Inversion Reaction Research Articles

In vivo assay of protein-protein interactions in Hin-mediated DNA inversion.

In order to form the catalytic nucleoprotein complex called the invertasome in the Hin-mediated DNA inversion reaction, interactions of the DNA-binding proteins Hin and Fis are required. Assays for these protein-protein interactions have been exploited with protein cross-linkers in vitro. In this study, an in vivo assay system that probes protein-protein interactions was developed. The formation of a DNA loop generated by protein interactions resulted in transcriptional repression of an artificially designed operon, which in turn increased the chance of survival of Escherichia coli host cells in a streptomycin-containing medium. Using this system, we were able to assay the Hin-Hin interaction that results in the pairing of the two recombination sites and protein interactions that result in the formation of the invertasome. This assay system also led us to find that an individual Hin dimer bound on a recombination site can form a stable complex with Fis bound on the recombinational enhancer; this finding has never been observed in in vitro studies. Possible pathways toward the formation of the invertasome are discussed based on the assay results for a previously reported Hin mutant.

Identification and characterization of the fis operon in enteric bacteria.

The small DNA binding protein Fis is involved in several different biological processes in Escherichia coli. It has been shown to stimulate DNA inversion reactions mediated by the Hin family of recombinases, stimulate integration and excision of phage lambda genome, regulate the transcription of several different genes including those of stable RNA operons, and regulate the initiation of DNA replication at oriC. fis has also been isolated from Salmonella typhimurium, and the genomic sequence of Haemophilus influenzae reveals its presence in this bacteria. This work extends the characterization of fis to other organisms. Very similar fis operon structures were identified in the enteric bacteria Klebsiella pneumoniae, Serratia marcescens, Erwinia carotovora, and Proteus vulgaris but not in several nonenteric bacteria. We found that the deduced amino acid sequences for Fis are 100% identical in K. pneumoniae, S. marcescens, E. coli, and S. typhimurium and 96 to 98% identical when E. carotovora and P. vulgaris Fis are considered. The deduced amino acid sequence for H. influenzae Fis is about 80% identical and 90% similar to Fis in enteric bacteria. However, in spite of these similarities, the E. carotovora, P. vulgaris, and H. influenzae Fis proteins are not functionally identical. An open reading frame (ORF1) preceding fis in E. coli is also found in all these bacteria, and their deduced amino acid sequences are also very similar. The sequence preceding ORF1 in the enteric bacteria showed a very strong similarity to the E. coli fis P region from -53 to +27 and the region around -116 containing an ihf binding site. Both beta-galactosidase assays and primer extension assays showed that these regions function as promoters in vivo and are subject to growth phase-dependent regulation. However, their promoter strengths vary, as do their responses to Fis autoregulation and integration host factor stimulation.

Quantum scattering on SN2 reactions: Influence of azimuthal rotations

Time independent quantum scattering calculations have been carried out on the SN2 Walden inversion reaction Cl−+CH3Cl(v,k)→ClCH3(v′,k′)+Cl−. The two C–Cl stretching degrees of freedom (quantum numbers v and v′) and the azimuthal angle describing the rotation of the CH3 group (quantum numbers k and k′) are treated explicitly. An infinite order sudden approximation has been introduced using Radau coordinates for the stretching modes. The potential energy surface of Vande Linde and Hase is used. The scattering problem is formulated in hyperspherical coordinates. For the reaction (k=0→k′=0) substitution is observed for initial vibrational excitation with v⩾2. If the system departs from the collinear reaction pathway (initial rotational excitation) the substitution cross sections are strongly decreased. The state-to-state cross sections σvk→v′k′ are large only for transitions with Δk=0. The total reaction cross sections σvk for given v vary only slightly at low values of the azimuthal quantum number k and rise for larger values of k. This is explained by multiple (avoided) crossings of the hyperspherical adiabats.

The saddle point of the nucleophilic substitution reaction Cl − + CH 3 Cl: results of large-scale coupled cluster calculations

On the basis of large-scale coupled cluster calculations including connectedz triple substitutions in a perturbative way, the geometrical parameters of the D3h saddle point of the Walden inversion reaction Cl− + CH3Cl′→ ClCH3 + Cl′− are predicted to be Rs (C—Cl) = 2.301 A and rs (C—H) = 1.069 A. The barrier height with respect to the reactants is recommended to be 11.5 ± 1.0 kJ mol−1. Connected triple substitutions lower the barrier height by almost a factor of 2, but have very little influence on the geometric structure of the saddle point.

