Characterization of the Vibrio alginolyticusfur Gene and Localization of Essential Amino Acid Sites in Fur by Site-Directed Mutagenesis

2007 ◽  
Vol 13 (1-3) ◽  
pp. 15-21 ◽  
Author(s):  
Qin Liu ◽  
Pengbo Wang ◽  
Yue Ma ◽  
Yuanxing Zhang
Keyword(s):  
Amino Acid ◽  
Essential Amino Acid ◽  
Acid Sites ◽  
Vibrio Alginolyticus ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
I Gene

scholarly journals Directed evolution and structural analysis of N-carbamoyl-D-amino acid amidohydrolase provide insights into recombinant protein solubility in Escherichia coli

2007 ◽  
Vol 402 (3) ◽  
pp. 429-437 ◽  
Author(s):  
Shimin Jiang ◽  
Chunhong Li ◽  
Weiwen Zhang ◽  
Yuanheng Cai ◽  
Yunliu Yang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Recombinant Protein ◽  
Protein Solubility ◽  
Acid Sites ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Triple Mutant ◽  
E Coli ◽  
Acid Amidohydrolase

One of the greatest bottlenecks in producing recombinant proteins in Escherichia coli is that over-expressed target proteins are mostly present in an insoluble form without any biological activity. DCase (N-carbamoyl-D-amino acid amidohydrolase) is an important enzyme involved in semi-synthesis of β-lactam antibiotics in industry. In the present study, in order to determine the amino acid sites responsible for solubility of DCase, error-prone PCR and DNA shuffling techniques were applied to randomly mutate its coding sequence, followed by an efficient screening based on structural complementation. Several mutants of DCase with reduced aggregation were isolated. Solubility tests of these and several other mutants generated by site-directed mutagenesis indicated that three amino acid residues of DCase (Ala18, Tyr30 and Lys34) are involved in its protein solubility. In silico structural modelling analyses suggest further that hydrophilicity and/or negative charge at these three residues may be responsible for the increased solubility of DCase proteins in E. coli. Based on this information, multiple engineering designated mutants were constructed by site-directed mutagenesis, among them a triple mutant A18T/Y30N/K34E (named DCase-M3) could be overexpressed in E. coli and up to 80% of it was soluble. DCase-M3 was purified to homogeneity and a comparative analysis with wild-type DCase demonstrated that DCase-M3 enzyme was similar to the native DCase in terms of its kinetic and thermodynamic properties. The present study provides new insights into recombinant protein solubility in E. coli.

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 The 2-Aminoethylphosphonate-Specific Transaminase of the 2-Aminoethylphosphonate Degradation Pathway

2002 ◽  
Vol 184 (15) ◽  
pp. 4134-4140 ◽  
Author(s):  
Alexander D. Kim ◽  
Angela S. Baker ◽  
Debra Dunaway-Mariano ◽  
W. W. Metcalf ◽  
B. L. Wanner ◽  
...  
Keyword(s):  
Amino Acid ◽  
Physiological Role ◽  
Degradation Pathway ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Purification And Characterization ◽  
Ph Optimum ◽  
Serovar Typhimurium ◽  
High Degree

ABSTRACT The 2-aminoethylphosphonate transaminase (AEPT; the phnW gene product) of the Salmonella enterica serovar Typhimurium 2-aminoethylphosphonate (AEP) degradation pathway catalyzes the reversible reaction of AEP and pyruvate to form phosphonoacetaldehyde (P-Ald) and l-alanine (l-Ala). Here, we describe the purification and characterization of recombinant AEPT. pH rate profiles (log Vm and log Vm /Km versus pH) revealed a pH optimum of 8.5. At pH 8.5, K eq is equal to 0.5 and the k cat values of the forward and reverse reactions are 7 and 9 s−1, respectively. The Km for AEP is 1.11 ± 0.03 mM; for pyruvate it is 0.15 ± 0.02 mM, for P-Ald it is 0.09 ± 0.01 mM, and for l-Ala it is 1.4 ± 0.03 mM. Substrate specificity tests revealed a high degree of discrimination, indicating a singular physiological role for the transaminase in AEP degradation. The 40-kDa subunit of the homodimeric enzyme is homologous to other members of the pyridoxalphosphate-dependent amino acid transaminase superfamily. Catalytic residues conserved within well-characterized members are also conserved within the seven known AEPT sequences. Site-directed mutagenesis demonstrated the importance of three selected residues (Asp168, Lys194, and Arg340) in AEPT catalysis.

Chitosanase from Streptomyces sp. strain N174: a comparative review of its structure and function

1997 ◽  
Vol 75 (6) ◽  
pp. 687-696 ◽  
Author(s):  
Tamo Fukamizo ◽  
Ryszard Brzezinski
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Substrate Binding ◽  
Structure And Function ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Streptomyces Sp ◽  
Binding Cleft ◽  
And Function

Novel information on the structure and function of chitosanase, which hydrolyzes the beta -1,4-glycosidic linkage of chitosan, has accumulated in recent years. The cloning of the chitosanase gene from Streptomyces sp. strain N174 and the establishment of an efficient expression system using Streptomyces lividans TK24 have contributed to these advances. Amino acid sequence comparisons of the chitosanases that have been sequenced to date revealed a significant homology in the N-terminal module. From energy minimization based on the X-ray crystal structure of Streptomyces sp. strain N174 chitosanase, the substrate binding cleft of this enzyme was estimated to be composed of six monosaccharide binding subsites. The hydrolytic reaction takes place at the center of the binding cleft with an inverting mechanism. Site-directed mutagenesis of the carboxylic amino acid residues that are conserved revealed that Glu-22 and Asp-40 are the catalytic residues. The tryptophan residues in the chitosanase do not participate directly in the substrate binding but stabilize the protein structure by interacting with hydrophobic and carboxylic side chains of the other amino acid residues. Structural and functional similarities were found between chitosanase, barley chitinase, bacteriophage T4 lysozyme, and goose egg white lysozyme, even though these proteins share no sequence similarities. This information can be helpful for the design of new chitinolytic enzymes that can be applied to carbohydrate engineering, biological control of phytopathogens, and other fields including chitinous polysaccharide degradation. Key words: chitosanase, amino acid sequence, overexpression system, reaction mechanism, site-directed mutagenesis.

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