scholarly journals Flavinylation in wild-type trimethylamine dehydrogenase and differentially charged mutant enzymes: a study of the protein environment around the N1 of the flavin isoalloxazine

1996 ◽  
Vol 317 (1) ◽  
pp. 267-272 ◽  
Author(s):  
Martin MEWIES ◽  
Leonard C. PACKMAN ◽  
F. Scott MATHEWS ◽  
Nigel S. SCRUTTON
Keyword(s):  
Escherichia Coli ◽  
Wild Type ◽  
Post Translational Modification ◽  
Mutant Proteins ◽  
Trimethylamine Dehydrogenase ◽  
Guanidino Group ◽  
Protein Environment ◽  
Mutagenic Changes ◽  
Positively Charged ◽  
Significant Mass

In wild-type trimethylamine dehydrogenase, residue Arg-222 is positioned close to the isoalloxazine N1/C2 positions of the 6S-cysteinyl FMN. The positively charged guanidino group of Arg-222 is thought to stabilize negative charge as it develops at the N1 position of the flavin during flavinylation of the enzyme. Three mutant trimethylamine dehydrogenases were constructed to alter the nature of the charge at residue 222. The amount of active flavinylated enzyme produced in Escherichia coli is reduced when Arg-222 is replaced by lysine (mutant R222K). Removal or reversal of the charge at residue 222 (mutants R222V and R222E, respectively) leads to the production of inactive enzymes that are totally devoid of flavin. A comparison of the CD spectra for the wild-type and mutant enzymes revealed no major structural change following mutagenesis. Like the wild-type protein, each mutant enzyme contained stoichiometric amounts of the 4Fe-4S cluster and ADP. Electrospray MS also indicated that the native and recombinant wild-type enzymes were isolated as a mixture of deflavo and holo enzyme, but that each of the mutant enzymes have masses expected for deflavo trimethylamine dehydrogenase. The MS data indicate that the lack of assembly of the mutant proteins with FMN is not due to detectable levels of post-translational modification of significant mass. The experiments reported here indicate that simple mutagenic changes in the FMN-binding site can reduce the proportion of flavinylated enzyme isolated from Escherichia coli and that positive charge is required at residue 222 if flavinylation is to proceed.

scholarly journals Computational redesign of the Escherichia coli ribose-binding protein ligand binding pocket for 1,3-cyclohexanediol and cyclohexanol

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Diogo Tavares ◽  
Artur Reimer ◽  
Shantanu Roy ◽  
Aurélie Joublin ◽  
Vladimir Sentchilo ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Ligand Binding ◽  
Binding Proteins ◽  
Binding Pocket ◽  
Wild Type ◽  
Ligand Binding Pocket ◽  
Mutant Proteins ◽  
Periplasmic Binding Proteins ◽  
Natural Ligands ◽  
Ribose Binding Protein

AbstractBacterial periplasmic-binding proteins have been acclaimed as general biosensing platform, but their range of natural ligands is too limited for optimal development of chemical compound detection. Computational redesign of the ligand-binding pocket of periplasmic-binding proteins may yield variants with new properties, but, despite earlier claims, genuine changes of specificity to non-natural ligands have so far not been achieved. In order to better understand the reasons of such limited success, we revisited here the Escherichia coli RbsB ribose-binding protein, aiming to achieve perceptible transition from ribose to structurally related chemical ligands 1,3-cyclohexanediol and cyclohexanol. Combinations of mutations were computationally predicted for nine residues in the RbsB binding pocket, then synthesized and tested in an E. coli reporter chassis. Two million variants were screened in a microcolony-in-bead fluorescence-assisted sorting procedure, which yielded six mutants no longer responsive to ribose but with 1.2–1.5 times induction in presence of 1 mM 1,3-cyclohexanediol, one of which responded to cyclohexanol as well. Isothermal microcalorimetry confirmed 1,3-cyclohexanediol binding, although only two mutant proteins were sufficiently stable upon purification. Circular dichroism spectroscopy indicated discernable structural differences between these two mutant proteins and wild-type RbsB. This and further quantification of periplasmic-space abundance suggested most mutants to be prone to misfolding and/or with defects in translocation compared to wild-type. Our results thus affirm that computational design and library screening can yield RbsB mutants with recognition of non-natural but structurally similar ligands. The inherent arisal of protein instability or misfolding concomitant with designed altered ligand-binding pockets should be overcome by new experimental strategies or by improved future protein design algorithms.

