scholarly journals Expression of two different forms of cDNA for thromboxane synthase in insect cells and site-directed mutagenesis of a critical cysteine residue

1993 ◽  
Vol 295 (2) ◽  
pp. 457-461 ◽  
Author(s):  
Z Xia ◽  
R F Shen ◽  
S J Baek ◽  
H H Tai
Keyword(s):  
Cdna Library ◽  
Insect Cells ◽  
Specific Activity ◽  
Short Form ◽  
Expression System ◽  
Cysteine Residue ◽  
Site Directed Mutagenesis ◽  
Mutant Enzyme ◽  
Directed Mutagenesis ◽  
Thromboxane Synthase

cDNA coding for human placental thromboxane synthase (EC 5.3.99.5) was amplified by PCR from a human placental cDNA library and sequenced. This cDNA and a shorter cDNA isolated from a human lung cDNA library with a deletion of 163 bp near the 3′ end were expressed in Spodoptera frugiperda (Sf9) insect cells using a baculovirus expression system. The cDNA from human placenta was expressed as an active enzyme (60 kDa) with a specific activity higher than those reported from other cell types, whereas the shorter cDNA was expressed in an inactive form (52 kDa). The active recombinant enzyme appeared to be unglycosylated as the molecular mass and the enzyme activity were not altered in the presence of tunicamycin. Site-directed mutagenesis was performed to convert a cysteine at position 480 in thromboxane synthase to a serine. This cysteine is found to be highly conserved in related cytochrome P-450 enzymes. The mutant enzyme was found to be inactive, although Western blot, immunoprecipitation and SDS/PAGE analysis indicated that the mutant enzyme was expressed at a level comparable with the wild-type enzyme. These results suggest that Cys-480 is essential for the enzyme catalytic activity and that the short-form cDNA may be a non-functional transcript.

scholarly journals Mammalian inositol monophosphatase: the identification of residues important for the binding of Mg2+ and Li+ ions using fluorescence spectroscopy and site-directed mutagenesis

1993 ◽  
Vol 296 (3) ◽  
pp. 811-815 ◽  
Author(s):  
M G Gore ◽  
P Greasley ◽  
G McAllister ◽  
C I Ragan
Keyword(s):  
Fluorescence Spectroscopy ◽  
Specific Activity ◽  
Fluorescence Emission ◽  
Site Directed Mutagenesis ◽  
Native Enzyme ◽  
Mutant Enzyme ◽  
Directed Mutagenesis ◽  
Inositol Monophosphatase ◽  
Fluorescence Emission Intensity ◽  
Ph Dependent

The fluorescence properties of residue Trp-219 in inositol monophosphatase are sensitive to the ionization of neighbouring groups. The pH-dependent changes in the fluorescence emission intensity and wavelength of maximum emission appear to arise as the result of two separate ionizations in the proximity of Trp-219, namely due to the ionization of His-217 and Cys-218. By studying the curve of fluorescence intensity against pH, given by the mutants Cys-218→Ala or His-217→Gln, the pK of His-217 was determined to be 7.54 and the pK of Cys-218 was estimated to be about 8.2. These mutants have altered kinetic parameters for catalytic Mg2+ ions and inhibitory Mg2+ and Li+ ions. The Cys-218→Ala mutant enzyme is not subject to inhibition by concentrations of Mg2+ ions up to 400 mM and has a specific activity of 156% of the maximum obtainable activity of the native enzyme. The His-217→Gln mutant enzyme shows reduced sensitivity to inhibition by Mg2+ and Li+ ions, and has a specific activity of 110% of that obtainable for the native enzyme.

scholarly journals Expression and site-directed mutagenesis of mouse prostaglandin E2 receptor EP3 subtype in insect cells

1995 ◽  
Vol 307 (2) ◽  
pp. 493-498 ◽  
Author(s):  
C Huang ◽  
H H Tai
Keyword(s):  
Ligand Binding ◽  
Prostaglandin E2 ◽  
Insect Cells ◽  
Expression System ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Binding Studies ◽  
Comparable Amount ◽  
Ep3 Receptor

