PspGI, a Type II Restriction Endonuclease from the Extreme Thermophile Pyrococcus sp.: Structural and Functional Studies to Investigate an Evolutionary Relationship with Several Mesophilic Restriction Enzymes

Pingoud, Vera; Conzelmann, Charlotte; Kinzebach, Steffen; Sudina, Anna; Metelev, Valeri; Кубарева, Е. А.; Bujnicki, Janusz M.; Lurz, Rudi; Lüder, Gerhild; Xu, Shuang-yong; Pingoud, Alfred

doi:10.1016/s0022-2836(03)00523-0

Cited by 50 publications

(67 citation statements)

References 61 publications

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“…However, with few exceptions concerning isoschizomers, restriction enzymes do not show sufficient sequence similarities to infer phylogenetic relationships without additional structural and/or functional information. Based on sequence alignments, we previously proposed that SsoII, PspGI, and EcoRII belong to the EcoRI branch and are structurally related to Bse634I, Cfr10I, and NgoMIV (23,25). This hypothesis was supported by a detailed experimental analysis and homology modeling, which demonstrated amino acid residues of SsoII and PspGI that align with functionally important residues in Bse634I, Cfr10I, and NgoMIV to be indeed essential for DNA binding and catalysis by SsoII and PspGI.…”

Section: Fig 3 Gel Electrophoretic Mobility Shift Experiments To Commentioning

confidence: 89%

“…The involvement of this region in DNA binding and cleavage by Cfr10I and EcoRII is also apparent from crystal structures (32,33). For SsoII and PspGI, mutational analysis and protein-DNA cross-linking determined this region to be likewise important (23,25).…”

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confidence: 88%

“…This has been interpreted as the enzymes having diverged early in evolution, presumably from a Type IIP enzyme that recognized 2CCXGG or X2CCGGX. It is intriguing to note that the sequence similarity over an interrupted stretch of ϳ70 amino acid residues can be extended to enzymes such as MboI (2GATC) that recognize unrelated sequences (25). Showing that MboI uses secondary structure elements composed of these ϳ70 amino acid residues for DNA binding and cleavage would be the first example of an evolutionary relationship between two subfamilies of restriction endonucleases with widely differing specificities within the EcoRI branch.…”

Section: Fig 3 Gel Electrophoretic Mobility Shift Experiments To Commentioning

confidence: 99%

“…That these five enzymes (and their close relatives, see above) belong to different families (Type IIP, Type IIE, and Type IIF) is interpreted as their having a common ancestor, presumably a homodimeric enzyme that already had the variant active site and that acquisition of an effector domain (Type IIE) or generation of a dimer-dimer interface (Type IIF) is a more recent event in evolution (23). Although this evolutionary relationship is well supported by a previous structure-function relationship study of SsoII and PspGI, the suggestion that other Type II restriction enzymes that recognize unrelated sequences, such as 2GATC, G2GYRCC, and GG2CC (25), are evolutionarily related to SsoII, PspGI, EcoRII, NgoMIV, and Cfr10I has not yet been explored experimentally.…”

mentioning

confidence: 98%

“…Cfr10I (R2CCGGY) (23)(24)(25). It is noteworthy that this group of related enzymes and their close relatives (SsoII: Kpn2kI/ Ecl18kI/StyD4I/SenPI; Cfr10I: Bse634I/BsrFI) comprises: (i) Type IIP enzymes such as SsoII and PspGI, homodimeric enzymes that recognize palindromic sequences; (ii) Type IIE enzymes such as EcoRII, homodimeric enzymes that need two recognition sequences for DNA cleavage, one sequence being cleaved and the other serving as an effector (26 -29); and (iii) Type IIF enzymes such as NgoMIV and Cfr10I, homotetrameric enzymes that cleave, in a concerted manner, DNA with two recognition sites (26,27,29,30).…”

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confidence: 99%

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Specificity Changes in the Evolution of Type II Restriction Endonucleases

