Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Expression of rat liver S-adenosylmethionine synthetase in Escherichia coli results in two active oligomeric forms.

ABSTRACT: A cDNA containing the complete coding sequence for rat liver S-adenosylmethionine synthetase was cloned into the prokaryotic expression vector pT7-7 and expressed in Escherichia coli BL21(DE3). A major additional band corresponding to a protein of 48 kDa was detected on SDS/PAGE after induction with isopropyl beta-D-thiogalactopyranoside. This protein was distributed in both the soluble and insoluble fractions and accounted for approx. 30% of the total bacterial protein. The soluble enzyme was fully active, as revealed by assays in vitro of S-adenosylmethionine synthetase activity. In addition, transformed bacteria exhibited highly increased levels of intracellular S-adenosylmethionine. Two active forms of the recombinant enzyme, with apparent molecular masses of 210 kDa and 110 kDa, were detected when cytosolic extracts of the transformed cells were fractionated by gel-filtration chromatography. It is concluded that the expressed S-adenosylmethionine synthetase polypeptide assemble as tetramers and dimers.

SUBMITTER: Alvarez L

PROVIDER: S-EPMC1137117 | biostudies-other | 1994 Jul

REPOSITORIES: biostudies-other

ACCESS DATA

Json Xml

Similar Datasets

Structural analysis of the active site geometry of N5-carboxyaminoimidazole ribonucleotide synthetase from Escherichia coli.

Project description:N(5)-Carboxyaminoimidazole ribonucleotide synthetase (N(5)-CAIR synthetase) converts 5-aminoimidazole ribonucleotide (AIR), MgATP, and bicarbonate into N(5)-CAIR, MgADP, and P(i). The enzyme is required for de novo purine biosynthesis in microbes yet is not found in humans suggesting that it represents an ideal and unexplored target for antimicrobial drug design. Here we report the X-ray structures of N(5)-CAIR synthetase from Escherichia coli with either MgATP or MgADP/P(i) bound in the active site cleft. These structures, determined to 1.6-A resolution, provide detailed information regarding the active site geometry before and after ATP hydrolysis. In both structures, two magnesium ions are observed. Each of these is octahedrally coordinated, and the carboxylate side chain of Glu238 bridges them. For the structure of the MgADP/P(i) complex, crystals were grown in the presence of AIR and MgATP. No electron density was observed for AIR, and the electron density corresponding to the nucleotide clearly revealed the presence of ADP and P(i) rather than ATP. The bound P(i) shifts by approximately 3 A relative to the gamma-phosphoryl group of ATP and forms electrostatic interactions with the side chains of Arg242 and His244. Since the reaction mechanism of N(5)-CAIR synthetase is believed to proceed via a carboxyphosphate intermediate, we propose that the location of the inorganic phosphate represents the binding site for stabilization of this reactive species. Using the information derived from the two structures reported here, coupled with molecular modeling, we propose a catalytic mechanism for N(5)-CAIR synthetase.

| S-EPMC2677550 | biostudies-literature

Study of the rat liver S-adenosylmethionine synthetase active site with 8-azido ATP.

Project description:The active site of rat liver S-adenosylmethionine synthetase was studied using 8-azido ATP, a photolabile analogue of ATP. Both forms of the enzyme, tetramer and dimer, could be labelled by using concentrations of the analogue similar to the KmATP values for each form, 350 microM and 1 mM respectively. Labelling of both S-adenosylmethionine synthetase forms with 8-azido [alpha-32P]ATP, followed by tryptic digestion and purification by HPLC, afforded one specifically labelled peptide in each case. Identification of the labelled peptide by amino acid analysis and peptide sequencing, and comparison with the enzyme sequence, indicated that the same peptide (267-286) was modified in both enzyme forms. The results are discussed on the basis of the high degree of similarity that this peptide shows in all the known S-adenosylmethionine synthetase sequences.

| S-EPMC1136963 | biostudies-other

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

Project description: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.

| S-EPMC1217272 | biostudies-other

Substrate specificity and catalysis by the editing active site of Alanyl-tRNA synthetase from Escherichia coli.

