scholarly journals Active-Site Residues of Escherichia coli DNA Gyrase Required in Coupling ATP Hydrolysis to DNA Supercoiling and Amino Acid Substitutions Leading to Novobiocin Resistance

2003 ◽  
Vol 47 (3) ◽  
pp. 1037-1046 ◽  
Author(s):  
Christian H. Gross ◽  
Jonathan D. Parsons ◽  
Trudy H. Grossman ◽  
Paul S. Charifson ◽  
Steven Bellon ◽  
...  
Keyword(s):  
Amino Acid ◽  
Atp Hydrolysis ◽  
Dna Gyrase ◽  
Dna Supercoiling ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Mutant Proteins ◽  
E Coli ◽  
Novobiocin Resistance

ABSTRACT DNA gyrase is a bacterial type II topoisomerase which couples the free energy of ATP hydrolysis to the introduction of negative supercoils into DNA. Amino acids in proximity to bound nonhydrolyzable ATP analog (AMP · PNP) or novobiocin in the gyrase B (GyrB) subunit crystal structures were examined for their roles in enzyme function and novobiocin resistance by site-directed mutagenesis. Purified Escherichia coli GyrB mutant proteins were complexed with the gyrase A subunit to form the functional A2B2 gyrase enzyme. Mutant proteins with alanine substitutions at residues E42, N46, E50, D73, R76, G77, and I78 had reduced or no detectable ATPase activity, indicating a role for these residues in ATP hydrolysis. Interestingly, GyrB proteins with P79A and K103A substitutions retained significant levels of ATPase activity yet demonstrated no DNA supercoiling activity, even with 40-fold more enzyme than the wild-type enzyme, suggesting that these amino acid side chains have a role in the coupling of the two activities. All enzymes relaxed supercoiled DNA to the same extent as the wild-type enzyme did, implying that only ATP-dependent reactions were affected. Mutant genes were examined in vivo for their abilities to complement a temperature-sensitive E. coli gyrB mutant, and the activities correlated well with the in vitro activities. We show that the known R136 novobiocin resistance mutations bestow a significant loss of inhibitor potency in the ATPase assay. Four new residues (D73, G77, I78, and T165) that, when changed to the appropriate amino acid, result in both significant levels of novobiocin resistance and maintain in vivo function were identified in E. coli.

scholarly journals Impact of the E540V Amino Acid Substitution in GyrB ofMycobacterium tuberculosison Quinolone Resistance

2011 ◽  
Vol 55 (8) ◽  
pp. 3661-3667 ◽  
Author(s):  
Hyun Kim ◽  
Chie Nakajima ◽  
Kazumasa Yokoyama ◽  
Zeaur Rahim ◽  
Youn Uck Kim ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Substitution ◽  
Dna Cleavage ◽  
Recombinant Dna ◽  
Dna Gyrase ◽  
Dna Supercoiling ◽  
Quinolone Resistance ◽  
Wild Type ◽  
Wild Type Enzyme

ABSTRACTAmino acid substitutions conferring resistance to quinolones inMycobacterium tuberculosishave generally been found within the quinolone resistance-determining regions (QRDRs) in the A subunit of DNA gyrase (GyrA) rather than the B subunit of DNA gyrase (GyrB). To clarify the contribution of an amino acid substitution, E540V, in GyrB to quinolone resistance inM. tuberculosis, we expressed recombinant DNA gyrases inEscherichia coliand characterized themin vitro. Wild-type and GyrB-E540V DNA gyrases were reconstitutedin vitroby mixing recombinant GyrA and GyrB. Correlation between the amino acid substitution and quinolone resistance was assessed by the ATP-dependent DNA supercoiling assay, quinolone-inhibited supercoiling assay, and DNA cleavage assay. The 50% inhibitory concentrations of eight quinolones against DNA gyrases bearing the E540V amino acid substitution in GyrB were 2.5- to 36-fold higher than those against the wild-type enzyme. Similarly, the 25% maximum DNA cleavage concentrations were 1.5- to 14-fold higher for the E540V gyrase than for the wild-type enzyme. We further demonstrated that the E540V amino acid substitution influenced the interaction between DNA gyrase and the substituent(s) at R-7, R-8, or both in quinolone structures. This is the first detailed study of the contribution of the E540V amino acid substitution in GyrB to quinolone resistance inM. tuberculosis.

