1 research outputs found
Eukaryotic Assimilatory Nitrate Reductase Fractionates N and O Isotopes with a Ratio near Unity
In order to (i) establish the biological systematics
necessary
to interpret nitrogen (N) and oxygen (O) isotope ratios of nitrate
(<sup>15</sup>N/<sup>14</sup>N and <sup>18</sup>O/<sup>16</sup>O)
in the environment and (ii) investigate the potential for isotopes
to elucidate the mechanism of a key N cycle enzyme, we measured the
nitrate N and O isotope effects (<sup>15</sup>ε and <sup>18</sup>ε) for nitrate reduction by two assimilatory eukaryotic nitrate
reductase (eukNR) enzymes. The <sup>15</sup>ε for purified extracts
of NADPH eukNR from the fungus <i>Aspergillus niger</i> and
the <sup>15</sup>ε for NADH eukNR from cell homogenates of the
marine diatom <i>Thalassiosira weissflogii</i> were indistinguishable,
yielding a mean <sup>15</sup>ε for the enzyme of 26.6 ±
0.2‰. Both forms of eukNR imparted near equivalent fractionation
on N and O isotopes. The increase in <sup>18</sup>O/<sup>16</sup>O
versus the increase in <sup>15</sup>N/<sup>14</sup>N (relative to
their natural abundances) was 0.96 ± 0.01 for NADPH eukNR and
1.09 ± 0.03 for NADH eukNR. These results are the first reliable
measurements of the coupled N and O isotope effects for any form of
eukNR. They support the prevailing view that intracellular reduction
by eukNR is the dominant step in isotope fractionation during nitrate
assimilation and that it drives the <sup>18</sup>ε:<sup>15</sup>ε ≈ 1 observed in phytoplankton cultures, suggesting
that this O-to-N isotope signature will apply broadly in the environment.
Our measured <sup>15</sup>ε and <sup>18</sup>ε may represent
the intrinsic isotope effects for eukNR-mediated N–O bond rupture,
a potential constraint on the nature of the enzyme’s transition
state