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Heterologous expression and maturation of an NADP-dependent [NiFe]-hydrogenase: a key enzyme in biofuel production.

Sun J, Hopkins RC, Jenney FE, McTernan PM, Adams MW - PLoS ONE (2010)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia, United States of America.

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.

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Synthesis of P. furiosus soluble hydrogenase I (SHI) and the plasmid constructs used for recombinant production.(a) Maturation pathway for the [NiFe] catalytic site of P. furiosus SHI is based on that proposed for E. coli hydrogenase 3 [10]. HypE and HypF generate thiocyanate in an ATP-dependent process from carbamoyl phosphate (CP). The source of the CO ligand is unknown. The HypCD complex binds the iron atom containing the CN and CO ligands and donates it to the catalytic subunit (PF0894). The Ni atom is provided by the HypAB-SlyD complex in a GTP-dependent reaction. The Ni and Fe atoms in the [NiFe] site are coordinated to PF0894 by the S atoms of four of its Cys residues (indicated by S). Activation of PF0894 requires cleavage of four C-terminal residues (-VVRL) catalyzed by a newly identified peptidase, FrxA (and to a much lesser extent by HycI). It is not known how the biosynthesis of PF0894 is coordinated with that of the other three subunits (PF0891, PF0892 and PF0893) to give catalytically-active SHI. The predicted cofactor content of mature heterotetrameric SHI [11] is shown together with their roles in the reversible production of hydrogen gas from NADPH. The abbreviations used are: CP, carbamoyl phosphate; CO, carbonyl ligand; CN, cyano ligand; FAD, flavin adenine dinucleotide. (b) The expression plasmids utilized for production of SHI in E. coli strain MW4W. The antibiotics refer to the selective marker on each plasmid: Amp, Ampicillin; Sm, Streptomycin; Kan, Kanamycin; Cam, Chloramphenicol.
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pone-0010526-g001: Synthesis of P. furiosus soluble hydrogenase I (SHI) and the plasmid constructs used for recombinant production.(a) Maturation pathway for the [NiFe] catalytic site of P. furiosus SHI is based on that proposed for E. coli hydrogenase 3 [10]. HypE and HypF generate thiocyanate in an ATP-dependent process from carbamoyl phosphate (CP). The source of the CO ligand is unknown. The HypCD complex binds the iron atom containing the CN and CO ligands and donates it to the catalytic subunit (PF0894). The Ni atom is provided by the HypAB-SlyD complex in a GTP-dependent reaction. The Ni and Fe atoms in the [NiFe] site are coordinated to PF0894 by the S atoms of four of its Cys residues (indicated by S). Activation of PF0894 requires cleavage of four C-terminal residues (-VVRL) catalyzed by a newly identified peptidase, FrxA (and to a much lesser extent by HycI). It is not known how the biosynthesis of PF0894 is coordinated with that of the other three subunits (PF0891, PF0892 and PF0893) to give catalytically-active SHI. The predicted cofactor content of mature heterotetrameric SHI [11] is shown together with their roles in the reversible production of hydrogen gas from NADPH. The abbreviations used are: CP, carbamoyl phosphate; CO, carbonyl ligand; CN, cyano ligand; FAD, flavin adenine dinucleotide. (b) The expression plasmids utilized for production of SHI in E. coli strain MW4W. The antibiotics refer to the selective marker on each plasmid: Amp, Ampicillin; Sm, Streptomycin; Kan, Kanamycin; Cam, Chloramphenicol.

Mentions: All [NiFe]-hydrogenases are comprised of at least two subunits, one of which (the large subunit, or LSU) contains the [NiFe] catalytic site, while the other (the small subunit, or SSU) contains three iron-sulfur (FeS) clusters. These clusters transfer electrons from an external electron donor to the [NiFe] active site in the LSU for reduction of protons. The assembly of the [NiFe] site and maturation of the enzyme requires the concerted reactions of multiple gene products. The facultative anaerobe E. coli contains three membrane-bound hydrogenases, and elegant studies by Böck and coworkers have shown that the assembly of one of them (HYD3) requires the participation of eight accessory proteins (encoded by hypA-hypF, hycI, and slyD, Fig. 1a) [9]. The final step of hydrogenase maturation requires a specific protease (HycI) that removes a peptide of ≤20 residues from the C-terminal of the LSU, allowing the protein to fold around the [NiFe] active site [12]. In contrast, it appears that only three maturation proteins are required for the production of a functional [FeFe]-hydrogenase [13].


