scholarly journals Probing the NADPH-binding site of Escherichia coli flavodoxin oxidoreductase

2000 ◽  
Vol 352 (2) ◽  
pp. 257-266 ◽  
Author(s):  
Claire LEADBEATER ◽  
Lisa MCIVER ◽  
Dominic J. CAMPOPIANO ◽  
Scott P. WEBSTER ◽  
Robert L. BAXTER ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cytochrome C ◽  
Binding Site ◽  
Tertiary Structure ◽  
Reductase Activity ◽  
Close Approach ◽  
Pyridine Nucleotide ◽  
Phosphate Group ◽  
Site Directed Mutagenesis ◽  
Wild Type

The structure of the Escherichia coli flavodoxin NADP+ oxidoreductase (FLDR) places three arginines (R144, R174 and R184) in the proposed NADPH-binding site. Mutant enzymes produced by site-directed mutagenesis, in which each arginine was replaced by neutral alanine, were characterized. All mutants exhibited decreased NADPH-dependent cytochrome c reductase activity (R144A, 241.6min-1; R174A, 132.1min-1; R184A, 305.5min-1 versus wild type, 338.9min-1) and increased Km for NADPH (R144A, 5.3µM; R174A, 20.2µM; R184A, 54.4µM versus wild type, 3.9µM). The kcat value for NADH-dependent cytochrome c reduction was increased for R174A (42.3min-1) and R184A (50.4min-1) compared with the wild type (33.0min-1), consistent with roles for R174 and R184 in discriminating between NADPH/NADH by interaction with the adenosine ribose 2′-phosphate. Stopped-flow studies indicated that affinity (Kd) for NADPH was markedly reduced in mutants R144A (635µM) and R184A (2.3mM) compared with the wild type (< 5µM). Mutant R184A displays the greatest change in pyridine nucleotide preference, with the NADH/NADPH Kd ratio > 175-fold lower than for wild-type FLDR. The rate constant for hydride transfer from NADPH to flavin was lowest for R174A (kred = 8.82s-1 versus 22.63s-1 for the wild type), which also exhibited tertiary structure perturbation, as evidenced by alterations in CD and fluorescence spectra. Molecular modelling indicated that movement of the C-terminal tryptophan (W248) of FLDR is necessary to permit close approach of the nicotinamide ring of NADPH to the flavin. The positions of NADPH phosphates in the modelled structure are consistent with the kinetic data, with R174 and R184 located close to the adenosine ribose 2′-phosphate group, and R144 likely to interact with the nicotinamide ribose 5′-phosphate group.

scholarly journals Mutations in the Escherichia coli UvrB ATPase motif compromise excision repair capacity

1989 ◽  
Vol 86 (17) ◽  
pp. 6577-6581 ◽  
Author(s):  
T W Seeley ◽  
L Grossman
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Excision Repair ◽  
Site Directed Mutagenesis ◽  
Sequence Motif ◽  
Amino Acid Side Chain ◽  
Uv Resistance ◽  
Wild Type ◽  
General Applicability ◽  
Repair Capacity

The Escherichia coli UvrB protein possesses an amino acid sequence motif common to many ATPases. The role of this motif in UvrB has been investigated by site-directed mutagenesis. Three UvrB mutants, with amino acid replacements at lysine-45, failed to confer UV resistance when tested in the UV-sensitive strain N364 (delta uvrB), while five other mutants constructed near this region of UvrB confer wild-type levels of UV resistance. Because even the conservative substitution of arginine for lysine-45 in UvrB results in failure to confer UV resistance, we believe we have identified an amino acid side chain in UvrB essential to nucleotide excision repair in E. coli. The properties of two purified mutant UvrB proteins, lysine-45 to alanine (K45A) and asparagine-51 to alanine (N51A), were analyzed in vitro. While the K45A mutant is fully defective in incision of UV-irradiated DNA, K45A is capable of interaction with UvrA in forming an ATP-dependent nucleoprotein complex. The K45A mutant, however, fails to activate the characteristic increase in ATPase activity observed with the wild-type UvrB in the presence of UvrA and DNA. From these results we conclude that there is a second nucleotide-dependent step in incision following initial complex formation, which is defective in the K45A mutant. This experimental approach may prove of general applicability in the study of function and mechanism of other ATPase motif proteins.

