Engineering of an archaeal phosphodiesterase to trigger aggregation-induced emission (AIE) of synthetic substrates

Abstract

Aggregation-induced emission (AIE) probes that can be triggered by enzymatic activity are sought for applications across the life sciences. The compound 2-(2-hydroxyphenyl)benzothiazole (HBT) is non-fluorescent in solution but exhibits AIE. To adapt HBT for potential clinical applications, two phosphoester derivatives (1 and 2) were prepared. For extension to molecular brachytherapy (delivery of a radionuclide), one phosphoester derivative (3) was prepared wherein a bromine atom is incorporated as a site for subsequent radioiodide substitution. Each probe 1–3 requires phosphatase action to engender AIE. For specificity in diagnostic and therapeutic applications, a heterologous (i.e., foreign to humans) enzyme was sought for conversion of 1–3 from aqueous-soluble to aqueous-insoluble form. Here, an archaeal phosphodiesterase PDE-MJ0936 (wild-type) was systematically engineered for activity toward 1–3. PDE-MJ0936 is a hyperthermal enzyme (activity at 70 °C and pH 9.8) and exists as a dimer, which together limit life science applications. Ten PDE mutants were engineered by site-directed mutagenesis to achieve a monomeric structure, wider substrate scope, and elastic substrate docking under physiological conditions. Among the mutants, PDE-C141D exhibited efficient activity towards bis(4-nitrophenyl)phosphate (K_m = 0.28 mM, k_cat = 3.0 s⁻¹ and k_cat/K_m = 1.1 × 10⁴ M⁻¹ s⁻¹) and efficiently hydrolyzed 3 at 37 °C and pH 7.0, conditions under which PDE-MJ0936 is silent. The substrates 1–3 are relatively resistant to alkaline phosphatase, PDE-MJ0936 and fetal bovine serum. The development of enzymatically triggered substrates and the complementary heterologous enzymes may support diverse applications in the life sciences.