Crystal Structure of an Archaeal Intein-encoded Homing Endonuclease PI-PfuI

Ichiyanagi, Kenji; Ishino, Yuji; Ariyoshi, Mariko; Komori, Keishi; Morikawa, Kosuke

doi:10.1006/jmbi.2000.3873

Cited by 109 publications

(91 citation statements)

References 40 publications

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“…This inference is in agreement with some of the members of LAGLIDADG-type HEases such as PI-SceI 28,38,39 and PI-PfuI. 37,40 Several lines of evidence suggest that the conformational changes in the protein and/or DNA often accompany protein-DNA interaction. 41 Fluorescent base analogs, such as 2-AP, are often used to monitor such conformational changes involving proteinnucleic acid interaction.…”

Section: Dna-binding Analysis Of Pi-mlei and Its Variants By Electropsupporting

confidence: 81%

“…27 The absence of catalytic lysine in PI-MleI as judged from sequence alignments has also been seen earlier in other LAGLIDADG enzymes such as PI-PfuI, wherein its absence in one of the catalytic centre has been linked with the differential cutting activity of the enzyme on two strands of the substrate DNA. 40 The LAGLIDADG-type HEases have been the subject of intense study over the past 2 decades. A combination of genetic, biochemical, structural, and bioinformatics approaches have provided insights into the conserved DOD motifs and the importance of the acidic amino acid residues in HEase activity.…”

Section: Dna-binding Analysis Of Pi-mlei and Its Variants By Electropmentioning

confidence: 99%

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Mutational analysis of active‐site residues in the Mycobacterium leprae RecA intein, a LAGLIDADG homing endonuclease: Asp¹²² and Asp¹⁹³ are crucial to the double‐stranded DNA cleavage activity whereas Asp²¹⁸ is not

2009

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Mycobacterium leprae recA harbors an in-frame insertion sequence that encodes an intein homing endonuclease (PI-MleI). Most inteins (intein endonucleases) possess two conserved LAGLIDADG (DOD) motifs at their active center. A common feature of LAGLIDADG-type homing endonucleases is that they recognize and cleave the same or very similar DNA sequences. However, PI-MleI is distinctive from other members of the family of LAGLIDADG-type HEases for its modular structure with functionally separable domains for DNA-binding and cleavage, each with distinct sequence preferences. Sequence alignment analyses of PI-MleI revealed three putative LAGLIDADG motifs; however, there is conflicting bioinformatics data in regard to their identity and specific location within the intein polypeptide. To resolve this conflict and to determine the activesite residues essential for DNA target site recognition and double-stranded DNA cleavage, we performed site-directed mutagenesis of presumptive catalytic residues in the LAGLIDADG motifs. Analysis of target DNA recognition and kinetic parameters of the wild-type PI-MleI and its variants disclosed that the two amino acid residues, Asp 122 (in Block C) and Asp 193 (in functional Block E), are crucial to the double-stranded DNA endonuclease activity, whereas Asp 218 (in pseudo-Block E) is not. However, despite the reduced catalytic activity, the PI-MleI variants, like the wild-type PIMleI, generated a footprint of the same length around the insertion site. The D122T variant showed significantly reduced catalytic activity, and D122A and D193A mutations although failed to affect their DNA-binding affinities, but abolished the double-stranded DNA cleavage activity. On the other hand, D122C variant showed approximately twofold higher double-stranded DNA cleavage activity, compared with the wild-type PI-MleI. These results provide compelling evidence that Asp 122 and Additional Supporting Information may be found in the online version of this article.Abbreviations: 2-AP, 2 aminopurine; bp, base pair; BPB, bromophenol blue; EDTA, ethylene diamine tetraacetic acid; form I DNA, negatively supercoiled DNA; form II DNA, nicked circular DNA; form III DNA, linear double-stranded DNA; ODN, oligonucleotide; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; PI-MleI, M. leprae RecA intein; SDS, sodium dodecyl sulfate. Asp 193 in DOD motif I and II, respectively, are bona fide active-site residues essential for DNA cleavage activity. The implications of these results are discussed in this report.

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Section: Dna-binding Analysis Of Pi-mlei and Its Variants By Electropsupporting

confidence: 81%

Section: Dna-binding Analysis Of Pi-mlei and Its Variants By Electropmentioning

confidence: 99%

Mutational analysis of active‐site residues in the Mycobacterium leprae RecA intein, a LAGLIDADG homing endonuclease: Asp¹²² and Asp¹⁹³ are crucial to the double‐stranded DNA cleavage activity whereas Asp²¹⁸ is not

2009

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“…Superposition of the available crystal structures indicates that inteins share overall structural similarity. The root mean square difference for the C␣ atoms of the Hint domain is ϳ1.4 Å between the Ssp DnaB and Sce VMA inteins (21-24), 1.2 Å between the Ssp DnaB and Mxe GyrA inteins (26), and 1.2 Å between the Ssp DnaB and P. furiosus Pol inteins (25).…”

Section: Resultsmentioning

confidence: 99%

“…Blocks A, N2, B, and N4 (gray) in the N-terminal subdomain (white) and blocks F and G (orange) in C-terminal subdomain (yellow) are shared by the splicing domains; blocks C, D, E, and H are found within the endonuclease domain (green). Residues involved in The x-ray structures of the intein of the vacuolar ATPase subunit from Saccharomyces cerevisiae (Sce VMA intein) (21)(22)(23)(24), the archaeal PI-PfuI intein from Pyrococcus furiosus (PIPfuI intein) (25), and the mini-intein of the GyrA protein from Mycobacterium xenopi (Mxe GyrA intein) (26) have revealed that inteins contain a horseshoe-like ␤-strand scaffold termed the Hint (Hedgehog, intein) module (11). Mutation of catalytic residues at the N-and C-terminal splice junctions of the Sce VMA inteins resulted in the trapping of the inactive precursor proteins for crystallization studies (22,23).…”

