Structural and functional analyses of a novel manganese-catalase from Bacillus subtilis R5

Catalases catalyze the decomposition of hydrogen peroxide into water and oxygen. Limited reports are available on characterization of manganese-catalases. We describe here molecular cloning and expression in Escherichia coli of a putative manganese-catalase gene from mesophilic bacterium, Bacillus subtilis R5. The gene product, Cat_Bsu, produced as a soluble protein, was purified to apparent homogeneity and biochemically characterized. The absorption spectra and nonsignificant inhibition by sodium azide indicated that it is a manganese-catalase. The protein was in homohexameric form in solution, with a subunit molecular weight of 30 kDa, containing ~2 Mn²⁺ and ~1 Ca²⁺ per subunit. Cat_Bsu showed highest activity at pH 8.0 and 55 °C. It was found to be highly active with a specific activity of 25,290 μmol min^-1 mg^-1 and apparent K_m and k_cat values of 98 mM and 1.27 × 10⁴ s^-1 subunit^-1, respectively. Although from a mesophilic source, it exhibited a half-life of 2 h at 80 °C. Furthermore, the active site and metal binding residues in Cat_Bsu were predicted by homology modelling and molecular docking. To the best of our knowledge, this is the first characterization of a manganese-catalase from genus Bacillus.