Acyl Rearrangement Research Articles

Importance of acyl rearrangement in the acid-catalyzed reaction of ε-caprolactam with carboxylic acids

Importance of acyl rearrangement in the acid-catalyzed reaction of ε-caprolactam with carboxylic acids

Protein splicing In Cis and In Trans

Intein-mediated protein splicing is a self-catalytic process in which the intervening intein sequence is removed from a precursor protein and the flanking extein segments are ligated with a native peptide bond. Splice junction proximal residues and internal residues within the intein direct these reactions. The identity of these residues varies in each intein, as groups of related residues populate conserved motifs. Although the basics of the four-step protein splicing pathway are known, mechanistic details are still unknown. Structural and kinetic analyses are beginning to shed some light. Several structures were reported for precursor proteins with mutations in catalytic residues, which stabilize the precursors for crystallographic study. Progress is being made despite limitations inherent in using mutated precursors. However, no uniform mechanism has emerged. Kinetic parameters were determined using conditional trans-splicing (splicing of split precursor fragments after intein reassembly). Several groups concluded that the rate of the initial acyl rearrangement step is rapid and Asn cyclization (step 3) is slow, suggesting that this latter step is rate limiting. Understanding the protein splicing pathway has allowed scientists to harness inteins for numerous applications.

Photochromic properties of 2-(N-acetyl-N-arylaminomethylene)benzo[b]furan-, thiophene-, selenophene- and tellurophene-3(2H)-ones

Novel photochromic systems - (2Z)-2-(N-acetyl-N-arylaminomethylene)benzo(b)tellurophene- 3(2H)-ones have been synthesized. Their properties were studied in comparison with analogous benzo(b)furan, benzo(b)thiophene, and benzo(b)selenophene derivatives. The long-wave absorption band of these compounds shifts bathochromically in the following order of heteroatoms: O < S < Se < Te from 380 nm to 455 nm and the quantum yields of photoinitiated N→O acyl rearrangement (including Z/E-isomerization around the C=C bond and fast thermal migration of the acetyl group) increase from 0.60 (S) to 0.71 (Te). The acid catalyzed reverse O→N acyl rearrangement constant rates are 1.1 · 10 -4 s -1 (Te) < 4.2 10 -4 s -1 (Se) < 1.0 10 -3 s -1 (S).

Kinetic Analysis of the Individual Steps of Protein Splicing for the Pyrococcus abyssi PolII Intein

Protein splicing involves the excision of an intervening polypeptide, the intein, from flanking polypeptides, the exteins, concomitant with the specific ligation of the exteins. The intein that interrupts the DNA polymerase II DP2 subunit in Pyrococcus abyssi can be overexpressed and purified as an unspliced precursor, which allows for a detailed in vitro kinetic analysis of the individual steps of protein splicing. The first order rate constant for splicing of this intein, which has a non-canonical Gln at its C terminus, is 9.3 x 10(-6) s(-1) at 60 degrees C. The rate constant for splicing increases 3-fold with substitution of Asn for the C-terminal Gln. The pseudo first order rate constant of dithiothreitol-dependent N-terminal cleavage is 1 x 10(-4) s(-1). The first order rate constant of C-terminal cleavage is 1.2 x 10(-5) s(-1) with Gln at the C-terminal position, 2.8 x 10(-4) s(-1) with Asn, and decreases significantly with mutation of the penultimate His of the intein to Ala. N-terminal cleavage is most efficient between pH 7 and 7.5 and decreases at both more acidic and alkaline pH values, whereas C-terminal cleavage and splicing are both efficient over a broader range of pH values.

Rescue of protein splicing activity from a Magnetospirillum magnetotacticum intein-like element.

The self-catalytic protein splicing mechanism is mediated by the intein plus the first amino acid following the intein C-terminus (termed the +1 residue). Although polymorphisms of conserved residues elsewhere in inteins have been widely reported, no splicing-competent intein has been observed without a Ser, Thr or Cys in this functionally essential +1 position. This residue is the nucleophile in two steps of the protein splicing pathway: ligation of the extein fragments during transesterification and formation of a peptide bond between the exteins by an acyl rearrangement. An intein-like element in a hypothetical protein (gene Magn8951) from Magnetospirillum magnetotacticum has all intein signature sequences except the +1 residue, where it has a Tyr. Although the Tyr side-chain hydroxyl can potentially mediate the transesterification reaction, an acyl shift has never been observed with this residue. When the activities of this bacterial intein-like element were studied, protein splicing was not observed and N-terminal cleavage predominated. Mutation of Tyr+1 to Phe or Ala indicated that the Tyr side-chain hydroxyl was not necessary for N-terminal cleavage. Protein splicing activity could be rescued by "reversion" of Tyr+1 to Cys.

