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A membrane‐bound [NiFe]‐hydrogenase large subunit precursor whose C‐terminal extension is not essential for cofactor incorporation but guarantees optimal maturation
- Source :
- MicrobiologyOpen, Vol 9, Iss 6, Pp 1197-1206 (2020), MicrobiologyOpen
- Publication Year :
- 2020
- Publisher :
- Wiley, 2020.
-
Abstract
- [NiFe]‐hydrogenases catalyze the reversible conversion of molecular hydrogen into protons end electrons. This reaction takes place at a NiFe(CN)2(CO) cofactor located in the large subunit of the bipartite hydrogenase module. The corresponding apo‐protein carries usually a C‐terminal extension that is cleaved off by a specific endopeptidase as soon as the cofactor insertion has been accomplished by the maturation machinery. This process triggers complex formation with the small, electron‐transferring subunit of the hydrogenase module, revealing catalytically active enzyme. The role of the C‐terminal extension in cofactor insertion, however, remains elusive. We have addressed this problem by using genetic engineering to remove the entire C‐terminal extension from the apo‐form of the large subunit of the membrane‐bound [NiFe]‐hydrogenase (MBH) from Ralstonia eutropha. Unexpectedly, the MBH holoenzyme derived from this precleaved large subunit was targeted to the cytoplasmic membrane, conferred H2‐dependent growth of the host strain, and the purified protein showed exactly the same catalytic activity as native MBH. The only difference was a reduced hydrogenase content in the cytoplasmic membrane. These results suggest that in the case of the R. eutropha MBH, the C‐terminal extension is dispensable for cofactor insertion and seems to function only as a maturation facilitator.<br />The apo‐forms of [NiFe]‐hydrogenase large subunits are usually synthesized with a C‐terminal peptide extension that is proteolytically cleaved off upon incorporation of the catalytic metal center. Although to a limited amount, an artificially precleaved large subunit, which was deleted for the C‐terminal extension by genetic engineering, still received the active site components delivered by the dedicated maturation machinery. This suggests that the C‐terminal extension optimizes metal center incorporation, but is not essential for the formation of catalytically active [NiFe]‐hydrogenase.
- Subjects :
- Carbon Monoxide
Cyanides
metalloenzyme
Iron
lcsh:QR1-502
Membrane Proteins
Original Articles
cofactor assembly
lcsh:Microbiology
Protein Subunits
nickel
Hydrogenase
Catalytic Domain
hydrogen
Escherichia coli
541 Physikalische Chemie
Original Article
Cupriavidus necator
ddc:541
Tat transport
Genetic Engineering
Plasmids
chemolithotrophy
Subjects
Details
- Language :
- English
- ISSN :
- 20458827
- Volume :
- 9
- Issue :
- 6
- Database :
- OpenAIRE
- Journal :
- MicrobiologyOpen
- Accession number :
- edsair.doi.dedup.....dcf85fee353223e65f82b78e77ad1aca