scholarly journals A novel amphibian Pi-class glutathione transferase isoenzyme from Xenopus laevis: importance of phenylalanine 111 in the H-site

2003 ◽  
Vol 373 (2) ◽  
pp. 539-545 ◽  
Author(s):  
Antonella DE LUCA ◽  
Bartolo FAVALORO ◽  
Erminia CARLETTI ◽  
Paolo SACCHETTA ◽  
Carmine DI ILIO
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Xenopus Laevis ◽  
Cdna Clone ◽  
Cellular Proliferation ◽  
Glutathione Transferase ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Calculated Molecular Mass ◽  
Specific Activities

Screening of a liver tumour cDNA library from Xenopus laevis resulted in the isolation of a full-length cDNA clone encoding a novel Pi-class amphibian glutathione transferase (GST) isoenzyme (designated as XlGSTP1-1). The gene encodes a protein of 212 amino acids with a calculated molecular mass of 24428 Da. The product of the gene has been overexpressed in Escherichia coli and characterized. XlGSTP1-1 has one of the highest specific activities towards 1-chloro-2,4-dinitrobenzene (1310 μmol/min per mg of protein) obtained with any GST. A notable feature of XlGSTP1-1 is the presence in the H-site of Phe111 and Pro208 in place of tyrosine and glycine residues respectively, present in other mammalian Pi-class GSTs. Site-directed mutagenesis indicate that Phe111 is involved in substrate specificity of XlGSTP1-1. We provide evidence showing that XlGSTP1-1 is present only in the embryo and its expression might be associated with cellular proliferation.

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 Site-Directed Mutagenesis Analysis of Amino Acids Critical for Activity of the Type I Signal Peptidase of the Archaeon Methanococcus voltae

2005 ◽  
Vol 187 (3) ◽  
pp. 1188-1191 ◽  
Author(s):  
Sonia L. Bardy ◽  
Sandy Y. M. Ng ◽  
David S. Carnegie ◽  
Ken F. Jarrell
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Aspartic Acid ◽  
Site Directed Mutagenesis ◽  
Signal Peptidase ◽  
Directed Mutagenesis ◽  
Type I ◽  
Aspartic Acid Residue ◽  
Conserved Residues ◽  
Methanococcus Voltae

ABSTRACT Site-directed mutagenesis studies of the signal peptidase of the methanogenic archaeon Methanococcus voltae identified three conserved residues (Ser52, His122, and Asp148) critical for activity. The requirement for one conserved aspartic acid residue distinguishes the archaeal enzyme from both the Escherichia coli and yeast Sec11 enzymes.

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

2003 ◽  
Vol 419 (2) ◽  
pp. 214-221 ◽  
Author(s):  
Erminia Carletti ◽  
Antonella De Luca ◽  
Andrea Urbani ◽  
Paolo Sacchetta ◽  
Carmine Di Ilio
Keyword(s):  
Xenopus Laevis ◽  
Molecular Cloning ◽  
Glutathione Transferase ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis

scholarly journals Identification of two acidic residues involved in the catalysis of xylanase A from Streptomyces lividans

1994 ◽  
Vol 302 (1) ◽  
pp. 291-295 ◽  
Author(s):  
A Moreau ◽  
M Roberge ◽  
C Manin ◽  
F Shareck ◽  
D Kluepfel ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Enzyme Activity ◽  
Streptomyces Lividans ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Protein Mutation ◽  
Acidic Residues

On the basis of similarities between known xylanase sequences of the F family, three invariant acidic residues of xylanase A from Streptomyces lividans were investigated. Site-directed-mutagenesis experiments were carried out in Escherichia coli after engineering the xylanase A gene to allow its expression. Replacement of Glu-128 or Glu-236 by their isosteric form (Gln) completely abolished enzyme activity with xylan and p-nitrophenyl beta-D-cellobioside, indicating that the two substrates are hydrolysed at the same site. These two amino acids probably represent the catalytic residues. Immunological studies, which showed that the two mutants retained the same epitopes, indicate that the lack of activity is the result of the mutation rather than misfolding of the protein. Mutation D124E did not affect the kinetic parameters with xylan as substrate, but D124N reduced the Km 16-fold and the Vmax. 14-fold when compared with the wild-type enzyme. The mutations had a more pronounced effect with p-nitrophenyl beta-D-cellobioside as the substrate. Mutation D124E increased the Km and decreased the Vmax. 5-fold each, while D124N reduced the Km 4.5-fold and the Vmax. 75-fold. The mutations had no effect on the cleavage mode of xylopentaose.

scholarly journals Construction and Characterization of Mutations at Codon 751 of the Escherichia coli gyrB Gene That Confer Resistance to the Antimicrobial Peptide Microcin B17 and Alter the Activity of DNA Gyrase

2001 ◽  
Vol 183 (6) ◽  
pp. 2137-2140 ◽  
Author(s):  
Francisco J. del Castillo ◽  
Ignacio del Castillo ◽  
Felipe Moreno
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Dna Gyrase ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Peptide Antibiotic ◽  
Resistant Mutants ◽  
Different Levels ◽  
Microcin B17

ABSTRACT Microcin B17 is a peptide antibiotic that inhibits DNA replication in Escherichia coli by targeting DNA gyrase. Previously, two independently isolated microcin B17-resistant mutants were shown to harbor the same gyrB point mutation that results in the replacement of tryptophan 751 by arginine in the GyrB polypeptide. We used site-directed mutagenesis to construct mutants in which tryptophan 751 was deleted or replaced by other amino acids. These mutants exhibit altered DNA gyrase activity and different levels of resistance to microcin B17.

Sign in / Sign up

Export Citation Format

Share Document