Permutation of the Active Site Motif of Tryparedoxin 2

2000 ◽  
Vol 381 (3) ◽  
pp. 211-219 ◽  
Author(s):  
Peter Steinert ◽  
Karin Plank-Schumacher ◽  
Marisa Montemartini ◽  
Hans-Jürgen Hecht ◽  
Leopold Flohé
Keyword(s):  
Active Site ◽  
Active Sites ◽  
Antioxidant Defense ◽  
Positive Charge ◽  
Specific Activity ◽  
Molecular Targets ◽  
E Coli ◽  
Active Site Motif ◽  
Nickel Chelate ◽  
Sitedirected Mutagenesis

Abstract Tryparedoxins (TXN) are thioredoxinrelated proteins which, as trypanothione:peroxiredoxin oxidoreductases, constitute the trypanothionedependent antioxidant defense and may also serve as substrates for ribonucleotide reductase in trypanosomatids. The active site motif of TXN2, [40]WCPPCR[45], of Crithidia fasciculata was mutated by sitedirected mutagenesis and eight corresponding muteins were expressed in E. coli as terminally Histagged proteins, purified to homogeneity by nickel chelate chromatography, and characterized in terms of specific activity, specificity and, if possible, kinetics. Exchange of Cys41 and Cys44 by serine yielded inactive products confirming their presumed involvement in catalysis. Exchange of Arg45 by aspartate resulted in loss of activity, suggesting an activation of active site cysteines by the positive charge of Arg45. Substitution of Trp40 by phenylalanine or tyrosine resulted in moderate decrease of specific activity, as did exchange of Pro42 by glycine. Kinetic analysis of these three muteins revealed that primarily the reaction with trypanothione is affected by the mutations. Simulation of thioredoxin or glutaredoxin like active sites in TXN2 (P42G and W40T/P43Y, respectively) did not result in thioredoxin or glutaredoxin like activities. These data underscore that TXNs, although belonging to the thioredoxin superfamily, represent a group of enzymes distinct from thioredoxins and glutaredoxins in terms of specificity, and appear attractive as molecular targets for the design of trypanocidal compounds.

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.

Construction of a Novel Recombinant Escherichia coli Strain Capable of Producing 1,3–propanediol

2011 ◽  
Vol 396-398 ◽  
pp. 2499-2502 ◽  
Author(s):  
Xiang Hui Qi ◽  
Qi Guo ◽  
Yu Tuo Wei ◽  
Hong Xu ◽  
Ri Bo Huang
Keyword(s):  
Escherichia Coli ◽  
Specific Activity ◽  
Gel Filtration ◽  
Coli Strain ◽  
Optimal Temperature ◽  
Recombinant Enzymes ◽  
Microbial Conversion ◽  
Gene Encoding ◽  
E Coli ◽  
Nickel Chelate

1, 3-propanediol (1, 3-PD) is biologically synthesized by glycerol dehydratase (GDHt) and 1, 3-propanediol dehydrogenase (PDOR). In present study, the gldABC gene, encoding GDHt from Klebsiella pneumoniae and the yqhD gene, encoding PDOR isoenzyme from E.coli BL21 were cloned and co-expressed in E.coli JM109 using plasmid pSE380. The over-expressed recombinant enzymes were purified by nickel-chelate chromatography combined with gel filtration to study the properties. Optimal temperature and pH of recombinant GDHt with specific activity of 85.8 U/mg were 45 °C and 9.0; and optimal temperature and pH of recombinant YqhD with specific activity of 80.0 U/mg were 37 °C, 7.0. The microbial conversion of 1,3-PD from glycerol by this recombinant E. coli strain was studied and the production of 1,3-PD was about 28.0 g/l.

scholarly journals Identification of the active-site lysine residues of two biosynthetic 3-dehydroquinases

1991 ◽  
Vol 275 (1) ◽  
pp. 1-6 ◽  
Author(s):  
S Chaudhuri ◽  
K Duncan ◽  
L D Graham ◽  
J R Coggins
Keyword(s):  
Escherichia Coli ◽  
Schiff Base ◽  
Nucleotide Sequence ◽  
Active Site ◽  
Active Sites ◽  
Amino Acid Sequences ◽  
E Coli ◽  
Lysine Residues ◽  
Schiff Base Formation ◽  
Base Formation

