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.

scholarly journals Characterization of the σ-Pore in Mutant hKv1.3 Potassium Channels

2018 ◽  
Vol 46 (3) ◽  
pp. 1112-1121
Author(s):  
Pavel Tyutyaev ◽  
Stephan Grissmer
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Double Mutant ◽  
Chloride Ions ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Potential Range ◽  
Peptide Toxins ◽  
Ion Conducting

Background/Aims: The replacement of the amino acid valine at position 388 (Shaker position 438) in hKv1.3 channels or at the homologue position 370 in hKv1.2 channels resulted in a channel with two different ion conducting pathways: One pathway was the central, potassium-selective α-pore, that was sensitive to block by peptide toxins (CTX or KTX in the hKv1.3_V388C channel and CTX or MTX in the hKv1.2_V370C channel). The other pathway (σ-pore) was behind the central α-pore creating an inward current at potentials more negative than -100 mV, a potential range where the central α-pore was closed. In addition, current through the σ-pore could not be reduced by CTX, KTX or MTX in the hKv1.3_V388C or the hKv1.2_V370C channel, respectively. Methods: For a more detailed characterization of the σ-pore, we created a trimer consisting of three hKv1.3_V388C α-subunits linked together and characterized current through this trimeric hKv1.3_V388C channel. Additionally, we determined which amino acids line the σ-pore in the tetrameric hKv1.3_V388C channel by replacing single amino acids in the tetrameric hKv1.3_V388C mutant channel that could be involved in σ-pore formation. Results: Overexpression of the trimeric hKv1.3_V388C channel in COS-7 cells yielded typical σ-pore currents at potentials more negative than -100 mV similar to what was observed for the tetrameric hKv1.3_V388C channel. Electrophysiological properties of the trimeric and tetrameric channel were similar: currents could be observed at potentials more negative than -100 mV, were not carried by protons or chloride ions, and could not be reduced by peptide toxins (CTX, MTX) or TEA. The σ-pore was mostly permeable to Na+ and Li+. In addition, in our site-directed mutagenesis experiments, we created a number of new double mutant channels in the tetrameric hKv1.3_V388C background channel. Two of these tetrameric double mutant channels (hKv1.3_V388C_T392Y and hKv1.3_V388C_Y395W) did not show currents through the σ-pore. Conclusions: From our experiments with the trimeric hKv1.3_V388C channel we conclude that the σ-pore exists in hKv1.3_V388C channels independently of the α-pore. From our site-directed mutagenesis experiments in the tetrameric hKv1.3_V388C channel we conclude that amino acid position 392 and 395 (Shaker position 442 and 445) line the σ-pore.

scholarly journals Characterization of Alginate Lyase from Pseudomonas syringae pv. syringae

2000 ◽  
Vol 182 (21) ◽  
pp. 6268-6271 ◽  
Author(s):  
Lori A. Preston ◽  
T. Y. Wong ◽  
Carol L. Bender ◽  
Neal L. Schiller
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Syringae ◽  
Alginate Lyase ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Gene Encoding ◽  
Tryptophan Residues ◽  
Lyase Activity ◽  
Alginate Lyases

ABSTRACT The gene encoding alginate lyase (algL) inPseudomonas syringae pv. syringae was cloned, sequenced, and overexpressed in Escherichia coli. Alginate lyase activity was optimal when the pH was 7.0 and when assays were conducted at 42°C in the presence of 0.2 M NaCl. In substrate specificity studies, AlgL from P. syringae showed a preference for deacetylated polymannuronic acid. Sequence alignment with other alginate lyases revealed conserved regions within AlgL likely to be important for the structure and/or function of the enzyme. Site-directed mutagenesis of histidine and tryptophan residues at positions 204 and 207, respectively, indicated that these amino acids are critical for lyase activity.

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.

scholarly journals Substrate binding tunes the reactivity of hispidin 3-hydroxylase, a flavoprotein monooxygenase involved in fungal bioluminescence

2020 ◽  
Vol 295 (47) ◽  
pp. 16013-16022
Author(s):  
Yapei Tong ◽  
Milos Trajkovic ◽  
Simone Savino ◽  
Willem J. H. van Berkel ◽  
Marco W. Fraaije
Keyword(s):  
Escherichia Coli ◽  
Affinity Chromatography ◽  
Kinetic Analysis ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Cofactor Specificity ◽  
Class A ◽  
Modeled Structure ◽  
Aromatic Product

Fungal bioluminescence was recently shown to depend on a unique oxygen-dependent system of several enzymes. However, the identities of the enzymes did not reveal the full biochemical details of this process, as the enzymes do not bear resemblance to those of other luminescence systems, and thus the properties of the enzymes involved in this fascinating process are still unknown. Here, we describe the characterization of the penultimate enzyme in the pathway, hispidin 3-hydroxylase, from the luminescent fungus Mycena chlorophos (McH3H), which catalyzes the conversion of hispidin to 3-hydroxyhispidin. 3-Hydroxyhispidin acts as a luciferin substrate in luminescent fungi. McH3H was heterologously expressed in Escherichia coli and purified by affinity chromatography with a yield of 100 mg/liter. McH3H was found to be a single component monomeric NAD(P)H-dependent FAD-containing monooxygenase having a preference for NADPH. Through site-directed mutagenesis, based on a modeled structure, mutant enzymes were created that are more efficient with NADH. Except for identifying the residues that tune cofactor specificity, these engineered variants may also help in developing new hispidin-based bioluminescence applications. We confirmed that addition of hispidin to McH3H led to the formation of 3-hydroxyhispidin as sole aromatic product. Rapid kinetic analysis revealed that reduction of the flavin cofactor by NADPH is boosted by hispidin binding by nearly 100-fold. Similar to other class A flavoprotein hydroxylases, McH3H did not form a stable hydroperoxyflavin intermediate. These data suggest a mechanism by which the hydroxylase is tuned for converting hispidin into the fungal luciferin.

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