Identification of Asp218 and Asp326 as the principal Mg2+ binding ligands of the homing endonuclease PI-SceI.
The monomeric homing endonuclease PI-SceI harbors two catalytic centers which cooperate in the cleavage of the two strands of its extended recognition sequence. Structural and biochemical data suggest that catalytic center I contains Asp218, Asp229, and Lys403, while catalytic center II contains Asp326, Thr341, and Lys301. The analogy with I-CreI, for which the cocrystal structure with the DNA substrate has been determined, suggests that Asp218 and Asp229 in catalytic center I and Asp326 and Thr341 in catalytic center II serve as ligands for Mg(2+), the essential divalent metal ion cofactor which can be replaced by Mn(2+) in vitro. We have carried out a mutational analysis of these presumptive Mg(2+) ligands. The variants carrying an alanine or asparagine substitution bind DNA, but (with the exception of the D229N variant) are inactive in DNA cleavage in the presence of Mg(2+), demonstrating that these residues are important for cleavage. Our finding that the PI-SceI variants carrying single cysteine substitutions at these positions are inactive in the presence of the oxophilic Mg(2+) but active in the presence of the thiophilic Mn(2+) suggests that the amino acid residues at these positions are involved in cofactor binding. From the fact that in the presence of Mn(2+) the D218C and D326C variants are even more active than the wild-type enzyme, it is concluded that Asp218 and Asp326 are the principal Mg(2+) ligands of PI-SceI. On the basis of these findings and the available structural information, a model for the composition of the two Mg(2+) binding sites of PI-SceI is proposed.
Schöttler
S
S
Institut für Biochemie, FB 08, Justus-Liebig-Universität, Heinrich-Buff-Ring 58, D-35392 Giessen, Germany.
Wende
W
W
Pingoud
V
V
Pingoud
A
A
D019943:Amino Acid Substitution / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain / Q000235:genetics; D003432:Cross-Linking Reagents / Q000737:chemistry; D004271:DNA, Fungal / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis; D008024:Ligands; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism*; D008345:Manganese / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D010777:Photochemistry; D011485:Protein Binding / Q000235:genetics; D006180:Proton-Translocating ATPases; D029701:Saccharomyces cerevisiae Proteins
D003432:Cross-Linking Reagents; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D008024:Ligands; D009838:Oligodeoxyribonucleotides; D029701:Saccharomyces cerevisiae Proteins; D001224:Aspartic Acid; D008345:Manganese; D004706:Endodeoxyribonucleases; D006180:Proton-Translocating ATPases; C081687:VMA1 protein, S cerevisiae; D008274:Magnesium