Pingoud

Sudina

Geyer

et al. 2005

Journal of Biological Chemistry

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How restriction enzymes with their different specificities and mode of cleavage evolved has been a long standing question in evolutionary biology. We have recently shown that several Type II restriction endonucleases, namely SsoII (2CCNGG), PspGI (2CCWGG), Eco-RII (2CCWGG), NgoMIV (G2CCGGC), and Cfr10I (R2CCGGY), which recognize similar DNA sequences (as indicated, where the downward arrows denote cleavage position), share limited sequence similarity over an interrupted stretch of ϳ70 amino acid residues with MboI, a Type II restriction endonuclease from Moraxella bovis (Pingoud, V., Conzelmann, C., Kinzebach, S., Sudina, A., Metelev, V., Kubareva, E., Bujnicki, J. M., Lurz, R., Luder, G., Xu, S. Y., and Pingoud, A. (2003) J. Mol. Biol. 329, 913-929). Nevertheless, MboI has a dissimilar DNA specificity (2GATC) compared with these enzymes. In this study, we characterize MboI in detail to determine whether it utilizes a mechanism of DNA recognition similar to SsoII, PspGI, EcoRII, NgoMIV, and Cfr10I. Mutational analyses and photocross-linking experiments demonstrate that MboI exploits the stretch of ϳ70 amino acids for DNA recognition and cleavage. It is therefore likely that MboI shares a common evolutionary origin with SsoII, PspGI, EcoRII, NgoMIV, and Cfr10I. This is the first example of a relatively close evolutionary link between Type II restriction enzymes of widely different specificities.

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Section: Fig 3 Gel Electrophoretic Mobility Shift Experiments To Commentioning

confidence: 89%

mentioning

confidence: 88%

Section: Fig 3 Gel Electrophoretic Mobility Shift Experiments To Commentioning

confidence: 99%

mentioning

confidence: 98%

mentioning

confidence: 99%

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Specificity Changes in the Evolution of Type II Restriction Endonucleases

Pingoud

Sudina

Geyer

et al. 2005

Journal of Biological Chemistry

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Restriction Endonuclease and DNA-Modification Methyltransferases

Jeltsch

Gumport

2006

Encyclopedia of Molecular Cell Biology and Molecular Medicine

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Theoretical model of restriction endonuclease HpaI in complex with DNA, predicted by fold recognition and validated by site‐directed mutagenesis

2006

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Type II restriction enzymes are commercially important deoxyribonucleases and very attractive targets for protein engineering of new specificities. At the same time they are a very challenging test bed for protein structure prediction methods. Typically, enzymes that recognize different sequences show little or no amino acid sequence similarity to each other and to other proteins. Based on crystallographic analyses that revealed the same PD-(D/E)XK fold for more than a dozen case studies, they were nevertheless considered to be related until the combination of bioinformatics and mutational analyses has demonstrated that some of these proteins belong to other, unrelated folds PLD, HNH, and GIY-YIG. As a part of a large-scale project aiming at identification of a three-dimensional fold for all type II REases with known sequences (currently approximately 1000 proteins), we carried out preliminary structure prediction and selected candidates for experimental validation. Here, we present the analysis of HpaI REase, an ORFan with no detectable homologs, for which we detected a structural template by protein fold recognition, constructed a model using the FRankenstein monster approach and identified a number of residues important for the DNA binding and catalysis. These predictions were confirmed by site-directed mutagenesis and in vitro analysis of the mutant proteins. The experimentally validated model of HpaI will serve as a low-resolution structural platform for evolutionary considerations in the subgroup of blunt-cutting REases with different specificities. The research protocol developed in the course of this work represents a streamlined version of the previously used techniques and can be used in a high-throughput fashion to build and validate models for other enzymes, especially ORFans that exhibit no sequence similarity to any other protein in the database.

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PspGI, a Type II Restriction Endonuclease from the Extreme Thermophile Pyrococcus sp.: Structural and Functional Studies to Investigate an Evolutionary Relationship with Several Mesophilic Restriction Enzymes

Cited by 50 publications

References 61 publications

Specificity Changes in the Evolution of Type II Restriction Endonucleases

Specificity Changes in the Evolution of Type II Restriction Endonucleases

Restriction Endonuclease and DNA-Modification Methyltransferases

Theoretical model of restriction endonuclease HpaI in complex with DNA, predicted by fold recognition and validated by site‐directed mutagenesis

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