Translating the Concept of Bispecific Antibodies to Engineering Heterodimeric Phosphotriesterases with Broad Organophosphate Substrate Recognition

Abstract

We have adopted the concept of bispecific antibodies, which can simultaneously block or cross-link two different biomolecular targets, to create bispecific enzymes by exploiting the homodimeric quaternary structure of bacterial phosphotriesterases (PTEs). The PTEs from Brevundimonas diminuta and Agrobacterium radiobacter, whose engineered variants can efficiently hydrolyze organophosphorus (OP) nerve agents and pesticides, respectively, have attracted considerable interest for the treatment of the corresponding intoxications. OP nerve agents and pesticides still pose a severe toxicological threat in military conflicts, including acts of terrorism, as well as in agriculture, leading to >100000 deaths per year. In principle, engineered conventional homodimeric PTEs may provoke hydrolytic inactivation of individual OPs in vivo, and their application as catalytic bioscavengers via administration into the bloodstream has been proposed. However, their narrow substrate specificity would necessitate therapeutic application of a set or mixture of different enzymes, which complicates biopharmaceutical development. We succeeded in combining subunits from both enzymes and to stabilize their heterodimerization by rationally designing electrostatic steering mutations, thus breaking the natural C2 symmetry. The resulting bispecific enzyme from two PTEs with different bacterial origin exhibits an ultrabroad OP substrate profile and allows the efficient detoxification of both nerve agents and pesticides. Our approach of combining two active sites with distinct substrate specificities within one artificial dimeric biocatalystretaining the size and general properties of the original enzyme without utilizing protein mixtures or much larger fusion proteinsnot only should facilitate biological drug development but also may be applicable to oligomeric enzymes with other catalytic activities.

Catalytic activity and stereoselectivity of engineered phosphotriesterases towards structurally different nerve agents in vitro

“…To take benefit of this technology, a PAS tag with 200 residues was appended to BdPTE-3, yielding BdPTE-4, to potentially increase its circulation time in vivo. Moreover, introduction of the flexible PAS sequence as a linker proved useful in the recent investigation of Escher et al (2020) with the aim to generate a bispecific catalytic bioscavenger. The resulting heterodimer BdPTE-7 combines the substrate profiles of its constituent subunits, BdPTE-1 and BdPTE-6, without impairing their individual catalytic activities.…”

“…The SapI restriction site in front of the stop-codon of the variant BdPTE-3 was used to insert a PAS#1(200) gene cassette according to Schlapschy et al (2013), yielding BdPTE-4. The expression plasmid encoding the heterodimeric variant BdPTE-7 composed of BdPTE-1 and BdPTE-6 subunits, fused by a PAS(100) linker between the C-terminus of BdPTE-1 and the N-terminus of BdPTE-6 and complemented by electrostatic steering mutations R152E as well as E71K to stabilize the heterodimerization, was constructed according to Escher et al (2020). For the additional C-terminal PASylation, two inverted SapI restriction sites were inserted upfront of the stop-codon via PCR, followed by the seamless insertion of a PAS#1(200) gene cassette as above.…”

“…The BdPTE variants were produced in E. coli BL21 and purified as previously described (Escher et al 2020;Job et al 2020). Briefly, bacteria were cultivated in shake flasks with 2 L LB medium supplemented with 100 mg L −1 ampicillin and 0.2 mM ZnSO 4 and induced with 200 µg L −1 anhydrotetracycline.…”

“…Furthermore, Job et al (2020) increased the oxidation stability of BdPTE 10-2-C3 by replacing two free Cys to Val, resulting in the variant 10-2-C3(C59V/C227V). Recent investigations by Escher et al (2020) led to an enlarged substrate spectrum by creating bispecific enzymes, e.g., by fusing two different PTE monomers via a 100-residue Pro/Ala/Ser (PAS) linker (Schlapschy et al 2013) to obtain a heterodimeric singlechain PTE (scPTE).…”

Enzymes, Reacting with Organophosphorus Compounds as Detoxifiers: Diversity and Functions

“…with N-acyl homoserine lactones [108,109]. Thus, they can be rationally combined to obtain universal enzyme preparations [110] or even hybrid molecules with other enzymes [111] or with 'guide' DNA [112]. This opens up new promising perspectives for their practical application.…”