Mechanism of dephosphorylation of glucosyl-3-phosphoglycerate by a histidine phosphatase

Qianqian Zheng; Dunquan Jiang; Wei Zhang; Qingqing Zhang; Qi Zhao; Jin Jin; Xin Li; Haitao Yang; Mark Bartlam; Neil Shaw; Weihong Zhou; Zihe Rao

doi:10.1074/jbc.M114.569913

Mechanism of dephosphorylation of glucosyl-3-phosphoglycerate by a histidine phosphatase

J Biol Chem. 2014 Aug 1;289(31):21242-51. doi: 10.1074/jbc.M114.569913. Epub 2014 Jun 9.

Authors

Qianqian Zheng¹, Dunquan Jiang¹, Wei Zhang¹, Qingqing Zhang¹, Qi Zhao², Jin Jin¹, Xin Li¹, Haitao Yang³, Mark Bartlam¹, Neil Shaw⁴, Weihong Zhou⁵, Zihe Rao⁶

Affiliations

¹ From the College of Life Sciences, Nankai University, Tianjin 300071, China.
² the Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520-8114.
³ the College of Life Sciences, Tianjin University, Tianjin 300073, China.
⁴ From the College of Life Sciences, Nankai University, Tianjin 300071, China, the National Laboratory of Macromolecules, Institute of Biophysics, Chinese Academy of Science, Beijing 100101, China.
⁵ From the College of Life Sciences, Nankai University, Tianjin 300071, China, zhouwh@nankai.edu.cn.
⁶ From the College of Life Sciences, Nankai University, Tianjin 300071, China, the National Laboratory of Macromolecules, Institute of Biophysics, Chinese Academy of Science, Beijing 100101, China, the Laboratory of Structural Biology, School of Medicine, Tsinghua University, Beijing 100084, China, and raozh@xtal.tsinghua.edu.cn.

Abstract

Mycobacterium tuberculosis (Mtb) synthesizes polymethylated polysaccharides that form complexes with long chain fatty acids. These complexes, referred to as methylglucose lipopolysaccharides (MGLPs), regulate fatty acid biosynthesis in vivo, including biosynthesis of mycolic acids that are essential for building the cell wall. Glucosyl-3-phosphoglycerate phosphatase (GpgP, EC 5.4.2.1), encoded by Rv2419c gene, catalyzes the second step of the pathway for the biosynthesis of MGLPs. The molecular basis for this dephosphorylation is currently not understood. Here, we describe the crystal structures of apo-, vanadate-bound, and phosphate-bound MtbGpgP, depicting unliganded, reaction intermediate mimic, and product-bound views of MtbGpgP, respectively. The enzyme consists of a single domain made up of a central β-sheet flanked by α-helices on either side. The active site is located in a positively charged cleft situated above the central β-sheet. Unambiguous electron density for vanadate covalently bound to His(11), mimicking the phosphohistidine intermediate, was observed. The role of residues interacting with the ligands in catalysis was probed by site-directed mutagenesis. Arg(10), His(11), Asn(17), Gln(23), Arg(60), Glu(84), His(159), and Leu(209) are important for enzymatic activity. Comparison of the structures of MtbGpgP revealed conformational changes in a key loop region connecting β1 with α1. This loop regulates access to the active site. MtbGpgP functions as dimer. L209E mutation resulted in monomeric GpgP, rendering the enzyme incapable of dephosphorylation. The structures of GpgP reported here are the first crystal structures for histidine-phosphatase-type GpgPs. These structures shed light on a key step in biosynthesis of MGLPs that could be targeted for development of anti-tuberculosis therapeutics.

Keywords: Bacterial Protein Phosphatase; Crystal Structure; Enzymatic Mechanism; Enzyme Mechanism; Mutagenesis; Mycobacterium Tuberculosis.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Dimerization
Glyceric Acids / metabolism*
Models, Molecular
Open Reading Frames
Phosphoric Monoester Hydrolases / metabolism*
Phosphorylation

Substances

Glyceric Acids
3-phosphoglycerate
Phosphoric Monoester Hydrolases

Associated data

PDB/4PZ9
PDB/4PZA
PDB/4QIH