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 Site-directed mutagenesis at aspartate and glutamate residues of xylanase from Bacillus pumilus

1992 ◽  
Vol 288 (1) ◽  
pp. 117-121 ◽  
Author(s):  
E P Ko ◽  
H Akatsuka ◽  
H Moriyama ◽  
A Shinmyo ◽  
Y Hata ◽  
...  
Keyword(s):  
Amino Acids ◽  
Reaction Mechanism ◽  
Amino Acid ◽  
Bacillus Pumilus ◽  
Specific Activity ◽  
Sequence Similarity ◽  
Site Directed Mutagenesis ◽  
Dimensional Structure ◽  
Amino Acid Sequence Similarity ◽  
Directed Mutagenesis

To elucidate the reaction mechanism of xylanase, the identification of amino acids essential for its catalysis is of importance. Studies have indicated the possibility that the reaction mechanism of xylanase is similar to that of hen's egg lysozyme, which involves acidic amino acid residues. On the basis of this assumption, together with the three-dimensional structure of Bacillus pumilus xylanase and its amino acid sequence similarity to other xylanases of different origins, three acidic amino acids, namely Asp-21, Glu-93 and Glu-182, were selected for site-directed mutagenesis. The Asp residue was altered to either Ser or Glu, and the Glu residues to Ser or Asp. The purified mutant xylanases D21E, D21S, E93D, E93S, E182D and E182S showed single protein bands of about 26 kDa on SDS/PAGE. C.d. spectra of these mutant enzymes show no effect on the secondary structure of xylanase, except that of D21E, which shows a little variation. Furthermore, mutations of Glu-93 and Glu-182 resulted in a drastic decrease in the specific activity of xylanase as compared with mutation of Asp-21. On the basis of these results we propose that Glu-93 and Glu-182 are the best candidates for the essential catalytic residues of xylanase.

scholarly journals Introduction of H-2Dd determinants into the H-2Ld antigen by site-directed mutagenesis.

1987 ◽  
Vol 166 (3) ◽  
pp. 744-760 ◽  
Author(s):  
D Koeller ◽  
R Lieberman ◽  
J Miyazaki ◽  
E Appella ◽  
K Ozato ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Mhc Class I ◽  
Amino Acid Position ◽  
Amino Acid Sequences ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Antigenic Sites ◽  
Vesicular Stomatitis ◽  
Mhc Class I Antigen

We used site-directed mutagenesis to localize serologically defined (s) and CTL (c)-defined alloantigenic determinants to discrete amino acid sequences of a murine MHC class I antigen. Based on the prediction that amino acid position 63-73 of the H-2Dd antigen forms s-allodeterminants, the H-2Ld gene was mutated in a sequential fashion to replace codons for amino acid positions 63, 65, 66, 70, and 73 with those of the H-2Dd amino acids. Epitopes of the mutant antigens expressed in L-cells were examined by the binding of a series of mAbs specific for the H-2Dd antigen. The mutant antigen M66 had substitutions at residues 63, 65, and 66, and resulted in the acquisition of a number of H-2Dd-specific s-epitopes. Mutant M70 had an additional substitution at residue 70, which led to the gain of multiple additional H-2Dd s-epitopes. Together, more than half of all the relevant H-2Dd s-epitopes were mapped into amino acid position 63-70 of the H-2Dd molecule, which was expressed in the mutant H-2Ld gene. The final mutation at residue 73 (M73) caused no new epitope gains, rather, a few Dd s-epitopes acquired by the preceding mutations were lost. All of the H-2Ld-specific s-determinants were retained in the mutant molecules, as were H-2Dd s-determinants specific for the alpha-2 or alpha-3 domains. Changes of these residues affected c-determinants defined by CTL. Anti-H-2Dd CTL cultures and an anti-H-2Dd CTL clone recognized the mutant H-2Ld molecules, M66 and M70. Some CTL clones generated against the Q10d molecule, which has an identical sequence to H-2Dd between residues 61 and 73, failed to recognize native H-2Dd or Ld but did crossreact with mutant Ld. While bulk-cultured anti-H-2Ld CTL cultures reacted strongly against M73, bulk-cultured H-2Ld restricted anti-vesicular stomatitis virus CTL did not. Finally, at the clonal level two of three anti-H-2Ld CTL clones lost reactivity with some or all of these mutant molecules. From these results we conclude that a stretch of amino acids from position 63 to 70 of the alpha-1 domain controls major s- and c-antigenic sites on the H-2Dd antigen and c-sites on H-2Ld antigen.