Photochromic 2-(N-acyl-N-arylaminomethylene)benzo[b]thiophene-3(2H)-ones with fluorescent labels and/or crown-ether receptors

Novel N-acylaminovinyl ketones of the benzo[b]thiophene series (AAB) capable of molecular switching of fluorescence or complexation with metal ions have been synthesized and their photochromic behavior studied using UV-Vis absorption/emission and 1 H- / 7 Li- NMR spectroscopy. Under UV irradiation the AAB compounds 2a–c containing fluorescent labels as their N-aryl substituents undergo Z/E- photo-isomerization and N→O acyl rearrangement resulting in the formation of O-acyl isomers 3a–c. The photo-switching of fluorescence has been found in the 4-fluorenonyl derivative 2a. The benzo-15-crown-5- containing AAB 2d,e are also susceptible to the same type of photo-initiated rearrangement. The corresponding O-acyl isomers 3d–e represent a group of chemosensors which readily form stable complexes with alkali- and alkali-earth- metal cations. The AAB 2d exhibits specific selectivity for Li + ion in the presence of Na + , K + and Cs + .

Mechanism of human S-adenosylmethionine decarboxylase proenzyme processing as revealed by the structure of the S68A mutant.

S-Adenosylmethionine decarboxylase (AdoMetDC) is a pyruvoyl-dependent enzyme that catalyzes the formation of the aminopropyl group donor in the biosynthesis of the polyamines spermidine and spermine. The enzyme is synthesized as a protein precursor and is activated by an autocatalytic serinolysis reaction that creates the pyruvoyl group. The autoprocessing reaction proceeds via an N --> O acyl rearrangement, generating first an oxyoxazolidine anion intermediate followed by an ester intermediate. A similar strategy is utilized in self-catalyzed protein splicing reactions and in autoproteolytic activation of protein precursors. Mutation of Ser68 to alanine in human AdoMetDC prevents processing by removing the serine side chain necessary for nucleophilic attack at the adjacent carbonyl carbon atom. We have determined the X-ray structure of the S68A mutant and have constructed models of the proenzyme and the oxyoxazolidine intermediate. Formation of the oxyoxazolidine intermediate is promoted by a hydrogen bond from Cys82 and stabilized by a hydrogen bond from Ser229. These observations are consistent with mutagenesis studies, which show that the C82S and C82A mutants process slowly and that the S229A mutant does not process at all. Donation of a proton by His243 to the nitrogen atom of the oxyoxazolidine ring converts the oxyoxazolidine anion to the ester intermediate. The absence of a base to activate the hydroxyl group of Ser68 suggests that strain may play a role in the cleavage reaction. Comparison of AdoMetDC with other self-processing proteins shows no common structural features. Comparison to histidine decarboxylase and aspartate decarboxylase shows that these pyruvoyl-dependent enzymes evolved different catalytic strategies for forming the same cofactor.

Macrocyclic diterpenoids from Euphorbia hyberna L. subsp. insularis and their reaction with oxyphilic reagents

The aerial parts of Euphorbia hyberna subsp. insularis from Sardinia contain large amounts of macrocyclic diterpenoids of the jatrophane type, while phorboids could not be isolated. Treatment of the major constituent ( 1a) with various acidic and oxyphilic reagents was investigated, showing the surprising reluctance of the macrocyclic system to undergo transannular reactions. Dehydration, isomerization of the exocyclic double and acyl rearrangement were, however, observed after reduction of the 9-keto group.

