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 Inhibition of rabbit muscle aldolase by phosphorylated aromatic compounds

1997 ◽  
Vol 323 (1) ◽  
pp. 71-77 ◽  
Author(s):  
Casimir BLONSKI ◽  
Danielle DE MOISSAC ◽  
Jacques PÉRIÉ ◽  
Jurgen SYGUSCH
Keyword(s):  
Schiff Base ◽  
Active Site ◽  
Enzyme Inactivation ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Rabbit Muscle ◽  
Lysine Residues ◽  
Schiff Base Formation ◽  
Fructose 1,6 Bisphosphate ◽  
Base Formation

The interactions of the phosphorylated derivatives of hydroquinone (HQN-P2), resorcinol (RSN-P2), 4-hydroxybenzaldehyde (HBA-P) and 2,4-dihydroxybenzaldehyde (DHBA-P; phosphate group at position 4) with fructose bisphosphate aldolase were analysed by enzyme kinetics, UV/visible difference spectroscopy and site-directed mutagenesis. Enzyme activity was competitively inhibited in the presence of HQN-P2, RSN-P2 and HBA-P, whereas DHBA-P exhibited slow-binding inhibition. Inhibition by DHBA-P involved active-site Schiff-base formation and required a phenol group ortho to the aldehyde moiety. Rates of enzyme inactivation and of Schiff-base formation by DHBA-P were identical, and corresponded to 3.2-3.5 DHBA-P molecules covalently bound per aldolase tetramer at maximal inactivation. Site-directed mutagenesis of the active-site lysine residues at positions 107, 146 and 229 was found to be consistent with Schiff-base formation between DHBA-P and Lys-146, and this was promoted by Lys-229. Mutation of Glu-187, located vicinally between Lys-146 and Lys-229 in the active site, perturbed the rate of Schiff-base formation, suggesting a functional role for Glu-187 in Schiff-base formation and stabilization. The decreased cleavage activity of the active-site mutants towards fructose 1,6-bisphosphate is consistent with a proton-transfer mechanism involving Lys-229, Glu-187 and Lys-146.

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 Characterization of a 12-Kilodalton Rhodanese Encoded byglpE of Escherichia coli and Its Interaction with Thioredoxin

2000 ◽  
Vol 182 (8) ◽  
pp. 2277-2284 ◽  
Author(s):  
W. Keith Ray ◽  
Gang Zeng ◽  
M. Benjamin Potters ◽  
Aqil M. Mansuri ◽  
Timothy J. Larson
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Active Site ◽  
Amino Acid Sequences ◽  
E Coli ◽  
Active Site Cysteine ◽  
Clusters Of Orthologous Groups ◽  
Thioredoxin 1 ◽  
First Time

ABSTRACT Rhodaneses catalyze the transfer of the sulfane sulfur from thiosulfate or thiosulfonates to thiophilic acceptors such as cyanide and dithiols. In this work, we define for the first time the gene, and hence the amino acid sequence, of a 12-kDa rhodanese fromEscherichia coli. Well-characterized rhodaneses are comprised of two structurally similar ca. 15-kDa domains. Hence, it is thought that duplication of an ancestral rhodanese gene gave rise to the genes that encode the two-domain rhodaneses. The glpEgene, a member of the sn-glycerol 3-phosphate (glp) regulon of E. coli, encodes the 12-kDa rhodanese. As for other characterized rhodaneses, kinetic analysis revealed that catalysis by purified GlpE occurs by way of an enzyme-sulfur intermediate utilizing a double-displacement mechanism requiring an active-site cysteine. TheKm s for SSO3 2− and CN− were 78 and 17 mM, respectively. The apparent molecular mass of GlpE under nondenaturing conditions was 22.5 kDa, indicating that GlpE functions as a dimer. GlpE exhibited ak cat of 230 s−1. Thioredoxin 1 from E. coli, a small multifunctional dithiol protein, served as a sulfur acceptor substrate for GlpE with an apparentKm of 34 μM when thiosulfate was near itsKm , suggesting that thioredoxin 1 or related dithiol proteins could be physiological substrates for sulfurtransferases. The overall degree of amino acid sequence identity between GlpE and the active-site domain of mammalian rhodaneses is limited (∼17%). This work is significant because it begins to reveal the variation in amino acid sequences present in the sulfurtransferases. GlpE is the first among the 41 proteins in COG0607 (rhodanese-related sulfurtransferases) of the database Clusters of Orthologous Groups of proteins (http://www.ncbi.nlm.nih.gov/COG/ ) for which sulfurtransferase activity has been confirmed.

