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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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Analysis of the P. furiosus maturation proteins required to produce active rSHI in E. coli.The specific activities are shown for rSHI in cell extracts of E. coli resulting from the co-expression of different Pf processing genes in E. coli MW1001. Bars indicate MV-linked specific activity (µmol H2 evolved min−1 mg−1). All cell extracts were heated for 30 min at 80°C prior to assay.
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pone-0010526-g005: Analysis of the P. furiosus maturation proteins required to produce active rSHI in E. coli.The specific activities are shown for rSHI in cell extracts of E. coli resulting from the co-expression of different Pf processing genes in E. coli MW1001. Bars indicate MV-linked specific activity (µmol H2 evolved min−1 mg−1). All cell extracts were heated for 30 min at 80°C prior to assay.

Mentions: In order to determine the minimum number of Pf genes needed for assembly of an active form of SHI in E. coli, different Pf accessory genes and plasmids were omitted from the complete heterologous expression system (Fig. 4a and Fig. 5). The results show that maximal recombinant production of functional SHI, as measured by the specific activity in the cell-free extract at 80°C, requires, in addition to the structural genes (PF0891-PF0894), coexpression of only the plasmid containing frxA (PF0975) (Fig. 4a). FrxA is, therefore, the protease required to process SHI by removing the four C-terminal –VVRL residues (Fig. 1a). Low activity was detected if HycI was present, but there was no activity if both proteases were absent, showing that E. coli proteases cannot process SHI. The E. coli processing enzymes HypABCD and HypEF appear to assemble a functional SHI whose catalytic subunit (PF0894) lacks C-terminal processing. This was confirmed both in vivo (Fig. 5) and using an in vitro assay (Fig. 4b) where extracts from E. coli cells expressing either only the four structural genes for SHI or only frxA were mixed and SHI activity was measured after incubation at 80°C. As expected, much lower SHI activity was obtained if hycI replaced frxA. Surprisingly, unprocessed SHI (where PF0894 lacks C-terminal cleavage) appears to be stable in E. coli, which will be of great utility to those interested in studying the mechanism of assembly of the [NiFe] site. The minimal expression system in E. coli therefore contains five Pf genes encoding only SHI and FrxA.


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)

Analysis of the P. furiosus maturation proteins required to produce active rSHI in E. coli.The specific activities are shown for rSHI in cell extracts of E. coli resulting from the co-expression of different Pf processing genes in E. coli MW1001. Bars indicate MV-linked specific activity (µmol H2 evolved min−1 mg−1). All cell extracts were heated for 30 min at 80°C prior to assay.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2865534&req=5

pone-0010526-g005: Analysis of the P. furiosus maturation proteins required to produce active rSHI in E. coli.The specific activities are shown for rSHI in cell extracts of E. coli resulting from the co-expression of different Pf processing genes in E. coli MW1001. Bars indicate MV-linked specific activity (µmol H2 evolved min−1 mg−1). All cell extracts were heated for 30 min at 80°C prior to assay.
Mentions: In order to determine the minimum number of Pf genes needed for assembly of an active form of SHI in E. coli, different Pf accessory genes and plasmids were omitted from the complete heterologous expression system (Fig. 4a and Fig. 5). The results show that maximal recombinant production of functional SHI, as measured by the specific activity in the cell-free extract at 80°C, requires, in addition to the structural genes (PF0891-PF0894), coexpression of only the plasmid containing frxA (PF0975) (Fig. 4a). FrxA is, therefore, the protease required to process SHI by removing the four C-terminal –VVRL residues (Fig. 1a). Low activity was detected if HycI was present, but there was no activity if both proteases were absent, showing that E. coli proteases cannot process SHI. The E. coli processing enzymes HypABCD and HypEF appear to assemble a functional SHI whose catalytic subunit (PF0894) lacks C-terminal processing. This was confirmed both in vivo (Fig. 5) and using an in vitro assay (Fig. 4b) where extracts from E. coli cells expressing either only the four structural genes for SHI or only frxA were mixed and SHI activity was measured after incubation at 80°C. As expected, much lower SHI activity was obtained if hycI replaced frxA. Surprisingly, unprocessed SHI (where PF0894 lacks C-terminal cleavage) appears to be stable in E. coli, which will be of great utility to those interested in studying the mechanism of assembly of the [NiFe] site. The minimal expression system in E. coli therefore contains five Pf genes encoding only SHI and FrxA.

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