scholarly journals Identification of Essential Amino Acid Residues of the NorM Na+/Multidrug Antiporter in Vibrio parahaemolyticus

2005 ◽  
Vol 187 (5) ◽  
pp. 1552-1558 ◽  
Author(s):  
Masato Otsuka ◽  
Makoto Yasuda ◽  
Yuji Morita ◽  
Chie Otsuka ◽  
Tomofusa Tsuchiya ◽  
...  
Keyword(s):  
Amino Acid ◽  
Ethidium Bromide ◽  
Vibrio Parahaemolyticus ◽  
Drug Transport ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Acidic Amino Acid ◽  
Wild Type ◽  
Transmembrane Region ◽  
Mutant Proteins

ABSTRACT NorM is a member of the multidrug and toxic compound extrusion (MATE) family and functions as a Na+/multidrug antiporter in Vibrio parahaemolyticus, although the underlying mechanism of the Na+/multidrug antiport is unknown. Acidic amino acid residues Asp32, Glu251, and Asp367 in the transmembrane region of NorM are conserved in one of the clusters of the MATE family. In this study, we investigated the role(s) of acidic amino acid residues Asp32, Glu251, and Asp367 in the transmembrane region of NorM by site-directed mutagenesis. Wild-type NorM and mutant proteins with amino acid replacements D32E (D32 to E), D32N, D32K, E251D, E251Q, D367A, D367E, D367N, and D367K were expressed and localized in the inner membrane of Escherichia coli KAM32 cells, while the mutant proteins with D32A, E251A, and E251K were not. Compared to cells with wild-type NorM, cells with the mutant NorM protein exhibited reduced resistance to kanamycin, norfloxacin, and ethidium bromide, but the NorM D367E mutant was more resistant to ethidium bromide. The NorM mutant D32E, D32N, D32K, D367A, and D367K cells lost the ability to extrude ethidium ions, which was Na+ dependent, and the ability to move Na+, which was evoked by ethidium bromide. Both E251D and D367N mutants decreased Na+-dependent extrusion of ethidium ions, but ethidium bromide-evoked movement of Na+ was retained. In contrast, D367E caused increased transport of ethidium ions and Na+. These results suggest that Asp32, Glu251, and Asp367 are involved in the Na+-dependent drug transport process.

scholarly journals Rapid resensitization of ASIC2a is conferred by three amino acid residues in the N terminus

2019 ◽  
Vol 151 (7) ◽  
pp. 944-953
Author(s):  
Jae Seung Lee ◽  
Hae-Jin Kweon ◽  
Hyosang Lee ◽  
Byung-Chang Suh
Keyword(s):  
Amino Acid ◽  
De Novo ◽  
Amino Acid Level ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Transmembrane Region ◽  
Acid Sensing Ion Channels ◽  
Electrophysiological Responses ◽  
N Terminus ◽  
Key Residues

Acid-sensing ion channels (ASICs), sensory molecules that continuously monitor the concentration of extracellular protons and initiate diverse intracellular responses through an influx of cations, are assembled from six subtypes that can differentially combine to form various trimeric channel complexes and elicit unique electrophysiological responses. For instance, homomeric ASIC1a channels have been shown to exhibit prolonged desensitization, and acid-evoked currents become smaller when the channels are repeatedly activated by extracellular protons, whereas homomeric or heteromeric ASIC2a channels continue to respond to repetitive acidic stimuli without exhibiting such desensitization. Although previous studies have provided evidence that both the desensitization of ASIC1a and rapid resensitization of ASIC2a commonly require domains that include the N terminus and the first transmembrane region of these channels, the biophysical basis of channel gating at the amino acid level has not been clearly determined. Here, we confirm that domain-swapping mutations replacing the N terminus of ASIC2a with that of ASIC2b result in de novo prolonged desensitization in homomeric channels following activation by extracellular protons. Such desensitization of chimeric ASIC2a mutants is due neither to internalization nor to degradation of the channel proteins. We use site-directed mutagenesis to narrow down the relevant portion of the N terminus of ASIC2a, identifying three amino acid residues within the N terminus (T25, T39, and I40) whose mutation is sufficient to phenocopy the desensitization exhibited by the chimeric mutants. A similar desensitization is observed in heteromeric ASICs containing the mutant subunit. These results suggest that T25, T39, and I40 of ASIC2a are key residues determining the rapid resensitization of homomeric and heteromeric ASIC2a channels upon proton activation.

scholarly journals Expression of recombinant human serum amyloid A in mammalian cells and demonstration of the region necessary for high-density lipoprotein binding and amyloid fibril formation by site-directed mutagenesis

