scholarly journals Molecular cloning, expression and site-directed mutagenesis of glutathione S-transferase from Ochrobactrum anthropi

1998 ◽  
Vol 335 (3) ◽  
pp. 573-579 ◽  
Author(s):  
Bartolo FAVALORO ◽  
Antonio TAMBURRO ◽  
Stefania ANGELUCCI ◽  
Antonella DE LUCA ◽  
Sonia MELINO ◽  
...  
Keyword(s):  
Amino Acid ◽  
Cumene Hydroperoxide ◽  
Data Bank ◽  
Site Directed Mutagenesis ◽  
Ochrobactrum Anthropi ◽  
Serine Residue ◽  
Glutathione S Transferase ◽  
Calculated Molecular Mass ◽  
Gst Activity

The gene coding for a novel glutathione S-transferase (GST) has been isolated from the bacterium Ochrobactrum anthropi. A PCR fragment of 230 bp was obtained using oligonucleotide primers deduced from N-terminal and ‘internal ’ sequences of the purified enzyme. The gene was obtained by screening of a genomic DNA partial library from O. anthropi constructed in pBluescript with a PCR fragment probe. The gene encodes a protein (OaGST) of 201 amino acids with a calculated molecular mass of 21738 Da. The product of the gene was expressed and characterized; it showed GST activity with substrates 1-chloro-2,4-dinitrobenzene (CDNB), p-nitrobenzyl chloride and 4-nitroquinoline 1-oxide, and glutathione-dependent peroxidase activity towards cumene hydroperoxide. The overexpressed product of the gene was also confirmed to have in vivo GST activity towards CDNB. The interaction of the recombinant GST with several antibiotics indicated that the enzyme is involved in the binding of rifamycin and tetracycline. The OaGST amino acid sequence showed the greatest identity (45%) with a GST from Pseudomonas sp. strain LB400. A serine residue in the N-terminal region is conserved in almost all known bacterial GSTs, and it appears to be the counterpart of the catalytic serine residue present in Theta-class GSTs. Substitution of the Ser-11 residue resulted in a mutant OaGST protein lacking CDNB-conjugating activity; moreover the mutant enzyme was not able to bind Sepharose–GSH affinity matrices. The amino acid and nucleotide sequences reported in this paper have been submitted to the EMBL Data Bank with the accession number Y17279.

scholarly journals Characterization of Resistance to the Nonnucleoside NS5B Inhibitor Filibuvir in Hepatitis C Virus-Infected Patients

2011 ◽  
Vol 56 (3) ◽  
pp. 1331-1341 ◽  
Author(s):  
Philip J. F. Troke ◽  
Marilyn Lewis ◽  
Paul Simpson ◽  
Katrina Gore ◽  
Jennifer Hammond ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Amino Acid ◽  
Site Directed Mutagenesis ◽  
Phenotypic Resistance ◽  
Number Of Patients ◽  
Chronic Hcv ◽  
Selection Of

ABSTRACTFilibuvir (PF-00868554) is an investigational nonnucleoside inhibitor of the hepatitis C virus (HCV) nonstructural 5B (NS5B) RNA-dependent RNA polymerase currently in development for treating chronic HCV infection. The aim of this study was to characterize the selection of filibuvir-resistant variants in HCV-infected individuals receiving filibuvir as short (3- to 10-day) monotherapy. We identified amino acid M423 as the primary site of mutation arising upon filibuvir dosing. Through bulk cloning of clinical NS5B sequences into a transient-replicon system, and supported by site-directed mutagenesis of the Con1 replicon, we confirmed that mutations M423I/T/V mediate phenotypic resistance. Selection in patients of an NS5B mutation at M423 was associated with a reduced replicative capacityin vitrorelative to the pretherapy sequence; consistent with this, reversion to wild-type M423 was observed in the majority of patients following therapy cessation. Mutations at NS5B residues R422 and M426 were detected in a small number of patients at baseline or the end of therapy and also mediate reductions in filibuvir susceptibility, suggesting these are rare but clinically relevant alternative resistance pathways. Amino acid variants at position M423 in HCV NS5B polymerase are the preferred pathway for selection of viral resistance to filibuvirin vivo.

scholarly journals Identification and Analysis of Genes Involved in Anaerobic Toluene Metabolism by Strain T1: Putative Role of a Glycine Free Radical