Project description:Aminoacyl-tRNA synthetases (ARSs) enhance the fidelity of protein synthesis through multiple mechanisms, including hydrolysis of the adenylate and cleavage of misacylated tRNA. Alanyl-tRNA synthetase (AlaRS) limits misacylation with glycine and serine by use of a dedicated editing domain, and a mutation in this activity has been genetically linked to a mouse model of a progressive neurodegenerative disease. Using the free-standing Pyrococcus horikoshii AlaX editing domain complexed with serine as a model and both Ser-tRNA(Ala) and Ala-tRNA(Ala) as substrates, the deacylation activities of the wild type and five different Escherichia coli AlaRS editing site substitution mutants were characterized. The wild-type AlaRS editing domain deacylated Ser-tRNA(Ala) with a k(cat)/K(M) of 6.6 × 10(5) M(-1) s(-1), equivalent to a rate enhancement of 6000 over the rate of enzyme-independent deacylation but only 12.2-fold greater than the rate with Ala-tRNA(Ala). While the E664A and T567G substitutions only minimally decreased k(cat)/K(M,) Q584H, I667E, and C666A AlaRS were more compromised in activity, with decreases in k(cat)/K(M) in the range of 6-, 6.6-, and 15-fold. C666A AlaRS was 1.7-fold more active on Ala-tRNA(Ala) relative to Ser-tRNA(Ala), providing the only example of a true reversal of substrate specificity and highlighting a potential role of the coordinated zinc in editing substrate specificity. Along with the potentially serious physiological consequences of serine misincorporation, the relatively modest specificity of the AlaRS editing domain may provide a rationale for the widespread phylogenetic distribution of AlaX free-standing editing domains, thereby contributing a further mechanism to lower concentrations of misacylated tRNA(Ala).

| S-EPMC3069921 | biostudies-literature

Two forms of ribosomal protein L2 of Escherichia coli that inhibit DnaA in DNA replication.

Project description:We purified an inhibitor of oriC plasmid replication and determined that it is a truncated form of ribosomal protein L2 evidently lacking 59 amino acid residues from the C-terminal region encoded by rplB. We show that this truncated form of L2 or mature L2 physically interacts with the N-terminal region of DnaA to inhibit initiation from oriC by apparently interfering with DnaA oligomer formation, and the subsequent assembly of the prepriming complex on an oriC plasmid. Both forms of L2 also inhibit the unwinding of oriC by DnaA. These in vitro results raise the possibility that one or both forms of L2 modulate DnaA function in vivo to regulate the frequency of initiation.

| S-EPMC3105425 | biostudies-literature

Escherichia coli Alanyl-tRNA synthetase oxidation

Project description:LC MS/MS analysis (Fred Hutchinson Cancer Research Center, Seattle, Washington)

2021-12-20 | MSV000088593 | MassIVE

Regulation of acetyl coenzyme A synthetase in Escherichia coli.

Project description:Cells of Escherichia coli growing on sugars that result in catabolite repression or amino acids that feed into glycolysis undergo a metabolic switch associated with the production and utilization of acetate. As they divide exponentially, these cells excrete acetate via the phosphotransacetylase-acetate kinase pathway. As they begin the transition to stationary phase, they instead resorb acetate, activate it to acetyl coenzyme A (acetyl-CoA) by means of the enzyme acetyl-CoA synthetase (Acs) and utilize it to generate energy and biosynthetic components via the tricarboxylic acid cycle and the glyoxylate shunt, respectively. Here, we present evidence that this switch occurs primarily through the induction of acs and that the timing and magnitude of this induction depend, in part, on the direct action of the carbon regulator cyclic AMP receptor protein (CRP) and the oxygen regulator FNR. It also depends, probably indirectly, upon the glyoxylate shunt repressor IclR, its activator FadR, and many enzymes involved in acetate metabolism. On the basis of these results, we propose that cells induce acs, and thus their ability to assimilate acetate, in response to rising cyclic AMP levels, falling oxygen partial pressure, and the flux of carbon through acetate-associated pathways.

| S-EPMC101899 | biostudies-literature

A bifunctional salvage pathway for two distinct S-adenosylmethionine by-products that is widespread in bacteria, including pathogenic Escherichia coli.