scholarly journals Catalytic and structural contributions for glutathione-binding residues in a Delta class glutathione S-transferase

2004 ◽  
Vol 382 (2) ◽  
pp. 751-757 ◽  
Author(s):  
Pakorn WINAYANUWATTIKUN ◽  
Albert J. KETTERMAN
Keyword(s):  
Binding Site ◽  
Active Site ◽  
Structural Integrity ◽  
Catalytic Mechanism ◽  
Tertiary Structure ◽  
Site Directed Mutagenesis ◽  
Active Site Residues ◽  
Anopheles Dirus ◽  
Steady State Kinetics ◽  
Cellular Detoxification

Glutathione S-transferases (GSTs) are dimeric proteins that play a major role in cellular detoxification. The GSTs in mosquito Anopheles dirus species B, an important malaria vector in South East Asia, are of interest because they can play an important role in insecticide resistance. In the present study, we characterized the Anopheles dirus (Ad)GST D3-3 which is an alternatively spliced product of the adgst1AS1 gene. The data from the crystal structure of GST D3-3 shows that Ile-52, Glu-64, Ser-65, Arg-66 and Met-101 interact directly with glutathione. To study the active-site function of these residues, alanine substitution site-directed mutagenesis was performed resulting in five mutants: I52A (Ile-52→Ala), E64A, S65A, R66A and M101A. Interestingly, the E64A mutant was expressed in Escherichia coli in inclusion bodies, suggesting that this residue is involved with the tertiary structure or folding property of this enzyme. However, the I52A, S65A, R66A and M101A mutants were purified by glutathione affinity chromatography and the enzyme activity characterized. On the basis of steady-state kinetics, difference spectroscopy, unfolding and refolding studies, it was concluded that these residues: (1) contribute to the affinity of the GSH-binding site (‘G-site’) for GSH, (2) influence GSH thiol ionization, (3) participate in kcat regulation by affecting the rate-limiting step of the reaction, and in the case of Ile-52 and Arg-66, influenced structural integrity and/or folding of the enzyme. The structural perturbations from these mutants are probably transmitted to the hydrophobic-substrate-binding site (‘H-site’) through changes in active site topology or through effects on GSH orientation. Therefore these active site residues appear to contribute to various steps in the catalytic mechanism, as well as having an influence on the packing of the protein.

scholarly journals 16S rRNA Mutations That Confer Tetracycline Resistance in Helicobacter pylori Decrease Drug Binding in Escherichia coli Ribosomes

2005 ◽  
Vol 187 (11) ◽  
pp. 3708-3712 ◽  
Author(s):  
Lisa Nonaka ◽  
Sean R. Connell ◽  
Diane E. Taylor
Keyword(s):  
Escherichia Coli ◽  
Helicobacter Pylori ◽  
16S Rrna ◽  
Binding Site ◽  
Tetracycline Resistance ◽  
Drug Binding ◽  
Multicopy Plasmid ◽  
Wild Type ◽  
E Coli ◽  
H Pylori

