Homologous Expression of a Subcomplex of Pyrococcus furiosus Hydrogenase that Interacts with Pyruvate Ferredoxin Oxidoreductase

Hydrogen gas is an attractive alternative fuel as it is carbon neutral and has higher energy content per unit mass than fossil fuels. The biological enzyme responsible for utilizing molecular hydrogen is hydrogenase, a heteromeric metalloenzyme requiring a complex maturation process to assemble its...

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Main Authors:	Hopkins, R. Christopher, Sun, Junsong, Jenney, Francis E., Chandrayan, Sanjeev K., McTernan, Patrick M., Adams, Michael W. W.
Format:	Online
Language:	English
Published:	Public Library of Science 2011
Online Access:	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3200332/

id	pubmed-3200332
recordtype	oai_dc
spelling	pubmed-32003322011-10-28 Homologous Expression of a Subcomplex of Pyrococcus furiosus Hydrogenase that Interacts with Pyruvate Ferredoxin Oxidoreductase Hopkins, R. Christopher Sun, Junsong Jenney, Francis E. Chandrayan, Sanjeev K. McTernan, Patrick M. Adams, Michael W. W. Research Article Hydrogen gas is an attractive alternative fuel as it is carbon neutral and has higher energy content per unit mass than fossil fuels. The biological enzyme responsible for utilizing molecular hydrogen is hydrogenase, a heteromeric metalloenzyme requiring a complex maturation process to assemble its O2-sensitive dinuclear-catalytic site containing nickel and iron atoms. To facilitate their utility in applied processes, it is essential that tools are available to engineer hydrogenases to tailor catalytic activity and electron carrier specificity, and decrease oxygen sensitivity using standard molecular biology techniques. As a model system we are using hydrogen-producing Pyrococcus furiosus, which grows optimally at 100°C. We have taken advantage of a recently developed genetic system that allows markerless chromosomal integrations via homologous recombination. We have combined a new gene marker system with a highly-expressed constitutive promoter to enable high-level homologous expression of an engineered form of the cytoplasmic NADP-dependent hydrogenase (SHI) of P. furiosus. In a step towards obtaining ‘minimal’ hydrogenases, we have successfully produced the heterodimeric form of SHI that contains only two of the four subunits found in the native heterotetrameric enzyme. The heterodimeric form is highly active (150 units mg−1 in H2 production using the artificial electron donor methyl viologen) and thermostable (t1/2 ∼0.5 hour at 90°C). Moreover, the heterodimer does not use NADPH and instead can directly utilize reductant supplied by pyruvate ferredoxin oxidoreductase from P. furiosus. The SHI heterodimer and POR therefore represent a two-enzyme system that oxidizes pyruvate and produces H2 in vitro without the need for an intermediate electron carrier. Public Library of Science 2011-10-24 /pmc/articles/PMC3200332/ /pubmed/22039508 http://dx.doi.org/10.1371/journal.pone.0026569 Text en Hopkins et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
repository_type	Open Access Journal
institution_category	Foreign Institution
institution	US National Center for Biotechnology Information
building	NCBI PubMed
collection	Online Access
language	English
format	Online
author	Hopkins, R. Christopher Sun, Junsong Jenney, Francis E. Chandrayan, Sanjeev K. McTernan, Patrick M. Adams, Michael W. W.
spellingShingle	Hopkins, R. Christopher Sun, Junsong Jenney, Francis E. Chandrayan, Sanjeev K. McTernan, Patrick M. Adams, Michael W. W. Homologous Expression of a Subcomplex of Pyrococcus furiosus Hydrogenase that Interacts with Pyruvate Ferredoxin Oxidoreductase
author_facet	Hopkins, R. Christopher Sun, Junsong Jenney, Francis E. Chandrayan, Sanjeev K. McTernan, Patrick M. Adams, Michael W. W.
author_sort	Hopkins, R. Christopher
title	Homologous Expression of a Subcomplex of Pyrococcus furiosus Hydrogenase that Interacts with Pyruvate Ferredoxin Oxidoreductase
title_short	Homologous Expression of a Subcomplex of Pyrococcus furiosus Hydrogenase that Interacts with Pyruvate Ferredoxin Oxidoreductase
title_full	Homologous Expression of a Subcomplex of Pyrococcus furiosus Hydrogenase that Interacts with Pyruvate Ferredoxin Oxidoreductase
title_fullStr	Homologous Expression of a Subcomplex of Pyrococcus furiosus Hydrogenase that Interacts with Pyruvate Ferredoxin Oxidoreductase
title_full_unstemmed	Homologous Expression of a Subcomplex of Pyrococcus furiosus Hydrogenase that Interacts with Pyruvate Ferredoxin Oxidoreductase
title_sort	homologous expression of a subcomplex of pyrococcus furiosus hydrogenase that interacts with pyruvate ferredoxin oxidoreductase
description	Hydrogen gas is an attractive alternative fuel as it is carbon neutral and has higher energy content per unit mass than fossil fuels. The biological enzyme responsible for utilizing molecular hydrogen is hydrogenase, a heteromeric metalloenzyme requiring a complex maturation process to assemble its O2-sensitive dinuclear-catalytic site containing nickel and iron atoms. To facilitate their utility in applied processes, it is essential that tools are available to engineer hydrogenases to tailor catalytic activity and electron carrier specificity, and decrease oxygen sensitivity using standard molecular biology techniques. As a model system we are using hydrogen-producing Pyrococcus furiosus, which grows optimally at 100°C. We have taken advantage of a recently developed genetic system that allows markerless chromosomal integrations via homologous recombination. We have combined a new gene marker system with a highly-expressed constitutive promoter to enable high-level homologous expression of an engineered form of the cytoplasmic NADP-dependent hydrogenase (SHI) of P. furiosus. In a step towards obtaining ‘minimal’ hydrogenases, we have successfully produced the heterodimeric form of SHI that contains only two of the four subunits found in the native heterotetrameric enzyme. The heterodimeric form is highly active (150 units mg−1 in H2 production using the artificial electron donor methyl viologen) and thermostable (t1/2 ∼0.5 hour at 90°C). Moreover, the heterodimer does not use NADPH and instead can directly utilize reductant supplied by pyruvate ferredoxin oxidoreductase from P. furiosus. The SHI heterodimer and POR therefore represent a two-enzyme system that oxidizes pyruvate and produces H2 in vitro without the need for an intermediate electron carrier.
publisher	Public Library of Science
publishDate	2011
url	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3200332/
_version_	1611483144199864320

Homologous Expression of a Subcomplex of Pyrococcus furiosus Hydrogenase that Interacts with Pyruvate Ferredoxin Oxidoreductase

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