scholarly journals Structure-Based Engineering of an Artificially Generated NADP+-Dependent d-Amino Acid Dehydrogenase

2017 ◽  
Vol 83 (11) ◽  
Author(s):  
Junji Hayashi ◽  
Tomonari Seto ◽  
Hironaga Akita ◽  
Masahiro Watanabe ◽  
Tamotsu Hoshino ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Substrate Specificity ◽  
Specific Activity ◽  
High Specific Activity ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
High Catalytic Activity ◽  
Acid Dehydrogenase ◽  
Amino Acid Dehydrogenase

ABSTRACT A stable NADP+-dependent d-amino acid dehydrogenase (DAADH) was recently created from Ureibacillus thermosphaericus meso-diaminopimelate dehydrogenase through site-directed mutagenesis. To produce a novel DAADH mutant with different substrate specificity, the crystal structure of apo-DAADH was determined at a resolution of 1.78 Å, and the amino acid residues responsible for the substrate specificity were evaluated using additional site-directed mutagenesis. By introducing a single D94A mutation, the enzyme's substrate specificity was dramatically altered; the mutant utilized d-phenylalanine as the most preferable substrate for oxidative deamination and had a specific activity of 5.33 μmol/min/mg at 50°C, which was 54-fold higher than that of the parent DAADH. In addition, the specific activities of the mutant toward d-leucine, d-norleucine, d-methionine, d-isoleucine, and d-tryptophan were much higher (6 to 25 times) than those of the parent enzyme. For reductive amination, the D94A mutant exhibited extremely high specific activity with phenylpyruvate (16.1 μmol/min/mg at 50°C). The structures of the D94A-Y224F double mutant in complex with NADP+ and in complex with both NADPH and 2-keto-6-aminocapronic acid (lysine oxo-analogue) were then determined at resolutions of 1.59 Å and 1.74 Å, respectively. The phenylpyruvate-binding model suggests that the D94A mutation prevents the substrate phenyl group from sterically clashing with the side chain of Asp94. A structural comparison suggests that both the enlarged substrate-binding pocket and enhanced hydrophobicity of the pocket are mainly responsible for the high reactivity of the D94A mutant toward the hydrophobic d-amino acids with bulky side chains. IMPORTANCE In recent years, the potential uses for d-amino acids as source materials for the industrial production of medicines, seasonings, and agrochemicals have been growing. To date, several methods have been used for the production of d-amino acids, but all include tedious steps. The use of NAD(P)+-dependent d-amino acid dehydrogenase (DAADH) makes single-step production of d-amino acids from oxo-acid analogs and ammonia possible. We recently succeeded in creating a stable DAADH and demonstrated that it is applicable for one-step synthesis of d-amino acids, such as d-leucine and d-isoleucine. As the next step, the creation of an enzyme exhibiting different substrate specificity and higher catalytic efficiency is a key to the further development of d-amino acid production. In this study, we succeeded in creating a novel mutant exhibiting extremely high catalytic activity for phenylpyruvate amination. Structural insight into the mutant will be useful for further improvement of DAADHs.

scholarly journals Identification of the Dimerization Domain of Dehalogenase IVa of Burkholderia cepacia MBA4

2000 ◽  
Vol 66 (8) ◽  
pp. 3180-3186 ◽  
Author(s):  
Jimmy S. H. Tsang ◽  
Benjamin C. M. Pang
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Burkholderia Cepacia ◽  
Recombinant Dna ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Dimerization Domain ◽  
Aggregation State ◽  
Catalytic Function ◽  
Recombinant Molecule

ABSTRACT Haloacid dehalogenases are enzymes that catalyze the hydrolytic removal of halogens from haloalkanoic acids. Dehalogenase IVa (DehIVa) from Burkholderia cepacia MBA4 and dehalogenase CI (DehCI) from Pseudomonas sp. strain CBS3 exhibit 68% identity. Despite their similarity DehIVa is a dimeric enzyme while DehCI is a monomer. In this work, we describe the identification of the domain that confers the dimerization function of DehIVa. Recombinant DNA molecules were constructed by fusion of the respective dehalogenase genes hdlIVa and dehCI. When amino acids 73 to 89 of DehCI were replaced by amino acids 74 to 90 of DehIVa, the recombinant molecule migrated like that of DehIVa in a nondenaturing activity-stained gel. Similarly, when residues 73 to 89 of DehIVa were replaced by the corresponding residues of DehCI, the chimera migrated as a monomer. These 17 amino acid changes were able to determine the aggregation states of the molecules. The retention of the catalytic function in these chimeras indicated that the overall folding of these proteins was not affected. Site-directed mutagenesis onhdlIVa however indicated that amino acids Phe58, Thr65, Leu78, and Phe92 of DehIVa are also important for the aggregation state of the protein. This indicates that the 17 residues are not sufficient for the dimerization of the protein.

