Interconversion of a pair of active-site residues in Escherichia coli cystathionine γ-synthase, E. coli cystathionine β-lyase, and Saccharomyces cerevisiae cystathionine γ-lyase and development of tools for the investigation of their mechanisms and reaction specificity

2009 ◽  
Vol 87 (2) ◽  
pp. 445-457 ◽  
Author(s):  
Ali Farsi ◽  
Pratik H. Lodha ◽  
Jennifer E. Skanes ◽  
Heidi Los ◽  
Navya Kalidindi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Active Site ◽  
Affinity Purification ◽  
Catalytic Efficiency ◽  
Active Site Residues ◽  
E Coli ◽  
Transsulfuration Pathway ◽  
Reaction Specificity

Cystathionine γ-synthase (CGS) and cystathionine β-lyase (CBL), which comprise the transsulfuration pathway of bacteria and plants, and cystathionine γ-lyase (CGL), the second enzyme of the fungal and animal reverse transsulfuration pathway, share ∼30% sequence identity and are almost indistinguishable in overall structure. One difference between the active site of Escherichia coli CBL and those of E. coli CGS and Saccharomyces cerevisiae CGL is the replacement of a pair of aromatic residues, F55 and Y338, of the former by acidic residues in CGS (D45 and E325) and CGL (E48 and E333). A series of interconverting, site-directed mutants of these 2 residues was constructed in CBL (F55D, Y338E, F55D/Y338E), CGS (D45F, E325Y and D45F/E325Y) and CGL (E48A,D and E333A,D,Y) to probe the role of these residues as determinants of reaction specificity. Mutation of either position results in a reduction in catalytic efficiency, as exemplified by the 160-fold reduction in the kcat/Kml-Cys of eCGS-D45F and the 2850- and 30-fold reductions in the kcat/Kml-Cth of the eCBL-Y338E and the yCGL-E333A,Y mutants, respectively. However, the in vivo reaction specificity of the mutants was not altered, compared with the corresponding wild-type enzymes. The ΔmetB and ΔmetC strains, the optimized CBL and CGL assay conditions, and the efficient expression and affinity purification systems described provide the necessary tools to enable the continued exploration of the determinants of reaction specificity in the enzymes of the transsulfuration pathways.

scholarly journals FKBP12 physically and functionally interacts with aspartokinase in Saccharomyces cerevisiae.

1997 ◽  
Vol 17 (10) ◽  
pp. 5968-5975 ◽  
Author(s):  
C M Alarcón ◽  
J Heitman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Conformational Changes ◽  
Active Site ◽  
Feedback Inhibition ◽  
Immunosuppressive Drugs ◽  
Intermediate Step ◽  
Active Site Residues ◽  
Peptidyl Prolyl Isomerase

The peptidyl-prolyl isomerase FKBP12 was originally identified as the intracellular receptor for the immunosuppressive drugs FK506 (tacrolimus) and rapamycin (sirolimus). Although peptidyl-prolyl isomerases have been implicated in catalyzing protein folding, the cellular functions of FKBP12 in Saccharomyces cerevisiae and other organisms are largely unknown. Using the yeast two-hybrid system, we identified aspartokinase, an enzyme that catalyzes an intermediate step in threonine and methionine biosynthesis, as an in vivo binding target of FKBP12. Aspartokinase also binds FKBP12 in vitro, and drugs that bind the FKBP12 active site, or mutations in FKBP12 surface and active site residues, disrupt the FKBP12-aspartokinase complex in vivo and in vitro.fpr1 mutants lacking FKBP12 are viable, are not threonine or methionine auxotrophs, and express wild-type levels of aspartokinase protein and activity; thus, FKBP12 is not essential for aspartokinase activity. The activity of aspartokinase is regulated by feedback inhibition by product, and genetic analyses reveal that FKBP12 is important for this feedback inhibition, possibly by catalyzing aspartokinase conformational changes in response to product binding.

scholarly journals Structure ofEscherichia coliGrx2 in complex with glutathione: a dual-function hybrid of glutaredoxin and glutathioneS-transferase