1998 ◽  
Vol 64 (5) ◽  
pp. 1650-1656 ◽  
Author(s):  
Peter W. Coschigano ◽  
Thomas S. Wehrman ◽  
L. Y. Young
Keyword(s):  
Free Radical ◽  
Amino Acid ◽  
Molecular Mass ◽  
Cysteine Residue ◽  
Open Reading Frames ◽  
Site Directed Mutagenesis ◽  
Pyruvate Formate Lyase ◽  
Calculated Molecular Mass ◽  
Acid Protein ◽  
Amino Acid Protein

ABSTRACT The denitrifying strain T1 is able to grow with toluene serving as its sole carbon source. Two mutants which have defects in this toluene utilization pathway have been characterized. A clone has been isolated, and subclones which contain tutD and tutE, two genes in the T1 toluene metabolic pathway, have been generated. ThetutD gene codes for an 864-amino-acid protein with a calculated molecular mass of 97,600 Da. The tutE gene codes for a 375-amino-acid protein with a calculated molecular mass of 41,300 Da. Two additional small open reading frames have been identified, but their role is not known. The TutE protein has homology to pyruvate formate-lyase activating enzymes. The TutD protein has homology to pyruvate formate-lyase enzymes, including a conserved cysteine residue at the active site and a conserved glycine residue that is activated to a free radical in this enzyme. Site-directed mutagenesis of these two conserved amino acids shows that they are also essential for the function of TutD.

scholarly journals Characterization of a glutathione S-transferase and a related glutathione-binding protein from gill of the blue mussel, Mytilus edulis

1995 ◽  
Vol 305 (1) ◽  
pp. 145-150 ◽  
Author(s):  
P J Fitzpatrick ◽  
T O B Krag ◽  
P Højrup ◽  
D Sheehan
Keyword(s):  
Amino Acid ◽  
Mytilus Edulis ◽  
Blue Mussel ◽  
Binding Protein ◽  
Protein A ◽  
Gel Filtration ◽  
Cumene Hydroperoxide ◽  
Gill Tissue ◽  
Glutathione S Transferase ◽  
Sequencing Studies

The major isoenzyme of glutathione S-transferase (GST 1) was purified to homogeneity from cytosolic extracts of Mytilus edulis gill tissue by GSH-agarose affinity chromatography followed by Mono Q ion-exchange f.p.l.c. This enzyme was particularly active with 1-chloro-2,4-dinitrobenzene, ethacrynic acid and cumene hydroperoxide as substrates. Immunoblotting and amino acid sequencing studies indicate that the enzyme belongs to the Pi class of GSTs. A related protein which binds to GSH-agarose was also purified. This GSH-binding protein did not immunoblot with GST antisera and showed no detectable catalytic activity with GST substrates although its N-terminal sequence was similar to Mu-class GSTs. Gel-filtration chromatography indicated that GST 1 is a dimer and the GSH-binding protein a monomer. Mass spectrometry and SDS/PAGE indicate subunit molecular masses of 24 kDa (GST 1) and 25 kDa (GSH-binding protein), respectively. Both proteins have amino acid compositions typical of GSTs.

scholarly journals Characterization of a Thermoacidophilic l-Arabinose Isomerase from Alicyclobacillus acidocaldarius: Role of Lys-269 in pH Optimum

2005 ◽  
Vol 71 (12) ◽  
pp. 7888-7896 ◽  
Author(s):  
Sang-Jae Lee ◽  
Dong-Woo Lee ◽  
Eun-Ah Choe ◽  
Young-Ho Hong ◽  
Seong-Bo Kim ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequences ◽  
Site Directed Mutagenesis ◽  
Bacillus Halodurans ◽  
Wild Type ◽  
Ph Optimum ◽  
Calculated Molecular Mass ◽  
Alicyclobacillus Acidocaldarius ◽  
Arabinose Isomerase