1996 ◽  
Vol 318 (3) ◽  
pp. 1041-1049 ◽  
Author(s):  
Himakshi PATEL ◽  
Jo BRAMALL ◽  
Helen WATERS ◽  
Maria C. DE BEER ◽  
Patricia WOO
Keyword(s):  
Amino Acid ◽  
Serum Amyloid A ◽  
Amyloid Fibrils ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Amyloid A ◽  
Amyloid Fibril Formation

Site-directed mutagenesis of the acute-phase human serum amyloid A (SAA1α) protein was used to evaluate the importance of the N-terminal amino acid residues, namely RSFFSFLGEAF. The full-length cDNA clone of SAA1α (pA1.mod.) was used to create two mutations, namely Gly-8 to Asp-8 and an 11 amino acid truncation between Arg-1 and Phe-11 respectively. Wild-type and mutant cDNAs were expressed in Chinese hamster ovary (CHO) cells under the control of the human cytomegalovirus promoter, which resulted in the secretion of the processed proteins into the culture media. Wild-type recombinant human SAA (rSAA) protein was shown to have pI values of 6.0 and 6.4, similar to the human SAA isoform SAA1α and SAA1α desArg found in acute-phase plasma. N-terminal sequencing of 56 residues confirmed its identity with human SAA1α. The total yield of wild-type rSAA measured by ELISA was between 3.5 and 30 mg/l. The two mutations resulted in reduced expression levels of the mutant SAA proteins (3–10 mg/l). Further measurements of rSAA concentration in lipid fractions of culture medium collected at a density of 1.21 g/ml (high-density lipoprotein; HDL) and 1.063–1.18 g/ml (very-low-density lipoprotein/low-density lipoprotein; VLDL/LDL) showed that 76% of the wild-type protein was found in the HDL fraction and the remaining 24% in the infranatant non-lipid fraction. In contrast the relative concentration of mutant rSAA in HDL and infranatant fractions was reversed. This is consistent with the previously proposed involvement of the 11 amino acid peptide in anchoring SAA protein on to HDL3 [Turnell, Sarra, Glover, Baum, Caspi, Baltz and Pepys (1986) Mol. Biol. Med.3, 387–407]. Wild-type rSAA protein was shown to form amyloid fibrils in vitro under acidic conditions as shown by electron microscopy, and stained positive with Congo Red and exhibited apple-green birefringence when viewed under polarized light. Under the same conditions mutSAA(G8D) and mutSAAΔ1–11 did not form amyloid fibrils. In conclusion, replacement of Gly-8 by Asp-8 or deletion of the first 11 amino acid residues at the N-terminus of rSAA diminishes its capacity to bind to HDL and decreases amyloid fibril formation.

scholarly journals Basic amino acid residue cluster within nuclear targeting sequence motif is essential for cytoplasmic plectin-vimentin network junctions.

1996 ◽  
Vol 134 (6) ◽  
pp. 1455-1467 ◽  
Author(s):  
B Nikolic ◽  
E Mac Nulty ◽  
B Mir ◽  
G Wiche
Keyword(s):  
Amino Acid ◽  
Intermediate Filaments ◽  
Basic Amino Acid ◽  
Site Directed Mutagenesis ◽  
Sequence Motif ◽  
Amino Acid Residues ◽  
Nuclear Targeting ◽  
Mutant Proteins ◽  
Carboxy Terminal

We have generated a series of plectin deletion and mutagenized cDNA constructs to dissect the functional sequences that mediate plectin's interaction with intermediate filament (IF) networks, and scored their ability to coalign or disrupt intermediate filaments when ectopically expressed in rat kangaroo PtK2 cells. We show that a stretch of approximately 50 amino acid residues within plectin's carboxy-terminal repeat 5 domain serves as a unique binding site for both vimentin and cytokeratin IF networks of PtK2 cells. Part of the IF-binding domain was found to constitute a functional nuclear localization signal (NLS) motif, as demonstrated by nuclear import of cytoplasmic proteins linked to this sequence. Site directed mutagenesis revealed a specific cluster of four basic amino acid residues (arg4277-arg4280) residing within the NLS sequence motif to be essential for IF binding. When mutant proteins corresponding to those expressed in PtK2 cells were expressed in bacteria and purified proteins subjected to a sensitive quantitative overlay binding assay using Eu3+-labeled vimentin, the relative binding capacities of mutant proteins measured were fully consistent with the mutant's phenotypes observed in living cells. Using recombinant proteins we also show by negative staining and rotary shadowing electron microscopy that in vitro assembled vimentin intermediate filaments become packed into dense aggregates upon incubation with plectin repeat 5 domain, in contrast to repeat 4 domain or a mutated repeat 5 domain.

