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

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.

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

Biochemical Journal ◽

10.1042/bj3150761 ◽

1996 ◽

Vol 315 (3) ◽

pp. 761-766 ◽

Cited By ~ 42

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.

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

Biochemical Journal ◽

10.1042/bj3070493 ◽

1995 ◽

Vol 307 (2) ◽

pp. 493-498 ◽

Cited By ~ 27

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.

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Cysteine residues in the Na+/dicarboxylate co-transporter, NaDC-1

Biochemical Journal ◽

10.1042/bj3440205 ◽

1999 ◽

Vol 344 (1) ◽

pp. 205-209 ◽

Cited By ~ 9

Author(s):

Ana M. PAJOR ◽

Sally J. KRAJEWSKI ◽

Nina SUN ◽

Rama GANGULA

Keyword(s):

Protein Trafficking ◽

Direct Relationship ◽

Transmembrane Domain ◽

Cysteine Residue ◽

Site Directed Mutagenesis ◽

Cysteine Residues ◽

Directed Mutagenesis ◽

Transport Activity ◽

Specific Reagent

The role of cysteine residues in the Na+/dicarboxylate co-transporter (NaDC-1) was tested using site-directed mutagenesis. The transport activity of NaDC-1 was not affected by mutagenesis of any of the 11 cysteine residues, indicating that no individual cysteine residue is necessary for function. NaDC-1 is sensitive to inhibition by the impermeant cysteine-specific reagent, p-chloromercuribenzenesulphonate (pCMBS). The pCMBS-sensitive residues in NaDC-1 are Cys-227, found in transmembrane domain 5, and Cys-476, located in transmembrane domain 9. Although cysteine residues are not required for function in NaDC-1, their presence appears to be important for protein stability or trafficking to the plasma membrane. There was a direct relationship between the number of cysteine residues, regardless of location, and the transport activity and expression of NaDC-1. The results indicate that mutagenesis of multiple cysteine residues in NaDC-1 may alter the shape or configuration of the protein, leading to alterations in protein trafficking or stability.

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The role of cysteine residues 129 and 329 in Escherichia coli K1 CMP-NeuAc synthase

Biochemical Journal ◽

10.1042/bj2950485 ◽

1993 ◽

Vol 295 (2) ◽

pp. 485-491 ◽

Cited By ~ 4

Author(s):

G Zapata ◽

P P Roller ◽

J Crowley ◽

W F Vann

Keyword(s):

Escherichia Coli ◽

Specific Activity ◽

Site Directed Mutagenesis ◽

Cysteine Residues ◽

Directed Mutagenesis ◽

Wild Type ◽

Acetylneuraminic Acid ◽

Denatured States ◽

Escherichia Coli K1

N-Acetylneuraminic acid cytidyltransferase (CMP-NeuAc synthase) of Escherichia coli K1 is sensitive to mercurials and has cysteine residues only at positions 129 and 329. The role of these residues in the catalytic activity and structure of the protein has been investigated by site-directed mutagenesis and chemical modification. The enzyme is inactivated by the thiol-specific reagent dithiodipyridine. Inactivation by this reagent is decreased in the presence of the nucleotide substrate CTP, suggesting that a thiol residue is at or near the active site. Site-directed mutagenesis of either residue Cys-129 to serine or Cys-329 to selected amino acids has minor effects on the specific activity of the enzyme, suggesting that cysteine is not essential for catalysis and that a disulphide bond is not an essential structural component. The limited reactivity of the enzyme to other thiol-blocking reagents suggests that its cysteine residues are partially exposed. The accessibility and role of the cysteine residues in enzyme structure were investigated by fluorescence, c.d. and denaturation studies of wild-type and mutant enzymes. The mutation of Cys-129 to serine makes the enzyme more sensitive to heat and chemical denaturation, but does not cause gross changes in the protein structure as judged by the c.d. spectrum. The mutant containing Ser-129 instead of Cys-129 had a complex denaturation pathway similar to that of wild-type E. coli K1 CMP-NeuAc synthase consisting of several partially denatured states. Cys-329 reacts more readily with N-[14C]ethylmaleimide when the enzyme is in a heat-induced relaxed state. Cys-129 is less reactive and is probably a buried residue.

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

Biochemical Journal ◽

10.1042/bj2950457 ◽

1993 ◽

Vol 295 (2) ◽

pp. 457-461 ◽

Cited By ~ 16

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.