scholarly journals Genetic and Functional Analyses of the Conserved C-Terminal Core Domain of Escherichia coli FtsZ

1999 ◽  
Vol 181 (24) ◽  
pp. 7531-7544 ◽  
Author(s):  
Xiaolan Ma ◽  
William Margolin
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Immunoblot Analysis ◽  
Conserved Residues ◽  
Core Domain ◽  
Mutant Proteins ◽  
E Coli ◽  
Stable Mutant ◽  
Functional Analyses

ABSTRACT In Escherichia coli, FtsZ is required for the recruitment of the essential cell division proteins FtsA and ZipA to the septal ring. Several C-terminal deletions of E. coliFtsZ, including one of only 12 amino acids that removes the highly conserved C-terminal core domain, failed to complement chromosomalftsZ mutants when expressed on a plasmid. To identify key individual residues within the core domain, six highly conserved residues were replaced with alanines. All but one of these mutants (D373A) failed to complement an ftsZ chromosomal mutant. Immunoblot analysis demonstrated that whereas I374A and F377A proteins were unstable in the cell, L372A, D373A, P375A, and L378A proteins were synthesized at normal levels, suggesting that they were specifically defective in some aspect of FtsZ function. In addition, all four of the stable mutant proteins were able to localize and form rings at potential division sites in chromosomal ftsZ mutants, implying a defect in a function other than localization and multimerization. Because another proposed function of FtsZ is the recruitment of FtsA and ZipA, we tested whether the C-terminal core domain was important for interactions with these proteins. Using two different in vivo assays, we found that the 12-amino-acid truncation of FtsZ was defective in binding to FtsA. Furthermore, two point mutants in this region (L372A and P375A) showed weakened binding to FtsA. In contrast, ZipA was capable of binding to all four stable point mutants in the FtsZ C-terminal core but not to the 12-amino-acid deletion.

scholarly journals Structural Maintenance of Chromosomes Protein of Bacillussubtilis Affects Supercoiling In Vivo

2002 ◽  
Vol 184 (19) ◽  
pp. 5317-5322 ◽  
Author(s):  
Janet C. Lindow ◽  
Robert A. Britton ◽  
Alan D. Grossman
Keyword(s):  
Topoisomerase I ◽  
Dna Gyrase ◽  
Dna Supercoiling ◽  
Null Mutant ◽  
Wild Type ◽  
Mutant Plasmid ◽  
Chromatid Cohesion ◽  
Structural Maintenance Of Chromosomes ◽  
Gyrase Inhibitors

ABSTRACT Structural maintenance of chromosomes (SMC) proteins are found in nearly all organisms. Members of this protein family are involved in chromosome condensation and sister chromatid cohesion. Bacillus subtilis SMC protein (BsSMC) plays a role in chromosome organization and partitioning. To better understand the function of BsSMC, we studied the effects of an smc null mutation on DNA supercoiling in vivo. We found that an smc null mutant was hypersensitive to the DNA gyrase inhibitors coumermycin A1 and norfloxacin. Furthermore, depleting cells of topoisomerase I substantially suppressed the partitioning defect of an smc null mutant. Plasmid DNA isolated from an smc null mutant was more negatively supercoiled than that from wild-type cells. In vivo cross-linking experiments indicated that BsSMC was bound to the plasmid. Our results indicate that BsSMC affects supercoiling in vivo, most likely by constraining positive supercoils, an activity which contributes to chromosome compaction and organization.

scholarly journals Investigation of the in vitro and in vivo activity of the type IV pilus extension ATPase BfpD

2020 ◽  
Author(s):  
Jinlei Zhao ◽  
Shahista Nisa ◽  
Michael S. Donnenberg
Keyword(s):  
Atpase Activity ◽  
Loss Of Function ◽  
Wild Type ◽  
Multifunctional Protein ◽  
Mutant Proteins ◽  
E Coli ◽  
Type Iv ◽  
Kinetics Of

