Unique Biogenesis of High-Molecular Mass Multimeric Metalloenzyme Nitrile Hydratase: Intermediates and a Proposed Mechanism for Self-Subunit Swapping Maturation

摘要

Rhodococcus rhodochrous J1 produces high- and low-molecular mass nitrile hydratases (H-NHase and L-NHase, respectively), depending on the inducer. The incorporation of cobalt into L-NHase has been found to depend on the alpha-subunit exchange between cobalt-free L-NHase (apo-L-NHase) and its cobalt-containing mediator, NhIAE (holo-NhIAE), this novel mode of post-translational maturation having been named self-subunit swapping and NhIE having been recognized as a self-subunit swapping chaperone. We discovered an H-NHase maturation mediator. NhhAG, consisting of NhhG and the alpha-subunit of H-NHase. The incorporation of cobalt into H-NHase was confirmed to be dependent on self-subunit swapping. For the first time, particles larger than apo-H-NHase were observed during the swapping process via dynamic light scattering measurements, suggesting the formation of intermediate complexes. On the basis of these findings, we initially proposed a possible mechanism for self-subunit swapping. Electron paramagnetic resonance analysis demonstrated that the coordination environment of a cobalt ion in holo-NhhAG is subtly different from that in H-NHase. Cobalt is inserted into cobalt-free NhhAG (apo-NhhAG) but not into apo-H-NHase, suggesting that NhhG functions not only as a self-subunit swapping chaperone but also as a metallochaperone. In addition, alpha-subunit swapping did not occur between apo-L-NHase and holo-NhhAG or between apo-H-NHase and holo-NhIAE in vitro. These findings revealed that self-subunit swapping is a subunit-specific reaction.