X-Ray Crystal Structure of a Mutant Assimilatory Nitrite Reductase That Shows Sulfite Reductase-Like Activity

Assimilatory nitrite reductase (aNiR) reduces nitrite ions (NO2-) to ammonium ions (NH4+), whereas assimilatory sulfite reductase reduces sulfite (SO32-) to hydrogen sulfide (HS-). Although aNiR can also reduce SO32-, its activity is much lower than when NO2- is reduced as the substrate. To increase the SO32--reduction activity of aNiR, we performed a N226K mutation of Nii3, a representative aNiR. The resulting Nii3-N226K variant could bind non-native targets, SO32-, and HCO3-, in addition to its native target, i.e., NO2-. We have determined the high-resolution structure of Nii3-N226K in its apo-state and in complex with SO32-, NO2-, and HCO3-. This analysis revealed conformational changes of Lys226 and the adjacent Lys224 upon binding of SO32-, but not NO2-. In contrast, HCO3- binding induced a conformational change at Arg179. After replacing Asn226 with a positively charged Lys, aNiR showed affinity for several anions. A comparison of all ligand-bound structures for Nii3-N226K revealed that structural changes in the active site depend on the size of the substrate.