scholarly journals Analysis of ftsQ Mutant Alleles in Escherichia coli: Complementation, Septal Localization, and Recruitment of Downstream Cell Division Proteins

2002 ◽  
Vol 184 (3) ◽  
pp. 695-705 ◽  
Author(s):  
Joseph C. Chen ◽  
Michael Minev ◽  
Jon Beckwith
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Random Mutagenesis ◽  
Dominant Negative ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Wild Type ◽  
Negative Effects ◽  
Mutant Proteins ◽  
Division Site

ABSTRACT FtsQ, a 276-amino-acid, bitopic membrane protein, is one of the nine proteins known to be essential for cell division in gram-negative bacterium Escherichia coli. To define residues in FtsQ critical for function, we performed random mutagenesis on the ftsQ gene and identified four alleles (ftsQ2, ftsQ6, ftsQ15, and ftsQ65) that fail to complement the ftsQ1(Ts) mutation at the restrictive temperature. Two of the mutant proteins, FtsQ6 and FtsQ15, are functional at lower temperatures but are unable to localize to the division site unless wild-type FtsQ is depleted, suggesting that they compete poorly with the wild-type protein for septal targeting. The other two mutants, FtsQ2 and FtsQ65, are nonfunctional at all temperatures tested and have dominant-negative effects when expressed in an ftsQ1(Ts) strain at the permissive temperature. FtsQ2 and FtsQ65 localize to the division site in the presence or absence of endogenous FtsQ, but they cannot recruit downstream cell division proteins, such as FtsL, to the septum. These results suggest that FtsQ2 and FtsQ65 compete efficiently for septal targeting but fail to promote the further assembly of the cell division machinery. Thus, we have separated the localization ability of FtsQ from its other functions, including recruitment of downstream cell division proteins, and are beginning to define regions of the protein responsible for these distinct capabilities.

scholarly journals Conversion of Escherichia coli pyruvate oxidase to an ‘α-ketobutyrate oxidase’

2000 ◽  
Vol 352 (3) ◽  
pp. 717-724 ◽  
Author(s):  
Ying-Ying CHANG ◽  
John E. CRONAN
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Random Mutagenesis ◽  
Fold Increase ◽  
Natural Substrate ◽  
Wild Type ◽  
Pyruvate Oxidase ◽  
Mutant Proteins ◽  
Essentially Normal

Escherichia coli pyruvate oxidase (PoxB), a lipid-activated homotetrameric enzyme, is active on both pyruvate and 2-oxobutanoate (‘α-ketobutyrate’), although pyruvate is the favoured substrate. By localized random mutagenesis of residues chosen on the basis of a modelled active site, we obtained several PoxB enzymes that had a markedly decreased activity with the natural substrate, pyruvate, but retained full activity with 2-oxobutanoate. In each of these mutant proteins Val-380had been replaced with a smaller residue, namely alanine, glycine or serine. One of these, PoxB V380A/L253F, was shown to lack detectable pyruvate oxidase activity in vivo; this protein was purified, studied and found to have a 6-fold increase in Km for pyruvate and a 10-fold lower Vmax with this substrate. In contrast, the mutant had essentially normal kinetic constants with 2-oxobutanoate. The altered substrate specificity was reflected in a decreased rate of pyruvate binding to the latent conformer of the mutant protein owing to the V380A mutation. The L253F mutation alone had no effect on PoxB activity, although it increased the activity of proteins carrying substitutions at residue 380, as it did that of the wild-type protein. The properties of the V380A/L253F protein provide new insights into the mode of substrate binding and the unusual activation properties of this enzyme.

scholarly journals Exploring by Pulsed EPR the Electronic Structure of Ubisemiquinone Bound at the QH Site of Cytochrome bo3 from Escherichia coli with in Vivo13C-Labeled Methyl and Methoxy Substituents

2011 ◽  
Vol 286 (12) ◽  
pp. 10105-10114 ◽  
Author(s):  
Myat T. Lin ◽  
Alexander A. Shubin ◽  
Rimma I. Samoilova ◽  
Kuppala V. Narasimhulu ◽  
Amgalanbaatar Baldansuren ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Wild Type ◽  
Mutant Proteins ◽  
Pulsed Epr ◽  
Cytochrome Bo3 ◽  
Methoxy Groups ◽  
Unpaired Spin ◽  
In The Wild ◽  
Electron Reduction ◽  
The One

