[FeFe]-Hydrogenase: Defined Lysate-Free Maturation Reveals a Key Role for Lipoyl-H-Protein in DTMA Ligand Biosynthesis

Adrien Pagnier; Batuhan Balci; Eric M. Shepard; Hao Yang; Douglas M. Warui; Stella Impano; Squire J. Booker; Brian M. Hoffman; William E. Broderick; Joan B. Broderick

doi:10.1002/anie.202203413

[FeFe]-Hydrogenase: Defined Lysate-Free Maturation Reveals a Key Role for Lipoyl-H-Protein in DTMA Ligand Biosynthesis

Adrien Pagnier, Batuhan Balci, Eric M. Shepard, Hao Yang, Douglas M. Warui, Stella Impano, Squire J. Booker, Brian M. Hoffman, William E. Broderick, Joan B. Broderick^*

^*Corresponding author for this work

Chemistry

Research output: Contribution to journal › Article › peer-review

17 Scopus citations

Abstract

Maturation of [FeFe]-hydrogenase (HydA) involves synthesis of a CO, CN⁻, and dithiomethylamine (DTMA)-coordinated 2Fe subcluster that is inserted into HydA to make the active hydrogenase. This process requires three maturation enzymes: the radical S-adenosyl-l-methionine (SAM) enzymes HydE and HydG, and the GTPase HydF. In vitro maturation with purified maturation enzymes has been possible only when clarified cell lysate was added, with the lysate presumably providing essential components for DTMA synthesis and delivery. Here we report maturation of [FeFe]-hydrogenase using a fully defined system that includes components of the glycine cleavage system (GCS), but no cell lysate. Our results reveal for the first time an essential role for the aminomethyl-lipoyl-H-protein of the GCS in hydrogenase maturation and the synthesis of the DTMA ligand of the H-cluster. In addition, we show that ammonia is the source of the bridgehead nitrogen of DTMA.

Original language	English (US)
Article number	e202203413
Journal	Angewandte Chemie - International Edition
Volume	61
Issue number	22
DOIs	https://doi.org/10.1002/anie.202203413
State	Published - May 23 2022

Funding

This work was funded by the U.S. DOE (DE\u2010SC0005404 to J.B.B. and E.M.S.). S.J.B acknowledges funding from the NSF (MCB\u20101716686), and B.M.H from the NIH GM 111097. The Mass Spectrometry Facility was in part funded by the MJ Murdock Charitable Trust and the NIH (P20GM103474 and S10OD28650). The authors thank Prof. Inger Andersson (Uppsala U.) for the gift of pBAD\u2010HisA\u2010slr0293, and Juan Fontecilla\u2010Camps, Roman Rohac, and Yvain Nicolet for helpful discussions.

Keywords

Biosynthesis
Dithiomethylamine
Glycine Cleavage System
Hydrogenase Maturation
Lipoyl-H-Protein

ASJC Scopus subject areas

Catalysis
General Chemistry

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10.1002/anie.202203413

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Pagnier, A., Balci, B., Shepard, E. M., Yang, H., Warui, D. M., Impano, S., Booker, S. J., Hoffman, B. M., Broderick, W. E., & Broderick, J. B. (2022). [FeFe]-Hydrogenase: Defined Lysate-Free Maturation Reveals a Key Role for Lipoyl-H-Protein in DTMA Ligand Biosynthesis. Angewandte Chemie - International Edition, 61(22), Article e202203413. https://doi.org/10.1002/anie.202203413

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abstract = "Maturation of [FeFe]-hydrogenase (HydA) involves synthesis of a CO, CN−, and dithiomethylamine (DTMA)-coordinated 2Fe subcluster that is inserted into HydA to make the active hydrogenase. This process requires three maturation enzymes: the radical S-adenosyl-l-methionine (SAM) enzymes HydE and HydG, and the GTPase HydF. In vitro maturation with purified maturation enzymes has been possible only when clarified cell lysate was added, with the lysate presumably providing essential components for DTMA synthesis and delivery. Here we report maturation of [FeFe]-hydrogenase using a fully defined system that includes components of the glycine cleavage system (GCS), but no cell lysate. Our results reveal for the first time an essential role for the aminomethyl-lipoyl-H-protein of the GCS in hydrogenase maturation and the synthesis of the DTMA ligand of the H-cluster. In addition, we show that ammonia is the source of the bridgehead nitrogen of DTMA.",

keywords = "Biosynthesis, Dithiomethylamine, Glycine Cleavage System, Hydrogenase Maturation, Lipoyl-H-Protein",

author = "Adrien Pagnier and Batuhan Balci and Shepard, {Eric M.} and Hao Yang and Warui, {Douglas M.} and Stella Impano and Booker, {Squire J.} and Hoffman, {Brian M.} and Broderick, {William E.} and Broderick, {Joan B.}",

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doi = "10.1002/anie.202203413",

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AU - Shepard, Eric M.

AU - Yang, Hao

AU - Warui, Douglas M.

AU - Impano, Stella

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AB - Maturation of [FeFe]-hydrogenase (HydA) involves synthesis of a CO, CN−, and dithiomethylamine (DTMA)-coordinated 2Fe subcluster that is inserted into HydA to make the active hydrogenase. This process requires three maturation enzymes: the radical S-adenosyl-l-methionine (SAM) enzymes HydE and HydG, and the GTPase HydF. In vitro maturation with purified maturation enzymes has been possible only when clarified cell lysate was added, with the lysate presumably providing essential components for DTMA synthesis and delivery. Here we report maturation of [FeFe]-hydrogenase using a fully defined system that includes components of the glycine cleavage system (GCS), but no cell lysate. Our results reveal for the first time an essential role for the aminomethyl-lipoyl-H-protein of the GCS in hydrogenase maturation and the synthesis of the DTMA ligand of the H-cluster. In addition, we show that ammonia is the source of the bridgehead nitrogen of DTMA.

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[FeFe]-Hydrogenase: Defined Lysate-Free Maturation Reveals a Key Role for Lipoyl-H-Protein in DTMA Ligand Biosynthesis

Abstract

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Keywords

ASJC Scopus subject areas

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