AbstractType IV pili (T4Ps) are multifunctional protein fibers found in many bacteria and archaea. All T4P systems have an extension ATPase, which provides the energy required to push structural subunits out of the membrane. We previously reported that the BfpD T4P ATPase from enteropathogenic E. coli (EPEC) has the expected hexameric structure and ATPase activity, the latter enhanced by the presence of the N-terminal cytoplasmic domains of its partner proteins BfpC and BfpE. In this study, we further investigated the kinetics of the BfpD ATPase. Despite high purity of the proteins, the reported enhanced ATPase activity was found to be from (an) ATPase(s) contaminating the N-BfpC preparation. Furthermore, although two mutations in highly conserved bfpD sites led to loss of function in vivo, the purified mutant proteins retained some ATPase activity, albeit less than the wild-type protein. Therefore, the observed ATPase activity of BfpD was also affected by (a) contaminating ATPase(s). Expression of the mutant bfpD alleles did not interfere with BfpD function in bacteria that also expressed wild-type BfpD. However, a similar mutation of bfpF, which encodes the retraction ATPase, blocked the function of wild-type BfpF when both were present. These results highlight similarities and differences in function and activity of T4P extension and retraction ATPases in EPEC.

scholarly journals Identifying the catalytic residue of the ATPase reaction of DNA gyrase

1993 ◽  
Vol 90 (23) ◽  
pp. 11232-11236 ◽  
Author(s):  
A P Jackson ◽  
A Maxwell
Keyword(s):  
Dna Cleavage ◽  
Atp Hydrolysis ◽  
Dna Gyrase ◽  
Site Directed Mutagenesis ◽  
Catalytic Residue ◽  
Wild Type ◽  
Mutant Proteins ◽  
General Base ◽  
Atpase Reaction ◽  
Hydrolysis Of

We propose a mechanism for the hydrolysis of ATP by the DNA gyrase B protein in which Glu42 acts as a general base and His38 has a role in aligning and polarizing the glutamate residue. We have tested this mechanism by site-directed mutagenesis, converting Glu42 to Ala, Asp, and Gln, and His38 to Ala. In the presence of wild-type A protein, B proteins bearing the mutations Ala42 and Gln42 show no detectable supercoiling or ATPase activities, while Asp42 and Ala38 proteins have reduced activities. In the DNA cleavage and relaxation reactions of gyrase, which do not require ATP hydrolysis, wild-type and mutant proteins have similar activities. When the 43-kDa N-terminal fragment of the gyrase B protein (which hydrolyzes ATP) contained the mutations Ala42 or Gln42, ATP was bound but not hydrolyzed, supporting the idea that Glu42 is involved in hydrolysis but not nucleotide binding.

scholarly journals Thermostabilization of Bacterial Fructosyl-Amino Acid Oxidase by Directed Evolution

2003 ◽  
Vol 69 (1) ◽  
pp. 139-145 ◽  
Author(s):  
Ryoichi Sakaue ◽  
Naoki Kajiyama
Keyword(s):  
Amino Acid ◽  
Directed Evolution ◽  
Amino Acid Substitutions ◽  
Acid Oxidase ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Amino Acid Oxidase ◽  
Fourth Round ◽  
Fructosyl Amino Acid Oxidase

ABSTRACT We succeeded in isolating several thermostable mutant fructosyl-amino acid oxidase (FAOX; EC 1.5.3) without reduction of productivity by directed evolution that combined an in vivo mutagenesis and membrane assay screening system. Five amino acid substitutions (T60A, A188G, M244L, N257S, and L261M) occurred in the most thermostable mutant obtained by a fourth round of directed evolution. This altered enzyme, FAOX-TE, was stable at 45°C, whereas the wild-type enzyme was not stable above 37°C. The Km values of FAOX-TE for d-fructosyl-l-valine and d-fructosyl-glycine were 1.50 and 0.58 mM, respectively, in contrast with corresponding values of 1.61 and 0.74 mM for the wild-type enzyme. This altered FAOX-TE will be useful in the diagnosis of diabetes.

scholarly journals Cryo-EM structure of the complete E. coli DNA Gyrase nucleoprotein complex

2019 ◽  
Author(s):  
Arnaud Vanden Broeck ◽  
Julio Ortiz ◽  
Valérie Lamour
Keyword(s):  
Spatial Organization ◽  
Atp Hydrolysis ◽  
Function Analysis ◽  
Bacterial Genome ◽  
Dna Gyrase ◽  
Strand Break ◽  
Dna Supercoiling ◽  
Vital Role ◽  
Phase Plate ◽  
E Coli

