『Abstract
We have studied the oxygen isotope signature of inorganic phosphate
(Pi) generated by hydrolysis of nucleic acid
phosphodiester (P-diester) compounds by cell-free enzymes (Deoxyribonuclease
1, Phosphodiesterase a, 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 Pi for a total of two O atoms
from water incorporated into Pi released
from P-diesters. In the presence of Phosphodiesterase a, 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.』
1. Introduction
1.1. Structure-reaction-based model for O-isotope effects of
phosphoester hydrolysis
2. Materials and methods
2.1. Cell-free enzymatic degradation experiments
2.2. E. coli phosphodiester degradation experiments
2.3. Analytical methods
3. Results
3.1. Cell-free enzymatic DNA degradation experiments
3.2. Cell-free enzymatic RNA degradation experiments
3.3. E. coli phosphodiester degradation experiments
4. Discussion
4.1. Cell-free DNA degradation experiments
4.2. Cell-free RNA degradation experiments
4.3. Parameter determination for the phosphoester hydrolysis
model: O-isotope fractionation factors associated with diester
bond cleavage
4.4. Prediction of δ18O signatures of regenerated
Pi
4.5. Testing the phosphoester hydrolysis model using intact microbial
cells: experiments with E. Coli
4.6. Interpretation of measured DIP δ18O values from
natural systems
5. Conclusions
Acknowledgment
References