2014 ◽  
Vol 70 (7) ◽  
pp. 1907-1913 ◽  
Author(s):  
Jun Ye ◽  
S. Venkadesh Nadar ◽  
Jiaojiao Li ◽  
Barry P. Rosen
Keyword(s):  
Escherichia Coli ◽  
Electron Donor ◽  
Active Site ◽  
Active Sites ◽  
Catalytic Cycle ◽  
Dual Function ◽  
Active Site Residues ◽  
E Coli ◽  
Arsenate Reduction ◽  
Gst Activity

The structure of glutaredoxin 2 (Grx2) fromEscherichia colico-crystallized with glutathione (GSH) was solved at 1.60 Å resolution. The structure of a mutant with the active-site residues Cys9 and Cys12 changed to serine crystallized in the absence of glutathione was solved to 2.4 Å resolution. Grx2 has an N-terminal domain characteristic of glutaredoxins, and the overall structure is congruent with the structure of glutathioneS-transferases (GSTs). Purified Grx2 exhibited GST activity. Grx2, which is the physiological electron donor for arsenate reduction byE. coliArsC, was docked with ArsC. The docked structure could be fitted with GSH bridging the active sites of the two proteins. It is proposed that Grx2 is a novel Grx/GST hybrid that functions in two steps of the ArsC catalytic cycle: as a GST it catalyzes glutathionylation of the ArsC–As(V) intermediate and as a glutaredoxin it catalyzes deglutathionylation of the ArsC–As(III)–SG intermediate.

scholarly journals Directed evolution induces tributyrin hydrolysis in a virulence factor of Xylella fastidiosa using a duplicated gene as a template

F1000Research ◽  
2014 ◽  
Vol 3 ◽  
pp. 215 ◽  
Author(s):  
Hossein Gouran ◽  
Sandeep Chakraborty ◽  
Basuthkar J. Rao ◽  
Bjarni Asgeirsson ◽  
Abhaya M. Dandekar
Keyword(s):  
Directed Evolution ◽  
Active Site ◽  
Xylella Fastidiosa ◽  
Catalytic Efficiency ◽  
Resistance Mechanisms ◽  
Loss Of Function ◽  
Active Site Residues ◽  
Protein Protein Interaction ◽  
Moonlighting Functions

Duplication of genes is one of the preferred ways for natural selection to add advantageous functionality to the genome without having to reinvent the wheel with respect to catalytic efficiency and protein stability. The duplicated secretory virulence factors ofXylella fastidiosa(LesA, LesB and LesC), implicated in Pierce's disease of grape and citrus variegated chlorosis of citrus species, epitomizes the positive selection pressures exerted on advantageous genes in such pathogens. A deeper insight into the evolution of these lipases/esterases is essential to develop resistance mechanisms in transgenic plants. Directed evolution, an attempt to accelerate the evolutionary steps in the laboratory, is inherently simple when targeted for loss of function. A bigger challenge is to specify mutations that endow a new function, such as a lost functionality in a duplicated gene. Previously, we have proposed a method for enumerating candidates for mutations intended to transfer the functionality of one protein into another related protein based on the spatial and electrostatic properties of the active site residues (DECAAF). In the current work, we presentin vivovalidation of DECAAF by inducing tributyrin hydrolysis in LesB based on the active site similarity to LesA. The structures of these proteins have been modeled using RaptorX based on the closely related LipA protein fromXanthomonas oryzae. These mutations replicate the spatial and electrostatic conformation of LesA in the modeled structure of the mutant LesB as well, providingin silicovalidation before proceeding to the laboriousin vivowork. Such focused mutations allows one to dissect the relevance of the duplicated genes in finer detail as compared to gene knockouts, since they do not interfere with other moonlighting functions, protein expression levels or protein-protein interaction.

