Structural and Functional Analysis of a Highly Active Designed Phosphotriesterase for the Detoxification of Organophosphate Nerve Agents Reveals an Unpredicted Conformation of the Active Site Loop

Abstract

Neurotoxic organophosphorus compounds (OPs) pose a severe threat if misused in military conflicts or by terrorists. Administration of a hydrolytic enzyme that can decompose the circulating nerve agent into non-toxic metabolites in vivo offers a potential treatment. A promising candidate is the homo-dimeric phosphotriesterase originating from the bacterium Brevundimonas diminuta (BdPTE), which has been subject to several rational and combinatorial protein design studies. A series of engineered versions with much improved catalytic efficiencies toward medically relevant nerve agents was described, carrying up to 22 mutations per enzyme subunit. To provide a basis for further rational design, we have determined the crystal structure of the highly active variant 10-2-C3(C59V/C227V)� stabilized against oxidation by substitution of two unpaired Cys residues�in complex with a substrate analogue at 1.5 Å resolution. Unexpectedly, the long loop segment (residues 253−276) that covers the active site shows a totally new conformation, with drastic structural deviations up to 19 Å, which was neither predicted in any of the preceding protein design studies nor seen in previous crystallographic analyses of less far evolved enzyme versions. Inspired by this structural insight, additional amino acid exchanges were introduced and their effects on protein stability as well as on the catalytic efficiency toward several neurotoxic OPs were investigated. Somewhat surprisingly, our results suggest that the presently available engineered version of BdPTE, in spite of its design on the basis of partly false structural assumptions, constitutes a fairly optimized enzyme for the detoxification of relevant OP nerve agents.

The impact of molecular variants, crystallization conditions and the space group on ligand–protein complexes: a case study on bacterial phosphotriesterase

“…Notably, in both structures the phosphonyl O atoms were observed to be at distances of 4.7 and 5.0 A ˚, respectively, from the buried �-Zn 2+ ion. The experimentally determined 3D structures of the OP acid products observed in the present study are consistent with the reported 3D structures of the complexes of PTEs with two other acid products: O,O-diethylphosphoric acid (DEP; PDB entry 3cak; Table 1; Kim et al, 2008) and ethyl-4-methylbenzylphosphonate (mEBP; PDB entry 7p85; Table 1; Job et al, 2023). The P-O O atoms of PTE-DEP and PTE-mEBP are placed symmetrically at 2.0-2.2 A ˚away from the two Zn 2+ ions, whereas in the case of the complex of the triester substrate analogue (C 2 H 5 O) 2 P(O)CH 2 phenyl-pCH 3 the P O O atom is 3.5 and 4.7 A ˚from the exposed �-Zn 2+ and the buried �-Zn 2+ , respectively (Vanhooke et al, 1996).…”

“…11). Together with the similar short distances (2.0-2.2 A ˚) reported for PTE complexes with acidic OP products such as O,O-diethylphosphoric acid (Kim et al, 2008) and O-ethyl-4-benzylphosphonic acid (Job et al, 2023), it is suggested that, regardless of the size of the substituent, the enzyme is sufficiently flexible to utilize the stabilization machinery offered by the two Zn 2+ ions. Accordingly, the buried and exposed Zn 2+ ions are proposed to stabilize the developing high-energy charged TS, thereby lowering its energy content, with concomitant acceleration of the reaction relative to water alone.…”

“…Furthermore, our results highlight the importance of removing residual tags used to increase expression and purification levels before attempting to study the 3D structures of complexes and conjugates of proteins. In fact, when one overlays the recently determined structure of PTE complexed with ethyl-4-methylbenzylphosphonate (mEBP; PDB entry 7p85; Table 1; Job et al, 2023) on that of A53_3, the benzyl group of mEBP is seen to be very near the side chain of Ile26 of the octapeptide tag. This is strong supporting evidence that the octapeptide binds in a position similar to that of the large leaving group of the OP.…”

Changes in Active Site Loop Conformation Relate to the Transition toward a Novel Enzymatic Activity

“…PLL and PTE share a (β/α) 8 fold with highly mobile loops that contribute to broadening their promiscuity to other substrates such as aryl-esters . Bacterial PTEs, although naturally efficient for the degradation of OP insecticides, have been further engineered in vitro for the development of biocatalysts close to catalytic perfection for decontaminating insecticides or chemical warfare agents. − These variants may find application in bioremediation of agricultural contaminations or for prophylaxis protection against OP poisoning. , However, most PTEs have been isolated from mesophilic microorganisms and show moderate stability limiting their potential for bioremediation or development of medical countermeasures to face OP poisoning. , A subfamily of PLL, namely, PLL-A, is composed of enzymes isolated from thermostable and hyperthermostable bacteria or archaea. Special attention has thus been paid to PLL-A for developing high-potential biocatalysts as robust enzymes that may offer numerous biotechnological advantages including resistance to high temperatures, tolerance to solvents and denaturants, and long-term storage .…”

“… 15 Bacterial PTEs, although naturally efficient for the degradation of OP insecticides, have been further engineered in vitro for the development of biocatalysts close to catalytic perfection for decontaminating insecticides or chemical warfare agents. 16 − 23 These variants may find application in bioremediation of agricultural contaminations or for prophylaxis protection against OP poisoning. 24 , 25 However, most PTEs have been isolated from mesophilic microorganisms and show moderate stability limiting their potential for bioremediation or development of medical countermeasures to face OP poisoning.…”