Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Institute of Biotechnology, Shanxi University, Taiyuan030006, China.
School of Life Science, Shanxi University, Taiyuan 030006, China.
J Agric Food Chem. 2021 Jun 9;69(22):6251-6259. doi: 10.1021/acs.jafc.1c01606. Epub 2021 May 28.
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.
黍米过氧化物酶（PmPOD）属于 III 类植物过氧化物酶，这类酶的典型特征是含有血红素辅基。PmPOD 不仅表现出依赖血红素的过氧化物酶活性，还表现出非依赖血红素的磷酸酶活性。晶体结构分析和序列比对表明，PmPOD 的β-结构域中含有一个磷酸酶催化环 CXXXXXR，类似于双特异性磷酸酶的活性位点。含有磷酸酶保守催化环 CXXXXXR 的重组截短黍米过氧化物酶（tPmPOD）被发现具有磷酸酶活性。5 个在磷酸酶活性位点含有 5 个不同突变的 tPmPOD 突变体与 tPmPOD 相比，表现出显著降低的磷酸酶活性，表明这 5 个氨基酸在 tPmPOD 的磷酸酶活性中起着重要作用。最后，亲核氨基酸 Cys192 与 Cys219 形成二硫键，以保护巯基的稳定性；因此，它可能在 PmPOD 的磷酸酶活性中起决定性作用。
