Characterization of a Naturally Occurring Trans-Splicing Intein from<i>Synechocystis</i>sp. PCC6803

Martin, Deana D.; Xu, Ming‐Qun; Evans, Thomas C.

doi:10.1021/bi001786g

Cited by 126 publications

(220 citation statements)

References 33 publications

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“…The results of these experiments are summarized in Table 1. For the wild-type intein, the apparent first order rate constant for cleavage was Ϸ1 ϫ 10 Ϫ3 s Ϫ1 , which is in excellent agreement with similar measurements made on other inteins (18,19). Mutation of Thr-72 and Asn-74 reduced the cleavage rate by 5-and 10-fold, respectively, indicating that these residues are important, but not essential for splicing activity.…”

Section: Resultssupporting

confidence: 85%

“…Our view is that insights into autocatalysis will require the structural analysis of active protein splicing precursors involving, as far as possible, wild-type inteins and exteins. Such an approach is greatly complicated by the short half-life of extein-intein fusions (18,19). Indeed, we have measured the half-life of an N-extein-GyrA fusion to be Ϸ12 min in the presence thiol cleaving agents (Table 1).…”

Section: Discussionmentioning

confidence: 99%

“…This has proven difficult because of the short half-life of splicing precursors, on the order of a few hours (18,19). Indeed, an active S. cerevisiae vacuolar ATPase (Sce VMA) intein has been shown to catalyze protein splicing within the crystalline lattice (15).…”

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confidence: 99%

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Semisynthesis of a segmental isotopically labeled protein splicing precursor: NMR evidence for an unusual peptide bond at the N-extein–intein junction

Romanelli

Shekhtman

Cowburn

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

158

128

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Protein splicing is a posttranslational autocatalytic process in which an intervening sequence, termed an intein, is removed from a host protein, the extein. Although we have a reasonable picture of the basic chemical steps in protein splicing, our knowledge of how these are catalyzed and regulated is less well developed. In the current study, a combination of NMR spectroscopy and segmental isotopic labeling has been used to study the structure of an active protein splicing precursor, corresponding to an N-extein fusion of the Mxe GyrA intein. The 1 JNC coupling constant for the (؊1) scissile peptide bond at the N-extein-intein junction was found to be Ϸ12 Hz, which indicates that this amide is highly polarized, perhaps because of nonplanarity. Additional mutagenesis and NMR studies indicate that conserved box B histidine residue is essential for catalysis of the first step of splicing and for maintaining the (؊1) scissile bond in its unusual conformation. Overall, these studies support the ''ground-state destabilization'' model as part of the mechanism of catalysis.

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Section: Resultssupporting

confidence: 85%

Section: Discussionmentioning

confidence: 99%

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Semisynthesis of a segmental isotopically labeled protein splicing precursor: NMR evidence for an unusual peptide bond at the N-extein–intein junction

Romanelli

Shekhtman

Cowburn

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

158

128

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“…Previously known split inteins, naturally occurring or artificial, all have a similar split site corresponding to or inside the endonuclease domain. They have shown variability and limitations in solubility, reaction kinetics, and compatibility with certain extein sequences (20,(25)(26)(27)(28)30). New split sites identified in this study that produced intein fragments of various lengths and interacting ␤-strands can be useful for comparative studies of split intein properties such as fragment binding and reaction kinetics; they can also expand the toolbox of protein trans-splicing in biotechnology.…”

