Journal: Molecular Microbiology

Article Title: Mutants of phage bIL67 RuvC with enhanced Holliday junction binding selectivity and resolution symmetry

doi: 10.1111/mmi.12343

Figure Lengend Snippet: Comparison of phage and cellular RuvC resolving enzymes. A. Alignment of selected phage RuvC proteins. Structural elements of phage bIL67, Escherichia coli ( Eco ) and Thermus thermophilus ( Tth ) proteins are indicated above and below the aligned sequences (pink bars, α-helix; cyan arrows, β-sheet). Conserved residues are highlighted in red (acidic; active site), blue (basic), cyan (others) and orange (F69 in Ec RuvC and F73 in Tth RuvC). Every ten residues are labelled as filled grey circles above the bIL67 RuvC sequence. Filled magenta circles indicate the location of bIL67 RuvC S10A, T11A, K40A, R46A, S109A, K110A, K120A, R121A, R124A, K125A and R124A+K125A substitution mutants. Sequence conservation in E. coli and T. thermophilus RuvC is taken from the Pfam database entry, PF02075, highlighting the most highly conserved residues among bacterial RuvC family proteins. Conserved residues in phage RuvC are based on 24 orthologues using a similar approach. The percentage identity refers to homology of each protein with bIL67 RuvC. Green arrows indicate the positions of insertions to generate N- and C-terminal 67- Ec RuvC and Ec -67RuvC hybrids (see ). Accession numbers of selected lactococcal phage RuvC proteins from bIL67 (NP_042322), bIL66 (AAA99046), Q54 (YP_762587) and Streptococcus pyogenes inducible phage EJ-1 (NP_945263). B. Structural superimposition based upon their central β-sheets of monomers of 67RuvC (cyan) and Ec RuvC (dark magenta) shown in cartoon representation as in but with α-helices shown as cylinders. Helices and termini are labelled. C. Superimpostion of the catalytically critical residues at the active sites of 67RuvC (cyan), Ec RuvC (dark magenta) and Tth RuvC (olive green). Side-chains are shown in stick representation with the rest of the structure in cartoon form. The 67RuvC residue labelled D8 is an asparagine in the crystal structure shown. The Mg 2+ cation (green sphere) and solvent ligands (red spheres) from the 67RuvC structure are shown for reference. D. Alignment of dimers of 67RuvC and Ec RuvC based upon superimposition of the αB dimer interface helices. Structures are represented and coloured as in (B).

Article Snippet: An E. coli codon-optimized version ( ) of the Lactococcus lactis phage bIL67 ruvC ( ORF23 ) gene (Schouler et al ., ) was synthesized and inserted into the T7 expression vector pET24a(+) at NdeI and BamHI sites by GenScript to create p67RuvCwt.

Techniques: Comparison, Sequencing, Residue, Solvent