Dominant form of cationic peroxidase from sorghum roots

Abstract

A dominant form of cationic peroxidase (PO-2) was isolated from sorghum (Sorghum bicolor L. Moench) roots and purified to electrophoretically homogeneous state. The enzyme is a monomer with mol wt of 49.7 kD. The optimum pH and the main catalytic constants (K_M, V_max, k_cat) were determined for oxidation of the main substrates including Н₂О₂, 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonate) (ABTS), 2,7-diaminofluorene, syringaldazine, 2,6-dimethoxyphenol, and o-dianisidine. The K_M values increased in the sequence: H₂O₂ < 2,7-diaminofluorene < ABTS < o-dianisidine, whereas the maximum turnover number (93.9 s^–1) was found for 2,7-diaminofluorene. Based on the analysis of molecular and catalytic properties of the enzyme, it was proven that PO-2 is a typical cationic plant peroxidase. Polycyclic aromatic hydrocarbons (phenanthrene, anthracene, fluorene), 2,2'-diphenic acid, and Ni ions had no significant influence on the activity of PO-2. The enzyme was inhibited by p-aminobenzoic acid, NaN₃, 1-naphthol, 9,10-anthraquinone, and 9,10-phenanthrenequinone. In the presence of NaN₃, 1-naphthol, and 9,10-phenanthrenequinone, a mixed competitive/noncompetitive type of inhibition was noted. The peroxidase PO-2 was found to oxidize synthetic anthraquinone dyes, phenanthrene, and some oxygenated derivatives of polycyclic aromatic hydrocarbons (9-phenanthrol; 1-naphthol; and 1-hydroxy-2-naphthoic, salicylic, and 2,2'-diphenic acids), which indirectly confirms the coupled plant–microbial metabolism of these compounds in the root zone of sorghum. The results indicate that 9,10-phenanthrenequinone and 2,2'-diphenic acid are the products of peroxidase-catalyzed oxidation of 9-phenanthrol.