[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

4 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

Keywords

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

ASJC Scopus subject areas

Catalysis
Chemistry(all)

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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), [e202203413]. https://doi.org/10.1002/anie.202203413

@article{c1d8e302e4a74b75b6b1844e2d82cfb4,

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

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.}",

note = "Funding Information: This work was funded by the U.S. DOE (DE‐SC0005404 to J.B.B. and E.M.S.). S.J.B acknowledges funding from the NSF (MCB‐1716686), 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‐HisA‐slr0293, and Juan Fontecilla‐Camps, Roman Rohac, and Yvain Nicolet for helpful discussions. Publisher Copyright: {\textcopyright} 2022 Wiley-VCH GmbH.",

year = "2022",

month = may,

day = "23",

doi = "10.1002/anie.202203413",

language = "English (US)",

volume = "61",

journal = "Angewandte Chemie - International Edition",

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publisher = "John Wiley and Sons Ltd",

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T1 - [FeFe]-Hydrogenase

T2 - Defined Lysate-Free Maturation Reveals a Key Role for Lipoyl-H-Protein in DTMA Ligand Biosynthesis

AU - Pagnier, Adrien

AU - Balci, Batuhan

AU - Shepard, Eric M.

AU - Yang, Hao

AU - Warui, Douglas M.

AU - Impano, Stella

AU - Booker, Squire J.

AU - Hoffman, Brian M.

AU - Broderick, William E.

AU - Broderick, Joan B.

N1 - Funding Information: This work was funded by the U.S. DOE (DE‐SC0005404 to J.B.B. and E.M.S.). S.J.B acknowledges funding from the NSF (MCB‐1716686), 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‐HisA‐slr0293, and Juan Fontecilla‐Camps, Roman Rohac, and Yvain Nicolet for helpful discussions. Publisher Copyright: © 2022 Wiley-VCH GmbH.

PY - 2022/5/23

Y1 - 2022/5/23

N2 - 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.

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.

KW - Biosynthesis

KW - Dithiomethylamine

KW - Glycine Cleavage System

KW - Hydrogenase Maturation

KW - Lipoyl-H-Protein

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U2 - 10.1002/anie.202203413

DO - 10.1002/anie.202203413

M3 - Article

C2 - 35319808

AN - SCOPUS:85127945307

SN - 1433-7851

VL - 61

JO - Angewandte Chemie - International Edition

JF - Angewandte Chemie - International Edition

IS - 22

M1 - e202203413

ER -

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

Abstract

Keywords

ASJC Scopus subject areas

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