title: Homologous and heterologous overexpression in Clostridium acetobutylicum and characterization of purified clostridial and algal Fe-only hydrogenases with high specific activities.
abstract: Clostridium acetobutylicum ATCC 824 was selected for the homologous overexpression of its Fe-only hydrogenase and for the heterologous expressions of the Chlamydomonas reinhardtii and Scenedesmus obliquus HydA1 Fe-only hydrogenases. The three Strep tag II-tagged Fe-only hydrogenases were isolated with high specific activities by two-step column chromatography. The purified algal hydrogenases evolve hydrogen with rates of around 700 micromol H(2) min(-1) mg(-1), while HydA from C. acetobutylicum (HydA(Ca)) shows the highest activity (5,522 micromol H(2) min(-1) mg(-1)) in the direction of hydrogen uptake. Further, kinetic parameters and substrate specificity were reported. An electron paramagnetic resonance (EPR) analysis of the thionin-oxidized HydA(Ca) protein indicates a characteristic rhombic EPR signal that is typical for the oxidized H cluster of Fe-only hydrogenases.
authors.affiliation: Laboratoire de Biotechnologie-Bioprocédés, UMR CNRS 5504, UMR INRA 792, INSA, 135 Avenue de Rangueil, 31077 Toulouse Cédex 4, France. girbal@insa-toulouse.fr
authors.forename: Laurence
authors.initials: L
authors.lastname: Girbal
authors.forename: Gregory
authors.initials: G
authors.lastname: von Abendroth
authors.forename: Martin
authors.initials: M
authors.lastname: Winkler
authors.forename: Paul M C
authors.initials: PM
authors.lastname: Benton
authors.forename: Isabelle
authors.initials: I
authors.lastname: Meynial-Salles
authors.forename: Christian
authors.initials: C
authors.lastname: Croux
authors.forename: John W
authors.initials: JW
authors.lastname: Peters
authors.forename: Thomas
authors.initials: T
authors.lastname: Happe
authors.forename: Philippe
authors.initials: P
authors.lastname: Soucaille
mesh_terms: D000460:Chlorophyta / Q000201:enzymology*; D046969:Clostridium acetobutylicum / Q000235:genetics*; D004578:Electron Spin Resonance Spectroscopy; D006864:Hydrogenase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D007506:Iron-Sulfur Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis*
chemical_list: D007506:Iron-Sulfur Proteins; D011994:Recombinant Proteins; C118830:iron hydrogenase; D006864:Hydrogenase