Michaelis-Menten parameters of Mca for <i>N</i>-deacetylation of GlcNAc and for amidase activity of MSmB.

a<p>Assays were performed using 10 µM enzyme and 0–10 mM MSmB in 50 mM HEPES (pH 7.5) at 37°C.</p>b<p>Assays were performed in the presence of 10 µM enzyme and 0–5.0 mM GlcNAc in 50 mM HEPES (pH 7.5) at 30°C.</p><p>Michaelis-Menten parameters of Mca for <i>N</i>-deacetylation of GlcNAc and for amidase activity of MSmB.</p

Sensitivity of <i>C</i>. <i>glutamicum</i> strains to alkylating agents tested by disk diffusion assay.

<p>mBBr, monobromobimane; IAM, iodoacetamide; NEM, <i>N</i>-ethylmaleimide; CDNB, 1-chloro-2,4-dinitrobenzene MG, methylglyoxal. *<i>P</i>≤0.05 or **<i>P</i>≤0.01 versus wild type for the mutants.</p>a<p>The values are mean±SD for three independent determinations.</p><p>Sensitivity of <i>C</i>. <i>glutamicum</i> strains to alkylating agents tested by disk diffusion assay.</p

Sensitivity of <i>C. glutamicum</i> strains to various classes of antibiotics tested by disk diffusion assay.

<p>**<i>P</i>≤0.01 versus wild type for the mutants.</p>a<p>The values are mean±SD for three independent determinations.</p><p>Sensitivity of <i>C. glutamicum</i> strains to various classes of antibiotics tested by disk diffusion assay.</p

Steady-state kinetic parameters of <i>C. glutamicum</i> Mca mutants for <i>N</i>-deacetylation of <i>N</i>-acetyl-D-glucosamine (GlcNAc).

<p>Assays were performed in the presence of 10 µM enzyme and 0–5.0 mM GlcNAc in 50 mM HEPES (pH 7.5) at 30°C.</p><p>Steady-state kinetic parameters of <i>C. glutamicum</i> Mca mutants for <i>N</i>-deacetylation of <i>N</i>-acetyl-D-glucosamine (GlcNAc).</p

Sensitivity of <i>C</i>. <i>glutamicum</i> strains to oxidizing and reducing agents tested by disk diffusion assay.

<p>**<i>P</i>≤0.01 versus wild type for the mutant.</p>a<p>The values are mean±SD for three independent determinations.</p><p>Sensitivity of <i>C</i>. <i>glutamicum</i> strains to oxidizing and reducing agents tested by disk diffusion assay.</p

Effects of divalent metal cations and pH on <i>C. glutamicum</i> Mca activity.

<p><b>A–E.</b> Catalytic activity of Mca in the presence of Co²⁺(A), Mn²⁺(B), Ni²⁺(C), Zn²⁺(D) and Fe²⁺(E), respectively, was analyzed with GlcNAc or MSmB as substrates. Apo-Mca was incubated with stoichiometric amounts of metal ions. After 30 min, the enzyme was diluted into assay buffer containing the substrate GlcNAc (5 mM) or MSmB (1 mM). The amidase activity (Left Y axis) and deacetylase activity (Right Y axis) were measured as described in “Materials and Methods”. <b>F.</b> Deacetylation of GlcNAc and amidase activity of MSmB by Zn²⁺-Mca at different pH levels. The <i>V/K</i> values were measured with 5 mM GlcNAc as substrate for deacetylase activity (Left Y axis) or 1 mM MSmB as substrate for amidase activity (Right Y axis) under six different pH values. <i>pK</i>_a values of 6.5 and 9.5 were determined by fitting <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115075#pone.0115075.e001" target="_blank">Equation 1</a> to the data (bars represent standard error of the mean).</p

The minimum inhibitory concentrations (MICs) of various antibiotics for <i>C</i>. <i>glutamicum</i> strains.

<p>**<i>P</i>≤0.01 versus wild type for the mutants.</p>a<p>The values are mean±SD for three independent determinations.</p><p>The minimum inhibitory concentrations (MICs) of various antibiotics for <i>C</i>. <i>glutamicum</i> strains.</p

Steady-state kinetic parameters of <i>C. glutamicum</i> Mca mutants for amidase activity of mycothiol bimane (MSmB).

<p>Assays were performed using 10 µM enzyme and 0–10 mM MSmB in 50 mM HEPES (pH 7.5) at 37°C.</p><p>Steady-state kinetic parameters of <i>C. glutamicum</i> Mca mutants for amidase activity of mycothiol bimane (MSmB).</p