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Updated: Nov 4, 2025
Identification of Post-translational Modifications of Plant Protein Complexes
Published on: February 22, 2014
Crystal Structure Analysis of Cationic Peroxidase from Proso Millet and Identification of Its Phosphatase Active
Xiaodong Cui1, Ke Wang1, Tingfen Wang1
1Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Institute of Biotechnology, Shanxi University, Taiyuan030006, China.
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.
Insights
Proso millet peroxidase (PmPOD) shows dual activity, acting as both a heme-dependent peroxidase and a heme-independent phosphatase. Key amino acids, particularly Cys192, are crucial for its phosphatase function and enzyme stability.
Area of Science:
- Biochemistry
- Enzymology
- Plant Science
Background:
- Class III plant peroxidases are heme-containing enzymes.
- Proso millet peroxidase (PmPOD) exhibits unusual dual enzymatic activity.
Purpose of the Study:
- To investigate the structural basis of PmPOD's dual activity.
- To identify key residues responsible for its phosphatase function.
Main Methods:
- Crystal structure analysis and sequence alignment of PmPOD.
- Expression and activity assays of recombinant truncated PmPOD (tPmPOD) and its mutants.
Main Results:
- PmPOD possesses a conserved phosphatase catalytic loop (CXXXXXR) in its β-domain.
- tPmPOD demonstrated significant phosphatase activity.
- Mutagenesis studies revealed critical roles for five specific amino acids in phosphatase activity.
- Cys192 forms a disulfide bond with Cys219, stabilizing the sulfhydryl group and potentially determining phosphatase activity.
Conclusions:
- PmPOD's phosphatase activity is heme-independent and linked to a specific catalytic loop.
- Specific amino acids, notably Cys192, are essential for PmPOD's phosphatase function and enzyme stability.
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