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201018_manuscript_nf.pdf (2.08 MB)

Biochemical Characterization of a Group-5 Soluble Diiron Monooxygenase Hydroxylase and Related Chaperonin-like Component

preprint
submitted on 19.10.2020, 15:20 and posted on 20.10.2020, 09:36 by Chihiro Inoue, Yoshitaka Abe, Nobutaka Fujieda

Recently, the functional expression of group-5 hydroxylase component (MimA and MimC) in Escherichia coli along with its related chaperonin-like component (MimG) was reported by Furuya and Kino. In this study, we report the purification via a heterologous expression system and the biochemical characterization of MimAC, the complex of MimA and MimC and MimG to understand their exact roles. MimAC and MimG were fused with His-tags and purified using affinity chromatography in a homogenous state on SDS-PAGE. Blue native PAGE demonstrated that the quaternary structure of MimG was almost identical to that of chaperonin GroEL, indicating that its function was also similar to GroEL. Size-exclusion chromatography and ICP-AES analysis demonstrated that MimAC was assembled in the dimer of two sort of subunits and exhibited two iron atoms and at least one zinc atom per two subunits. This result indicated that MimAC possessed a dinuclear iron center, similar to other soluble diiron monooxygenase hydroxylases.

Funding

JST PRESTO (JPMJPR16S9)

Tonen General Sekiyu Research/Development Encouragement & Scholarship Foundation

History

Email Address of Submitting Author

fujieda@biochem.osakafu-u.ac.jp

Institution

Osaka Prefecture University

Country

Japan

ORCID For Submitting Author

0000-0003-1045-6063

Declaration of Conflict of Interest

The authors have declared that no competing interests exist.

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