ABSTRACT Tetracycline resistance in clinical isolates of Helicobacter pylori has been associated with nucleotide substitutions at positions 965 to 967 in the 16S rRNA. We constructed mutants which had different sequences at 965 to 967 in the 16S rRNA gene present on a multicopy plasmid in Escherichia coli strain TA527, in which all seven rrn genes were deleted. The MICs for tetracycline of all mutants having single, double, or triple substitutions at the 965 to 967 region that were previously found in highly resistant H. pylori isolates were higher than that of the mutant exhibiting the wild-type sequence of tetracycline-susceptible H. pylori. The MIC of the mutant with the 965TTC967 triple substitution was 32 times higher than that of the E. coli mutant with the 965AGA967 substitution present in wild-type H. pylori. The ribosomes extracted from the tetracycline-resistant E. coli 965TTC967 variant bound less tetracycline than E. coli with the wild-type H. pylori sequence at this region. The concentration of tetracycline bound to the ribosome was 40% that of the wild type. The results of this study suggest that tetracycline binding to the primary binding site (Tet-1) of the ribosome at positions 965 to 967 is influenced by its sequence patterns, which form the primary binding site for tetracycline.

scholarly journals Effects of mutating Asn-52 to isoleucine on the haem-linked properties of cytochrome c

1994 ◽  
Vol 302 (1) ◽  
pp. 95-101 ◽  
Author(s):  
A Schejter ◽  
T I Koshy ◽  
T L Luntz ◽  
R Sanishvili ◽  
I Vig ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
General Increase ◽  
Reduction Potentials ◽  
Cytochromes C ◽  
In The Wild ◽  
Order Of Magnitude ◽  
The Stability ◽  
Haem Iron

Asn-52 of rat cytochrome c and baker's yeast iso-1-cytochrome c was changed to isoleucine by site-directed mutagenesis and the mutated proteins expressed in and purified from cultures of transformed yeast. This mutation affected the affinity of the haem iron for the Met-80 sulphur in the ferric state and the reduction potential of the molecule. The yeast protein, in which the sulphur-iron bond is distinctly weaker than in vertebrate cytochromes c, became very similar to the latter: the pKa of the alkaline ionization rose from 8.3 to 9.4 and that of the acidic ionization decreased from 3.4 to 2.8. The rates of binding and dissociation of cyanide became markedly lower, and the affinity was lowered by half an order of magnitude. In the ferrous state the dissociation of cyanide from the variant yeast cytochrome c was three times slower than in the wild-type. The same mutation had analogous but less pronounced effects on rat cytochrome c: it did not alter the alkaline ionization pKa nor its affinity for cyanide, but it lowered its acidic ionization pKa from 2.8 to 2.2. These results indicate that the mutation of Asn-52 to isoleucine increases the stability of the cytochrome c closed-haem crevice as observed earlier for the mutation of Tyr-67 to phenylalanine [Luntz, Schejter, Garber and Margoliash (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 3524-3528], because of either its effects on the hydrogen-bonding of an interior water molecule or a general increase in the hydrophobicity of the protein in the domain occupied by the mutated residues. The reduction potentials were affected in different ways; the Eo of rat cytochrome c rose by 14 mV whereas that of the yeast iso-1 cychrome c was 30 mV lower as a result of the change of Asn-52 to isoleucine.

scholarly journals Mutations in the cyclic AMP binding site of the cyclic AMP receptor protein of Escherichia coli

1988 ◽  
Vol 253 (3) ◽  
pp. 801-807 ◽  
Author(s):  
A M Gronenborn ◽  
R Sandulache ◽  
S Gärtner ◽  
G M Clore
Keyword(s):  
Escherichia Coli ◽  
Cyclic Amp ◽  
Binding Site ◽  
Cyclic Nucleotides ◽  
Binding Pocket ◽  
Receptor Protein ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Cyclic Amp Receptor Protein ◽  
Better Than

Mutants in the cyclic AMP binding site of the cyclic AMP receptor protein (CRP) of Escherichia coli have been constructed by oligonucleotide-directed mutagenesis. They have been phenotypically characterized and their ability to enhance the expression of catabolite-repressible operons has been tested. In addition, the binding of cyclic nucleotides to the mutants has been investigated. It is shown that the six mutants made fall into one of three classes: (i) those that bind cyclic AMP better than the wild type protein (Ser-62→Ala) and result in greater transcription enhancement; (ii) those that bind cyclic AMP similarly to wild type (Ser-83→Ala, Ser-83→Lys, Thr-127→Ala, Ser-129→Ala); and (iii) those that do not bind cyclic AMP at all (Arg-82→Leu). Implications of these findings with respect to present models of the cyclic nucleotide binding pocket of CRP are discussed.

