Current standard assays using artificial substrates overestimate phosphodiesterase activity

Phosphodiesterases (PDE) are important enzymes in environments with significant proportion of phosphodiesters. The breakdown of phosphodiesters is a two step process, initiated by PDE hydrolyzing an ester-P bond on the diester molecule, breaking it into an organic moiety and a phosphomonoester. In the second step, the phosphomonoester is further hydrolyzed by phosphomonoesterase (PME) into the second organic moiety and free phosphate, Pi. The methods to assess extracellular PDE activities commonly use the substrate analogues bis-(p-nitrophenyl) phosphate (bis-pNPP) or bis-(4-methylumbelliferyl) phosphate (bis-MUFP), measure the resulting concentration of p-nitrophenol (pNP) or 4-methylumbelliferon (MUF), and assume the contribution from phosphomonoester hydrolysis by PME to be insignificant. To verify this, we measured concentrations of MUF and Pi at the end of the 30- and 60-min incubation of wetland plant root segments with a phosphodiester substrate. We found that the hydrolysis was complete (MUF:Pi ratio ∼ 2) confirming the importance of step 2. We suggest that a correction for additional hydrolysis by PME should be employed in samples where this is possible, such as plant roots or microbial cultures. Here, the MUF:Pi should be verified for each new sample type tested for its PDE activity, and values obtained from the PDE assay should be lowered accordingly. We do not recommend using the standard artificial substrates for PDE activity measurements in more heterogeneous samples, for example soils, where these assays have low reliability and can produce erroneous results.