Heterologous expression and maturation of an NADP-dependent [NiFe]-hydrogenase: a key enzyme in biofuel production.

Sun J, Hopkins RC, Jenney FE, McTernan PM, Adams MW - PLoS ONE (2010)

Synthesis of P. furiosus soluble hydrogenase I (SHI) and the plasmid constructs used for recombinant production.(a) Maturation pathway for the [NiFe] catalytic site of P. furiosus SHI is based on that proposed for E. coli hydrogenase 3 [10]. HypE and HypF generate thiocyanate in an ATP-dependent process from carbamoyl phosphate (CP). The source of the CO ligand is unknown. The HypCD complex binds the iron atom containing the CN and CO ligands and donates it to the catalytic subunit (PF0894). The Ni atom is provided by the HypAB-SlyD complex in a GTP-dependent reaction. The Ni and Fe atoms in the [NiFe] site are coordinated to PF0894 by the S atoms of four of its Cys residues (indicated by S). Activation of PF0894 requires cleavage of four C-terminal residues (-VVRL) catalyzed by a newly identified peptidase, FrxA (and to a much lesser extent by HycI). It is not known how the biosynthesis of PF0894 is coordinated with that of the other three subunits (PF0891, PF0892 and PF0893) to give catalytically-active SHI. The predicted cofactor content of mature heterotetrameric SHI [11] is shown together with their roles in the reversible production of hydrogen gas from NADPH. The abbreviations used are: CP, carbamoyl phosphate; CO, carbonyl ligand; CN, cyano ligand; FAD, flavin adenine dinucleotide. (b) The expression plasmids utilized for production of SHI in E. coli strain MW4W. The antibiotics refer to the selective marker on each plasmid: Amp, Ampicillin; Sm, Streptomycin; Kan, Kanamycin; Cam, Chloramphenicol.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2865534&req=5

pone-0010526-g001: Synthesis of P. furiosus soluble hydrogenase I (SHI) and the plasmid constructs used for recombinant production.(a) Maturation pathway for the [NiFe] catalytic site of P. furiosus SHI is based on that proposed for E. coli hydrogenase 3 [10]. HypE and HypF generate thiocyanate in an ATP-dependent process from carbamoyl phosphate (CP). The source of the CO ligand is unknown. The HypCD complex binds the iron atom containing the CN and CO ligands and donates it to the catalytic subunit (PF0894). The Ni atom is provided by the HypAB-SlyD complex in a GTP-dependent reaction. The Ni and Fe atoms in the [NiFe] site are coordinated to PF0894 by the S atoms of four of its Cys residues (indicated by S). Activation of PF0894 requires cleavage of four C-terminal residues (-VVRL) catalyzed by a newly identified peptidase, FrxA (and to a much lesser extent by HycI). It is not known how the biosynthesis of PF0894 is coordinated with that of the other three subunits (PF0891, PF0892 and PF0893) to give catalytically-active SHI. The predicted cofactor content of mature heterotetrameric SHI [11] is shown together with their roles in the reversible production of hydrogen gas from NADPH. The abbreviations used are: CP, carbamoyl phosphate; CO, carbonyl ligand; CN, cyano ligand; FAD, flavin adenine dinucleotide. (b) The expression plasmids utilized for production of SHI in E. coli strain MW4W. The antibiotics refer to the selective marker on each plasmid: Amp, Ampicillin; Sm, Streptomycin; Kan, Kanamycin; Cam, Chloramphenicol.
Mentions: All [NiFe]-hydrogenases are comprised of at least two subunits, one of which (the large subunit, or LSU) contains the [NiFe] catalytic site, while the other (the small subunit, or SSU) contains three iron-sulfur (FeS) clusters. These clusters transfer electrons from an external electron donor to the [NiFe] active site in the LSU for reduction of protons. The assembly of the [NiFe] site and maturation of the enzyme requires the concerted reactions of multiple gene products. The facultative anaerobe E. coli contains three membrane-bound hydrogenases, and elegant studies by Böck and coworkers have shown that the assembly of one of them (HYD3) requires the participation of eight accessory proteins (encoded by hypA-hypF, hycI, and slyD, Fig. 1a) [9]. The final step of hydrogenase maturation requires a specific protease (HycI) that removes a peptide of ≤20 residues from the C-terminal of the LSU, allowing the protein to fold around the [NiFe] active site [12]. In contrast, it appears that only three maturation proteins are required for the production of a functional [FeFe]-hydrogenase [13].

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia, United States of America.

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.

Show MeSH
Related in: MedlinePlus