A cDNA encoding for mouse prostaglandin E2 (PGE2) receptor EP3 subtype was cloned from a mouse kidney cDNA library by PCR using terminal primers derived from the known sequence of mouse lung EP3 receptor cDNA. The cloned cDNA was confirmed by sequencing and was expressed in Trichoplusia ni (MG1) insect cells using a baculovirus expression system. A specific protein of 60 kDa was detected by immunoblot with antibodies generated against a unique decapeptide sequence present in the second extracellular loop of the EP3 receptor. Specific binding of [3H]PGE2 with a Kd of 3 nM was also found in the membrane fraction of the insect cells. Ligand binding of the receptor was further studied by site-directed mutagenesis. Arg-309 of the receptor was separately mutated to lysine, glutamate and valine. cDNAs of the wild-type and mutant EP3 receptors were respectively expressed and studied in MG1 insect cells. Binding studies indicated that both glutamate and valine mutant EP3 receptors had no binding of [3H]PGE2. On the contrary, the lysine mutant receptor exhibited an even tighter binding (Kd = 1.3 nM) than the wild-type EP3 receptor. Immunoblot studies indicated that these receptors were expressed in a comparable amount in MG1 insect cells. These results suggest that Arg-309 of EP3 receptor may be essential in ligand binding through ionic interaction.

scholarly journals Identification of an important cysteine residue in human glutamate–cysteine ligase catalytic subunit by site-directed mutagenesis

1998 ◽  
Vol 336 (3) ◽  
pp. 675-680 ◽  
Author(s):  
Zhongheng TU ◽  
M. W. ANDERS
Keyword(s):  
Expression System ◽  
Baculovirus Expression System ◽  
Cysteine Residue ◽  
Catalytic Subunit ◽  
Site Directed Mutagenesis ◽  
Regulatory Subunit ◽  
Cysteine Residues ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Glutamate Cysteine Ligase

Glutamate–cysteine ligase (GLCL) catalyses the rate-limiting step in glutathione biosynthesis. To identify cysteine residues in GLCL that are involved in its activity, eight conserved cysteine residues in human GLCL catalytic subunit (hGLCLC) were replaced with glycine residues by PCR-based site-directed mutagenesis. Both recombinant hGLCLC and hGLCL holoenzyme were expressed and purified with a baculovirus expression system. The activity of purified hGLCL holoenzyme with the mutant hGLCLC-C553G was 110±12 µmol/h per mg of protein compared with 370±20 µmol/h per mg of protein for the wild-type. Holoenzymes with hGLCLC-C52G, -C248G, -C249G, -C295G, -C491G, -C501G or -C605G showed activities similar to the wild type. The Km values of hGLCL containing hGLCLC-C553G were slightly lower than those of the wild type, indicating that the replacement of cysteine-553 with Gly in hGLCLC did not significantly affect substrate binding by the enzyme. hGLCLC-C553G was more easily dissociated from hGLCLR than the wild-type hGLCLC. GLCL activity increased by 11% after hGLCLC-C553G was incubated with an equimolar amount of purified hGLCL regulatory subunit (hGLCLR) at room temperature for 30 min, but increased by 110% after wild-type hGLCLC was incubated with hGLCLR for 10 min. These results indicate that cysteine-553 in hGLCLC is involved in heterodimer formation between hGLCLC and hGLCLR.

scholarly journals Site-directed mutagenesis of rat liver S-adenosylmethionine synthetase. Identification of a cysteine residue critical for the oligomeric state

1996 ◽  
Vol 315 (3) ◽  
pp. 761-766 ◽  
Author(s):  
J Mingorance ◽  
L Alvarez ◽  
E Sánchez-Góngora ◽  
J M Mato ◽  
M A Pajares
Keyword(s):  
Rat Liver ◽  
Specific Activity ◽  
Expression System ◽  
Site Directed Mutagenesis ◽  
Cysteine Residues ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Oligomeric State ◽  
Protein Ratio ◽  
Adenosylmethionine Synthetase

We have examined the functional importance of the cysteine residues of rat liver S-adenosylmethionine synthetase. For this purpose the ten cysteine residues of the molecule were changed to serines by site-directed mutagenesis. Ten recombinant enzyme mutants were obtained by using a bacterial expression system. The same level of expression was obtained for the wild type and mutants, but the ratio of S-adenosylmethionine synthetase between soluble and insoluble fractions differed for some of the mutant forms. The immunoreactivity against an anti-(rat liver S-adenosylmethionine synthetase) antibody was equivalent in all the cases. Effects on S-adenosylmethionine synthetase activities were also measured. Mutants C57S, C69S, C105S and C121S showed decreased relative specific activity of 68, 85, 63 and 29%, respectively, compared with wild-type, whereas C312S resulted in an increase of 1.6-fold. Separation of tetramer and dimer forms for wild type and mutants was carried out by using phenyl-Sepharose columns. The dimer/tetramer ratio was calculated based on the activity and on the protein level estimated by immunoblotting. No monomeric forms of the enzyme were detected in any case. Comparison of dimer/tetramer ratios indicates the importance of cysteine-69 (dimer/tetramer protein ratio of 88 versus 10.2 in the wild type) in maintaining the oligomeric state of rat liver S-adenosylmethionine synthetase. Moreover, all the mutations carried out of cysteine residues between cysteine-35 and cysteine-105 altered the ratio between oligomeric forms.