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.

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 Amino Acids in Positions 48, 52, and 73 Differentiate the Substrate Specificities of the Highly Homologous Chlorocatechol 1,2-Dioxygenases CbnA and TcbC

2005 ◽  
Vol 187 (15) ◽  
pp. 5427-5436 ◽  
Author(s):  
Shenghao Liu ◽  
Naoto Ogawa ◽  
Toshiya Senda ◽  
Akira Hasebe ◽  
Kiyotaka Miyashita
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Ralstonia Eutropha ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Sequence Comparisons ◽  
Substrate Specificities ◽  
High Conservation ◽  
The Difference

ABSTRACT Chlorocatechol 1,2-dioxygenase (CCD) is the first-step enzyme of the chlorocatechol ortho-cleavage pathway, which plays a central role in the degradation of various chloroaromatic compounds. Two CCDs, CbnA from the 3-chlorobenzoate-degrader Ralstonia eutropha NH9 and TcbC from the 1,2,4-trichlorobenzene-degrader Pseudomonas sp. strain P51, are highly homologous, having only 12 different amino acid residues out of identical lengths of 251 amino acids. But CbnA and TcbC are different in substrate specificities against dichlorocatechols, favoring 3,5-dichlorocatechol (3,5-DC) and 3,4-dichlorocatechol (3,4-DC), respectively. A study of chimeric mutants constructed from the two CCDs indicated that the N-terminal parts of the enzymes were responsible for the difference in the substrate specificities. Site-directed mutagenesis studies further identified the amino acid in position 48 (Leu in CbnA and Val in TcbC) as critical in differentiating the substrate specificities of the enzymes, which agreed well with molecular modeling of the two enzymes. Mutagenesis studies also demonstrated that Ile-73 of CbnA and Ala-52 of TcbC were important for their high levels of activity towards 3,5-DC and 3,4-DC, respectively. The importance of Ile-73 for 3,5-DC specificity determination was also shown with other CCDs such as TfdC from Burkholderia sp. NK8 and TfdC from Alcaligenes sp. CSV90 (identical to TfdC from R. eutropha JMP134), which convert 3,5-DC preferentially. Together with amino acid sequence comparisons indicating high conservation of Leu-48 and Ile-73 among CCDs, these results suggested that TcbC of strain P51 had diverged from other CCDs to be adapted to conversion of 3,4-DC.

scholarly journals Asymmetry in catalysis: ‘unidirectional’ amino acid racemases

2021 ◽  
Vol 43 (1) ◽  
pp. 28-34
Author(s):  
Stephen L. Bearne
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Active Site ◽  
Site Directed Mutagenesis ◽  
Hydroxyl Group ◽  
Directed Mutagenesis ◽  
Biotechnological Applications ◽  
Enzyme Substrate ◽  
Brønsted Base ◽  
Catalytic Dyad

d-Amino acids play widespread structural, functional and regulatory roles in organisms. These d-amino acids often arise through the stereoinversion of the more plentiful l-amino acids catalysed by amino acid racemases and epimerases. Such enzymes are of interest since many are recognized targets for the development of drugs or may be employed industrially in biotransformation reactions. Despite their enzyme–substrate complexes being diastereomers, some racemases and epimerases exhibit a kinetic pseudo-symmetry, binding their enantiomeric or epimeric substrate pairs with roughly equal affinities and catalyzing their stereoinversion with similar turnover numbers. In other cases, this kinetic pseudo-symmetry is absent or may be ‘broken’ by substitution of a catalytic Cys by Ser at the active site of cofactor-independent racemases and epimerases, or by altering the Brønsted base of the catalytic dyad that facilitates deprotonation of the Cys residue. Moreover, a natural Thr-containing l-Asp/Glu racemase was discovered that catalyses ‘unidirectional’ substrate turnover, unlike the typical bidirectional racemases and epimerases. These observations suggest that bidirectional Cys–Cys racemases may be re-engineered into ‘unidirectional’ racemases through substitution of the thiol by a hydroxyl group. Catalysis by such ‘unidirectional’ racemase precursors could then be optimized further by site-directed mutagenesis and directed evolution to furnish useful enzymes for biotechnological applications.

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