Section: Discussionmentioning

confidence: 89%

Synthetic Two-piece and Three-piece Split Inteins for Protein trans-Splicing

Sun

Yang

Liu

2004

Journal of Biological Chemistry

111

109

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Inteins are protein-intervening sequences that can self-excise and concomitantly splice together the flanking polypeptides. Two-piece split inteins capable of protein trans-splicing have been found in nature and engineered in laboratories, but they all have a similar split site corresponding to the endonuclease domain of the intein. Can inteins be split at other sites and do transsplicing? After testing 13 split sites engineered into a Ssp DnaB mini-intein, we report the finding of three new split sites that each produced a two-piece split intein capable of protein trans-splicing. These three functional split sites are located in different loop regions between ␤-strands of the intein structure, and one of them is just 11 amino acids from the beginning of the intein. Because different inteins have similar structures and similar ␤-strands, these new split sites may be generalized to other inteins. We have also demonstrated for the first time that a three-piece split intein could function in protein trans-splicing. These findings have implications for intein structure-function, evolution, and uses in biotechnology.An intein is a protein-intervening sequence that catalyzes a protein-splicing reaction in which the intein sequence is precisely excised and its flanking sequences (N-and C-exteins) join with a peptide bond to produce the mature host protein (spliced protein) (1). The mechanism of protein splicing typically has four steps: two acyl rearrangements at the two splicing junctions, a trans-esterification between the two junctions, and a cyclization of the Asn residue at the C-terminal junction (2-4). Crystal structures of inteins revealed a splicing domain consisting of 11-12 ␤-strands and forming a compact horseshoe shape with the splicing junctions located in the central cleft (5-11). A majority of inteins also have a homing endonuclease domain inserted in the splicing domain sequence (12). These bifunctional inteins are ϳ350 -550 amino acids (aa) 1 long, although some extra large inteins are up to 1650 aa long and also contain tandem repeats (13,14). Nearly 200 intein and inteinlike sequences have been found in a wide variety of host proteins and in microorganisms belonging to bacteria, Archaea, and eukaryotes (12,15). Their sporadic phylogenetic distributions suggest lateral gene transfer through intein homing (16,17). Inteins generally share only low levels of sequence similarity, but they share striking similarities in structure, reaction mechanism, and evolution (4,18,19,21). It is thought that inteins first originated with just the splicing domain and then acquired the endonuclease domain, with the latter conferring genetic mobility to the intein. During intein evolution, however, some inteins lost their endonuclease domain to become mini-inteins consisting of just the ϳ130-aa protein-splicing domain plus a linker sequence of various lengths in place of the endonuclease domain (12,22).An interesting event of intein evolution is the loss of sequence continuity in some inteins, which apparently produced ...

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“…This use of temperatureinducible splicing of inteins isolated from extreme thermophiles was also useful in the discovery of the chemical mechanism of splicing (6, 10 -13). Previously, Evans and coworkers (9,14,15) have described the kinetics of the trans-splicing of the intein that interrupts the DnaE protein of Synechocystis sp. PCC6803 (Ssp).…”

Section: Step 5)mentioning

confidence: 99%

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

Mills

Dorval²,

Lewandowski³

2005

Journal of Biological Chemistry

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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 noncanonical Gln at its C terminus, is 9.3 ؋ 10 ؊6 s ؊1 at 60°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 ؋ 10 ؊4 s ؊1 . The first order rate constant of C-terminal cleavage is 1.2 ؋ 10 ؊5 s ؊1 with Gln at the C-terminal position, 2.8 ؋ 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.Protein splicing involves the post-translational, self-directed excision of an intervening polypeptide, or intein, from flanking polypeptides, or exteins. The splicing process also involves the specific ligation of the extein segments by an amide bond, concomitant with the excision of the intein (1).The canonical mechanism of protein splicing consists of four separate but coordinated chemical steps each directed by the intein without the assistance of other proteins or cofactors (1). The first step of splicing is an N-S or N-O acyl rearrangement of the amide bond linking the N-extein and intein (see Fig. 1, step 1). This is followed by a transesterification that transfers the N-extein to the side chain of the N-terminal Cys, Ser, or Thr of the C-extein (see Fig. 1, step 2). The third step of splicing couples the cyclization of the C-terminal Asn of the intein to peptide bond cleavage, and excises the intein from the newly ligated exteins (see Fig. 1, step 3). Finally, the ester bond linking the exteins is rearranged to an amide (see Fig. 1, step 4), and the C-terminal aminosuccinimide of the intein is presumably hydrolyzed to either Asn or iso-Asn (see Fig. 1, step 5).Inteins that splice via variations of the canonical mechanism have previously been described. For instance, our group recently described the protein splicing of the intein that interrupts the DNA polymerase II DP2 subunit in Pyrococcus abyssi (Pab), 1 which is able to facilitate protein splicing with a Cterminal Gln residue (2). These results are complementary to those of Pietrokovski and coworkers (3), who demonstrated that the intein that interrupts the ribonucleotide reductase (RNR) of the Chilo iridescent virus can facilitate protein splicing with a C-terminal Gln and that the intein interrupting the RNR of Carboxydothermus hydrogenoformans can facilitate protein splicing w...

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Characterization of a Naturally Occurring Trans-Splicing Intein fromSynechocystissp. PCC6803

Cited by 126 publications

References 33 publications

Semisynthesis of a segmental isotopically labeled protein splicing precursor: NMR evidence for an unusual peptide bond at the N-extein–intein junction

Semisynthesis of a segmental isotopically labeled protein splicing precursor: NMR evidence for an unusual peptide bond at the N-extein–intein junction

Synthetic Two-piece and Three-piece Split Inteins for Protein trans-Splicing

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

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