A Thermostable Bacterial Metallohydrolase that Degrades Organophosphate Plasticizers

Abstract

A cyclase-phosphotriesterase (C-PTE) from Ruegeria pomeroyi DSS-3 has recently been identified for its capacity to detoxify several organophosphate compounds. However, several aspects of this enzyme remain unexplored, such as its activity with industrial organophosphates, its molecular structure, and its thermostability. In this work, the crystal structure of C-PTE is reported, which is solved to 2.3 Å resolution, providing insight into the enzyme's mechanism of action, revealing a binuclear Zn²⁺ active site and distant similarity to other phosphotriesterases from the amidohydrolase superfamily. It is shown that C-PTE catalyzes the hydrolysis of the OP plasticizers triphenyl phosphate (TPhP) and tris(2-chloropropyl) phosphate (TCPP), albeit with low efficiency, but not the sterically bulkier tri-o-tolyl phosphate (ToTP). Finally, it is demonstrated that, even though Ruegeria pomeroyi DSS-3 is not a thermophile, C-PTE exhibits remarkable thermostability and retains structure up to 90 °C. Overall, these findings advance the understanding of C-PTE, suggesting that it is a good candidate for engineering owing to its thermostability and that it could contribute to bioremediation strategies to reduce the impact of pollution by industrial organophosphates.

Keywords: bioremediation; cyclase; organophosphosphate compounds; plasticizers.

Figure 1

X‐ray crystal structure of C‐PTE from Ruegeria pomeroyi DSS‐3. A) The asymmetric unit of the crystal structure, containing five domain‐swapped dimers arranged in a twisted crescent. The structure is colored by chain. B) The domain‐swapped dimer; one protomer is shown in dark blue and the second protomer in light blue. The zinc atoms are shown in spheres. C) Zinc coordination within the active site. Coordinating side chains are shown as sticks, with zinc shown in spheres. Coordinate covalent bonds are represented as black dashes. D) Comparison of C‐PTE active site (blue) with the active site of isatin hydrolase (beige, PDB 5NNB) shows high conservation of active site geometry, despite isatin hydrolase displaying mononuclear metal binding. E) The active site of an example PTE (2R1N) is similar to C‐PTE in terms of composition.

Figure 2

Bioinformatic and phylogenetic analysis of C‐PTE. A) Sequence similarity to other TIM amidohydrolase superfamily members in the ESM‐1b embedding space. Nodes represent a 2D embedding of sequences projected from the ESM‐1b representation space with tSNE. Each node is colored according to the highest shared sequence identity with representatives from the 9 other TIM amidohydrolase families. Sequences included in the phylogenetic tree are labeled and highlighted in bold. B) Phylogenetic reconstruction of the C‐PTE. Clades with >5 tips are collapsed and labeled. Isatin hydrolase and C‐PTE tips are highlighted for clarity. Ultrafast bootstrap support values are plotted at the corresponding nodes. The tree is rooted on a kynurenine formamidase outgroup that includes solved structures 4COG, 4COB, and 4CO9, designated Clade 0. Characterized isatin hydrolases (PDB IDs: 5MNP, 5NNB, 4J0N) and C‐PTE are labeled in both the tSNE projection of A) the ESM‐1b representation space and B) the phylogenetic tree.

Figure 3

Docking of isatin and methyl paraoxon reveals a sterically complementary binding pocket. Chain I of C‐PTE is shown in gray surface and sticks, with isatin shown in pink sticks and methyl paraoxon in green sticks. The active site metal ions are shown as spheres.

Figure 4

CD of C‐PTE reveals high thermal stability. A) Secondary structure characterization in solution. B) Thermal melting curve reveals C‐PTE undergoes little change in secondary structure up to 95 °C.