In vivo identification of intermediate stages of the DNA inversion reaction catalyzed by the Salmonella Hin recombinase.

The Hin recombinase catalyzes a site-specific recombination reaction that results in the reversible inversion of a 1-kbp segment of the Salmonella chromosome. The DNA inversion reaction catalyzed by the Salmonella Hin recombinase is a dynamic process proceeding through many intermediate stages, requiring multiple DNA sites and the Fis accessory protein. Biochemical analysis of this reaction has identified intermediate steps in the inversion reaction but has not yet revealed the process by which transition from one step to another occurs. Because transition from one reaction step to another proceeds through interactions between specific amino acids, and between amino acids and DNA bases, it is possible to study these transitions through mutational analysis of the proteins involved. We isolated a large number of mutants in the Hin recombinase that failed to carry out the DNA exchange reaction. We generated genetic tools that allowed the assignment of these mutants to specific transition steps in the recombination reaction. This genetic analysis, combined with further biochemical analysis, allowed us to define contributions by specific amino acids to individual steps in the DNA inversion reaction. Evidence is also presented in support of a model that Fis protein enhances the binding of Hin to the hixR recombination site. These studies identified regions within the Hin recombinase involved in specific transition steps of the reaction and provided new insights into the molecular details of the reaction mechanism.

Additions and corrections: Transverse compression and the secondary H/D isotope effects in intramolecular SN2 methyl-transfer reactions

Using ab initio molecular orbital theory mainly at the 3-21+G level, intramolecular SN2 methyl transfer between two oxygens confined within a rigid template is found to proceed exclusively by a high energy retention mechanism when the oxygens are separated by three or four bonds, and by a high energy inversion mechanism when the oxygens are separated by six bonds. Both mechanisms exist when the oxygens are separated by five bonds. The CH3/CD3 kinetic isotope effects are normal (1.21-1.34) in the retention processes and inverse (0.66-0.81) in the inversion reactions. In the case of inversion, compression of C-H bonds of the transition state by structural effects in the plane perpendicular to the O-C-O plane increases the inverse isotope effect. The retention barriers are high because retention is inherently unfavorable, even when pericyclic stabilization of the transition state is possible. The inversion barriers are high because a rigid template cannot accommodate a linear O-CH3 -O structure, and the O-C-O bending vibration is stiff (the Eschenmoser effect). Using a novel design strategy, a nonrigid template has been found in which the barrier and the CH3/CD3 kinetic isotope effect are the same as in an intermolecular reaction.Key words: Eschenmoser effect, isotope effect, compression, SN2, sigmatropic rearrangement.

Transverse compression and the secondary H/D isotope effects in intramolecular SN2 methyl-transfer reactions

Using ab initio molecular orbital theory mainly at the 3-21 + G level, intramolecular SN2 methyl transfer between two oxygens confined within a rigid template is found to proceed exclusively by a high energy retention mechanism when the oxygens are separated by three or four bonds, and by a high energy inversion mechanism when the oxygens are separated by six bonds. Both mechanisms exist when the oxygens are separated by five bonds. The CH3/CD3 kinetic isotope effects are normal (1.21-1.34) in the retention processes and inverse (0.66-0.81) in the inversion reactions. In the case of inversion, compression of C-H bonds of the transition state by structural effects in the plane perpendicular to the O-C-O plane increases the inverse isotope effect. The retention barriers are high because retention is inherently unfavorable, even when pericyclic stabilization of the transition state is possible. The inversion barriers are high because a rigid template cannot accommodate a linear O-CH3-O structure, and the O-C-O bending vibration is stiff (the Eschenmoser effect). Using a novel design strategy, a nonrigid template has been found in which the barrier and the CH3/CD3 kinetic isotope effect are the same as in an intermolecular reaction.Key words: Eschenmoser effect, isotope effect, compression, SN2, sigmatropic rearrangement.

