FIGURE 3.

Designing a gain-of-function lysin (amidase) catalytic domain, XlyA+5K^CAT. A, bactericidal activities of wild-type lysins and a chimera (XlyA^CAT:PlyL^CBD) against B. cereus (Bcer) or B. subtilis (Bsub). B, sequence alignment and secondary structure assignments of XlyA^CAT and PlyL^CAT. Residues involved in catalysis and substrate recognition are boxed in red. Residue pairs in blue boxes indicate sites mutated to lysine in XlyA^CAT to create XlyA^CAT+5K. Below the sequences, asterisks indicate identity; and colons indicate close similarity. C, stereo Cα overlay of XlyA^CAT (pink), Xly A+ 5K^CAT (green), and PlyL^CAT (cyan). XlyA mutated residues are labeled, and their side chains shown as sticks. Orientation is the same as in Fig. 2B. D, electrostatic surfaces of wild-type XlyA^CAT and XlyA+5K^CAT, viewed as in A, showing that mutations do not affect the active site face. E, bactericidal activities (against B. subtilis) of wild-type and mutant XlyA catalytic domains, compared with full-length enzyme. XlyA+4K^CAT is similar to XlyA+5K^CAT but has one less residue changed to lysine. F, pulldown (PD) binding assays (Western blots using anti-His tag antibody) using whole cells (B. cereus, B. subtilis, or B. anthracis Sterne) and catalytically inactivated His-tagged mutants of PlyL^CAT, XlyA^CAT, and XlyA5K^CAT. LC = loading control. The input protein concentration was 1 μm or (for B. anthracis) 10 μm.