Section: Figmentioning

confidence: 99%

Crystal Structure of a Mini-intein Reveals a Conserved Catalytic Module Involved in Side Chain Cyclization of Asparagine during Protein Splicing

Ding

Ghosh

et al. 2003

Journal of Biological Chemistry

134

186

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We have determined the crystal structure of a 154-residue intein derived from the dnaB gene of Synechocystis sp. strain PCC6803 and refined it to a 2.0-Å resolution. The x-ray structure suggests that this intein possesses two catalytic sites that appear to be separately responsible for splicing and cleavage of the Nand C-terminal scissile bonds. The conserved intein block F residues are the important components of a catalytic site for side chain cyclization of the last intein residue, Asn-154. The data suggest that the imidazole ring of His-143 is involved in the activation of the side chain N␦ atom of Asn-154, leading to a nucleophilic attack on the carbonyl carbon of Asn-154. Substitution of His-143 with Ala or Gln resulted in the inhibition of C-terminal cleavage. His-153, Asp-136, and a water molecule appear to constitute an oxyanion binding site by contacting the carbonyl oxygen of Asn-154 to stabilize the transition state. The structure and mutagenesis data also support that the close contact between the hydroxyl groups of Thr-138 and Ser-155, whose side chain participates in an S 3 O acyl shift, plays an important role in the nucleophile orientation. Our structural modeling suggests that this catalytic module is conserved in the C-terminal subdomains of inteins from diverse organisms.Protein splicing is a posttranslational processing event that involves the precise removal of an intervening sequence, an intein, from a protein precursor with concomitant ligation of the flanking protein sequences (N and C exteins) via a native peptide bond (1-3). In vitro splicing of inteins in heterologous proteins suggests that inteins with the first C-extein residue contain sufficient information needed for the autocatalytic splicing process without the requirement of exogenous energy or protein co-factor (4, 5).Among the more than 130 inteins identified so far, the majority contain two discrete functional domains, the homing endonuclease domain and the splicing domain (Fig. 1A) (InBase, the New England Biolabs Intein Data Base (6)). The protein splicing function of the intein does not depend on their homing endonuclease activity since splicing-proficient minimal inteins (mini-inteins) occur naturally (7-9) and have been generated by deleting the centrally located endonuclease domain. The splicing domain of a dual function intein appears to be split by the endonuclease domain into two segments, the N-and C-terminal subdomains. The N-terminal subdomain (in the range of 100 -150 residues) contains the conserved intein blocks A, B, N2, and N4, whereas the C-terminal subdomain of ϳ35-50 residues contains blocks F and G (6, 10). The N-terminal subdomain of inteins not only exhibits sequence homology with the hedgehog proteins of eucaryotes but also shares an acyl rearrangement mechanism of breaking the peptide bond preceding a nucleophile-containing residue (11). This raises the question of whether inteins share a common ancestor with other autoprocessing proteins and have evolved by acquiring a new catalytic capacity imbedde...

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“…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)(3)(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)(6)(7)(8)(9)(10)(11). A majority of inteins also have a homing endonuclease domain inserted in the splicing domain sequence (12).…”

mentioning

confidence: 99%

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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Crystal Structure of an Archaeal Intein-encoded Homing Endonuclease PI-PfuI

Cited by 109 publications

References 40 publications

Mutational analysis of active‐site residues in the Mycobacterium leprae RecA intein, a LAGLIDADG homing endonuclease: Asp¹²² and Asp¹⁹³ are crucial to the double‐stranded DNA cleavage activity whereas Asp²¹⁸ is not

Mutational analysis of active‐site residues in the Mycobacterium leprae RecA intein, a LAGLIDADG homing endonuclease: Asp¹²² and Asp¹⁹³ are crucial to the double‐stranded DNA cleavage activity whereas Asp²¹⁸ is not

Crystal Structure of a Mini-intein Reveals a Conserved Catalytic Module Involved in Side Chain Cyclization of Asparagine during Protein Splicing

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

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Crystal Structure of an Archaeal Intein-encoded Homing Endonuclease PI-PfuI

Cited by 109 publications

References 40 publications

Mutational analysis of active‐site residues in the Mycobacterium leprae RecA intein, a LAGLIDADG homing endonuclease: Asp122 and Asp193 are crucial to the double‐stranded DNA cleavage activity whereas Asp218 is not

Mutational analysis of active‐site residues in the Mycobacterium leprae RecA intein, a LAGLIDADG homing endonuclease: Asp122 and Asp193 are crucial to the double‐stranded DNA cleavage activity whereas Asp218 is not

Crystal Structure of a Mini-intein Reveals a Conserved Catalytic Module Involved in Side Chain Cyclization of Asparagine during Protein Splicing

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

Contact Info

Product

Resources

About

Mutational analysis of active‐site residues in the Mycobacterium leprae RecA intein, a LAGLIDADG homing endonuclease: Asp¹²² and Asp¹⁹³ are crucial to the double‐stranded DNA cleavage activity whereas Asp²¹⁸ is not

Mutational analysis of active‐site residues in the Mycobacterium leprae RecA intein, a LAGLIDADG homing endonuclease: Asp¹²² and Asp¹⁹³ are crucial to the double‐stranded DNA cleavage activity whereas Asp²¹⁸ is not