Chemical Transformation of a 1-Deoxynojirimycin Derivative into 1-Deoxymannojirimycin and 1-Deoxygalactostatin

Treatment of 2,3-di-O-benzyl-N-benzyloxycarbonyl-6-O-t-butyldiphenylsilyl-1,5-dideoxy-1,5-imino-D-glucitol (4) with sodium hydride resulted in an intramolecular cyclization concomitant with silyl migration, giving the carbamate derivative 5 in good yield. This was efficiently converted into 1-deoxymannojirimycin (2) via regioselective p-toluenesulfonylation followed by an inversion reaction at the C-2 position. On the other hand, the monochloromethylsulfonate 10 obtained from 4 underwent configurational change at the C-4 position by the action of sodium benzoate. The resulting benzoate 11 was deprotected to afford 1-deoxygalactostatin (3).

Morphology development in epoxy/polymer systems: thermosetting epoxy micro particles with a thermoplastic shell

A novel method based on phase inversion and inter-boundary reaction in a reactive system comprising a thermoplastic phenoxy and a thermosetting epoxy has been proposed to design micro epoxy particles with a thermoplastic polymer shell. Due to the hydroxyl group in phenoxy, inter-domain reactions were found to lead to a chemical link between the polymer and the epoxy network. As cure progressed, the phenoxy component was first expelled out of the epoxy spheres at initial stages of cure and a phase-separated morphology was set. Subsequently, the pendant -OH groups in the phenoxy shells were grafted onto the outer surfaces of the epoxy core particles (3–4 μm) at high enough temperatures (187°C or higher) during later stages of cure. This paper describes the mechanisms and fundamental phenomenon under which this unique method of preparing polymer/epoxy spheres (epoxy cores/phenoxy shell) of controlled sizes can be realized. Several factors have been found to affect the geometry of the epoxy spheres and the chemical bondings between the polymer and epoxy.

Stimulation of DNA inversion by FIS: evidence for enhancer-independent contacts with the Gin-gix complex.

Efficient DNA inversion catalysed by the invertase Gin requires the cis-acting recombinational enhancer and the Escherichia coliFIS protein. Binding of FIS bends the enhancer DNA and, on a negatively supercoiled DNA inversion substrate, facilitates the formation of a synaptic complex with specific topology. Previous studies have indicated that FIS-independent Gin mutants can be isolated which have lost the topological constraints imposed on the inversion reaction yet remain sensitive to the stimulatory effect of FIS. Whether the effect of FIS is purely architectural, or whether in addition direct protein contacts between Gin and FIS are required for efficient catalysis has remained an unresolved question. Here we show that FIS mutants impaired in DNA binding are capable of either positively or negatively affecting the inversion reaction both in vivo and in vitro. We further demonstrate that the mutant protein FIS K25E/V66A/M67T dramatically enhances the cleavage of recombination sites by FIS-independent Gin in an enhancer-independent manner. Our observations suggest that FIS plays a dual role in the inversion reaction and stimulates both the assembly of the synaptic complex as well as DNA strand cleavage.

Hin-mediated inversion on positively supercoiled DNA.

Hin recombinase requires negatively supercoiled DNA for an efficient inversion. We have generated positively supercoiled plasmid DNA using reverse gyrase from Sulfolobus shibatae and subjected it to the Hin-mediated inversion reaction. Both Hin and Fis showed the same DNA binding activity regardless of the superhelical handedness of the substrate plasmid. However, inversion activity on positively supercoiled DNA was less than 1% of negatively supercoiled DNA. Assays designed to probe steps in inversion, showed that on positively supercoiled DNA, Hin was able to cleave the recombination sites with the same efficiency shown on negatively supercoiled DNA but was not able to exchange the cleaved DNA. Based on the theoretical differences between positive and negative supercoiling, our data may suggest that unwinding of the double helix at recombination sites is needed after DNA cleavage for strand exchange to occur.

Synthesis and stereochemical nonrigidity of isomeric Zn(ii)bis-[N-isopropyloxy(mercapto)naphthaldiminates

Kinetics and reaction mechanisms governing inversion of the tetrahedral configuration at the metal center in the series ofbis-chelate Zn(II) complexes of 3,2-, 1,2-, and 2,1-oxy(mercapto)naphthaldimines, respectively4–6, have been studied with the use of dynamic1H NMR spectroscopy. A polytopal rearrangement of the diagonal twist type has been found to be an energetically preferable pathway of the inversion reaction for complexes4 and5 with a ZnN2O2 coordination site, whereas the inversion reaction for complexes with a ZnN2S2 coordination site occurs by an intramolecular dissociation-recombination pathway that involves cleavage of a Zn-N coordination bond. In the case of complexes6, the inversion reaction is governed mainly by intramolecular degenerate ligand exchange reactions.