The lysine residues involved in Schiff-base formation at the active sites of both the 3-dehydroquinase component of the pentafunctional arom enzyme of Neurospora crassa and of the monofunctional 3-dehydroquinase of Escherichia coli were labelled by treatment with 3-dehydroquinate in the presence of NaB3H4. Radioactive peptides were isolated by h.p.l.c. following digestion with CNBr (and in one case after further digestion with trypsin). The sequence established for the N. crassa peptide was ALQHGDVVKLVVGAR, and that for the E. coli peptide was QSFDADIPKIA. An amended nucleotide sequence for the E. coli gene (aroD) that encode 3-dehydroquinase is also presented, along with a revised alignment of the deduced amino acid sequences for the biosynthetic enzymes.

scholarly journals Identification of Arg-12 in the active site of Escherichia coli K1 CMP-sialic acid synthetase

1999 ◽  
Vol 343 (2) ◽  
pp. 397-402 ◽  
Author(s):  
Daniel M. STOUGHTON ◽  
Gerardo ZAPATA ◽  
Robert PICONE ◽  
Willie F. VANN
Keyword(s):  
Escherichia Coli ◽  
Sialic Acid ◽  
Active Site ◽  
Positive Charge ◽  
Enzymic Activity ◽  
Site Directed Mutagenesis ◽  
E Coli ◽  
Steady State Kinetic ◽  
State Kinetic ◽  
Escherichia Coli K1

Escherichia coli K1 CMP-sialic acid synthetase catalyses the synthesis of CMP-sialic acid from CTP and sialic acid. The active site of the 418 amino acid E. coli enzyme was localized to its N-terminal half. The bacterial CMP-sialic acid synthetase enzymes have a conserved motif, IAIIPARXXSKGLXXKN, at their N-termini. Several basic residues have been identified at or near the active site of the E. coli enzyme by chemical modification and site-directed mutagenesis. Only one of the lysines in the N-terminal motif, Lys-21, appears to be essential for activity. Mutation of Lys-21 in the N-terminal motif results in an inactive enzyme. Furthermore, Arg-12 of the N-terminal motif appears to be an active-site residue, based on the following evidence. Substituting Arg-12 with glycine or alanine resulted in inactive enzymes, indicating that this residue is required for enzymic activity. The Arg-12 → Lys mutant was partially active, demonstrating that a positive charge is required at this site. Steady-state kinetic analysis reveals changes in kcat, Km and Ks for CTP, which implicates Arg-12 in catalysis and substrate binding.

scholarly journals Temperature-dependence of intramolecular coupling of active sites in pyruvate dehydrogenase multienzyme complexes

1983 ◽  
Vol 213 (2) ◽  
pp. 331-338 ◽  
Author(s):  
L C Packman ◽  
C J Stanley ◽  
R N Perham
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Active Site ◽  
Active Sites ◽  
Bacillus Stearothermophilus ◽  
Enzyme Complex ◽  
E Coli ◽  
Multienzyme Complexes ◽  
Enzyme Subunits ◽  
Polypeptide Chains ◽  
Core Migration

Intramolecular coupling of active sites in the pyruvate dehydrogenase multienzyme complexes of Escherichia coli, ox heart and Bacillus stearothermophilus was measured at various temperatures. As the temperature was raised, the extent of active-site coupling was found to increase, approaching a maximum near the physiological growth temperature of the organism. Under these conditions, a single pyruvate dehydrogenase (lipoamide) dimer appeared able to cause a rapid (20s) reductive acetylation of probably all 24 polypeptide chains in the dihydrolipoamide acetyltransferase core of the enzyme complex from E. coli at 37 degrees C, and of most if not all of the 60 polypeptide chains in the dihydrolipoamide acetyltransferase cores of the enzymes from ox heart and B. stearothermophilus at 37 degrees C and 60 degrees C respectively. Experiments designed to measure the inter-core and intra-core migration of enzyme subunits suggested that, in the bacterial enzymes at least, this was not a major contributor to active-site coupling.

scholarly journals Structures of Mammalian and Bacterial Fructose-1,6-bisphosphatase Reveal the Basis for Synergism in AMP/Fructose 2,6-Bisphosphate Inhibition