Zinc Inhibition of Protein trans-Splicing and Identification of Regions Essential for Splicing and Association of a Split Intein*

Two important aspects of protein splicing were investigated by employing the trans-splicing intein from the dnaE gene of Synechocystis sp. PCC6803. First, we demonstrated that both protein splicing and cleavage at the N-terminal splice junction were inhibited in the presence of zinc ion. The trans-splicing reaction was partially blocked at a concentration of 1-10 microm Zn(2+) and completely inhibited at 100 microm Zn(2+); the inhibition by zinc was reversed in the presence of ethylenediaminetetraacetic acid. We propose that inactivation of Cys(160) at the C-terminal splice junction by the chelation of zinc affects both the N-S acyl rearrangement and the transesterification steps in the splicing pathway. Furthermore, in vivo and in vitro assays were established for the determination of intein residues and regions required for splicing or association between the N- and C-terminal intein halves. N-terminal truncation of the intein C-terminal segment inhibited both splicing and association activities, suggesting this region is crucial for the formation of an interface between the two intein halves. The replacement of conserved residues in blocks B and F with alanine abolished splicing but allowed for association. This is the first evidence showing that the conserved residues in block F are required for protein splicing.

An alternative protein splicing mechanism for inteins lacking an N-terminal nucleophile.

Variations in the intein-mediated protein splicing mechanism are becoming more apparent as polymorphisms in conserved catalytic residues are identified. The conserved Ser or Cys at the intein N-terminus and the conserved intein penultimate His are absent in the KlbA family of inteins. These inteins were predicted to be inactive, since an N-terminal Ala cannot perform the initial reaction of the standard protein splicing pathway to yield the requisite N-terminal splice junction (thio)ester. Despite the presence of an N-terminal Ala and a penultimate Ser, the KlbA inteins splice efficiently using an alternative protein splicing mechanism. In this non-canonical pathway, the C-extein nucleophile attacks a peptide bond at the N-terminal splice junction rather than a (thio)ester bond, alleviating the need to form the initial (thio)ester at the N-terminal splice junction. The remainder of the two pathways is the same: branch resolution by Asn cyclization is followed by an acyl rearrangement to form a native peptide bond between the ligated exteins.

Reactivity of the Cysteine Residues in the Protein Splicing Active Center of the Mycobacterium tuberculosis RecA Intein

Protein splicing involves the self-catalyzed excision of an intervening polypeptide segment, an intein, from a precursor protein. The first two steps in the protein splicing process lead to the formation of ester intermediates through nucleophilic attacks by the side chains of cysteine, serine, or threonine residues adjacent to the splice junctions. Since both nucleophilic residues in the Mycobacterium tuberculosis RecA intein are cysteine, their reactivities could be compared by sulfhydryl group titration. This was accomplished by using fusion proteins containing a truncated RecA intein modified by mutation to prevent protein splicing, in which the cysteines at the splice junctions were the only sulfhydryl groups. The ability to undergo hydroxylamine-induced cleavage at the upstream splice junction showed that the modified intein was not impaired in the ability to form ester intermediates. Sulfhydryl titration with iodoacetamide, monitored by quantitating the residual thiols after reaction with a maleimide derivative of biotin, revealed a striking difference in the apparent pKa values of the cysteines at the two splice junctions. The apparent pKa of the cysteine at the upstream splice junction, which initiates the N–S acyl rearrangement leading to the linear ester intermediate, was approximately 8.2, whereas that of the cysteine residue at the downstream splice junction, which initiates the transesterification reaction converting the linear ester to the branched ester intermediate, was about 5.8. This suggests that the transesterification step is facilitated by an unusually low pKa of the attacking thiol group. Comparison of the rates of cleavage of the linear ester intermediates derived from the M. tuberculosis RecA and the Saccharomyces cerevisiae VMA inteins by dithiothreitol and hydroxylamine revealed that the former reacted relatively more slowly with dithiothreitol, suggesting that the RecA intein has diverged in the course of evolution to react preferentially with thiolate anions and thus lacks the basic groups that may facilitate nucleophilic attack by thiols in other inteins.

Self-catalyzed Cleavage of the Yeast Nucleoporin Nup145p Precursor

Nup145p is a component of the nuclear pore complex of Saccharomyces cerevisiae and is essential for mRNA export. Nup145p and its apparent vertebrate homologue are the only known nucleoporins to be composed of two functionally independent peptide moieties resulting from the post-translational cleavage of a large precursor molecule. In this study, the proteolytic cleavage site of Nup145p has been mapped upstream of an evolutionary conserved serine residue. Cleavage occurs at the same site when a precursor is artificially expressed in Escherichia coli. A hydroxyl-containing residue is critical for the reaction, although a thiol-containing residue offers an acceptable replacement. In vitro kinetics experiments using a purified precursor molecule demonstrate that the cleavage is self-catalyzed and that the catalytic domain lies within the N-terminal moiety. Taken altogether, our data are consistent with a proteolytic mechanism involving an N>O acyl rearrangement and a subsequent ester intermediate uncovered in other self-processing proteins.