ABSTRACT The araA gene encoding l-arabinose isomerase (AI) from the thermoacidophilic bacterium Alicyclobacillus acidocaldarius was cloned, sequenced, and expressed in Escherichia coli. Analysis of the sequence revealed that the open reading frame of the araA gene consists of 1,491 bp that encodes a protein of 497 amino acid residues with a calculated molecular mass of 56,043 Da. Comparison of the deduced amino acid sequence of A. acidocaldarius AI (AAAI) with other AIs demonstrated that AAAI has 97% and 66% identities (99% and 83% similarities) to Geobacillus stearothermophilus AI (GSAI) and Bacillus halodurans AI (BHAI), respectively. The recombinant AAAI was purified to homogeneity by heat treatment, ion-exchange chromatography, and gel filtration. The purified enzyme showed maximal activity at pH 6.0 to 6.5 and 65°C under the assay conditions used, and it required divalent cations such as Mn2+, Co2+, and Mg2+ for its activity. The isoelectric point (pI) of the enzyme was about 5.0 (calculated pI of 5.5). The apparent Km values of the recombinant AAAI for l-arabinose and d-galactose were 48.0 mM (V max, 35.5 U/mg) and 129 mM (V max, 7.5 U/mg), respectively, at pH 6 and 65°C. Interestingly, although the biochemical properties of AAAI are quite similar to those of GSAI and BHAI, the three AIs from A. acidocaldarius (pH 6), G. stearothermophilus (pH 7), and B. halodurans (pH 8) exhibited different pH activity profiles. Based on alignment of the amino acid sequences of these homologous AIs, we propose that the Lys-269 residue of AAAI may be responsible for the ability of the enzyme to act at low pH. To verify the role of Lys-269, we prepared the mutants AAAI-K269E and BHAI-E268K by site-directed mutagenesis and compared their kinetic parameters with those of wild-type AIs at various pHs. The pH optima of both AAAI-K269E and BHAI-E268K were rendered by 1.0 units (pH 6 to 7 and 8 to 7, respectively) compared to the wild-type enzymes. In addition, the catalytic efficiency (k cat/Km ) of each mutant at different pHs was significantly affected by an increase or decrease in V max. From these results, we propose that the position corresponding to the Lys-269 residue of AAAI could play an important role in the determination of the pH optima of homologous AIs.

Site-directed mutagenesis of the cysteinyl residues and the active-site serine residue of bacterial D-amino acid transaminase

Biochemistry ◽  
1989 ◽  
Vol 28 (2) ◽  
pp. 505-509 ◽  
Author(s):  
M. Merola ◽  
A. Martinez del Pozo ◽  
H. Ueno ◽  
P. Recsei ◽  
A. Di Donato ◽  
...  
Keyword(s):  
Amino Acid ◽  
Active Site ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Serine Residue

scholarly journals Additional Determinants within Escherichia coli FNR Activating Region 1 and RNA Polymerase α Subunit Required for Transcription Activation

2005 ◽  
Vol 187 (5) ◽  
pp. 1724-1731 ◽  
Author(s):  
K. Derek Weber ◽  
Owen D. Vincent ◽  
Patricia J. Kiley
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Rna Polymerase ◽  
Class Ii ◽  
Site Directed Mutagenesis ◽  
Class I ◽  
Side Chains ◽  
Alanine Scanning Mutagenesis ◽  
Α Subunit

ABSTRACT The global anaerobic regulator FNR is a DNA binding protein that activates transcription of genes required for anaerobic metabolism in Escherichia coli through interactions with RNA polymerase (RNAP). Alanine-scanning mutagenesis of FNR amino acid residues 181 to 193 of FNR was utilized to determine which amino acid side chains are required for transcription of both class II and class I promoters. In vivo assays of FNR function demonstrated that a core of residues (F181, R184, S187, and R189) was required for efficient activation of class II promoters, while at a class I promoter, FF(−61.5), only S187 and R189 were critical for FNR activation. Site-directed mutagenesis of positions 184, 187, and 189 revealed that the positive charge contributes to the function of the side chain at positions 184 and 189 while the serine hydroxyl is critical for the function of position 187. Subsequent analysis of the carboxy-terminal domain of the α subunit (αCTD) of RNAP, using an alanine library in single copy, revealed that in addition to previously characterized side chains (D305, R317, and L318), E286 and E288 contributed to FNR activation of both class II and class I promoters, suggesting that αCTD region 285 to 288 also participates in activation by FNR. In conclusion, this study demonstrates that multiple side chains within region 181 to 192 are required for FNR activation and the surface of αCTD required for FNR activation is more extensive than previously observed.

scholarly journals On the catalytic mechanism of prokaryotic leader peptidase 1

1992 ◽  
Vol 282 (2) ◽  
pp. 539-543 ◽  
Author(s):  
M T Black ◽  
J G R Munn ◽  
A E Allsop
Keyword(s):  
Catalytic Mechanism ◽  
Serine Proteinase ◽  
Site Directed Mutagenesis ◽  
Cysteine Residues ◽  
Calculated Molecular Mass ◽  
E Coli ◽  
Leader Peptidase ◽  
Mutant Forms ◽  
Thiol Proteinases

