Fig. 4: Enzyme assay of DslA, related enzymes, and general lysozymes.

Enzymes were incubated with acetylated peptidoglycan (GlcNAc, blue) and peptidoglycan partly deacetylated via treatment with Bdellovibrio Bd0468 and Bd3279 (GlcN, red). Fluorescence reading monitors the hydrolytic release of FITC-labeled peptidoglycan fragments into solution. Left grouping: comparison of specificity of DslA, hen egg-white lysozyme (HEWL), and Streptomyces globisporus mutanolysin. The non-specific mutanolysin hydrolyzes both acetylated- and deacetylated peptidoglycan equally, but HEWL is specific for the acetylated form (with residual activity on GlcN sample indicative of mixed material, partial N-deacetylation¹⁵). DslA displays strong activity for the deacetylated GlcN sample and is unable to turnover acetylated GlcNAc peptidoglycan. Middle grouping: relative low activity of DslA putative catalytic E to Q mutants, and reduced activity for Y228A variant. Right grouping: homologous DslB and DslC proteins display a similar preference for a deacetylated substrate. Significance analysis uses two-tailed t-test, ***p < 0.0005, *p < 0.05. n = 12 independent samples for HEWL, Mutanolysin, and DlsA experiments. n = 3 independent samples for DlsA mutants and homologs (DlsB and DlsC) experiments. Data are represented as the mean ± standard deviation.