Synthesis of Wasabidienone A via a Novel Acyl Rearrangement Reaction from Carbon to Oxygen

Synthesis of Wasabidienone A via a Novel Acyl Rearrangement Reaction from Carbon to Oxygen

Modulation of Protein Splicing of the Saccharomyces cerevisiae Vacuolar Membrane ATPase Intein

Protein splicing of the Saccharomyces cerevisiae vacuolar membrane ATPase intein involves four highly coordinated reactions that result in precise cleavage and formation of peptide bonds. In this study, we investigated the roles of the last N-extein residue (-1 residue) and the intein penultimate residue in modulating splicing reactions. Most of the 20 amino acid substitutions at the -1 position had no effect on overall protein splicing but could lead to significant accumulation of thioester intermediates when splicing was blocked by mutation. A subset of -1 substitutions attenuated the initiation of protein splicing and enabled us to demonstrate in vitro splicing of a mesophilic intein containing all wild-type catalytic residues. Substitutions involving the intein penultimate residue allowed modulation of the branch resolution and C-terminal cleavage reaction. Our data suggest that the N-S acyl rearrangement, which initiates splicing, may also serve as the rate-limiting step. Through appropriate amino acid substitutions, we were able to modulate splicing reactions in vitro by change in pH or temperature or addition of thiol reagents. Both insertion and deletion were tolerated in the central region of the intein although splicing or structure of the intein may have been affected.

Characterization and Functional Analysis of the Cis-autoproteolysis Active Center of Glycosylasparaginase

Glycosylasparaginase is an N-terminal nucleophile hydrolase and is activated by intramolecular autoproteolytic processing. This cis-autoproteolysis possesses unique kinetics characterized by a reversible N-O acyl rearrangement step in the processing. Arg-180 and Asp-183, involved in binding of the substrate in the mature enzyme, are also involved in binding of free amino acids in the partially formed substrate pocket on certain mutant precursors. This binding site is sequestered in the wild-type precursor. Binding of free amino acids on mutant precursors can either inhibit or accelerate their processing, depending on the individual mutants and amino acids. The polypeptide sequence at the processing site, which is highly conserved, adopts a special conformation. Asp-151 is essential for maintaining this conformation, possibly by anchoring its side chain into the partially formed substrate pocket through interaction with Arg-180. The reactive nucleophile Thr-152 is activated not only by deprotonation by His-150 but also by interaction with Thr-170, suggesting a His-Thr-Thr active triad for the autoproteolysis.

Protein splicing: a novel form of gene expression and paradigm for self-catalzyed protein rearrangements

Abstract

Protein Splicing of Inteins and Hedgehog Autoproteolysis: Structure, Function, and Evolution