AbstractDNA Gyrase is an essential enzyme involved in the homeostatic control of DNA supercoiling and the target of successful antibacterial compounds. Despite extensive studies, the detailed architecture of DNA Gyrase from the model genetic organism E. coli, is still missing, impeding structure-function analysis of E. coli-specific catalytic regulation and limiting the study of conformational intermediates of this highly flexible macromolecule. Herein, we determined the complete molecular structure of the E. coli DNA Gyrase bound to a 180 bp DNA and the antibiotic Gepotidacin, using phase-plate single-particle cryo-electron microscopy. Our data unveil with unprecedented details the structural and spatial organization of the functional domains, their connections and the position of the conserved GyrA-box motif. The deconvolution of closed and pre-opening states of the DNA-binding domain provides a better understanding of the allosteric movements of the enzyme complex. In this region, the local atomic resolution reaching up to 3.0 Å enables the identification of the antibiotic density in the DNA complex. Altogether, this study paves the way for the cryo-EM determination of gyrase complexes with antibiotics and opens perspectives for targeting conformational intermediates. The type 2A DNA topoisomerases (Top2) are nanomachines that control DNA topology during multiple cellular processes such as replication, transcription and cell division 1-4. These enzymes catalyze the transport of a DNA duplex through a double strand break to perform DNA relaxation, decatenation and unknotting. DNA Gyrase plays a vital role in the compaction of the bacterial genome and is the sole type 2 topoisomerase able to introduce negative supercoils into DNA, a reaction coupled to ATP hydrolysis 5.

scholarly journals Disulfide bond engineering of AppA phytase for increased thermostability requires co-expression of protein disulfide isomerase in Pichia pastoris

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Laura Navone ◽  
Thomas Vogl ◽  
Pawarisa Luangthongkam ◽  
Jo-Anne Blinco ◽  
Carlos H. Luna-Flores ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Phytic Acid ◽  
Disulfide Bond ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
E Coli ◽  
Biochemical Characterisation ◽  
Disulfide Bond Isomerase ◽  
Microbial Systems ◽  
Protein Disulfide

Abstract Background Phytases are widely used commercially as dietary supplements for swine and poultry to increase the digestibility of phytic acid. Enzyme development has focused on increasing thermostability to withstand the high temperatures during industrial steam pelleting. Increasing thermostability often reduces activity at gut temperatures and there remains a demand for improved phyases for a growing market. Results In this work, we present a thermostable variant of the E. coli AppA phytase, ApV1, that contains an extra non-consecutive disulfide bond. Detailed biochemical characterisation of ApV1 showed similar activity to the wild type, with no statistical differences in kcat and KM for phytic acid or in the pH and temperature activity optima. Yet, it retained approximately 50% activity after incubations for 20 min at 65, 75 and 85 °C compared to almost full inactivation of the wild-type enzyme. Production of ApV1 in Pichia pastoris (Komagataella phaffi) was much lower than the wild-type enzyme due to the presence of the extra non-consecutive disulfide bond. Production bottlenecks were explored using bidirectional promoters for co-expression of folding chaperones. Co-expression of protein disulfide bond isomerase (Pdi) increased production of ApV1 by ~ 12-fold compared to expression without this folding catalyst and restored yields to similar levels seen with the wild-type enzyme. Conclusions Overall, the results show that protein engineering for enhanced enzymatic properties like thermostability may result in folding complexity and decreased production in microbial systems. Hence parallel development of improved production strains is imperative to achieve the desirable levels of recombinant protein for industrial processes.

scholarly journals Arabidopsis Disulfide Reductase, Trx-h2, Functions as an RNA Chaperone under Cold Stress

2021 ◽  
Vol 11 (15) ◽  
pp. 6865
Author(s):  
Eun Seon Lee ◽  
Joung Hun Park ◽  
Seong Dong Wi ◽  
Ho Byoung Chae ◽  
Seol Ki Paeng ◽  
...  
Keyword(s):  
Cold Stress ◽  
Wild Type ◽  
Rna Chaperone ◽  
E Coli ◽  
Cold Sensitive ◽  
Thioredoxin H ◽  
Functional Rnas

