Crystal structure and initial characterization of a novel archaeal-like Holliday junction-resolving enzyme from Thermus thermophilus phage Tth15-6

This study describes the production, characterization and structure determination of a novel Holliday junction-resolving enzyme. The enzyme, termed Hjc_15-6, is encoded in the genome of phage TthlS-6, which infects Thermus thermophilus. Hjc_15-6 was heterologously produced in Escherichia colt and high yields of soluble and biologically active recombinant enzyme were obtained in both complex and defined media Amino-acid sequence and structure comparison suggested that the enzyme belongs to a group of enzymes classified as archaeal Holliday junction-resolving enzymes, which are typically divalent metal ion-binding dimers that are able to cleave X-shaped dsDNA-Holliday junctions (Hjs). The crystal structure of Hjc_15-6 was determined to 2.5 angstrom resolution using the selenomethionine single-wavelength anomalous dispersion method. To our knowledge, this is the first crystal structure of an Hj-resolving enzyme originating from a bacteriophage that can be classified as an archaeal type of Hj-resolving enzyme. As such, it represents a new fold for Hj-resolving enzymes from phages. Characterization of the structure of Hjc_15-6 suggests that it may form a dimer, or even a homodimer of dimers, and activity studies show endonuclease activity towards Hjs. Furthermore, based on sequence analysis it is proposed that Hjc_15-6 has a three-part catalytic motif corresponding to E-SD-EVK, and this motif may be common among other Hj-resolving enzymes originating from thermophilic bacteriophages.

Automated summary

This study describes the production, characterization, and structure determination of a novel Holliday junction-resolving enzyme called Hjc_15-6. The enzyme belongs to a group of archaeal enzymes and is capable of cleaving X-shaped dsDNA-Holliday junctions. The crystal structure of Hjc_15-6 was determined, and it represents a new fold for Holliday junction-resolving enzymes. It shows endonuclease activity towards Hjs and may form a dimer or a homodimer of dimers.