A bacterial amber suppressor in Saccharomyces cerevisiae is selectively recognized by a bacterial aminoacyl-tRNA synthetase

1990 ◽  
Vol 10 (4) ◽  
pp. 1633-1641
Author(s):  
H Edwards ◽  
P Schimmel
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
High Specificity ◽  
Trna Synthetase ◽  
Aminoacyl Trna Synthetase ◽  
Cell Compartment ◽  
E Coli ◽  
Bacterial Enzyme ◽  
Amber Suppressor

Little is known about the conservation of determinants for the identities of tRNAs between organisms. We showed previously that Escherichia coli tyrosine tRNA synthetase can charge the Saccharomyces cerevisiae mitochondrial tyrosine tRNA in vivo, even though there are substantial sequence differences between the yeast mitochondrial and bacterial tRNAs. The S. cerevisiae cytoplasmic tyrosine tRNA differs in sequence from both its yeast mitochondrial and E. coli counterparts. To test whether the yeast cytoplasmic tyrosyl-tRNA synthetase recognizes the E. coli tRNA, we expressed various amounts of an E. coli tyrosine tRNA amber suppressor in S. cerevisiae. The bacterial tRNA did not suppress any of three yeast amber alleles, suggesting that the yeast enzymes retain high specificity in vivo for their homologous tRNAs. Moreover, the nucleotides in the sequence of the E. coli suppressor that are not shared with the yeast cytoplasmic tyrosine tRNA do not create determinants which are efficiently recognized by other yeast charging enzymes. Therefore, at least some of the determinants that influence in vivo recognition of the tyrosine tRNA are specific to the cell compartment and organism. In contrast, expression of the cognate bacterial tyrosyl-tRNA synthetase together with the bacterial suppressor tRNA led to suppression of all three amber alleles. The bacterial enzyme recognized its substrate in vivo, even when the amount of bacterial tRNA was less than about 0.05% of that of the total cytoplasmic tRNA.

Photosensitization of Escherichia coli and Saccharomyces cerevisiae by phenylheptatriyne from Bidens pilosa

1980 ◽  
Vol 26 (6) ◽  
pp. 698-705 ◽  
Author(s):  
Thor Arnason ◽  
Chi-Kit Wat ◽  
Kelsey Downum ◽  
Etsuo Yamamoto ◽  
Elizabeth Graham ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Singlet Oxygen ◽  
Uv Radiation ◽  
Theory Model ◽  
Survival Curves ◽  
Bidens Pilosa ◽  
E Coli ◽  
Test Organisms

The photosensitizing action of 7-phenylhepta-2,4,6-triyne (PHT), a polyacetylenic compound isolated from Bidens pilosa L. (Asteraceae), has been studied using Escherichia coli and Saccharomyces cerevisiae as the test organisms. The survival curves for E. coli treated with PHT and ultraviolet (UV) radiation were obtained and have been interpreted quantitatively on the basis of a target theory model. The number of "targets" in the cell that must be destroyed before cell death occurs was estimated at six, whereas the dose, D0, that reduced the surviving fraction of the population to 1/e of its value was estimated to be 280 J/m2.Survival was enhanced in aerobic conditions as compared with anaerobic conditions, which is strong evidence that PHT does not behave as a photodynamic sensitizer in vivo. This view was confirmed by work with azide (a quencher of singlet oxygen), D2O (which increases the lifetime of singlet oxygen), and superoxide dismutase (which scavenges superoxide radicals). None of these treatments modified the survival curves significantly, indicating that activated species of O2 are probably not involved in photosensitizations with PHT in vivo.Cell respiration was found to be rapidly inhibited by mild treatments of PHT and UV radiation, suggesting that nuclear metabolism is not the primary target of photosensitization as is the case with another group of photosensitizers, the furanocoumarins. The available evidence indicates that PHT is a representative of a new class of phototoxic compounds. A mechanism of action involving production of free radicals is proposed.

RAD4 gene of Saccharomyces cerevisiae: molecular cloning and partial characterization of a gene that is inactivated in Escherichia coli

1987 ◽  
Vol 7 (3) ◽  
pp. 1180-1192
Author(s):  
R Fleer ◽  
C M Nicolet ◽  
G A Pure ◽  
E C Friedberg
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Insertional Mutagenesis ◽  
Genomic Library ◽  
Excision Repair ◽  
Essential Gene ◽  
Yeast Cells ◽  
E Coli ◽  
Uv Sensitivity

