4O98

Crystal structure of Pseudomonas oleovorans PoOPH mutant H250I/I263W

Summary for 4O98

• Entry DOI: 10.2210/pdb4o98/pdb
• Descriptor: organophosphorus hydrolase, ZINC ION (3 entities in total)
• Functional Keywords: alphabeta/betaalpha sandwich, organophophorus hydrolase, beta-lactamase superfamily, hydrolase, zinc binding
• Biological source: Pseudomonas oleovorans
• Total number of polymer chains: 2
• Total formula weight: 73350.54

Authors

Luo, X.J.,Kong, X.D.,Zhao, J.,Chen, Q.,Zhou, J.H.,Xu, J.H. (deposition date: 2014-01-02, release date: 2014-12-03, Last modification date: 2024-10-30)

Primary citation

Luo, X.J.,Kong, X.D.,Zhao, J.,Chen, Q.,Zhou, J.,Xu, J.H.
Switching a newly discovered lactonase into an efficient and thermostable phosphotriesterase by simple double mutations His250Ile/Ile263Trp
Biotechnol.Bioeng., 111:1920-1930, 2014

PubMed Abstract: OPHC2 is a thermostable organophosphate (OP) hydrolase in the β-lactamase superfamily. OPs are highly toxic synthetic chemicals with no natural analogs. How did OPHC2 acquire phosphotriesterase (PTE) activity remained unclear. In this study, an OPHC2 analogue, PoOPH was discovered from Pseudomonas oleovorans exhibiting high lactonase and esterase activities and latent PTE activity. Sequence analysis revealed conserved His250 and Ile263 and site-directed mutagenesis at these crucial residues enhanced PTE activity. The best variant PoOPHM2 carrying H250I/I263W mutations displayed 6,962- and 106-fold improvements in catalytic efficiency for methyl-parathion and ethyl-paraoxon degradation, whereas the original lactonase and esterase activities decreased dramatically. A 1.4 × 10(7) -fold of specificity inversion was achieved by only two residue substitutions. Significantly, thermostability of the variants was not compromised. Crystal structure of PoOPHM2 was determined at 2.25 Å resolution and docking studies suggested that the two residues in the binding pocket determine substrate recognition. Lastly, new organophosphorus hydrolases (OPHs) were discovered using simple double mutations. Among them, PpOPHM2 from Pseudomonas putida emerged as a new promising OPH with very high activity (41.0 U mg(-1) ) toward methyl-parathion. Our results offer a first scrutiny to PTE activity evolution of OPHs in β-lactamase superfamily and provide efficient and robust enzymes for OP detoxification.

PubMed: 24771278
DOI: 10.1002/bit.25272

Experimental method

X-RAY DIFFRACTION (2.251 Å)