Crystal Structure Analysis of Cationic Peroxidase from Proso Millet and Identification of Its Phosphatase Active Sites.

Abstract

Proso millet peroxidase (PmPOD) belongs to class III plant peroxidases, which are enzymes typically characterized by their heme coenzymes. PmPOD exhibits not only heme-dependent peroxidase activity but also heme-independent phosphatase activity. Crystal structure analysis and sequence alignment showed that PmPOD contained a phosphatase catalytic loop CXXXXXR in its β-domain that is similar to the active site of a dual-specific phosphatase. Recombinant truncated proso millet peroxidase (tPmPOD), which contained only a conserved catalytic loop CXXXXXR of phosphatase, was found to exhibit phosphatase activity. Five tPmPOD mutants containing five different mutations in the phosphatase active sites exhibited significantly lower phosphatase activity compared to that of tPmPOD, indicating that the five amino acids play important roles in the phosphatase activity of tPmPOD. Finally, nucleophilic amino acid Cys192 formed a disulfide bond with Cys219 to protect the stability of a sulfhydryl group; thus, it may play a decisive role in the phosphatase activity of PmPOD.