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

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, O-16 is preferentially incorporated into nucleotides released from DNA; whereas O-18 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 O-18 abundance levels provide a new tool, enzyme-specific O-isotope fractionation, for investigations of organophosphate metabolism and phosphorus cycling pathways in natural aquatic systems. (C) 2009 Elsevier Ltd. All rights reserved.