2007 ◽  
Vol 282 (49) ◽  
pp. 36121-36131 ◽  
Author(s):  
Justin K. Hines ◽  
Xiaoming Chen ◽  
Jay C. Nix ◽  
Herbert J. Fromm ◽  
Richard B. Honzatko
Keyword(s):  
Conformational Change ◽  
Conformational Changes ◽  
Active Site ◽  
Active Sites ◽  
Control Point ◽  
Type I ◽  
Allosteric Site ◽  
E Coli ◽  
Fructose 1,6 Bisphosphatase ◽  
A Subunit

Fructose-1,6-bisphosphatase (FBPase) operates at a control point in mammalian gluconeogenesis, being inhibited synergistically by fructose 2,6-bisphosphate (Fru-2,6-P2) and AMP. AMP and Fru-2,6-P2 bind to allosteric and active sites, respectively, but the mechanism responsible for AMP/Fru-2,6-P2 synergy is unclear. Demonstrated here for the first time is a global conformational change in porcine FBPase induced by Fru-2,6-P2 in the absence of AMP. The Fru-2,6-P2 complex exhibits a subunit pair rotation of 13° from the R-state (compared with the 15° rotation of the T-state AMP complex) with active site loops in the disengaged conformation. A three-state thermodynamic model in which Fru-2,6-P2 drives a conformational change to a T-like intermediate state can account for AMP/Fru-2,6-P2 synergism in mammalian FBPases. AMP and Fru-2,6-P2 are not synergistic inhibitors of the Type I FBPase from Escherichia coli, and consistent with that model, the complex of E. coli FBPase with Fru-2,6-P2 remains in the R-state with dynamic loops in the engaged conformation. Evidently in porcine FBPase, the actions of AMP at the allosteric site and Fru-2,6-P2 at the active site displace engaged dynamic loops by distinct mechanisms, resulting in similar quaternary end-states. Conceivably, Type I FBPases from all eukaryotes may undergo similar global conformational changes in response to Fru-2,6-P2 ligation.

Properties of phosphorylated protein intermediates of the bacterial phosphoenolpyruvate:sugar phosphotransferase system

1992 ◽  
Vol 70 (3-4) ◽  
pp. 242-246 ◽  
Author(s):  
J. W. Anderson ◽  
E. B. Waygood ◽  
M. H. Saier Jr. ◽  
J. Reizer
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Active Site ◽  
Active Sites ◽  
Sugar Transport ◽  
Wild Type ◽  
Phosphotransferase System ◽  
E Coli ◽  
Phosphorylated Protein ◽  
Enzyme I

The phosphohydrolysis properties of the following phosphoprotein intermediates of the bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) were investigated: enzyme I, HPr, and the IIAGlc domain of the glucose enzyme II of Bacillus subtilis; and IIAGlc (fast and slow forms) of Escherichia coli. The phosphohydrolysis properties were also studied for the site-directed mutant H68A of B. subtilis IIAGlc. Several conclusions were reached. (i) The phosphohydrolysis properties of the homologous phosphoprotein intermediates of B. subtilis and E. coli are similar. (ii) These properties deviate from those of isolated Nδ1- and Nε2-phosphohistidine indicating the participation of neighbouring residues at the active sites of these proteins. (iii) The rates of phosphohydrolysis of the H68A mutant of B. subtilis IIAGlc were reduced compared with the wild-type protein, suggesting that both His-83 and His-68 are present at the active site of wild-type IIAGlc. (iv) The removal of seven N-terminal residues of E. coli IIAGlc reduced the rates of phosphohydrolysis between pH 5 and 8.Key words: phosphoenolpyruvate:sugar phosphotransferase system, phosphoproteins, phosphohistidine, phosphorylation, sugar transport.

