title: High yield purification of a tagged cytoplasmic [NiFe]-hydrogenase and a catalytically-active nickel-free intermediate form.
abstract: The cytoplasmic [NiFe]-hydrogenase I (SHI) of the hyperthermophile Pyrococcus furiosus evolves hydrogen gas (H2) from NADPH. It has been previously used for biohydrogen production from sugars using a mixture of enzymes in an in vitro cell-free synthetic pathway. The theoretical yield (12 H2/glucose) is three times greater than microbial fermentation (4 H2/glucose), making the in vitro approach very promising for large scale biohydrogen production. Further development of this process at an industrial scale is limited by the availability of the H2-producing SHI. To overcome the obstacles of the complex biosynthetic and maturation pathway for the [NiFe] site of SHI, the four gene operon encoding the enzyme was overexpressed in P. furiosus and included a polyhistidine affinity tag. The one-step purification resulted in a 50-fold increase in yield compared to the four-step purification procedure for the native enzyme. A trimeric form was also identified that lacked the [NiFe]-catalytic subunit but catalyzed NADPH oxidation with a specific activity similar to that of the tetrameric form. The presence of an active trimeric intermediate confirms the proposed maturation pathway where, in the terminal step, the NiFe-containing catalytic subunit assembles with NADPH-oxidizing trimeric form to give the active holoenzyme.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602, USA.
authors.forename: Sanjeev K
authors.initials: SK
authors.lastname: Chandrayan
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602, USA.
authors.forename: Chang-Hao
authors.initials: CH
authors.lastname: Wu
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602, USA.
authors.forename: Patrick M
authors.initials: PM
authors.lastname: McTernan
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602, USA. Electronic address: adams@bmb.uga.edu.
authors.forename: Michael W W
authors.initials: MW
authors.lastname: Adams
mesh_terms: D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D055162:Biocatalysis; D020134:Catalytic Domain; D003593:Cytoplasm / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D006864:Hydrogenase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D009249:NADP / Q000378:metabolism; D009532:Nickel / Q000378:metabolism; D055503:Protein Multimerization; D020027:Pyrococcus furiosus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics
chemical_list: D019843:Archaeal Proteins; D009249:NADP; D009532:Nickel; C043296:nickel-iron hydrogenase; D006864:Hydrogenase
keywords: Affinity purification; Enzyme maturation; Hydrogen; Hydrogenase; Pyrococcus