scholarly journals Molecular and Functional Analyses of Kunjin Virus Infectious cDNA Clones Demonstrate the Essential Roles for NS2A in Virus Assembly and for a Nonconservative Residue in NS3 in RNA Replication

2003 ◽  
Vol 77 (14) ◽  
pp. 7804-7813 ◽  
Author(s):  
Wen Jun Liu ◽  
Hua Bo Chen ◽  
Alexander A. Khromykh
Keyword(s):  
Amino Acid ◽  
Virus Assembly ◽  
Nonstructural Protein ◽  
Rna Replication ◽  
Site Directed Mutagenesis ◽  
Single Amino Acid ◽  
Cdna Clones ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Common Amino Acid

ABSTRACT A number of full-length cDNA clones of Kunjin virus (KUN) were previously prepared; it was shown that two of them, pAKUN and FLSDX, differed in specific infectivities of corresponding in vitro transcribed RNAs by ∼100,000-fold (A. A. Khromykh et al., J. Virol. 72:7270-7279, 1998). In this study, we analyzed a possible genetic determinant(s) of the observed differences in infectivity initially by sequencing the entire cDNAs of both clones and comparing them with the published sequence of the parental KUN strain MRM61C. We found six common amino acid residues in both cDNA clones that were different from those in the published MRM61C sequence but were similar to those in the published sequences of other flaviviruses from the same subgroup. pAKUN clone had four additional codon changes, i.e., Ile59 to Asn and Arg175 to Lys in NS2A and Tyr518 to His and Ser557 to Pro in NS3. Three of these substitutions except the previously shown marker mutation, Arg175 to Lys in NS2A, reverted to the wild-type sequence in the virus eventually recovered from pAKUN RNA-transfected BHK cells, demonstrating the functional importance of these residues in viral replication and/or viral assembly. Exchange of corresponding DNA fragments between pAKUN and FLSDX clones and site-directed mutagenesis revealed that the Tyr518-to-His mutation in NS3 was responsible for an ∼5-fold decrease in specific infectivity of transcribed RNA, while the Ile59-to-Asn mutation in NS2A completely blocked virus production. Correction of the Asn59 in pAKUN NS2A to the wild-type Ile residue resulted in complete restoration of RNA infectivity. Replication of KUN replicon RNA with an Ile59-to-Asn substitution in NS2A and with a Ser557-to-Pro substitution in NS3 was not affected, while the Tyr518-to-His substitution in NS3 led to severe inhibition of RNA replication. The impaired function of the mutated NS2A in production of infectious virus was complemented in trans by the helper wild-type NS2A produced from the KUN replicon RNA. However, replicon RNA with mutated NS2A could not be packaged in trans by the KUN structural proteins. The data demonstrated essential roles for the KUN nonstructural protein NS2A in virus assembly and for NS3 in RNA replication and identified specific single-amino-acid residues involved in these functions.

scholarly journals Effects of Specific Amino Acid Substitutions on Activities of Dinitrogenase Reductase-Activating Glycohydrolase fromRhodospirillum rubrum

2001 ◽  
Vol 183 (19) ◽  
pp. 5743-5746 ◽  
Author(s):  
Babu S. Antharavally ◽  
Russell R. Poyner ◽  
Yaoping Zhang ◽  
Gary P. Roberts ◽  
Paul W. Ludden
Keyword(s):  
Amino Acid ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Paramagnetic Resonance ◽  
Specific Amino Acid ◽  
Dinitrogenase Reductase ◽  
Electron Paramagnetic ◽  
Hydrolysis Of

ABSTRACT Site-directed mutagenesis of the draG gene was used to generate altered forms of dinitrogenase reductase-activating glycohydrolase (DRAG) with D123A, H142L, H158N, D243G, and E279R substitutions. The amino acid residues H142 and E279 are not required either for the coordination to the metal center or for catalysis since the variants H142L and E279R retained both catalytic and electron paramagnetic resonance spectral properties similar to those of the wild-type enzyme. Since DRAG-H158N and DRAG-D243G variants lost their ability to bind Mn(II) and to catalyze the hydrolysis of the substrate, H158 and D243 residues could be involved in the coordination of the binuclear Mn(II) center in DRAG.

scholarly journals The C-Terminal Region of Bacteroides fragilis Toxin Is Essential to Its Biological Activity

