Metal chaperone, NhpC, involved in the metallocenter biosynthesis of nitrile hydratase

The Journal of General and Applied Microbiology

article

Yoshiteru Hashimoto¹  Yuko Ube¹  Shiori Doi¹  Takuto Kumano¹  Michihiko Kobayashi¹ 

[1] Institute of Applied Biochemistry and Graduate School of Life and Environmental Sciences, The University of Tsukuba;Microbiology Research Center for Sustainability (MiCS), The University of Tsukuba

关键词: apoenzyme;    holoenzyme;    iron;   

DOI  :  10.2323/jgam.2020.03.003

学科分类：微生物学和免疫学

来源: Applied Microbiology, Molecular and Cellulrar Biosciences Research Foundation

【 摘 要 】

Pseudomonas chlororaphis B23 yields nitrile hydratase (NHase) used for the production of 5-cyanovaleramide at the industrial level. Although the nhpC gene (known as P47K ) located just downstream of the NHase structural genes ( nhpAB ) has been important for efficient NHase expression, the key role of nhpC remains poorly studied. Here, we purified two NHases expressed in the presence and absence of nhpC , respectively, and characterized them. The purified NHase expressed with nhpC proved to be an iron-containing holo-NHase, while the purified one expressed without nhpC was identified as an apo-NHase, which was iron-deficient. These findings indicated that nhpC would play a crucial role in the post-translational incorporation of iron into the NHase active site as a metal chaperone. In the overall amino acid sequence of NhpC, only the N -terminus exhibited similarities to the CobW protein involved in cobalamin biosynthesis, the UreG and HypB proteins essential for the metallocenter biosynthesis of urease and hydrogenase, respectively. NhpC contains a P-loop motif known as a nucleotide-binding site, and Lys23 and Thr24 are conserved in the P-loop motif in NhpC. Expression analysis of NHase formed in the presence of each mutant NhpC (i.e., K23A and T24A) resulted in immunodetectable production of a mutant NhpC and remarkable expression of NHase lacking the enzyme activity. These findings suggested that an intact P-loop containing Lys23 and Thr24 would be essential for the NhpC function in vivo for the post-translational metallocenter assembly of NHase.

【 授权许可】

Unknown