scholarly journals Role of αArg145 and βArg263 in the active site of penicillin acylase of Escherichia coli

2002 ◽  
Vol 365 (1) ◽  
pp. 303-309 ◽  
Author(s):  
Wynand B.L. ALKEMA ◽  
Antoon K. PRINS ◽  
Erik de VRIES ◽  
Dick B. JANSSEN
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Penicillin Acylase ◽  
Precursor Protein ◽  
Site Directed Mutagenesis ◽  
Penicillin G ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Arginine Residues

The active site of penicillin acylase of Escherichia coli contains two conserved arginine residues. The function of these arginines, αArg145 and βArg263, was studied by site-directed mutagenesis and kinetic analysis of the mutant enzymes. The mutants αArg145→Leu (αArg145Leu), αArg145Cys and αArg145Lys were normally processed and exported to the periplasm, whereas expression of the mutants βArg263Leu, βArg263Asn and βArg263Lys yielded large amounts of precursor protein in the periplasm, indicating that βArg263 is crucial for efficient processing of the enzyme. Either modification of both arginine residues by 2,3-butanedione or replacement by site-directed mutagenesis yielded enzymes with a decreased specificity (kcat/Km) for 2-nitro-5-[(phenylacetyl)amino]benzoic acid, indicating that both residues are important in catalysis. Compared with the wild type, the αArg145 mutants exhibited a 3–6-fold-increased preference for 6-aminopenicillanic acid as the deacylating nucleophile compared with water. Analysis of the steady-state parameters of these mutants for the hydrolysis of penicillin G and phenylacetamide indicated that destabilization of the Michaelis—Menten complex accounts for the improved activity with β-lactam substrates. Analysis of pH—activity profiles of wild-type enzyme and the βArg263Lys mutant showed that βArg263 has to be positively charged for catalysis, but is not involved in substrate binding. The results provide an insight into the catalytic mechanism of penicillin acylase, in which αArg145 is involved in binding of β-lactam substrates and βArg263 is important both for stabilizing the transition state in the reaction and for correct processing of the precursor protein.

scholarly journals Genetic and Biochemical Analysis of Phosphatase Activity of Escherichia coli NRII (NtrB) and Its Regulation by the PII Signal Transduction Protein

2003 ◽  
Vol 185 (4) ◽  
pp. 1299-1315 ◽  
Author(s):  
Augen A. Pioszak ◽  
Alexander J. Ninfa
Keyword(s):  
Escherichia Coli ◽  
Signal Transduction ◽  
Binding Site ◽  
Phosphatase Activity ◽  
Atp Binding ◽  
Wild Type ◽  
Small Surface ◽  
Central Domain ◽  
Atp Binding Site ◽  
Terminal Domain

ABSTRACT Mutant forms of Escherichia coli NRII (NtrB) were isolated that retained wild-type NRII kinase activity but were defective in the PII-activated phosphatase activity of NRII. Mutant strains were selected as mimicking the phenotype of a strain (strain BK) that lacks both of the related PII and GlnK signal transduction proteins and thus has no mechanism for activation of the NRII phosphatase activity. The selection and screening procedure resulted in the isolation of numerous mutants that phenotypically resembled strain BK to various extents. Mutations mapped to the glnL (ntrB) gene encoding NRII and were obtained in all three domains of NRII. Two distinct regions of the C-terminal, ATP-binding domain were identified by clusters of mutations. One cluster, including the Y302N mutation, altered a lid that sits over the ATP-binding site of NRII. The other cluster, including the S227R mutation, defined a small surface on the “back” or opposite side of this domain. The S227R and Y302N proteins were purified, along with the A129T (NRII2302) protein, which has reduced phosphatase activity due to a mutation in the central domain of NRII, and the L16R protein, which has a mutation in the N-terminal domain of NRII. The S227R, Y302N, and L16R proteins were specifically defective in the PII-activated phosphatase activity of NRII. Wild-type NRII, Y302N, A129T, and L16R proteins bound to PII, while the S227R protein was defective in binding PII. This suggests that the PII-binding site maps to the “back” of the C-terminal domain and that mutation of the ATP-lid, central domain, and N-terminal domain altered functions necessary for the phosphatase activity after PII binding.

