Cleavage experiments with deoxythymidine 3′,5′-bis-(p-nitrophenyl phosphate) suggest that the homing endonuclease I-PpoI follows the same mechanism of phosphodiester bond hydrolysis as the non-specific Serratia nuclease1Dedicated to Prof. Dr. Günter Maass on the occasion of his 65th birthday.1

Institut für Biochemie (FB 15), Justus-Liebig-Universität, Giessen, Germany.
FEBS Letters (Impact Factor: 3.17). 02/1999; 443(2):209-14. DOI: 10.1016/S0014-5793(98)01660-3
Source: PubMed

ABSTRACT We show here that two nucleases, Serratia nuclease and I-PpoI, with contrasting specificities, i.e. non-specific vs. highly sequence specific, share a structurally similar active site region with conservation of the catalytically relevant histidine and asparagine residues. On the basis of a comparison of the available structures and biochemical data for wild type and mutant variants of Serratia nuclease and I-PpoI we propose that both enzymes have a common catalytic mechanism, a proposition that is supported by our finding that both enzymes accept deoxythymidine 3',5'-bis-(p-nitrophenyl phosphate) as a substrate and cleave it in an identical manner. According to this mechanism a histidine residue functions as a general base and Mg2+ bound to an asparagine residue as a Lewis acid in phosphodiester bond cleavage.

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Available from: Kurt L. Krause, Feb 19, 2014
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    • "The main difference in the action of the nucleases analyzed has been found in transition state stabilization. For instance, Arg57 has been supposed to be required for neutralization of the extra negative charge of the transition state in SmNase (Friedhoff et al., 1999), while non-conserved Asp95 plays this role in Anasp_NucA (Meiss et al., 2000). Moreover, In the case of endoG Cys115 located in the position corresponding to Arg57 seems to be tot well suited for transition state stabilization (Schafer et al., 2004). "
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    ABSTRACT: Kamchatka crab duplex-specific nuclease (Par_DSN) has been classified as a member of the family of DNA/RNA non-specific beta-beta-alpha metal finger (bba-Me-finger) nucleases, the archetype of which is the nuclease from Serratia marcescens. Although the enzyme under investigation seems to belong to the family of S. marcescens nucleases, Par_DSN exhibits a marked preference for double-stranded DNA as a substrate and this property is unusual for other members of this family. We have searched other Arthropod species and identified a number of novel Par_DSN homologs. A phylogenetic analysis demonstrates that the Par_DSN-like enzymes constitute a separate branch in the evolutionary tree of bba-Me-finger nucleases. Combining sequence analysis and site-directed mutagenesis, we found that Par_DSN and its homologs possess the nuclease domain that is slightly longer than that of classic Serratia relatives. The active site composition of Par_DSN is similar but not identical to that of classic Serratia nucleases. Based on these findings, we proposed a new classification of Par_DSN-like nucleases.
    Gene 08/2008; 418(1-2):41-8. DOI:10.1016/j.gene.2008.04.005 · 2.14 Impact Factor
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    • "What is the general acid in Vvn that protonates the leaving 3¢- oxygen group? The magnesium ion in I-PpoI has been shown biochemically (Friedhoff et al., 1999a; Mannino et al., 1999) and structurally (Galburt et al., 1999) to Fig. 6. The superimposition of the endonuclease active sites of Vvn with thèbba±metal' fold found in several endonucleases. "
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    The EMBO Journal 09/2003; 22(15):4014-25. DOI:10.1093/emboj/cdg377 · 10.43 Impact Factor
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    • "These results suggest that H-N-H colicin proteins are zinc-dependent enzymes in vivo, and that zinc plays a structural role in stabilizing the enzyme (Pommer et al. 1999). However, structural comparison (Friedhoff et al. 1999b; Miller et al. 1999; Grishin 2001) has revealed that the H-N-H motif has a ␤␤␣-Metal fold similar to the active sites of several other nucleases, including the homing endonuclease I-PpoI, Serratia nuclease, and Phage T4 Endonuclease VII (Friedhoff et al. 1999a; Kuhlmann et al. 1999; Raaijmakers et al. 1999). The divalent metal ions in these enzymes have been suggested to play catalytic roles (Lunin et al. 1997; Galburt et al. 1999; Miller et al. 1999), implying that the zinc ion in the H-N-H motif could be involved in DNA hydrolysis. "
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    Protein Science 01/2003; 11(12):2947-57. DOI:10.1110/ps.0220602 · 2.85 Impact Factor
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