Generation of various amino acids mutants in the trpR gene of Escherichia coli by site-directed mutagenesis

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.

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

Biochemical Journal ◽

10.1042/bj3020291 ◽

1994 ◽

Vol 302 (1) ◽

pp. 291-295 ◽

Cited By ~ 25

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.

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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

Journal of Bacteriology ◽

10.1128/jb.183.6.2137-2140.2001 ◽

2001 ◽

Vol 183 (6) ◽

pp. 2137-2140 ◽

Cited By ~ 42

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.

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

Biochemical Journal ◽

10.1042/bj20030261 ◽

2003 ◽

Vol 373 (2) ◽

pp. 539-545 ◽

Cited By ~ 3

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.

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