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-NHasenand L-NHase, respectively), depending on the inducer. The incorporation of cobalt into L-NHase has beennfound to depend on the R-subunit exchange between cobalt-free L-NHase (apo-L-NHase) and its cobalt-ncontaining mediator, NhlAE (holo-NhlAE), this novel mode of post-translational maturation having beennnamed self-subunit swapping and NhlE having been recognized as a self-subunit swapping chaperone. Wendiscovered an H-NHase maturation mediator, NhhAG, consisting of NhhG and the R-subunit of H-NHase.nThe incorporation of cobalt into H-NHase was confirmed to be dependent on self-subunit swapping. For thenfirst time, particles larger than apo-H-NHase were observed during the swapping process via dynamic lightnscattering measurements, suggesting the formation of intermediate complexes. On the basis of these findings,nwe initially proposed a possible mechanism for self-subunit swapping. Electron paramagnetic resonancenanalysis demonstrated that the coordination environment of a cobalt ion in holo-NhhAG is subtly differentnfrom that inH-NHase. Cobalt is inserted into cobalt-free NhhAG(apo-NhhAG) but not into apo-H-NHase,nsuggesting that NhhG functions not only as a self-subunit swapping chaperone but also as a metallochaper-none. In addition, R-subunit swapping did not occur between apo-L-NHase and holo-NhhAGor between apo-nH-NHase and holo-NhlAE in vitro. These findings revealed that self-subunit swapping is a subunit-specificnreaction.