P77: A single amino acid substitution improves the manganese peroxidase activity of DypB from the ligninolytic bacterium Rhodococcus jostii RHA1

The lignin-degrading Dyp-type peroxidase DypB from Rhodococcus jostii RHA1 catalyzes the peroxide-dependent oxidation of manganese (Mn²⁺), albeit 10⁴-fold less efficiently than fungal manganese peroxidase. Substitution of Asn246, a distal heme residue, with alanine, increased the enzyme’s apparent k_cat and k_cat/K_m values for Mn²⁺ by 80- and 15-fold, respectively. A 2.2 Å resolution X-ray crystal structure of the DypB:Mn²⁺ complex revealed the divalent cation to be bound in a pocket at the heme edge of N246A surrounded by acidic residues. However, the first coordination sphere was entirely comprised of solvent, consistent with the variant’s high K_m for Mn²⁺ (17 ± 2 mM). N246A catalyzed the manganese-dependent transformation of hard wood Kraft lignin and its solvent-extracted fractions. Gel permeation and high pressure liquid chromatography established that lower molecular weight fractions were transformed more efficiently than the higher molecular weight fractions. The N246A DypB variant is most efficient bacterial manganese peroxidase described to date. The structural data should facilitate further engineering of this system for biocatalytic applications.