Project description:S-adenosyl-l-methionine (SAM) is a necessary cosubstrate for numerous essential enzymatic reactions including protein and nucleotide methylations, secondary metabolite synthesis and radical-mediated processes. Radical SAM enzymes produce 5'-deoxyadenosine, and SAM-dependent enzymes for polyamine, neurotransmitter and quorum sensing compound synthesis produce 5'-methylthioadenosine as by-products. Both are inhibitory and must be addressed by all cells. This work establishes a bifunctional oxygen-independent salvage pathway for 5'-deoxyadenosine and 5'-methylthioadenosine in both Rhodospirillum rubrum and Extraintestinal Pathogenic Escherichia coli. Homologous genes for this pathway are widespread in bacteria, notably pathogenic strains within several families. A phosphorylase (Rhodospirillum rubrum) or separate nucleoside and kinase (Escherichia coli) followed by an isomerase and aldolase sequentially function to salvage these two wasteful and inhibitory compounds into adenine, dihydroxyacetone phosphate and acetaldehyde or (2-methylthio)acetaldehyde during both aerobic and anaerobic growth. Both SAM by-products are metabolized with equal affinity during aerobic and anaerobic growth conditions, suggesting that the dual-purpose salvage pathway plays a central role in numerous environments, notably the human body during infection. Our newly discovered bifunctional oxygen-independent pathway, widespread in bacteria, salvages at least two by-products of SAM-dependent enzymes for carbon and sulfur salvage, contributing to cell growth.

| S-EPMC7237333 | biostudies-literature

Protein kinase C phosphorylation of rat liver S-adenosylmethionine synthetase: dissociation and production of an active monomer.

Project description:The regulation of rat liver S-adenosylmethionine synthetase (AdoMet synthetase), a key enzyme in methionine metabolism, by protein kinase C (PKC) phosphorylation has been studied. Both enzyme forms, tetramer and dimer, are phosphorylated by this kinase in the same residue, Thr-342, of the sequence. Phosphorylation of the dimer leads to its dissociation, with production of a fully-active monomer. The kinetics of the monomer have been studied, and a KmMet of 931.9 microM, a KmATP of 708 microM and a Vmax of 66.8 nmol/min/mg have been calculated. Alkaline phosphatase treatment of both enzyme forms (tetramer and dimer) produces a reduction in their activity with no change in the oligomeric state. On the other hand, PKC phosphorylation of the alkaline phosphatase-treated AdoMet synthetase forms leads to the dissociation of the dimer to produce a monomer. Rephosphorylation occurs again in the same residue, Thr-342, of the sequence. The significance of AdoMet synthetase regulation by PKC phosphorylation is further discussed.

| S-EPMC1137638 | biostudies-other

Factors defining the functional oligomeric state of Escherichia coli DegP protease.

Project description:Escherichia coli DegP protein is a periplasmic protein that functions both as a protease and as a chaperone. In the absence of substrate, DegP oligomerizes as a hexameric cage but in its presence DegP reorganizes into 12 and 24-mer cages with large chambers that house the substrate for degradation or refolding. Here, we studied the factors that determine the oligomeric state adopted by DegP in the presence of substrate. Using size exclusion chromatography and electron microscopy, we found that the size of the substrate molecule is the main factor conditioning the oligomeric state adopted by the enzyme. Other factors such as temperature, a major regulatory factor of the activity of this enzyme, did not influence the oligomeric state adopted by DegP. In addition, we observed that substrate concentration exerted an effect only when large substrates (full-length proteins) were used. However, small substrate molecules (peptides) always triggered the same oligomeric state regardless of their concentration. These results clarify important aspects of the regulation of the oligomeric state of DegP.

| S-EPMC3081313 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data