Compound- and enzyme-specific phosphodiester hydrolysis mechanisms revealed by δ18O of dissolved inorganic phosphate: Implications for marine P cycling

Abstract

We have studied the oxygen isotope signature of inorganic phosphate (P i) generated by hydrolysis of nucleic acid phosphodiester (P-diester) compounds by cell-free enzymes (Deoxyribonuclease 1, Phosphodiesterase 1, Alkaline phosphatase) and microbial cultures at natural isotopic abundances. We demonstrate that the diesterase-catalyzed hydrolytic step leads to incorporation of at least one water O into released P i for a total of two O atoms from water incorporated into P i released from P-diesters. In the presence of Phosphodiesterase 1, 16O is preferentially incorporated into nucleotides released from DNA; whereas 18O is preferentially incorporated into nucleotides released from RNA. A strong consistency between predicted O-isotope regeneration signatures based on results of cell-free enzyme experiments and measured isotopic signatures from independent experiments with E. coli cultures was observed and confirms proposed models for phosphoester hydrolysis. Results from these studies made at natural 18O abundance levels provide a new tool, enzyme-specific O-isotope fractionation, for investigations of organophosphate metabolism and phosphorus cycling pathways in natural aquatic systems.