A membrane‐bound [NiFe]‐hydrogenase large subunit precursor whose C‐terminal extension is not essential for cofactor incorporation but guarantees optimal maturation
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 extensi...
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doaj-442e21d0a70a4dd0817519a13edb085c2020-11-25T03:31:11ZengWileyMicrobiologyOpen2045-88272020-06-01961197120610.1002/mbo3.1029A membrane‐bound [NiFe]‐hydrogenase large subunit precursor whose C‐terminal extension is not essential for cofactor incorporation but guarantees optimal maturationSven Hartmann0Stefan Frielingsdorf1Giorgio Caserta2Oliver Lenz3Institut für Chemie Physikalische Chemie Technische Universität Berlin Berlin GermanyInstitut für Chemie Physikalische Chemie Technische Universität Berlin Berlin GermanyInstitut für Chemie Physikalische Chemie Technische Universität Berlin Berlin GermanyInstitut für Chemie Physikalische Chemie Technische Universität Berlin Berlin GermanyAbstract [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.https://doi.org/10.1002/mbo3.1029chemolithotrophycofactor assemblyhydrogenmetalloenzymenickelTat transport |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Sven Hartmann Stefan Frielingsdorf Giorgio Caserta Oliver Lenz |
spellingShingle |
Sven Hartmann Stefan Frielingsdorf Giorgio Caserta Oliver Lenz A membrane‐bound [NiFe]‐hydrogenase large subunit precursor whose C‐terminal extension is not essential for cofactor incorporation but guarantees optimal maturation MicrobiologyOpen chemolithotrophy cofactor assembly hydrogen metalloenzyme nickel Tat transport |
author_facet |
Sven Hartmann Stefan Frielingsdorf Giorgio Caserta Oliver Lenz |
author_sort |
Sven Hartmann |
title |
A membrane‐bound [NiFe]‐hydrogenase large subunit precursor whose C‐terminal extension is not essential for cofactor incorporation but guarantees optimal maturation |
title_short |
A membrane‐bound [NiFe]‐hydrogenase large subunit precursor whose C‐terminal extension is not essential for cofactor incorporation but guarantees optimal maturation |
title_full |
A membrane‐bound [NiFe]‐hydrogenase large subunit precursor whose C‐terminal extension is not essential for cofactor incorporation but guarantees optimal maturation |
title_fullStr |
A membrane‐bound [NiFe]‐hydrogenase large subunit precursor whose C‐terminal extension is not essential for cofactor incorporation but guarantees optimal maturation |
title_full_unstemmed |
A membrane‐bound [NiFe]‐hydrogenase large subunit precursor whose C‐terminal extension is not essential for cofactor incorporation but guarantees optimal maturation |
title_sort |
membrane‐bound [nife]‐hydrogenase large subunit precursor whose c‐terminal extension is not essential for cofactor incorporation but guarantees optimal maturation |
publisher |
Wiley |
series |
MicrobiologyOpen |
issn |
2045-8827 |
publishDate |
2020-06-01 |
description |
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. |
topic |
chemolithotrophy cofactor assembly hydrogen metalloenzyme nickel Tat transport |
url |
https://doi.org/10.1002/mbo3.1029 |
work_keys_str_mv |
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