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Characterization of the restriction enzyme‐like endonuclease encoded by the Entamoeba histolytica non‐long terminal repeat retrotransposon EhLINE1
Author(s) -
Yadav Vijay Pal,
Mandal Prabhat Kumar,
Rao Desirazu N,
Bhattacharya Sudha
Publication year - 2009
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2009.07419.x
Subject(s) - endonuclease , biology , restriction enzyme , exonuclease , dna , microbiology and biotechnology , enzyme , entamoeba histolytica , biochemistry , deoxyribonuclease i , reverse transcriptase , retrotransposon , genetics , gene , genome , polymerase , polymerase chain reaction , transposable element , base sequence
The genome of the human pathogen Entamoeba histolytica , a primitive protist, contains non‐long terminal repeat retrotransposable elements called EhLINEs. These encode reverse transcriptase and endonuclease required for retrotransposition. The endonuclease shows sequence similarity with bacterial restriction endonucleases. Here we report the salient enzymatic features of one such endonuclease. The kinetics of an EhLINE1‐encoded endonuclease catalyzed reaction, determined under steady‐state and single‐turnover conditions, revealed a significant burst phase followed by a slower steady‐state phase, indicating that release of product could be the slower step in this reaction. For circular supercoiled DNA the K m was 2.6 × 10 −8 m and the k cat was 1.6 × 10 −2 sec −1 . For linear E. histolytica DNA substrate the K m and k cat values were 1.3 × 10 −8 m and 2.2 × 10 −4 sec −1 respectively. Single‐turnover reaction kinetics suggested a noncooperative mode of hydrolysis. The enzyme behaved as a monomer. While Mg 2+ was required for activity, 60% activity was seen with Mn 2+ and none with other divalent metal ions. Substitution of PDX 12‐14 D (a metal‐binding motif) with PAX 12‐14 D caused local conformational change in the protein tertiary structure, which could contribute to reduced enzyme activity in the mutated protein. The protein underwent conformational change upon the addition of DNA, which is consistent with the known behavior of restriction endonucleases. The similarities with bacterial restriction endonucleases suggest that the EhLINE1‐encoded endonuclease was possibly acquired from bacteria through horizontal gene transfer. The loss of strict sequence specificity for nicking may have been subsequently selected to facilitate spread of the retrotransposon to intergenic regions of the E. histolytica genome.