In contrast to other Saccharomyces cerevisiae RAD genes involved in nucleotide excision repair of DNA, the RAD4 gene could not be isolated by screening a yeast genomic library for recombinant plasmids which complement the UV sensitivity of rad4 mutants (Pure et al., J. Mol. Biol. 183:31-42, 1985). We therefore attempted to walk to RAD4 from the neighboring SPT2 gene and obtained an integrating derivative of a plasmid isolated by Roeder et al. (Mol. Cell. Biol. 5:1543-1553, 1985) which contains a 4-kilobase fragment of yeast DNA including a mutant allele of SPT2. When integrated into several different rad4 mutant strains, this plasmid (pR169) complements UV sensitivity at a frequency of approximately 10%. However, a centromeric plasmid containing rescued sequences which include flanking yeast DNA no longer complements the phenotype of rad4 mutants. Complementing activity was restored by in vivo repair of a defined gap in the centromeric plasmid. The repaired plasmid fully complements the UV sensitivity of all rad4 mutants tested when isolated directly from yeast cells, but when this plasmid is propagated in Escherichia coli complementing activity is lost. We have mapped the physical location of the RAD4 gene by insertional mutagenesis and by transcript mapping. The gene is approximately 2.3 kilobases in size and is located immediately upstream of the SPT2 gene. Both genes are transcribed in the same direction. RAD4 is not an essential gene, and no increased transcription of this gene is observed in cells exposed to the DNA-damaging agent 4-nitroquinoline-1-oxide. The site of inactivation of RAD4 in a particular plasmid propagated in E. coli was localized to a 100-base-pair region by gene disruption and gap repair experiments. In addition, we have identified the approximate locations of the chromosomal rad4-2, rad4-3, and rad4-4 mutations.

scholarly journals A bacterial amber suppressor in Saccharomyces cerevisiae is selectively recognized by a bacterial aminoacyl-tRNA synthetase.

1990 ◽  
Vol 10 (4) ◽  
pp. 1633-1641 ◽  
Author(s):  
H Edwards ◽  
P Schimmel
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
High Specificity ◽  
Trna Synthetase ◽  
Aminoacyl Trna Synthetase ◽  
Cell Compartment ◽  
E Coli ◽  
Bacterial Enzyme ◽  
Amber Suppressor

Little is known about the conservation of determinants for the identities of tRNAs between organisms. We showed previously that Escherichia coli tyrosine tRNA synthetase can charge the Saccharomyces cerevisiae mitochondrial tyrosine tRNA in vivo, even though there are substantial sequence differences between the yeast mitochondrial and bacterial tRNAs. The S. cerevisiae cytoplasmic tyrosine tRNA differs in sequence from both its yeast mitochondrial and E. coli counterparts. To test whether the yeast cytoplasmic tyrosyl-tRNA synthetase recognizes the E. coli tRNA, we expressed various amounts of an E. coli tyrosine tRNA amber suppressor in S. cerevisiae. The bacterial tRNA did not suppress any of three yeast amber alleles, suggesting that the yeast enzymes retain high specificity in vivo for their homologous tRNAs. Moreover, the nucleotides in the sequence of the E. coli suppressor that are not shared with the yeast cytoplasmic tyrosine tRNA do not create determinants which are efficiently recognized by other yeast charging enzymes. Therefore, at least some of the determinants that influence in vivo recognition of the tyrosine tRNA are specific to the cell compartment and organism. In contrast, expression of the cognate bacterial tyrosyl-tRNA synthetase together with the bacterial suppressor tRNA led to suppression of all three amber alleles. The bacterial enzyme recognized its substrate in vivo, even when the amount of bacterial tRNA was less than about 0.05% of that of the total cytoplasmic tRNA.

scholarly journals The Active-Site Cysteines of the Periplasmic Thioredoxin-Like Protein CcmG of Escherichia coli Are Important but Not Essential for Cytochrome c Maturation In Vivo

1998 ◽  
Vol 180 (7) ◽  
pp. 1947-1950 ◽  
Author(s):  
Renata A. Fabianek ◽  
Hauke Hennecke ◽  
Linda Thöny-Meyer
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Low Molecular Weight ◽  
Thiol Compounds ◽  
Cytochrome C Maturation ◽  
E Coli ◽  
The Family ◽  
Membrane Bound ◽  
Hydrophilic Domain