The cytochrome bo3 ubiquinol oxidase from Escherichia coli resides in the bacterial cytoplasmic membrane and catalyzes the two-electron oxidation of ubiquinol-8 and four-electron reduction of O2 to water. The one-electron reduced semiquinone forms transiently during the reaction, and the enzyme has been demonstrated to stabilize the semiquinone. The semiquinone is also formed in the D75E mutant, where the mutation has little influence on the catalytic activity, and in the D75H mutant, which is virtually inactive. In this work, wild-type cytochrome bo3 as well as the D75E and D75H mutant proteins were prepared with ubiquinone-8 13C-labeled selectively at the methyl and two methoxy groups. This was accomplished by expressing the proteins in a methionine auxotroph in the presence of l-methionine with the side chain methyl group 13C-labeled. The 13C-labeled quinone isolated from cytochrome bo3 was also used for the generation of model anion radicals in alcohol. Two-dimensional pulsed EPR and ENDOR were used for the study of the 13C methyl and methoxy hyperfine couplings in the semiquinone generated in the three proteins indicated above and in the model system. The data were used to characterize the transferred unpaired spin densities on the methyl and methoxy substituents and the conformations of the methoxy groups. In the wild type and D75E mutant, the constraints on the configurations of the methoxy side chains are similar, but the D75H mutant appears to have altered methoxy configurations, which could be related to the perturbed electron distribution in the semiquinone and the loss of enzymatic activity.

Crystal engineering: deletion mutagenesis of the 24 kDa fragment of the DNA gyrase B subunit from Staphylococcus aureus

1999 ◽  
Vol 55 (9) ◽  
pp. 1626-1629 ◽  
Author(s):  
Glenn E. Dale ◽  
Dirk Kostrewa ◽  
Bernard Gsell ◽  
Martin Stieger ◽  
Allan D'Arcy
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Crystal Engineering ◽  
Dna Gyrase ◽  
Wild Type ◽  
Scanning Calorimetry ◽  
Mutant Proteins ◽  
B Subunit ◽  
Deletion Mutagenesis ◽  
Single Well

The 24 kDa fragment of DNA gyrase B from Staphylococcus aureus was expressed in Escherichia coli and purified for crystallization. Crystals of the wild-type protein grew in the presence of cyclothialidine but proved difficult to reproduce. In order to improve the crystallization, the flexible regions of the protein were deleted by mutagenesis. The mutant proteins were analyzed by differential scanning calorimetry and the most stable mutants produced crystals. It was possible to reproducibly grow in the microbatch system single well defined crystals which belonged to the space group C2 and diffracted isotropically to approximately 2 Å resolution.

scholarly journals Lysine Residue 117 of the FasG Adhesin of Enterotoxigenic Escherichia coli Is Essential for Binding of 987P Fimbriae to Sulfatide

1999 ◽  
Vol 67 (11) ◽  
pp. 5755-5761 ◽  
Author(s):  
Byung-Kwon Choi ◽  
Dieter M. Schifferli
Keyword(s):  
Escherichia Coli ◽  
Essential Role ◽  
Surface Display ◽  
Salt Bridge ◽  
Site Directed Mutagenesis ◽  
Enterotoxigenic Escherichia Coli ◽  
Wild Type ◽  
Binding Properties ◽  
Brush Borders ◽  
Positively Charged

ABSTRACT The FasG subunit of the 987P fimbriae of enterotoxigenic strains ofEscherichia coli was previously shown to mediate fimbrial binding to a glycoprotein and a sulfatide receptor on intestinal brush borders of piglets. Moreover, the 987P adhesin FasG is required for fimbrial expression, since fasG null mutants are nonfimbriated. In this study, fasG was modified by site-directed mutagenesis to study its sulfatide binding properties. Twenty single mutants were generated by replacing positively charged lysine (K) or arginine (R) residues with small, nonpolar alanine (A) residues. Reduced levels of binding to sulfatide-containing liposomes correlated with reduced fimbriation and FasG surface display in fourfasG mutants (R27A, R286A, R226A, and R368). Among the 16 remaining normally fimbriated mutants with wild-type levels of surface-exposed FasG, only one mutant (K117A) did not interact at all with sulfatide-containing liposomes. Four mutants (K117A, R116A, K118A, and R200A) demonstrated reduced binding to such liposomes. Since complete phenotypic dissociation between the structure and specific function of 987P was observed only with mutant K117A, this residue is proposed to play an essential role in the FasG-sulfatide interaction, possibly communicating with the sulfate group of sulfatide by hydrogen bonding and/or salt bridge formation. Residues K17, R116, K118, and R200 may stabilize this interaction.