Overexpression and Characterization of the Gene Encoding 1,3-Propanediol Oxidoreductase of Citrobacter freundii

2014 ◽  
Vol 636 ◽  
pp. 121-124
Author(s):  
Xiang Hui Qi ◽  
Jing Fei Zhu ◽  
Yan Luo ◽  
Jing Lin ◽  
Xu Wang ◽  
...  
Keyword(s):  
Specific Activity ◽  
Gel Filtration ◽  
Raw Material ◽  
Biological Synthesis ◽  
Citrobacter Freundii ◽  
Gene Encoding ◽  
E Coli ◽  
Nickel Chelate ◽  
Future Work

Glycerol can be biologically converted to 1,3-propanediol (1,3-PD), a key raw material required for the synthesis of polytrimethylene terephthalate and other polyester fibers. 1,3-propanediol oxidoreductase (PDOR) is the rate-limiting enzyme in 1,3-PD synthesis biologically pathway. In present study, the dhaT gene encoding PDOR was cloned from Citrobacter freundii and overexpressed in E. coli BL21. The recombinant enzyme was purified by nickel-chelate chromatography combined with gel filtration to study the enzymatic characterization. Specific activity of recombinant PDOR was 55.2 U/mg. Km and Vmax values were 8.9 mM, 40.2 U/mg respectively. Holoenzyme of PDOR maybe a decamer of which a monomer has a molecular mass of 43 kDa. This research may form a basis for the future work on biological synthesis of 1, 3-PD.

scholarly journals Inactivation of horse liver mitochondrial aldehyde dehydrogenase by disulfiram. Evidence that disulfiram is not an active-site-directed reagent

1987 ◽  
Vol 242 (2) ◽  
pp. 499-503 ◽  
Author(s):  
C G Sanny ◽  
H Weiner
Keyword(s):  
Catalytic Activity ◽  
Aldehyde Dehydrogenase ◽  
Active Site ◽  
Active Sites ◽  
Specific Activity ◽  
Cysteine Residue ◽  
Complete Inactivation ◽  
Horse Liver ◽  
Inactivation Mechanism ◽  
Liver Aldehyde Dehydrogenase

The inhibition of mitochondrial (pI 5) horse liver aldehyde dehydrogenase by disulfiram (tetraethylthiuram disulphide) was investigated to determine if the drug was an active-site-directed inhibitor. Stoichiometry of inhibition was determined by using an analogue, [35S]tetramethylthiuram disulphide. A 50% loss of the dehydrogenase activity was observed when only one site per tetrameric enzyme was modified, and complete inactivation was not obtained even after seven sites per tetramer were modified. Modification of only two sites accounted for a loss of 75% of the initial catalytic activity. The number of functioning active sites per tetrameric enzyme, as determined by the magnitude of the pre-steady-state burst of NADH formation, did not decrease until approx. 75% of the catalytic activity was lost. These data indicate that disulfiram does not modify the essential nucleophilic amino acid at the active site of the enzyme. The data support an inactivation mechanism involving the formation of a mixed disulphide with a non-essential cysteine residue, resulting in a lowered specific activity of the enzyme.

Expression of full-length human alkylglycerol monooxygenase and fragments in Escherichia coli

Pteridines ◽  
2013 ◽  
Vol 24 (1) ◽  
pp. 111-115 ◽  
Author(s):  
Matthias Mayer ◽  
Markus A. Keller ◽  
Katrin Watschinger ◽  
Gabriele Werner-Felmayer ◽  
Ernst R. Werner ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Active Sites ◽  
Gel Filtration ◽  
Full Length ◽  
Expression Vectors ◽  
Alkyl Ether ◽  
High Tendency ◽  
Hydrophobic Domain ◽  
E Coli

AbstractAlkylglycerol monooxygenase (AGMO; EC 1.14.16.5) is the only enzyme known to cleave the O-alkyl ether bond of alkylglycerols in humans. It is an integral membrane protein with nine predicted transmembrane domains. We attempted to express and purify full-length and truncated forms of AGMO in Escherichia coli. Full-length AGMO could not be expressed in three different E. coli expression strains, three different expression vectors and several induction systems. We succeeded, however, in expression of three N-terminally strep-tagged truncated forms, named active sites 1, 2 and 3, with 205, 134 and 61 amino acids, respectively. Active site 1 fragment, containing two predicted transmembrane regions, a membrane associated region and all known amino acid residues important for catalytic activity, was not fully soluble even in 8 M urea. Active site 2 containing only one predicted membrane associated domain required 8 M urea for solubilisation and eluted in gel filtration in 1 M urea as a trimer. Active site 3 with no hydrophobic domain eluted in gel filtration in 1 M urea as monomer and dimer. These results show that even truncated forms of AGMO are barely soluble when expressed in E. coli and show a high tendency for aggregation.

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