The Mxe GyrA mini-intein represents a splicing element without a DRR or core endonuclease domain. Larger inteins, like the Sce VMA intein, contain homing endonucleases. The structural similarity among splicing elements and Hh-C17 can be seen in the common positioning of β strands and the large number of residues with superimposable Cα atoms (the main chain carbon atom to which the side chain is attached) (Figure 3Figure 3). However, there is little sequence identity among the three proteins except for the residues involved in catalysis (Figure 1Figure 1 and Figure 3Figure 3). Because of the similarity of their core structures, Hall et al. 1997xHall, T.M.T, Porter, J.A, Young, K.E, Koonin, E.V, Beachy, P.A, and Leahy, D.J. Cell. 1997; 91: 85–97Abstract | Full Text | Full Text PDF | PubMed | Scopus (150)See all ReferencesHall et al. 1997 proposed that inteins and Hh-C17 evolved from a common precursor (Figure 4Figure 4). The Hint module mediates ester/thioester formation, activating the linkage between the element and a second protein domain. Other systems, such as the Ntn hydrolase family, employ an acyl rearrangement to activate catalysis but do not have structural similarity to the Hint module (Hall et al. 1997xHall, T.M.T, Porter, J.A, Young, K.E, Koonin, E.V, Beachy, P.A, and Leahy, D.J. Cell. 1997; 91: 85–97Abstract | Full Text | Full Text PDF | PubMed | Scopus (150)See all ReferencesHall et al. 1997). Inteins and Hh-C subsequently evolved separate methods of cleaving this bond. Inteins evolved the ability to ligate two exteins and acquired the DRR and core endonuclease. The endonuclease allowed inteins to spread by lateral transmission. Hh-C17 acquired a sterol recognition region (SRR) directing addition of cholesterol to the hedgehog protein signaling domain, anchoring the signaling domain to the cell surface. Alternatively, the Hint module could have invaded a preexisting signaling domain/SRR element. Several nematode Hh-C domains contain unrelated C-terminal extensions that may interact with molecules other than cholesterol and have been tentatively termed adduct recognition regions (Beachy et al. 1997xCold Spring Harbor Symp. Beachy, P.A, Cooper, M.K, Young, K.E, von Kessler, D.P, Park, W, Hall, T.M.T, Leahy, D.J, and Porter, J.A. Quant. Biol. 1997; in pressSee all ReferencesBeachy et al. 1997). The order of these events is speculative, including the order of module assembly. Each of the intein and hedgehog elements may have coevolved or associated after independent formation. Endonucleases may have also been lost from inteins. As we understand the mechanism of protein splicing and Hh-C autoproteolysis, we will begin to be able to harness these elements to cleave or splice any target protein at will.Figure 4A Scenario for Evolution of Inteins and Hh-CA primordial subdomain was duplicated and loop exchange occurred between subdomains to generate the Hint module. Inteins then acquired the DNA recognition region (DRR) and core endonuclease domain (ENDO) and Hh-C17 acquired a sterol recognition region (SRR). The order of these events is speculative. Adapted with permission from Hall et al., 1997.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Protein splicing and autoproteolysis mechanisms

It has generally been assumed that the conversion of all inactive protein precursors to biologically active proteins is mediated by specific processing enzymes. However, numerous examples of self-catalyzed protein rearrangements have recently been discovered, including protein splicing and autoproteolysis of hedgehog proteins, glycosylasparaginases and pyruvoyl enzyme precursors. The initial formation of an ester bond by the acyl rearrangement of a peptide bond is a common feature of all of these autoprocessing reactions, which manifest themselves in diverse biological functions ranging from protein splicing to protein targeting, proenzyme activation, and the generation of enzyme-bound prosthetic groups. Although such acyl rearrangements are thermodynamically unfavorable, their coupling to diverse types of self-catalyzed irreversible steps drives the protein rearrangements to completion.

Protein splicing: estimation of the rate of O-N and S-N acyl rearrangements, the last step of the splicing process.

The last step in the sequence of reactions that lead to protein splicing is the intramolecular O-N or S-N acyl rearrangement of the ester or thioester linkage, respectively, between the two exteins and hydrolysis of the aminosuccinimide residue at the C-terminus of intein. This paper presents data on the rates of O-N and S-N acyl rearrangements of two model depsipeptides as a function of pH and temperature. The rates of rearrangement of both the oxygen ester and the thioester depsipeptide increased strikingly with pH, with the thioester being about 10(3) times more reactive at pH 5.5, and had a relatively low dependence on temperature, indicative of a low activating energy. The rates of O-N and S-N acyl rearrangement of these two model depsipeptides greatly exceed the rate of protein splicing, explaining why the last step of protein splicing can occur without catalysis by the intein.

Biphotochromic Norbornadiene Systems

Novel biphotochromic systems containing two norbornadiene moieties in one molecule or one norbornadienyl group in 2-N-(3-phenylnorbornadiene-2-carbonyl)]-N-arylaminomethylene-(2H) benzo[b]thiophenones have been prepared. Under UV/VIS irradiation the latter compounds undergo Z/E-isomerktion acompanied by N → O acyl rearrangement and valence isomerization in the norbornadiene fragment. Bis-norbornadienyl derivatives upon irradiation convert through stepwise isomerization into corresponding quadricyclanes. The back reactions are catalyzed with trifluoroacetic acid at room temperature.