2006 ◽  
Vol 74 (10) ◽  
pp. 5595-5601 ◽  
Author(s):  
Cynthia L. Sears ◽  
Simy L. Buckwold ◽  
Jai W. Shin ◽  
Augusto A. Franco
Keyword(s):  
Biological Activity ◽  
Amino Acid ◽  
Point Mutations ◽  
Bacteroides Fragilis ◽  
Terminal Region ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Reverse Transcription Pcr ◽  
C Terminus

ABSTRACT To evaluate the role of the C-terminal region in Bacteroides fragilis toxin (BFT) activity, processing, and secretion, sequential C-terminal truncation and point mutations were created by site-directed mutagenesis. Determination of BFT activity on HT29/C1 cells, cleavage of E-cadherin, and the capacity to induce interleukin-8 secretion by wild-type BFT and C-terminal deletion mutants showed that deletion of only 2 amino acid residues at the C terminus significantly reduced BFT biological activity and deletion of eight or more amino acid residues obliterated BFT biologic activity. Western blot and reverse transcription-PCR analyses indicated that BFT mutants lacking seven or fewer amino acid residues in the C-terminal region are processed and expressed similar to wild-type BFT. However, BFT mutants lacking eight or more amino acids at the C terminus are expressed similar to wild-type BFT but are unstable. We concluded that the C terminus of BFT is not tolerant of modest amino acid deletions, suggesting that it is biologically important for BFT activity.

scholarly journals Site-directed mutagenesis of the putative active site of human 17β-hydroxysteroid dehydrogenase type 1

1994 ◽  
Vol 304 (1) ◽  
pp. 289-293 ◽  
Author(s):  
T J Puranen ◽  
M H Poutanen ◽  
H E Peltoketo ◽  
P T Vihko ◽  
R K Vihko
Keyword(s):  
Amino Acid ◽  
Catalytic Properties ◽  
Catalytic Efficiency ◽  
Hydroxysteroid Dehydrogenase ◽  
Site Directed Mutagenesis ◽  
Dimensional Structure ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Wild Type Enzyme

Several amino acid residues (Cys54, Tyr155, His210, His213 and His221) at a putative catalytic site of human 17 beta-hydroxysteroid dehydrogenase type 1 were mutated to Ala. Replacement of His221 by Ala remarkably reduced the catalytic activity, which resulted from a change of both the Km and the Vmax. values of the enzyme. Compared with the wild-type enzyme, the catalytic efficiency of the His221-->Ala mutant was reduced 20-fold for the oxidative reaction and 11-fold for the reductive reaction. With similar mutations at His210 or His213, no notable effects on the catalytic properties of the enzyme were detected. However, a simultaneous mutation of these amino acid residues decreased the Vmax. values of both oxidation and reduction by about 50% from those measured for the wild-type enzyme. Although Cys54 has been localized in the cofactor-binding region of the enzyme, a Cys54-->Ala mutation did not lead to changes in the enzymic activity. The most dramatic effects on the catalytic properties of the enzyme were achieved by mutating Tyr155, which resulted in an almost completely inactivation of the enzyme. The decreased enzymic activities of the Tyr155-->Ala, His210-->Ala + His213-->Ala and His221-->Ala mutations were also reflected in a reduced immunoreactivity of the enzymes. The results thus suggest that the lower catalytic efficiency of the mutant enzymes is due to an exchange of catalytically important amino acid residues and/or remarkable alterations in the three-dimensional structure of the enzyme. The recently detected polymorphisms (Ala237<-->Val and Ser312<-->Gly) were not found to affect either the catalytic or the immunological properties of the type 1 enzyme.

scholarly journals Overproduction of l-Cysteine andl-Cystine by Escherichia coli Strains with a Genetically Altered Serine Acetyltransferase

1998 ◽  
Vol 64 (5) ◽  
pp. 1607-1611 ◽  
Author(s):  
Shigeru Nakamori ◽  
Shin-ichiro Kobayashi ◽  
Chitose Kobayashi ◽  
Hiroshi Takagi
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Feedback Inhibition ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Serine Acetyltransferase ◽  
Methionine Residue ◽  
Cysteine Desulfhydrase ◽  
K 12