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Site-Directed Mutagenesis of the 1,3-β-Glucan Synthase Catalytic Subunit ofPneumocystis jiroveciiand Susceptibility Assays Suggest Its Sensitivity to Caspofungin

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01159-18 ◽

2018 ◽

Vol 62 (12) ◽

Cited By ~ 3

Author(s):

A. Luraschi ◽

S. Richard ◽

P. M. Hauser

Keyword(s):

Pneumocystis Carinii ◽

Culture Method ◽

Catalytic Subunit ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Wild Type ◽

Content Type ◽

Resistant Species ◽

Rats And Mice

ABSTRACTThe echinocandin caspofungin inhibits the catalytic subunit Gsc1 of the enzymatic complex synthesizing 1,3-β-glucan, an essential compound of the fungal wall. Studies with rodents showed that caspofungin is effective againstPneumocystisasci. However, its efficacy against asci ofPneumocystis jirovecii, the species infecting exclusively humans, remains controversial. The aim of this study was to assess the sensitivity to caspofungin of theP. jiroveciiGsc1 subunit, as well as of those ofPneumocystis cariniiandPneumocystis murinainfecting, respectively, rats and mice. In the absence of an establishedin vitroculture method forPneumocystisspecies, we used functional complementation of theSaccharomyces cerevisiaegsc1 deletant. In the fungal pathogenCandida albicans, mutations leading to amino acid substitutions in Gsc1 confer resistance to caspofungin. We introduced the corresponding mutations into thePneumocystis gsc1genes using site-directed mutagenesis. In spot dilution tests, the sensitivity to caspofungin of the complemented strains decreased with the number of mutations introduced, suggesting that the wild-type enzymes are sensitive. The MICs of caspofungin determined by Etest and YeastOne for strains complemented withPneumocystisenzymes (respectively, 0.125 and 0.12 μg/ml) were identical to those upon complementation with the enzyme ofC. albicans, for which caspofungin presents low MICs. However, they were lower than the MICs upon complementation with the enzyme of the resistant speciesCandida parapsilosis(0.19 and 0.25 μg/ml). Sensitivity levels of Gsc1 enzymes of the threePneumocystisspecies were similar. Our results suggest thatP. jiroveciiis sensitive to caspofungin during infections, as areP. cariniiandP. murina.

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Biologically active monomeric and heterodimeric recombinant human calpain I produced using the baculovirus expression system

Biochemical Journal ◽

10.1042/bj3140511 ◽

1996 ◽

Vol 314 (2) ◽

pp. 511-519 ◽

Cited By ~ 48

Author(s):

Sheryl L. MEYER ◽

Donna BOZYCZKO-COYNE ◽

Satish K. MALLYA ◽

Chrysanthe M. SPAIS ◽

Ron BIHOVSKY ◽

...

Keyword(s):

Expression System ◽

Baculovirus Expression System ◽

Cysteine Proteases ◽

Catalytic Subunit ◽

Biologically Active ◽

Regulatory Subunit ◽

Calcium Dependent ◽

Autocatalytic Cleavage ◽

Cell Extracts ◽

Baculovirus Expression

Calpain I is a heterodimeric protein that is part of a family of calcium-activated intracellular cysteine proteases presumed to play a role in mediating signals transduced by calcium. Expression of bioactive recombinant human calpain I has been achieved using the baculovirus expression system, by either co-infection with two viruses, each expressing one of the subunits, or infection with a single virus containing both subunits. The ~80 kDa catalytic subunit exhibited calcium-dependent proteolytic activity when expressed alone or with the ~30 kDa regulatory subunit. Baculoviral recombinant calpain I appeared fully active in that the catalytic subunit in unpurified cell extracts exhibited calcium-dependent autocatalytic cleavage at the correct locus. The amount of ~80 kDa subunit accumulated at steady state was greatly increased by co-expression of the ~30 kDa subunit, suggesting a possible role for enzyme stabilization by the latter subunit. The recombinant human calpain I was purified to near homogeneity and compared with purified native human erythrocyte calpain I. The recombinant and native enzymes had equivalent inhibition constants for structurally diverse calpain inhibitors, identical calcium activation profiles, and similar specific activities, demonstrating the suitability of using the recombinant protein for studies of the native enzyme.