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

1993 ◽  
Vol 295 (2) ◽  
pp. 485-491 ◽  
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.

scholarly journals OR03-06 NAD+ Availability Modulates 11β-HSD1-Mediated Glucocorticoid Regeneration in Mouse Skeletal Muscle

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Yasir S Elhassan ◽  
Ali Kabli ◽  
Thomas Nielsen ◽  
Rachel Fletcher ◽  
Lucy Oakey ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Dehydrogenase Activity ◽  
Reductase Activity ◽  
Wild Type Mouse ◽  
Pyridine Nucleotide ◽  
Wild Type ◽  
Parent Molecule ◽  
Nicotinamide Riboside ◽  
Glucocorticoid Metabolism

Abstract 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) is an NADPH-dependant reductase located in the sarcoplasmic reticulum (SR) lumen of skeletal muscle. It generates active glucocorticoids to regulate permissive and adaptive metabolism and contributes to the development of the Cushing’s syndrome phenotype in mice receiving oral corticosterone. The SR enzyme hexose-6-phosphate dehydrogenase (H6PDH) generates NADPH which supports 11β-HSD1 activity. H6PDH depletion disrupts the SR NADPH/NADP ratio leading 11β-HSD1 to assume glucocorticoid-inactivating dehydrogenase activity. Little is understood regarding routes to NAD(P)(H) biosynthesis and metabolism in the SR. Here we asked whether modulating cellular nicotinamide adenine dinucleotide (NAD+) availability (the parent molecule of NAD(P)(H)) would influence muscle 11β-HSD1 activity given its sensitivity to the SR NADPH/NADP ratio. We used FK866 to inhibit nicotinamide phospho-ribosyltransferase (NAMPT, rate-limiting enzyme in NAD+ biosynthesis) to deplete NAD(P)(H) in wild type mouse primary myotubes. FK866 treatment for 48h impaired cellular energetic status, reducing NAD+ (&gt;90%), NADP+ (&gt;50%) and ATP (&gt;30%) without limiting cell viability. 11β-HSD1 reductase activity was decreased to 30% that of untreated cells (152±18 vs. 512±44 pmol/mg protein/h respectively, p&lt;0.005). Employing H6PD knockout myotubes, NADP+-dependent 11β-HSD1 dehydrogenase activity was also impaired following NAMPT inhibition. The NAD+ precursor nicotinamide riboside (NR, 0.5mM), which bypasses NAMPT inhibition through the NR kinase pathway restored NAD+ levels and rapidly rescued 11β-HSD1 reductase activity in wild type and dehydrogenase activity in H6PD knockout myotubes. To assess this in vivo, we examined 11β-HSD1 reductase activity in muscle explants of inducible muscle-specific NAMPT knockout mice in which NAD+ levels are reduced by 90%, and show 40% lower activity compared to wild type explants (114±14 vs. 67±10 pmol/mg protein/h, p=0.04). These data suggest a novel level of redox-regulated 11β-HSD1-mediated glucocorticoid metabolism in skeletal muscle. These data also imply a pathway by which NAD+ status is communicated between the cytosol and the SR, which is contrary to the current belief that the pyridine nucleotide pool in these compartments is separate. NAMPT inhibition is being studied as a potential anti-cancer therapy and these data reveal hitherto unanticipated effects this therapy may have in a range of tissues.

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