Preparation of enantiomerically pure trans- and cis-2-(1-naphthyl)cyclohexan-1-ols

Abstract Lipase-mediated treatment of racemic trans -2-(1-naphthyl)cyclohexan-1-ol with vinyl acetate allows clear-cut enantiospecific kinetic acetylation to give (+)-(1 R ,2 S )-acetate in excellent chemical and enantiomeric excesses leaving (+)-(1 S ,2 R )-alcohol in an excellent enantiomeric purity and excellent recovery. The enantiomerically pure alcohol obtained is transformed into the diastereomeric alcohol neatly via the Mitsunobu inversion reaction.

DNA Looping by Saccharomyces cerevisiae High Mobility Group Proteins NHP6A/B: CONSEQUENCES FOR NUCLEOPROTEIN COMPLEX ASSEMBLY AND CHROMATIN CONDENSATION

The formation of higher order protein.DNA structures often requires bending of DNA strands between specific sites, a process that can be facilitated by the action of nonspecific DNA-binding proteins which serve as assembly factors. A model for this activity is the formation of the invertasome, an intermediate structure created in the Hin-mediated site-specific DNA inversion reaction, which is stimulated by the prokaryotic nucleoid-associated protein HU. Previously, we have shown that the mammalian HMG1/2 proteins substitute for HU in this system and display efficient DNA wrapping activity in vitro. In the present work, we isolate the primary sources of assembly factor activity in Saccharomyces cerevisiae, as measured by the ability to stimulate invertasome formation, and show that these are the previously identified NHP6A/B proteins. NHP6A/B have comparable or greater activity in DNA binding, bending, and supercoiling with respect to HU and HMG1 and appear to form more stable protein.DNA complexes. In addition, expression of NHP6A in mutant Escherichia coli cells lacking HU and Fis restores normal morphological appearance to these cells, specifically in nucleoid condensation and segregation. From these data we predict diverse architectural roles for NHP6A/B in manipulating chromosome structure and promoting the assembly of multicomponent protein.DNA complexes.

Activation energies for pyramidal inversion of sulfonium selenonium and telluronium ylides based on ab initio MO calculations

Ab initio MO calculations of sulfonium, selenonium and telluronium ylides R12X+–C–R22(X = S, Se, Te; R1= H, Me; R2= H, CN) have been performed to estimate their thermal stabilities toward the pyramidal inversion (vertex inversion) reaction. Geometrical parameters at both the global minima and the saddlepoints are independent of the levels of theory employed. However, the activation energies for the pyramidal inversion reaction depend on the levels of theory employed; a method superior to the Hartree–Fock level of theory was necessary for the present purposes. The activation energies for the pyramidal inversion were also found to depend on the substituents. The present computations clearly indicate that the telluronium ylides require higher activation energies for the pyramidal inversion reaction than those of the sulfonium and selenonium ylides.

Enzymatic Optical Resolution ofN-Benzyl-3-pyrrolidinol

The enzymatic optical resolution of racemic N-benzyl-3-pyrrolidinol (2) was investigated. It was found that only (R)-2 was acetylated by the lipase Amano P in yields of 50% and 100%e.e. Remaining (S)-alcohol 2 could be chemically inverted to (R)-acetate 3. A continuous reaction in a column reactor could be also applied to this optical resolution. After concentrating the eluted solution, the residue was subjected to the inversion reaction to provide (R)-acetate 3 in an 83% yield and 91%e.e. from the racemate.

Analysis of subunit interaction by introducing disulfide bonds at the dimerization domain of Hin recombinase.

