The crystal structure of the phosphotriesterase from M. tuberculosis, another member of phosphotriesterase-like lactonase family

https://doi.org/10.1016/j.bbrc.2019.01.069Get rights and content

Highlights

  • The 2.3 Å crystal structure of phosphotriesterase from M. tuberculosis was solved.

  • The mPHP is a dimer with a distorted (β/α)8 barrel structure.

  • The catalytic pocket of mPHP contains two Zinc atoms.

  • The mPHP is active form.

  • The structural comparison concluded mPHP belongs to PLL family.

Abstract

Organophosphates (OPs) have been used widely as insecticides for protecting the agricultural crops from the pests. These compounds are highly toxic because they can cause the irreversible damage to human nervous system. Phosphotriesterases (PTEs), widely exist in many different kinds of bacteria, insects and mammals, can hydrolyze phosphotriesters (one major kind of OP) sufficiently. The phosphotriesterase-activities of PTEs are considered to derive from the lactonase-activities during the evolution, and phosphotriesterase-like lactonase family (PLL), is the closest protein family to PTE family based on protein-protein blast results. But members of PLL family exhibit higher lactonase activities than the phosphotriesterase activities, while the best substrates for PTEs are phosphotriesters. In this paper, the X-ray crystal structure of phosphotriesterase from M. tuberculosis (mPHP) was solved at a resolution of 2.3 Å. The structure reveals that the mPHP is a dimer with a typical distorted (β/α)8 barrel structure like other structures of PLL family and PTE family. The architecture of active pocket of mPHP coordinates with 2 metal ions which is also similar to other PLLs and PTEs. The activity assay proved the mPHP is biological active form and the Atomic Absorption Spectroscopy assay gave the evidence that the two metal ions bound to the active pocket were Zinc cations. The structural comparison between mPHP and other homologues concluded that the mPHP should belong to PLL family, not PTE family.

Introduction

Organophosphates (OPs), a group of highly toxic compounds, have been used widely as insecticides for protecting the agricultural crops from the pests since 1940s. But these compounds cause the irreversible damage to human nervous system by inhibiting the activity of the enzyme acetylcholinesterase, and result in the paralysis and finally death continuously [1,2]. Every year, there are thousands of hazardous injuries caused by human- or animals-exposure to the OPs reported worldwide [2,3]. The typical chemical structure of the organophosphate is three ester linkages surround a phosphate center. Among these three linkages, one ester group is comparable less stable than the other two one. The cleavage of the labile bond, when an organophosphate molecule is hydrolyzed by acetylcholinesterase, inhibits the enzyme irreversibly. Therefore, the toxic effects of insecticides to human and animal cannot be ignored, even though there are the huge benefits of using the insecticides during the crop planting and the food production. Except developed as the pesticides, the organophosphates, especially the most toxic ones among them are expanded as the nerve agents for military purpose, such as Sarin, VX and Tabun [4]. Considering the health and safety, the environmental protection and chronic threaten by nerve agents, the development of enzymes that could hydrolyze and neutralize organophosphate compounds is required urgently [3].

There are few kinds of enzyme that can use these compounds as substrates because the organophosphates are not naturally occurring materials. Phosphotriesterases (PTEs), which have been identified in many different species, such as bacterium, insects and mammals, could hydrolyze these synthetic compounds sufficiently [5]. The phosphotriesterase-activities of such enzymes are considered to derive from the lactonases and take advantage of hydrolyzing the phosphotriesters during the evolution [[6], [7], [8], [9]]. The benefits of phosphotriesters-hydrolysis might be response to the limitation of inorganic phosphate in the soil, which is a normal limiting factor during the bacteria growth [10], like Pseudomonas aeruginosa which can grow in the soil without inorganic phosphate and use the organophosphates as the sole phosphorus source [11]. The closest protein super-family to PTE based on protein sequence blast is the phosphotriesterase-like lactonase family (PLL) (www.ncbi.nlm.nih.gov/BLAST). The PTEs, which can use phosphotriesters as substrates but have promiscuous lactonase activity for different lactones, with variable kcat/KM from 160 to 6.5 × 105 M−1s−1 [7]. While the PLLs exhibit much higher lactonase hydrolyzing activity than phosphotriesterase [12]. The first crystal structure from PLL family, the phosphotriesterase from sulfolobus solfataricus (SsoPox), was determinated in 2008 by Dr. Mikael H Elias [6].

The function of phosphotriesterase from M. tuberculosis H37Rv (mPHP) during the life cycle is still unclear, but it should be related to the bacteria growth in the extreme environment, especial the inorganic phosphate lacking condition. And according to the sequence analysis and the activity comparison, previous research indicated the mPHP should belongs to the PLLs [12]. In this article, we solved the crystal structure of mPHP with the resolution of 2.30 Å. The monomeric structure was a typical distorted (β/α)8 barrel like other PLLs and PTEs. Here, we concluded that mPHP belonged to the PLL family based on the structural alignments and comparisons with other enzymes from PTE, lactonase and PLL families.

Section snippets

Materials and methods

Gene cloning, protein expression, crystallization and data collection: The gene cloning, protein expression, crystallization and data collection was described in the previous article [13].

Structure determination: The phase of mPHP was determined by using the molecular replacement (MR) program PHASER [14] from CCP4 suite [15]. The structure of phosphotriesterase from sulfolobus solfataricus (SsoPox) (PDB code: 2VC5) [6] was used as the search model. The initial model was rebuilt by ARP/wARP [16,

Results and discussions

The Overall Characteristic of mPHP: The crystal belongs to the space group C2221, with unit-cell parameters of a = 68.1 Å, b = 150.8 Å, c = 74.5 Å, and α = β = γ = 90.0°. Each asymmetric unit contains only one mPHP molecule with 55% solvent content and the Matthews coefficient is 2.73. There are 324 amino acid residues (form Pro2 to Tyr325) except the N-terminal His-tag residues are found, and the first residue of the N-terminal (Met1) and the last residue of the C-terminal (Gln326) are missing

Acknowledgements

We are grateful thank Dr Xiuna Yang (Shanghai IBCB, China) for critical reading and idea discussion of the manuscript. We also thank the staff at the Shanghai Synchrotron Radiation Facility (China) their assistance in data collection. This work was supported by grants from the National Key Research and Development Program of China (Grant No. 15 2017YFC0840300), the Strategic Priority Research Program of the Chinese Academy of Sciences (Grant No. XDB08020200), the State Key Development Program

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