title: Unique biogenesis of high-molecular mass multimeric metalloenzyme nitrile hydratase: intermediates and a proposed mechanism for self-subunit swapping maturation.
abstract: 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 α-subunit exchange between cobalt-free L-NHase (apo-L-NHase) and its cobalt-containing mediator, NhlAE (holo-NhlAE), this novel mode of post-translational maturation having been named self-subunit swapping and NhlE having been recognized as a self-subunit swapping chaperone. We discovered an H-NHase maturation mediator, NhhAG, consisting of NhhG and the α-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, α-subunit swapping did not occur between apo-L-NHase and holo-NhhAG or between apo-H-NHase and holo-NhlAE in vitro. These findings revealed that self-subunit swapping is a subunit-specific reaction.
authors.affiliation: Institute of Applied Biochemistry and Graduate School of Life and Environmental Sciences, The University of Tsukuba, Ibaraki, Japan.
authors.forename: Zhemin
authors.initials: Z
authors.lastname: Zhou
authors.forename: Yoshiteru
authors.initials: Y
authors.lastname: Hashimoto
authors.forename: Tianwei
authors.initials: T
authors.lastname: Cui
authors.forename: Yumi
authors.initials: Y
authors.lastname: Washizawa
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Mino
authors.forename: Michihiko
authors.initials: M
authors.lastname: Kobayashi
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism; D003035:Cobalt / Q000378:metabolism; D006836:Hydro-Lyases / Q000737:chemistry* / Q000378:metabolism*; D008667:Metalloproteins / Q000378:metabolism; D008970:Molecular Weight; D055503:Protein Multimerization; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D012240:Rhodococcus / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D008667:Metalloproteins; D021122:Protein Subunits; D003035:Cobalt; D006836:Hydro-Lyases; C048831:nitrile hydratase