ABSTRACT Organisms that overproduced l-cysteine andl-cystine from glucose were constructed by usingEscherichia coli K-12 strains. cysE genes coding for altered serine acetyltransferase, which was genetically desensitized to feedback inhibition by l-cysteine, were constructed by replacing the methionine residue at position 256 of the serine acetyltransferase protein with 19 other amino acid residues or the termination codon to truncate the carboxy terminus from amino acid residues 256 to 273 through site-directed mutagenesis by using PCR. A cysteine auxotroph, strain JM39, was transformed with plasmids having these altered cysE genes. The serine acetyltransferase activities of most of the transformants, which were selected based on restored cysteine requirements and ampicillin resistance, were less sensitive than the serine acetyltransferase activity of the wild type to feedback inhibition by l-cysteine. At the same time, these transformants produced approximately 200 mg ofl-cysteine plus l-cystine per liter, whereas these amino acids were not detected in the recombinant strain carrying the wild-type serine acetyltransferase gene. However, the production ofl-cysteine and l-cystine by the transformants was very unstable, presumably due to a cysteine-degrading enzyme of the host, such as cysteine desulfhydrase. Therefore, mutants that did not utilize cysteine were derived from host strain JM39 by mutagenesis withN-methyl-N′-nitro-N-nitrosoguanidine. When a newly derived host was transformed with plasmids having the altered cysE genes, we found that the production ofl-cysteine plus l-cystine was markedly increased compared to production in JM39.

Effects of amino acid substitutions at the active site in Escherichia coli beta-galactosidase.

Genetics ◽  
1988 ◽  
Vol 120 (3) ◽  
pp. 637-644
Author(s):  
C G Cupples ◽  
J H Miller
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Amino Acid ◽  
Active Site ◽  
Site Directed Mutagenesis ◽  
Amino Acid Substitutions ◽  
Lacz Gene ◽  
Wild Type ◽  
Mutant Proteins ◽  
Beta Galactosidase

Abstract Forty-nine amino acid substitutions were made at four positions in the Escherichia coli enzyme beta-galactosidase; three of the four targeted amino acids are thought to be part of the active site. Many of the substitutions were made by converting the appropriate codon in lacZ to an amber codon, and using one of 12 suppressor strains to introduce the replacement amino acid. Glu-461 and Tyr-503 were replaced, independently, with 13 amino acids. All 26 of the strains containing mutant enzymes are Lac-. Enzyme activity is reduced to less than 10% of wild type by substitutions at Glu-461 and to less than 1% of wild type by substitutions at Tyr-503. Many of the mutant enzymes have less than 0.1% wild-type activity. His-464 and Met-3 were replaced with 11 and 12 amino acids, respectively. Strains containing any one of these mutant proteins are Lac+. The results support previous evidence that Glu-461 and Tyr-503 are essential for catalysis, and suggest that His-464 is not part of the active site. Site-directed mutagenesis was facilitated by construction of an f1 bacteriophage containing the complete lacZ gene on a single EcoRI fragment.

scholarly journals Lys-197 and Asp-414 are critical residues for binding of ATP/Mg2+ by rat brain inositol 1,4,5-trisphosphate 3-kinase

1993 ◽  
Vol 291 (3) ◽  
pp. 811-816 ◽  
Author(s):  
D Communi ◽  
K Takazawa ◽  
C Erneux
Keyword(s):  
Enzyme Activity ◽  
Amino Acid ◽  
Rat Brain ◽  
Catalytic Domain ◽  
Enzymic Activity ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Calmodulin Binding ◽  
Inositol 1,4,5 Trisphosphate

Rat brain inositol 1,4,5-trisphosphate (InsP3) 3-kinase A was expressed in Escherichia coli in order to identify the amino acid residues involved in substrate ATP/Mg2+ binding. Two amino acid regions that are conserved in the catalytic domain of InsP3 3-kinase isoenzymes A and B had characteristics consistent with two ATP/Mg(2+)-binding motives. Site-directed mutagenesis was performed on residues Lys-197, Lys-207 and Asp-414 to generate three mutant enzymes, referred to as C5 K197I, C5 K207I and C5 D414N. Comparison of the wild-type and mutant proteins with regard to enzymic activity revealed that C5 K197I exhibited 10% of control enzyme activity, C5 D414N was totally inactive and C5 K207I was fully active. The reduced levels of enzyme activity for C5 K197I and C5 D414N were correlated with an altered ability of the mutant enzymes to bind ATP/Mg2+, as determined by ATP-agarose affinity chromatography. Neither Ca2+/calmodulin binding nor InsP3 binding appeared to be affected. Mutant C5 K207I showed the same characteristics as the wild-type enzyme. Taken together, these results strongly indicated (i) that amino acid residues Lys-197 and Asp-414 are necessary for InsP3 3-kinase activity and form part of the ATP/Mg(2+)-binding domain, and (ii) that amino acid residues Lys-197, Lys-207 and Asp-414 are not involved in either InsP3 binding or enzyme stimulation by Ca2+/calmodulin.

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