Structural and biochemical characterization of active sites mutant in human inorganic pyrophosphatase

作者全名：Zheng, Shuping; Zheng, Chenhua; Chen, Sishi; Guo, Jianpeng; Huang, Lirui; Huang, Zhenhong; Xu, Sunting; Wu, Yihan; Li, Shunfa; Lin, Junjin; You, Yiqing; Hu, Fen

作者地址：[Zheng, Shuping; Chen, Sishi; Lin, Junjin] Fujian Med Univ, Publ Technol Serv Ctr, Fuzhou, Peoples R China; [Zheng, Chenhua] Fujian Med Univ, Sch Basic Med Sci, Expt Teaching Ctr Basic Med Sci, Fuzhou, Fujian, Peoples R China; [Guo, Jianpeng] Fujian Med Univ, Sch Pharm, Dept Pharmacol, Fuzhou, Peoples R China; [Guo, Jianpeng] Fujian Med Univ, Fujian Key Lab Drug Target Discovery & Struct & Fu, Fuzhou, Peoples R China; [Huang, Lirui; Huang, Zhenhong; Xu, Sunting; Wu, Yihan; Li, Shunfa; Hu, Fen] Fujian Med Univ, Sch Basic Med Sci, Key Lab Gastrointestinal Canc, Minist Educ, Fuzhou, Peoples R China; [You, Yiqing] Chongqing Med Univ, Key Lab Diagnost Med, Chinese Minist Educ, Chongqing, Peoples R China; [Hu, Fen] Fujian Med Univ, 1 Xue Yuan Rd,Univ Town, Fuzhou, Fujian, Peoples R China

通信作者：Hu, F (通讯作者)，Fujian Med Univ, 1 Xue Yuan Rd,Univ Town, Fuzhou, Fujian, Peoples R China.

来源：BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS

ESI学科分类：BIOLOGY & BIOCHEMISTRY

WOS号：WOS:001209314400001

JCR分区：Q2

影响因子：2.8

年份：2024

卷号：1868

期号：5

开始页：　

结束页：　

文献类型：Article

关键词：PPase; Crystal structure; Catalytic mechanism; Molecular docking; Molecular dynamics simulation

摘要：Inorganic pyrophosphatases (PPases) are enzymes that catalyze the conversion of inorganic pyrophosphate (PPi) into phosphate (Pi). Human inorganic pyrophosphatase 1 (Hu-PPase) exhibits high expression levels in a variety of tumors and plays roles in cell proliferation, apoptosis, invasion and metastasis, making it a promising prognostic biomarker and a target for cancer therapy. Despite its widespread presence, the catalytic mechanism of Hu-PPase in humans remains inadequately understood. The signature motif amino acid sequence (DXDPXD) within the active sites of PPases is preserved across different species. In this research, an enzymatic activity assay revealed that mutations led to a notable reduction in enzymatic function, although the impact of the four amino acids on the activity of the pocket varied. To investigate the influence of these residues on the substrate binding and enzymatic function of PPase, the crystal structure of the Hu-PPase-ED quadruple mutant (D116A/D118A/ P119A/D121A) was determined at 1.69 & Aring; resolution. The resulting structure maintained a barrel-like shape similar to that of the wild-type, albeit lacking Mg2+ ions. Molecular docking analysis demonstrated a decreased ability of Hu-PPase-ED to bind to PPi. Further, molecular dynamics simulation analysis indicated that the mutation rendered the loop of Mg2+ ion-binding residues less stable. Therefore, the effect on enzyme activity did not result from a change in the gross protein structure but rather from a mutation that abolished the Mg2+-coordinating groups, thereby eliminating Mg2+ binding and leading to the loss of enzyme activity.

基金机构：Natural Science Foundation of Fujian Province [2022 J01663, 2023 J01324]; Startup Fund for Scientific Research Project of Fujian Medical University [2019QH1015, 2023QH1005]; Chinese college students innovation and entrepreneurship training program [202310392015]

基金资助正文：We thank the staff at BL19U1 beamlines at SSRF of the National Facility for Protein Science in Shanghai (NFPS) , Shanghai Advanced Research Institute, Chinese Academy of Sciences, for providing technical support in X-ray diffraction data collection and analysis. This work was supported by the Natural Science Foundation of Fujian Province (Grant No. 2022 J01663, 2023 J01324) ; Startup Fund for Scientific Research Project of Fujian Medical University (Grant No. 2019QH1015, 2023QH1005) ; Chinese college students innovation and entrepreneurship training program (Grant No. 202310392015) .