title: Characterization of the restriction enzyme-like endonuclease encoded by the Entamoeba histolytica non-long terminal repeat retrotransposon EhLINE1.
abstract: 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 x 10(-8) M and the k(cat) was 1.6 x 10(-2) sec(-1). For linear E. histolytica DNA substrate the K(m) and k(cat) values were 1.3 x 10(-8) M and 2.2 x 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.
authors.affiliation: School of Environmental Sciences, Jawaharlal Nehru University, New Delhi, India.
authors.forename: Vijay Pal
authors.initials: VP
authors.lastname: Yadav
authors.forename: Prabhat Kumar
authors.initials: PK
authors.lastname: Mandal
authors.forename: Desirazu N
authors.initials: DN
authors.lastname: Rao
authors.forename: Sudha
authors.initials: S
authors.lastname: Bhattacharya
mesh_terms: D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004262:DNA Restriction Enzymes / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D004748:Entamoeba histolytica / Q000201:enzymology* / Q000378:metabolism; D018503:Genome, Protozoan; D007700:Kinetics; D017434:Protein Structure, Tertiary; D018626:Retroelements / Q000235:genetics*; D013379:Substrate Specificity; D020079:Terminal Repeat Sequences
chemical_list: D018626:Retroelements; D004247:DNA; D004262:DNA Restriction Enzymes