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Exploring the directionality of Escherichia coli formate hydrogenlyase: a membrane-bound enzyme capable of fixing carbon dioxide to organic acid.

ABSTRACT: During mixed-acid fermentation Escherichia coli produces formate, which is initially excreted out the cell. Accumulation of formate, and dropping extracellular pH, leads to biosynthesis of the formate hydrogenlyase (FHL) complex. FHL consists of membrane and soluble domains anchored within the inner membrane. The soluble domain comprises a [NiFe] hydrogenase and a formate dehydrogenase that link formate oxidation directly to proton reduction with the release of CO₂ and H₂ . Thus, the function of FHL is to oxidize excess formate at low pH. FHL subunits share identity with subunits of the respiratory Complex I. In particular, the FHL membrane domain contains subunits (HycC and HycD) that are homologs of NuoL/M/N and NuoH, respectively, which have been implicated in proton translocation. In this work, strain engineering and new assays demonstrate unequivocally the nonphysiological reverse activity of FHL in vivo and in vitro. Harnessing FHL to reduce CO₂ to formate is biotechnologically important. Moreover, assays for both possible FHL reactions provide opportunities to explore the bioenergetics using biochemical and genetic approaches. Comprehensive mutagenesis of hycC did not identify any single amino acid residues essential for FHL operation. However, the HycD E199, E201, and E203 residues were found to be critically important for FHL function.

SUBMITTER: Pinske C

PROVIDER: S-EPMC5061711 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

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Exploring the directionality of Escherichia coli formate hydrogenlyase: a membrane-bound enzyme capable of fixing carbon dioxide to organic acid.

Pinske Constanze C Sargent Frank F

MicrobiologyOpen 20160502 5

During mixed-acid fermentation Escherichia coli produces formate, which is initially excreted out the cell. Accumulation of formate, and dropping extracellular pH, leads to biosynthesis of the formate hydrogenlyase (FHL) complex. FHL consists of membrane and soluble domains anchored within the inner membrane. The soluble domain comprises a [NiFe] hydrogenase and a formate dehydrogenase that link formate oxidation directly to proton reduction with the release of CO<sub>2</sub> and H<sub>2</sub> . ...[more]

PMID: 27139710

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Project description:Formate dehydrogenase H (FDH-H) and [NiFe]-hydrogenase 3 (Hyd-3) form the catalytic components of the hydrogen-producing formate hydrogenlyase (FHL) complex, which disproportionates formate to H2 and CO2 during mixed acid fermentation in enterobacteria. FHL comprises minimally seven proteins and little is understood about how this complex is assembled. Early studies identified a ferredoxin-like protein, HydN, as being involved in FDH-H assembly into the FHL complex. In order to understand how FDH-H and its small subunit HycB, which is also a ferredoxin-like protein, attach to the FHL complex, the possible roles of HydN and its paralogue, YsaA, in FHL complex stability and assembly were investigated. Deletion of the hycB gene reduced redox dye-mediated FDH-H activity to approximately 10%, abolished FHL-dependent H2-production, and reduced Hyd-3 activity. These data are consistent with HycB being an essential electron transfer component of the FHL complex. The FDH-H activity of the hydN and the ysaA deletion strains was reduced to 59 and 57% of the parental, while the double deletion reduced activity of FDH-H to 28% and the triple deletion with hycB to 1%. Remarkably, and in contrast to the hycB deletion, the absence of HydN and YsaA was without significant effect on FHL-dependent H2-production or total Hyd-3 activity; FDH-H protein levels were also unaltered. This is the first description of a phenotype for the E. coli ysaA deletion strain and identifies it as a novel factor required for optimal redox dye-linked FDH-H activity. A ysaA deletion strain could be complemented for FDH-H activity by hydN and ysaA, but the hydN deletion strain could not be complemented. Introduction of these plasmids did not affect H2 production. Bacterial two-hybrid interactions showed that YsaA, HydN, and HycB interact with each other and with the FDH-H protein. Further novel anaerobic cross-interactions of 10 ferredoxin-like proteins in E. coli were also discovered and described. Together, these data indicate that FDH-H activity measured with the redox dye benzyl viologen is the sum of the FDH-H protein interacting with three independent small subunits and suggest that FDH-H can associate with different redox-protein complexes in the anaerobic cell to supply electrons from formate oxidation.

Project description:Trabulsiella guamensis is a nonpathogenic enterobacterium that was isolated from a vacuum cleaner on the island of Guam. It has one H2-oxidizing Hyd-2-type hydrogenase (Hyd) and encodes an H2-evolving Hyd that is most similar to the uncharacterized Escherichia coli formate hydrogenlyase (FHL-2 Ec ) complex. The T. guamensis FHL-2 (FHL-2 Tg ) complex is predicted to have 5 membrane-integral and between 4 and 5 cytoplasmic subunits. We showed that the FHL-2 Tg complex catalyzes the disproportionation of formate to CO2 and H2 FHL-2 Tg has activity similar to that of the E. coli FHL-1 Ec complex in H2 evolution from formate, but the complex appears to be more labile upon cell lysis. Cloning of the entire 13-kbp FHL-2 Tg operon in the heterologous E. coli host has now enabled us to unambiguously prove FHL-2 Tg activity, and it allowed us to characterize the FHL-2 Tg complex biochemically. Although the formate dehydrogenase (FdhH) gene fdhF is not contained in the operon, the FdhH is part of the complex, and FHL-2 Tg activity was dependent on the presence of E. coli FdhH. Also, in contrast to E. coli, T. guamensis can ferment the alternative carbon source cellobiose, and we further investigated the participation of both the H2-oxidizing Hyd-2 Tg and the H2-forming FHL-2 Tg under these conditions.IMPORTANCE Biological H2 production presents an attractive alternative for fossil fuels. However, in order to compete with conventional H2 production methods, the process requires our understanding on a molecular level. FHL complexes are efficient H2 producers, and the prototype FHL-1 Ec complex in E. coli is well studied. This paper presents the first biochemical characterization of an FHL-2-type complex. The data presented here will enable us to solve the long-standing mystery of the FHL-2 Ec complex, allow a first biochemical characterization of T. guamensis's fermentative metabolism, and establish this enterobacterium as a model organism for FHL-dependent energy conservation.

Dataset Information

Exploring the directionality of Escherichia coli formate hydrogenlyase: a membrane-bound enzyme capable of fixing carbon dioxide to organic acid.

Publications

Exploring the directionality of Escherichia coli formate hydrogenlyase: a membrane-bound enzyme capable of fixing carbon dioxide to organic acid.

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