Farmer, D.A., Brindley, A.A., Hitchcock, A. et al. (6 more authors) (2019) The ChlD subunit links the motor and porphyrin binding subunits of magnesium chelatase. Biochemical Journal, 476 (13). pp. 1875-1887. ISSN 0264-6021
Abstract
Magnesium chelatase initiates chlorophyll biosynthesis, catalysing the MgATP2- dependent insertion of a Mg2+ ion into protoporphyin IX. The catalytic core of this large enzyme complex consists of three subunits: Bch/ChlI, Bch/ChlD and Bch/ChlH (in bacteriochlorophyll and chlorophyll producing species respectively). The D and I subunits are members of the AAA+ (ATPases associated with various cellular activities) superfamily of enzymes, and they form a complex that binds to H, the site of metal ion insertion. In order to investigate the physical coupling between ChlID and ChlH in vivo and in vitro , ChlD was FLAG-tagged in the cyanobacterium Synechocystis sp. PCC 6803 and co-immunoprecipitation experiments showed interactions with both ChlI and ChlH. Co-production of recombinant ChlD and ChlH in Escherichia coli yielded a ChlDH. Quantitative analysis using microscale thermophoresis (MST) showed magnesium-dependent binding ( K d 331 ± 58 nM) between ChlD and H. The physical basis for a ChlD-H interaction was investigated using chemical crosslinking coupled with mass spectrometry (XL-MS), together with modifications that either truncate ChlD or modify single residues. We found that the C-terminal integrin I domain of ChlD governs association with ChlH, the Mg2+ dependence of which also mediates the cooperative response of the Synechocystis chelatase to magnesium. Our work, showing the interaction site between the AAA+ motor and the chelatase domain of magnesium chelatase, will be essential for understanding how free energy from the hydrolysis of ATP on the AAA+ ChlI subunit is transmitted via the bridging subunit ChlD to the active site on ChlH.
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Copyright, Publisher and Additional Information: | ©2019 The Author(s). This is an author-produced version of a paper subsequently published in Biochemical Journal. Uploaded in accordance with the publisher's self-archiving policy. | ||||||
Keywords: | magnesium chelatase; ATPase; ATPases associated with diverse cellular activities (AAA); enzyme catalysis; protein-protein interaction; porphyrin; thermophoresis; cross-linking | ||||||
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Institution: | The University of Sheffield | ||||||
Academic Units: | The University of Sheffield > Faculty of Engineering (Sheffield) > Department of Chemical and Biological Engineering (Sheffield) The University of Sheffield > Faculty of Science (Sheffield) > Department of Chemistry (Sheffield) The University of Sheffield > Faculty of Science (Sheffield) > School of Biosciences (Sheffield) > Department of Molecular Biology and Biotechnology (Sheffield) |
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Depositing User: | Symplectic Sheffield | ||||||
Date Deposited: | 12 Jun 2019 12:18 | ||||||
Last Modified: | 07 Dec 2021 09:11 | ||||||
Status: | Published | ||||||
Publisher: | Portland Press | ||||||
Refereed: | Yes | ||||||
Identification Number: | https://doi.org/10.1042/bcj20190095 | ||||||
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