title: Atypical effect of temperature tuning on the insertion of the catalytic iron-sulfur center in a recombinant [FeFe]-hydrogenase.
abstract: The expression of recombinant [FeFe]-hydrogenases is an important step for the production of large amount of these enzymes for their exploitation in biotechnology and for the characterization of the protein-metal cofactor interactions. The correct assembly of the organometallic catalytic site, named H-cluster, requires a dedicated set of maturases that must be coexpressed in the microbial hosts or used for in vitro assembly of the active enzymes. In this work, the effect of the post-induction temperature on the recombinant expression of CaHydA [FeFe]-hydrogenase in E. coli is investigated. The results show a peculiar behavior: the enzyme expression is maximum at lower temperatures (20°C), while the specific activity of the purified CaHydA is higher at higher temperature (30°C), as a consequence of improved protein folding and active site incorporation.
authors.affiliation: Department of Life Sciences and Systems Biology, University of Torino, via Accademia Albertina 13, Torino, 10123, Italy.
authors.forename: Simone
authors.initials: S
authors.lastname: Morra
authors.affiliation: Department of Life Sciences and Systems Biology, University of Torino, via Accademia Albertina 13, Torino, 10123, Italy.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Cordara
authors.affiliation: Department of Life Sciences and Systems Biology, University of Torino, via Accademia Albertina 13, Torino, 10123, Italy.
authors.forename: Gianfranco
authors.initials: G
authors.lastname: Gilardi
authors.affiliation: Department of Life Sciences and Systems Biology, University of Torino, via Accademia Albertina 13, Torino, 10123, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Valetti
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D046969:Clostridium acetobutylicum / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006864:Hydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007506:Iron-Sulfur Proteins / Q000737:chemistry; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D007506:Iron-Sulfur Proteins; D011994:Recombinant Proteins; D006864:Hydrogenase
keywords: [FeFe]-hydrogenases; bio-hydrogen; metalloenzyme; recombinant expression