The catalytic mechanism of leader peptidase 1 (LP1) of the bacterium Escherichia coli has been investigated by a combination of site-directed mutagenesis, assays of enzyme activity in vivo utilizing a strain of E. coli which has a conditional defect in LP1 activity, and gene cloning. The biological activity of mutant forms of E. coli LP1 demonstrates that this enzyme belongs to a novel class of proteinases. The possibility that LP1 may be an aspartyl proteinase has been excluded on the basis of primary sequence comparison and mutagenesis. Assignment of LP1 to one of the other three recognized classes of proteinases (metalloproteinases, thiol proteinases and the classical serine proteinases) can also be excluded, as it is clearly demonstrated that none of the histidine or cysteine residues within LP1 are required for catalytic activity. The Pseudomonas fluorescens lep gene has been cloned and sequenced and the corresponding amino acid sequence compared with that of E. coli LP1. The E. coli LP1 and P. fluorescens LP1 primary sequences are 50% identical after insertion of gaps. The P. fluorescens LP1 has 39 fewer amino acids, a calculated molecular mass of 31903 Da and functions effectively in vivo in E. coli. None of the cysteine residues and only one of the histidine residues which are present in E. coli LP1 are conserved in sequence position in the P. fluorescens LP1 enzyme. The possibility that LP1 is a novel type of serine proteinase is discussed.

scholarly journals Mu-class glutathione transferase from Xenopus laevis: molecular cloning, expression and site-directed mutagenesis

2002 ◽  
Vol 365 (3) ◽  
pp. 685-691 ◽  
Author(s):  
Antonella De LUCA ◽  
Bartolo FAVALORO ◽  
Stefania ANGELUCCI ◽  
Paolo SACCHETTA ◽  
Carmine Di ILIO
Keyword(s):  
Xenopus Laevis ◽  
Catalytic Mechanism ◽  
Glutathione Transferase ◽  
Structural Instability ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Calculated Molecular Mass

A cDNA encoding a Mu-class glutathione transferase (XlGSTM1-1) has been isolated from a Xenopus laevis liver library, and its nucleotide sequence has been determined. XlGSTM1-1 is composed of 219 amino acid residues with a calculated molecular mass of 25359Da. Unlike many mammalian Mu-class GSTs, XlGSTM1-1 has a narrow spectrum of substrate specificity and it is also less effective in conjugating 1-chloro-2,4-dinitrobenzene. A notable structural feature of XlGSTM1-1 is the presence of the Cys-139 residue in place of the Glu-139, as well as the absence of the Cys-114 residue, present in other Mu-class GSTs, which is replaced by Ala. Site-directed mutagenesis experiments indicate that Cys-139 is not involved in the catalytic mechanism of XlGSTM1-1 but may be in part responsible for its structural instability, and experiments in vivo confirmed the role of this residue in stability. Evidence indicating that Arg-107 is essential for the 1-chloro-2,4-dinitrobenzene conjugation capacity of XlGSTM1-1 is also presented.

scholarly journals Identification and properties of the Deinococcus grandis and Deinococcus proteolyticus single-stranded DNA binding proteins (SSB).

2007 ◽  
Vol 54 (1) ◽  
pp. 79-87 ◽  
Author(s):  
Paweł Filipkowski ◽  
Józef Kur
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Amino Acid Sequence ◽  
Deinococcus Radiodurans ◽  
Thermus Thermophilus ◽  
Biochemical Properties ◽  
Thermus Aquaticus ◽  
Single Stranded Dna ◽  
Calculated Molecular Mass

To study the biochemical properties of SSB's from Deinococcus grandis (DgrSSB) and Deinococcus proteolyticus (DprSSB), we have cloned the ssb genes obtained by PCR and have developed Escherichia coli overexpression systems. The genes consist of an open reading frame of 891 (DgrSSB) and 876 (DprSSB) nucleotides encoding proteins of 296 and 291 amino acids with a calculated molecular mass of 32.29 and 31.33 kDa, respectively. The amino-acid sequence of DgrSSB exhibits 45%, 44% and 82% identity and the amino-acid sequence of DprSSB reveals 43%, 43% and 69% identity with Thermus aquaticus (TaqSSB), Thermus thermophilus (TthSSB) and Deinococcus radiodurans SSBs, respectively. We show that DgrSSB and DprSSB are similar to Thermus/Deinococcus SSBs in their biochemical properties. They are functional as homodimers, with each monomer encoding two single-stranded DNA binding domains (OB-folds). In fluorescence titrations with poly(dT), both proteins bind single-stranded DNA with a binding site size of about 33 nt per homodimer. In a complementation assay in E. coli, DgrSSB and DprSSB took over the in vivo function of EcoSSB. Thermostability with half-lives of about 1 min at 65-67.5 degrees C make DgrSSB and DprSSB similar to the known SSB of Deinococcus radiodurans (DraSSB).

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