The Hin recombinase exists as a homodimer in solution and binds to its recombination site (hix) as a dimer (Glasgow, A. C., Bruist, M. F., and Simon M. I. (1989) J. Biol. Chem. 264, 10072-10082). Previous mutational and structural studies of related proteins suggested the location of a putative dimerization domain. In order to probe the function of this region of the protein, cysteine residues were introduced at each of the seven positions comprising the domain. Proteins containing cysteine substitutions at positions 101 and 104 were able to form disulfide bonds spontaneously in crude extracts. M101C showed wild type inversion activity only if the cysteine residue was not engaged in a disulfide bond. This mutant, which is cross-linked by a disulfide bond at the dimeric interface, was found to be defective in the DNA cleavage step during inversion. H107C displayed the wild type DNA cleavage activity, even though its DNA binding activity was not detected by three different binding assays. This suggests that it maintains specificity for DNA binding but dissociates rapidly after binding. The remaining inversion-defective mutants fell into two groups. One group lost its capacity to specifically bind to DNA (G102C, F105C, and F106C), and the other group (R103C and F104C), while able to bind to DNA, was defective in subsequent steps of the inversion reaction. Characteristics of these mutants led me to postulate that movement of subunits at the dimerization interface is critical during DNA binding and during formation of the invertasome, which is the recombination structure. Furthermore, the relative position of subunits within the dimer may be important for maintaining stable DNA binding.

IHF supresses the inhibitory effect of H-NS on HU function in the hin inversion system

In the hin-mediated DNA inversion system, HU facilitates formation of the synaptic complex composed of two recombination sites spaced 996 bp apart and of the enhancer situated between them, by looping the DNA as to promote interaction of Hin invertase with the Fis enhancer factor [Johnson et al., Nature 329 (1987) 462–465]. The HU requirement for the in vivo hin-mediated inversion was demonstrated previously [Wada et al. Gene 76 (1989) 345–352; Hillyard et al. J. Bacteriol. 172 (1990) 5402–5407; Haykinson and Johnson, EMBO J. 12 (1993) 2503–2512] and in the current experiments. This HU action, however, required IHF when H-NS was present in the cell; i.e., the inversion reaction of the hin-invertible DNA fragment carried by the ptCK1202R plasmid proceeded efficiently in host cells either deficient in H-NS or in the presence of both H-NS and IHF, but not in the cells depleted for IHF alone. The level of hin mRNA in mutant cells lacking HU or IHF, in which hin inversion did not occur, was normal or slightly increased. When IHF was absent, the stimulating effect of HU on in vitro DNA circle formation of a 125-bp hin fragment between hixL and the enhancer where Fis binds was inhibited by H-NS. The present study provides an example of a multi-component interaction between HU, H-NS and IHF on the hin DNA region, which contains three characteristic sites, a d(A/T) 4 stretch and bent DNA site, and two putative IHF-binding sites.

The role of coenzyme A in the biotransformation of 2-arylpropionic acids

A number of 2-arylpropionic acid non-steroidal anti-inflammatory drugs (‘profens’) undergo highly enantioselective inversion from the ( R)- to ( S)-enantiomer. The mechanism of this inversion reaction involves the initial enantioselective formation of a coenzyme A thioester followed by epimerization and finally hydrolysis to regenerate free acids. Long-chain fatty acyl-CoA synthetase appears to mediate the initial thioester formation and an epimerase of an unknown physiologic function effects the second step. The hydrolases mediating the final step are poorly defined. Available evidence suggests that the liver is quantitatively the most important tissue site of inversion but local tissue inversion may influence the pharmacological and toxicological response of a given organ. Data from isolated rat hepatocytes indicate that other xenobiotics can modulate the formation and hydrolysis of ibuprofenyl-CoA by influencing inversion pathways, non-inversion pathways or both. Interactions between xenobiotics may therefore accentuate inter-individual variability in response to 2-arylprpionic acids. The formation of 2-arylpropionyl-CoA thioesters in vivo has the potential to disrupt numerous biochemical pathways in addition to enhancing individual exposure to the potent anti-inflammatory ( S)-enantiomers.

Transition vector symmetry and the internal pseudo-rotation and inversion paths of CIF4 +

A new definition of the transition vector symmetry group is suggested. The utility of the definition is demonstrated by calculations of the pathways of the internal pseudo-rotation and inversion of CIF4+(DZP and DZP/MP2 levels). The lowest internal pseudo-rotation proceeds by a Berry mechanism with an activation barrier of 6.7 kcal mol–1. The lever mechanism has a higher barrier of 39.5 kcal mol–1 and would lead to more extensive fluorine scrambling. The inversion reaction that leads to enantiomerization corresponds to a planar D4h transition state with an activation barrier of 60 kcal mol–1.