The thioredoxin-h (Trx-h) family of Arabidopsis thaliana comprises cytosolic disulfide reductases. However, the physiological function of Trx-h2, which contains an additional 19 amino acids at its N-terminus, remains unclear. In this study, we investigated the molecular function of Trx-h2 both in vitro and in vivo and found that Arabidopsis Trx-h2 overexpression (Trx-h2OE) lines showed significantly longer roots than wild-type plants under cold stress. Therefore, we further investigated the role of Trx-h2 under cold stress. Our results revealed that Trx-h2 functions as an RNA chaperone by melting misfolded and non-functional RNAs, and by facilitating their correct folding into active forms with native conformation. We showed that Trx-h2 binds to and efficiently melts nucleic acids (ssDNA, dsDNA, and RNA), and facilitates the export of mRNAs from the nucleus to the cytoplasm under cold stress. Moreover, overexpression of Trx-h2 increased the survival rate of the cold-sensitive E. coli BX04 cells under low temperature. Thus, our data show that Trx-h2 performs function as an RNA chaperone under cold stress, thus increasing plant cold tolerance.

scholarly journals MetR-Regulated Vibrio cholerae Metabolism Is Required for Virulence

mBio ◽  
2012 ◽  
Vol 3 (5) ◽  
Author(s):  
Ryan W. Bogard ◽  
Bryan W. Davies ◽  
John J. Mekalanos
Keyword(s):  
Amino Acid ◽  
Vibrio Cholerae ◽  
Virulence Factor ◽  
Current Knowledge ◽  
Intestinal Colonization ◽  
Wild Type ◽  
Pathogenic Potential ◽  
Content Type ◽  
Mouse Intestine

ABSTRACTLysR-type transcriptional regulators (LTTRs) are the largest, most diverse family of prokaryotic transcription factors, with regulatory roles spanning metabolism, cell growth and division, and pathogenesis. Using a sequence-defined transposon mutant library, we screened a panel ofV. choleraeEl Tor mutants to identify LTTRs required for host intestinal colonization. Surprisingly, out of 38 LTTRs, only one severely affected intestinal colonization in the suckling mouse model of cholera: the methionine metabolism regulator, MetR. Genetic analysis of genes influenced by MetR revealed thatglyA1andmetJwere also required for intestinal colonization. Chromatin immunoprecipitation of MetR and quantitative reverse transcription-PCR (qRT-PCR) confirmed interaction with and regulation ofglyA1, indicating that misregulation ofglyA1is likely responsible for the colonization defect observed in themetRmutant. TheglyA1mutant was auxotrophic for glycine but exhibited wild-type trimethoprim sensitivity, making folate deficiency an unlikely cause of its colonization defect. MetJ regulatory mutants are not auxotrophic but are likely altered in the regulation of amino acid-biosynthetic pathways, including those for methionine, glycine, and serine, and this misregulation likely explains its colonization defect. However, mutants defective in methionine, serine, and cysteine biosynthesis exhibited wild-type virulence, suggesting that these amino acids can be scavenged in vivo. Taken together, our results suggest that glycine biosynthesis may be required to alleviate an in vivo nutritional restriction in the mouse intestine; however, additional roles for glycine may exist. Irrespective of the precise nature of this requirement, this study illustrates the importance of pathogen metabolism, and the regulation thereof, as a virulence factor.IMPORTANCEVibrio choleraecontinues to be a severe cause of morbidity and mortality in developing countries. Identification ofV. choleraefactors critical to disease progression offers the potential to develop or improve upon therapeutics and prevention strategies. To increase the efficiency of virulence factor discovery, we employed a regulator-centric approach to multiplex our in vivo screening capabilities and allow whole regulons inV. choleraeto be interrogated for pathogenic potential. We identified MetR as a new virulence regulator and serine hydroxymethyltransferase GlyA1 as a new MetR-regulated virulence factor, both required byV. choleraeto colonize the infant mouse intestine. Bacterial metabolism is a prerequisite to virulence, and current knowledge of in vivo metabolism of pathogens is limited. Here, we expand the known role of amino acid metabolism and regulation in virulence and offer new insights into the in vivo metabolic requirements ofV. choleraewithin the mouse intestine.

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