scholarly journals Deletion and Substitution Analysis of the Escherichia coli TonB Q160 Region

2007 ◽  
Vol 189 (13) ◽  
pp. 4662-4670 ◽  
Author(s):  
Hema Vakharia-Rao ◽  
Kyle A. Kastead ◽  
Marina I. Savenkova ◽  
Charles M. Bulathsinghala ◽  
Kathleen Postle
Keyword(s):  
Escherichia Coli ◽  
Conformational Changes ◽  
Vitamin B ◽  
Initial Contact ◽  
Wild Type ◽  
Carboxy Terminus ◽  
Mutant Proteins ◽  
Nested Deletions ◽  
Energy Dependent ◽  
Arginyl Residues

ABSTRACT The active transport of iron siderophores and vitamin B12 across the outer membrane (OM) of Escherichia coli requires OM transporters and the potential energy of the cytoplasmic membrane (CM) proton gradient and CM proteins TonB, ExbB, and ExbD. A region at the amino terminus of the transporter, called the TonB box, directly interacts with TonB Q160 region residues. R158 and R166 in the TonB Q160 region were proposed to play important roles in cocrystal structures of the TonB carboxy terminus with OM transporters BtuB and FhuA. In contrast to predictions based on the crystal structures, none of the single, double, or triple alanyl substitutions at arginyl residues significantly decreased TonB activity. Even the quadruple R154A R158A R166A R171A mutant TonB still retained 30% of wild-type activity. Up to five residues centered on TonB Q160 could be deleted without inactivating TonB or preventing its association with the OM. TonB mutant proteins with nested deletions of 7, 9, or 11 residues centered on TonB Q160 were inactive and appeared never to have associated with the OM. Because the 7-residue-deletion mutant protein (TonBΔ7, lacking residues S157 to Y163) could still form disulfide-linked dimers when combined with W213C or F202C in the TonB carboxy terminus, the TonBΔ7 deletion did not prevent necessary energy-dependent conformational changes that occur in the CM. Thus, it appeared that initial contact with the OM is made through TonB residues S157 to Y163. It is hypothesized that the TonB Q160 region may be part of a large disordered region required to span the periplasm and contact an OM transporter.

scholarly journals Characterization of Dominantly Negative Mutant ClyA Cytotoxin Proteins in Escherichia coli

2003 ◽  
Vol 185 (18) ◽  
pp. 5491-5499 ◽  
Author(s):  
Sun Nyunt Wai ◽  
Marie Westermark ◽  
Jan Oscarsson ◽  
Jana Jass ◽  
Elke Maier ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protein Interactions ◽  
Single Channel ◽  
Dominant Negative ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Protein Protein Interactions ◽  
Mutant Proteins ◽  
Negative Feature

ABSTRACT We report studies of the subcellular localization of the ClyA cytotoxic protein and of mutations causing defective translocation to the periplasm in Escherichia coli. The ability of ClyA to translocate to the periplasm was abolished in deletion mutants lacking the last 23 or 11 amino acid residues of the C-terminal region. A naturally occurring ClyA variant lacking four residues (183 to 186) in a hydrophobic subdomain was retained mainly in the cytosolic fraction. These mutant proteins displayed an inhibiting effect on the expression of the hemolytic phenotype of wild-type ClyA. Studies in vitro with purified mutant ClyA proteins revealed that they were defective in formation of pore assemblies and that their activity in hemolysis assays and in single-channel conductance tests was at least 10-fold lower than that of the wild-type ClyA. Tests with combinations of the purified proteins indicated that mutant and wild-type ClyA interacted and that formation of heteromeric assemblies affected the pore-forming activity of the wild-type protein. The observed protein-protein interactions were consistent with, and provided a molecular explanation for, the dominant negative feature of the mutant ClyA variants.

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