scholarly journals Site-Directed Mutagenesis of the Heterotrimeric Killer Toxin Zymocin Identifies Residues Required for Early Steps in Toxin Action

2014 ◽  
Vol 80 (20) ◽  
pp. 6549-6559 ◽  
Author(s):  
Sabrina Wemhoff ◽  
Roland Klassen ◽  
Friedhelm Meinhardt
Keyword(s):  
Kluyveromyces Lactis ◽  
Killer Toxin ◽  
Cysteine Residue ◽  
Site Directed Mutagenesis ◽  
Target Cells ◽  
Directed Mutagenesis ◽  
Content Type ◽  
Protein Toxin ◽  
Assisted Delivery

ABSTRACTZymocin is aKluyveromyces lactisprotein toxin composed of αβγ subunits encoded by the cytoplasmic virus-like element k1 and functions by αβ-assisted delivery of the anticodon nuclease (ACNase) γ into target cells. The toxin binds to cells' chitin and exhibits chitinase activityin vitrothat might be important during γ import.Saccharomyces cerevisiaestrains carrying k1-derived hybrid elements deficient in either αβ (k1ORF2) or γ (k1ORF4) were generated. Loss of either gene abrogates toxicity, and unexpectedly, Orf2 secretion depends on Orf4 cosecretion. Functional zymocin assembly can be restored by nuclear expression of k1ORF2 or k1ORF4, providing an opportunity to conduct site-directed mutagenesis of holozymocin. Complementation required active site residues of α's chitinase domain and the sole cysteine residue of β (Cys250). Since βγ are reportedly disulfide linked, the requirement for the conserved γ C231 was probed. Toxicity of intracellularly expressed γ C231A indicated no major defect in ACNase activity, while complementation of k1ΔORF4 by γ C231A was lost, consistent with a role of β C250 and γ C231 in zymocin assembly. To test the capability of αβ to carry alternative cargos, the heterologous ACNase fromPichia acaciae(P. acaciaeOrf2 [PaOrf2]) was expressed, along with its immunity gene, in k1ΔORF4. While efficient secretion of PaOrf2 was detected, suppression of the k1ΔORF4-derived k1Orf2 secretion defect was not observed. Thus, the dependency of k1Orf2 on k1Orf4 cosecretion needs to be overcome prior to studying αβ's capability to deliver other cargo proteins into target cells.

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.

Site-directed mutagenesis of human lysyl hydroxylase expressed in insect cells

1996 ◽  
Vol 15 (3) ◽  
pp. 196
Author(s):  
A. Pirskanen ◽  
A.-M. Kaimio ◽  
R. Myllylä ◽  
K.I. Kivirikko
Keyword(s):  
Insect Cells ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Lysyl Hydroxylase

scholarly journals Production of l-Ribose from l-Ribulose by a Triple-Site Variant of Mannose-6-Phosphate Isomerase from Geobacillus thermodenitrificans

2012 ◽  
Vol 78 (11) ◽  
pp. 3880-3884 ◽  
Author(s):  
Yu-Ri Lim ◽  
Soo-Jin Yeom ◽  
Deok-Kun Oh
Keyword(s):  
Specific Activity ◽  
Thermus Thermophilus ◽  
Catalytic Efficiency ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Geobacillus Thermodenitrificans ◽  
Wild Type Enzyme ◽  
Content Type ◽  
Mannose 6 Phosphate

ABSTRACTA triple-site variant (W17Q N90A L129F) of mannose-6-phosphate isomerase fromGeobacillus thermodenitrificanswas obtained by combining variants with residue substitutions at different positions after random and site-directed mutagenesis. The specific activity and catalytic efficiency (kcat/Km) forl-ribulose isomerization of this variant were 3.1- and 7.1-fold higher, respectively, than those of the wild-type enzyme at pH 7.0 and 70°C in the presence of 1 mM Co2+. The triple-site variant produced 213 g/literl-ribose from 300 g/literl-ribulose for 60 min, with a volumetric productivity of 213 g liter−1h−1, which was 4.5-fold higher than that of the wild-type enzyme. Thekcat/Kmand productivity of the triple-site variant were approximately 2-fold higher than those of theThermus thermophilusR142N variant of mannose-6-phosphate isomerase, which exhibited the highest values previously reported.

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