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

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 adaptHBTfor potential clinical applications, two phosphoester derivatives (1and2) 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 probe1-3requires phosphatase action to engender AIE. For specificity in diagnostic and therapeutic applications, a heterologous (i.e., foreign to humans) enzyme was sought for conversion of1-3from aqueous-soluble to aqueous-insoluble form. Here, an archaeal phosphodiesterase PDE-MJ0936 (wild-type) was systematically engineered for activity toward1-3. PDE-MJ0936 is a hyperthermal enzyme (activity at 70 degrees 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)andk(cat)/K-m= 1.1 x 10(4)M(-1)s(-1)) and efficiently hydrolyzed3at 37 degrees C and pH 7.0, conditions under which PDE-MJ0936 is silent. The substrates1-3are 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.