myristoylation site and a palmitoylation site were predicted. Recombinant NcIMP1 (rNcIMP1) was expressed in Escherichia coli and then purified rNcIMP1 was used to prepare specific antisera in mice. Mouse polyclonal antibodies raised against the rNcIMP1 recognized an approximate 43 kDa native IMP1 protein. Immunofluorescence analysis showed that NcIMP1 was localized on the membrane of N. caninum tachyzoites. The N-myristoylation site and the palmitoylation site were found to contribute to the localization of NcIMP1. Furthermore, the rNcIMP1-specific antibodies could inhibit cell invasion by N. caninum tachyzoites in vitro. All the results indicate that NcIMP1 is likely to be a membrane protein of N. caninum and may be involved in parasite invasion.

Characterization of the open reading frame 7a from Bombyx mori nucleopolyhedrovirus

2012 ◽  
Vol 40 (2) ◽  
pp. 865-873
Author(s):  
Yong Liu ◽  
Feng Yu ◽  
Huiling Wu ◽  
Qing Cao ◽  
Yu Wu ◽  
...  
Keyword(s):  
Bombyx Mori ◽  
Open Reading Frame ◽  
Bombyx Mori Nucleopolyhedrovirus ◽  
Reading Frame
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