title: Construction of a subunit-fusion nitrile hydratase and discovery of an innovative metal ion transfer pattern.
abstract: Metallochaperones are metal-binding proteins designed to deliver the appropriate metal to a target protein. The metal is usually transferred between different proteins. In this study, we discovered that metal was transferred between the same subunit of a mutant nitrile hydratase (NHase). Various "activator proteins" mediate the trafficking of metal ions into NHases. We constructed fusion NHases by fusing the β- and α-subunits and/or the "activator proteins" of the NHase from Pseudomonas putida. The fusion NHases exhibited higher thermostability and tolerance to high concentrations of the product amide. The mechanism of the cobalt incorporation changed from a self-subunit swapping pattern to an apoprotein-specific molecular chaperone pattern in vivo and a metallochaperone pattern in vitro. Notably, the cobalt transfer occurred between the same α-subunit in the metallochaperone pattern. These results not only demonstrated the superiority of fusion-type NHases, but also revealed an innovative metal ion transfer pattern in metalloprotein biosynthesis.
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China.
authors.forename: Yuanyuan
authors.initials: Y
authors.lastname: Xia
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China.
authors.forename: Wenjing
authors.initials: W
authors.lastname: Cui
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China.
authors.forename: Zhongmei
authors.initials: Z
authors.lastname: Liu
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China.
authors.forename: Li
authors.initials: L
authors.lastname: Zhou
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China.
authors.forename: Youtian
authors.initials: Y
authors.lastname: Cui
authors.affiliation: Institute of Applied Biochemistry, and Graduate School of Life and Environmental Sciences, The University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan.
authors.forename: Michihiko
authors.initials: M
authors.lastname: Kobayashi
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China.
authors.forename: Zhemin
authors.initials: Z
authors.lastname: Zhou
mesh_terms: D001692:Biological Transport; D003035:Cobalt; D004789:Enzyme Activation; D006836:Hydro-Lyases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D007477:Ions / Q000378:metabolism*; D013058:Mass Spectrometry; D008670:Metals / Q000378:metabolism*; D008954:Models, Biological; D008970:Molecular Weight; D011485:Protein Binding; D021122:Protein Subunits / Q000235:genetics* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins
chemical_list: D007477:Ions; D008670:Metals; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D003035:Cobalt; D006836:Hydro-Lyases; C048831:nitrile hydratase