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Topology of the Erwinia chrysanthemi oligogalacturonate porin KdgM

Biochemical Journal ◽

10.1042/bj20030027 ◽

2003 ◽

Vol 372 (2) ◽

pp. 329-334 ◽

Cited By ~ 15

Author(s):

Teijo PELLINEN ◽

Helena AHLFORS ◽

Nicolas BLOT ◽

Guy CONDEMINE

Keyword(s):

Sequence Similarity ◽

Site Directed Mutagenesis ◽

Erwinia Chrysanthemi ◽

Cysteine Residues ◽

Directed Mutagenesis ◽

Wild Type ◽

Transmembrane Segments

The Erwinia chrysanthemi oligogalacturonate-specific monomeric porin, KdgM, does not present homology with any porins of known structure. A model of this protein, based on sequence similarity and the amphipathy profile, was constructed. The model depicts a β-barrel composed of 14 antiparallel β-strands. The accuracy of this model was tested by the chemical labelling of cysteine residues introduced by site-directed mutagenesis. The protein has seven surface-exposed loops. They are rather small with the exception of one, loop L6. Deletion of this loop allowed the entry of maltopentaose into the bacteria, a molecule too large to enter through the wild-type KdgM. Loop L6 could fold back into the lumen of the pore and play the role of the constriction loop L3 of general porins. With 14 transmembrane segments, the KdgM porin family could represent the smallest porin characterized to date.

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Site-directed mutagenesis of the 1,3-β glucan synthase catalytic subunit ofPneumocystis jiroveciiand susceptibility assays suggest its sensitivity to caspofungin

10.1101/340281 ◽

2018 ◽

Author(s):

A. Luraschi ◽

S. Richard ◽

P. M. Hauser

Keyword(s):

Candida Parapsilosis ◽

Pneumocystis Carinii ◽

Culture Method ◽

Catalytic Subunit ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Wild Type ◽

Resistant Species ◽

Rats And Mice

AbstractThe echinocandin caspofungin inhibits the catalytic subunit Gsc1 of the enzymatic complex synthetizing 1,3-β glucan, an essential compound of the fungal wall. Studies in rodents showed that caspofungin can treatPneumocystisinfections. However, its efficacy againstPneumocystis jirovecii, the species infecting exclusively humans, remains controversial. The aim of this study was to assess the sensitivity to caspofungin of theP. jiroveciiGsc1 subunit, as well as of those ofPneumocystis cariniiandPneumocystis murinainfecting respectively rats and mice. In absence of an establishedin vitroculture method forPneumocystisspecies, we used functional complementation of theSaccharomyces cerevisiae gsc1deletant. In the fungal pathogenCandida albicans, mutations leading to amino acid substitutions in Gsc1 confer resistance to caspofungin. We introduced the corresponding mutations into thePneumocystis gsc1genes using site-directed mutagenesis. In spot dilution tests, the sensitivity to caspofungin of the complemented strains decreased with the number of mutations introduced, suggesting that the wild-type enzymes are sensitive. The minimum inhibitory concentrations of caspofungin determined by E-test®and Yeastone®for strains complemented withPneumocystisenzymes (respectively 0.125 and 0.12 microg/ml) were identical to those upon complementation with the enzyme ofC. albicansthat is sensitive to caspofungin. However, they were lower than the MICs upon complementation with the enzyme of the resistant speciesCandida parapsilosis(0.19 and 0.25). Sensitivity levels of Gsc1 enzymes of the threePneumocystisspecies were similar. Our results suggest thatP. jiroveciiis sensitive to caspofungin during infections, asP. cariniiandP. murina.

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Cysteine-86 is not needed for the enzymic activity of glutathione S-transferase 3-3

Biochemical Journal ◽

10.1042/bj2780293 ◽

1991 ◽

Vol 278 (1) ◽

pp. 293-297 ◽

Cited By ~ 18

Author(s):

J C Hsieh ◽

S C Huang ◽

W L Chen ◽

Y C Lai ◽

M F Tam

Keyword(s):

Spodoptera Frugiperda ◽

Expression System ◽

Baculovirus Expression System ◽

Cysteine Residue ◽

Enzymic Activity ◽

Site Directed Mutagenesis ◽

Terminal Sequence ◽

Glutathione S Transferase ◽

Baculovirus Expression ◽

Modified Peptides

Recombinant glutathione S-transferase 3-3 expressed in Spodoptera frugiperda (SF9) cells with the use of a baculovirus expression system was modified with 1 mM-iodoacetamide. Amino acid analysis indicated that 0.79 +/- 0.15 cysteine residue was modified per enzyme subunit. The S-carbaminomethylated protein retains the GSH-conjugating activity. Glutathione S-transferase 3-3 modified with iodo[14C]acetamide was digested with Achromobacter proteinase I and the resulting peptides were separated by h.p.l.c. The modified peptides were pooled and further digested with Staphylococcus aureus V8 proteinase. Isotope-labelled peptides were isolated and collected for N-terminal sequence analysis. By this procedure, cysteine-86 was identified as the major S-carbaminomethylated residue. Verification of this findings came from the use of site-directed mutagenesis in which this cysteine was replaced by serine (C86S mutant). The C86S mutant is enzymically active. Therefore cysteine-86 is not needed for the conjugation of GSH with electrophilic compounds on glutathione S-transferase 3-3.

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