title: Self-subunit swapping occurs in another gene type of cobalt nitrile hydratase.
abstract: Self-subunit swapping is one of the post-translational maturation of the cobalt-containing nitrile hydratase (Co-NHase) family of enzymes. All of these NHases possess a gene organization of <β-subunit> <α-subunit> <activator protein>, which allows the activator protein to easily form a mediatory complex with the α-subunit of the NHase after translation. Here, we discovered that the incorporation of cobalt into another type of Co-NHase, with a gene organization of <α-subunit> <β-subunit> <activator protein>, was also dependent on self-subunit swapping. We successfully isolated a recombinant NHase activator protein (P14K) of Pseudomonas putida NRRL-18668 by adding a Strep-tag N-terminal to the P14K gene. P14K was found to form a complex [α(StrepP14K)(2)] with the α-subunit of the NHase. The incorporation of cobalt into the NHase of P. putida was confirmed to be dependent on the α-subunit substitution between the cobalt-containing α(StrepP14K)(2) and the cobalt-free NHase. Cobalt was inserted into cobalt-free α(StrepP14K)(2) but not into cobalt-free NHase, suggesting that P14K functions not only as a self-subunit swapping chaperone but also as a metallochaperone. In addition, NHase from P. putida was also expressed by a mutant gene that was designed with a <β-subunit> <α-subunit> <P14K> order. Our findings expand the general features of self-subunit swapping maturation.
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Liu
authors.forename: Wenjing
authors.initials: W
authors.lastname: Cui
authors.forename: Yuanyuan
authors.initials: Y
authors.lastname: Xia
authors.forename: Youtian
authors.initials: Y
authors.lastname: Cui
authors.forename: Michihiko
authors.initials: M
authors.lastname: Kobayashi
authors.forename: Zhemin
authors.initials: Z
authors.lastname: Zhou
mesh_terms: D000595:Amino Acid Sequence; D001059:Apoproteins / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D001483:Base Sequence; D003035:Cobalt / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005798:Genes, Bacterial / Q000235:genetics*; D020035:Holoenzymes / Q000235:genetics / Q000378:metabolism; D006836:Hydro-Lyases / Q000737:chemistry / Q000235:genetics*; D056511:Metallochaperones / Q000378:metabolism; D008969:Molecular Sequence Data; D005810:Multigene Family / Q000235:genetics; D011499:Protein Processing, Post-Translational; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics*; D016958:Pseudomonas putida / Q000201:enzymology* / Q000235:genetics*; D013056:Spectrophotometry, Ultraviolet
chemical_list: D001059:Apoproteins; D001426:Bacterial Proteins; D020035:Holoenzymes; D056511:Metallochaperones; D021122:Protein Subunits; D003035:Cobalt; D006836:Hydro-Lyases; C048831:nitrile hydratase