ABSTRACT A new member of the family of periplasmic protein thiol:disulfide oxidoreductases, CcmG (also called DsbE), was characterized with regard to its role in cytochrome c maturation in Escherichia coli. The CcmG protein was shown to be membrane bound, facing the periplasm with its C-terminal, hydrophilic domain. A chromosomal, nonpolar in-frame deletion in ccmG resulted in the complete absence of all c-type cytochromes. Replacement of either one or both of the two cysteine residues of the predicted active site in CcmG (WCPTC) led to low but detectable levels ofBradyrhizobium japonicum holocytochromec 550 expressed in E. coli. This defect, but not that of the ccmG null mutant, could be complemented by adding low-molecular-weight thiol compounds to growing cells, which is in agreement with a reducing function for CcmG.

scholarly journals In vitro turnover numbers do not reflect in vivo activities of yeast enzymes

2021 ◽  
Vol 118 (32) ◽  
pp. e2108391118
Author(s):  
Yu Chen ◽  
Jens Nielsen
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Large Scale ◽  
Metabolic Model ◽  
Proteomics Data ◽  
Weak Correlation ◽  
E Coli ◽  
Activity Of Enzymes

Turnover numbers (kcat values) quantitatively represent the activity of enzymes, which are mostly measured in vitro. While a few studies have reported in vivo catalytic rates (kapp values) in bacteria, a large-scale estimation of kapp in eukaryotes is lacking. Here, we estimated kapp of the yeast Saccharomyces cerevisiae under diverse conditions. By comparing the maximum kapp across conditions with in vitro kcat we found a weak correlation in log scale of R2 = 0.28, which is lower than for Escherichia coli (R2 = 0.62). The weak correlation is caused by the fact that many in vitro kcat values were measured for enzymes obtained through heterologous expression. Removal of these enzymes improved the correlation to R2 = 0.41 but still not as good as for E. coli, suggesting considerable deviations between in vitro and in vivo enzyme activities in yeast. By parameterizing an enzyme-constrained metabolic model with our kapp dataset we observed better performance than the default model with in vitro kcat in predicting proteomics data, demonstrating the strength of using the dataset generated here.

scholarly journals RAD4 gene of Saccharomyces cerevisiae: molecular cloning and partial characterization of a gene that is inactivated in Escherichia coli.

1987 ◽  
Vol 7 (3) ◽  
pp. 1180-1192 ◽  
Author(s):  
R Fleer ◽  
C M Nicolet ◽  
G A Pure ◽  
E C Friedberg
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Insertional Mutagenesis ◽  
Genomic Library ◽  
Excision Repair ◽  
Essential Gene ◽  
Yeast Cells ◽  
E Coli ◽  
Uv Sensitivity

In contrast to other Saccharomyces cerevisiae RAD genes involved in nucleotide excision repair of DNA, the RAD4 gene could not be isolated by screening a yeast genomic library for recombinant plasmids which complement the UV sensitivity of rad4 mutants (Pure et al., J. Mol. Biol. 183:31-42, 1985). We therefore attempted to walk to RAD4 from the neighboring SPT2 gene and obtained an integrating derivative of a plasmid isolated by Roeder et al. (Mol. Cell. Biol. 5:1543-1553, 1985) which contains a 4-kilobase fragment of yeast DNA including a mutant allele of SPT2. When integrated into several different rad4 mutant strains, this plasmid (pR169) complements UV sensitivity at a frequency of approximately 10%. However, a centromeric plasmid containing rescued sequences which include flanking yeast DNA no longer complements the phenotype of rad4 mutants. Complementing activity was restored by in vivo repair of a defined gap in the centromeric plasmid. The repaired plasmid fully complements the UV sensitivity of all rad4 mutants tested when isolated directly from yeast cells, but when this plasmid is propagated in Escherichia coli complementing activity is lost. We have mapped the physical location of the RAD4 gene by insertional mutagenesis and by transcript mapping. The gene is approximately 2.3 kilobases in size and is located immediately upstream of the SPT2 gene. Both genes are transcribed in the same direction. RAD4 is not an essential gene, and no increased transcription of this gene is observed in cells exposed to the DNA-damaging agent 4-nitroquinoline-1-oxide. The site of inactivation of RAD4 in a particular plasmid propagated in E. coli was localized to a 100-base-pair region by gene disruption and gap repair experiments. In addition, we have identified the approximate locations of the chromosomal rad4-2, rad4-3, and rad4-4 mutations.

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