title: Heterologous expression and maturation of an NADP-dependent [NiFe]-hydrogenase: a key enzyme in biofuel production.
abstract: Hydrogen gas is a major biofuel and is metabolized by a wide range of microorganisms. Microbial hydrogen production is catalyzed by hydrogenase, an extremely complex, air-sensitive enzyme that utilizes a binuclear nickel-iron [NiFe] catalytic site. Production and engineering of recombinant [NiFe]-hydrogenases in a genetically-tractable organism, as with metalloprotein complexes in general, has met with limited success due to the elaborate maturation process that is required, primarily in the absence of oxygen, to assemble the catalytic center and functional enzyme. We report here the successful production in Escherichia coli of the recombinant form of a cytoplasmic, NADP-dependent hydrogenase from Pyrococcus furiosus, an anaerobic hyperthermophile. This was achieved using novel expression vectors for the co-expression of thirteen P. furiosus genes (four structural genes encoding the hydrogenase and nine encoding maturation proteins). Remarkably, the native E. coli maturation machinery will also generate a functional hydrogenase when provided with only the genes encoding the hydrogenase subunits and a single protease from P. furiosus. Another novel feature is that their expression was induced by anaerobic conditions, whereby E. coli was grown aerobically and production of recombinant hydrogenase was achieved by simply changing the gas feed from air to an inert gas (N2). The recombinant enzyme was purified and shown to be functionally similar to the native enzyme purified from P. furiosus. The methodology to generate this key hydrogen-producing enzyme has dramatic implications for the production of hydrogen and NADPH as vehicles for energy storage and transport, for engineering hydrogenase to optimize production and catalysis, as well as for the general production of complex, oxygen-sensitive metalloproteins.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia, United States of America.
authors.forename: Junsong
authors.initials: J
authors.lastname: Sun
authors.forename: Robert C
authors.initials: RC
authors.lastname: Hopkins
authors.forename: Francis E
authors.initials: FE
authors.lastname: Jenney
authors.forename: Patrick M
authors.initials: PM
authors.lastname: McTernan
authors.forename: Michael W W
authors.initials: MW
authors.lastname: Adams
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D056804:Biofuels / Q000382:microbiology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006864:Hydrogenase / Q000378:metabolism*; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism*; D010957:Plasmids / Q000737:chemistry / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D020027:Pyrococcus furiosus / Q000201:enzymology*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D001616:beta-Galactosidase / Q000235:genetics
chemical_list: D056804:Biofuels; D011994:Recombinant Proteins; D009249:NADP; C043296:nickel-iron hydrogenase; D006864:Hydrogenase; D001616:beta-Galactosidase
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