Structure, flexibility, and mechanism of the Bacillus stearothermophilus RecU Holliday junction resolvase.
Here we report a high resolution structure of RecU-Holliday junction resolvase from Bacillus stearothermophilus. The functional unit of RecU is a homodimer that contains a "mushroom" like structure with a rigid cap and two highly flexible loops extending outwards. These loops appear to be highly flexible/dynamic, and presumably are directly involved in DNA binding and holding it for catalysis. Structural modifications of both the protein and DNA upon their interaction are essential for catalysis. An Mg2+ ion is present in each of the two active sites in this homodimeric enzyme, and two water molecules are coordinated with each Mg2+ ion. Our data are consistent with one of these water molecules acting as a nucleophile and the other as a general acid. The identities of the general base and general acid involved in catalysis and the Lewis acid that stabilizes the pentacovalent transition state phosphate ion are proposed. A model for the RecU-Holliday junction DNA complex is also proposed and discussed in the context of DNA binding and cleavage.
Kelly
Stephen J
SJ
Children's Hospital Oakland Research Institute, Oakland, CA 94609, USA.
Li
Jie
J
Setlow
Peter
P
Jedrzejas
Mark J
MJ
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D045565:Holliday Junction Resolvases / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014961:X-Ray Diffraction
D001426:Bacterial Proteins; D045565:Holliday Junction Resolvases