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.

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.

scholarly journals Evidence that pyridoxal phosphate modification of lysine residues (Lys-55 and Lys-59) causes inactivation of hydroxymethylbilane synthase (porphobilinogen deaminase)

1989 ◽  
Vol 262 (1) ◽  
pp. 119-124 ◽  
Author(s):  
A D Miller ◽  
L C Packman ◽  
G J Hart ◽  
P R Alefounder ◽  
C Abell ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Recombinant Strain ◽  
Pyridoxal Phosphate ◽  
Porphobilinogen Deaminase ◽  
Wild Type ◽  
E Coli ◽  
Lysine Residues

A recombinant strain of Escherichia coli has been constructed that produces approx. 200 times the amount of hydroxymethylbilane synthase found in wild-type E. coli [Hart, Abell & Battersby (1986) Biochem. J. 240, 273-276]. Enzyme purified from this strain is shown to be permanently inactivated by pyridoxal 5′-phosphate/NaB1H3(3)H1. The inactivation is not complete despite the fact that approx. 1 mol of lysine residues is modified per mol of enzyme. Evidence is gained showing that (a) modification of one of two conserved lysine residues (Lys-55 or Lys-59) results in inactivation of hydroxymethylbilane synthase and (b) these lysine residues are present in or close to the active site.

scholarly journals Roles of the C-Terminal Amino Acids of Non-Hexameric Helicases: Insights from Escherichia coli UvrD

2021 ◽  
Vol 22 (3) ◽  
pp. 1018
Author(s):  
Hiroaki Yokota
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Amino Acid ◽  
Single Molecule ◽  
Underlying Mechanism ◽  
Amino Acid Sequences ◽  
E Coli ◽  
X Ray Crystallography ◽  
Terminal Amino

Helicases are nucleic acid-unwinding enzymes that are involved in the maintenance of genome integrity. Several parts of the amino acid sequences of helicases are very similar, and these quite well-conserved amino acid sequences are termed “helicase motifs”. Previous studies by X-ray crystallography and single-molecule measurements have suggested a common underlying mechanism for their function. These studies indicate the role of the helicase motifs in unwinding nucleic acids. In contrast, the sequence and length of the C-terminal amino acids of helicases are highly variable. In this paper, I review past and recent studies that proposed helicase mechanisms and studies that investigated the roles of the C-terminal amino acids on helicase and dimerization activities, primarily on the non-hexermeric Escherichia coli (E. coli) UvrD helicase. Then, I center on my recent study of single-molecule direct visualization of a UvrD mutant lacking the C-terminal 40 amino acids (UvrDΔ40C) used in studies proposing the monomer helicase model. The study demonstrated that multiple UvrDΔ40C molecules jointly participated in DNA unwinding, presumably by forming an oligomer. Thus, the single-molecule observation addressed how the C-terminal amino acids affect the number of helicases bound to DNA, oligomerization, and unwinding activity, which can be applied to other helicases.

Schiff Base Formation upon Complexation of Tris(acetylacetonato)iron with N[(CH2CH2NH2)(CH2CH2OH)(CH2CH2CH2OH)]

2004 ◽  
Vol 630 (13-14) ◽  
pp. 2558-2561 ◽  
Author(s):  
F. Ekkehardt Hahn ◽  
Christoph Jocher ◽  
Thomas L�gger ◽  
Tania Pape
Keyword(s):  
Schiff Base ◽  
Schiff Base Formation ◽  
Base Formation
Sign in / Sign up

Export Citation Format

Share Document