The Function of Two Radical-SAM Enzymes, HcgA and HcgG, in the Biosynthesis of the [Fe]-Hydrogenase Cofactor

Francisco J. Arriaza-Gallardo; Sebastian Schaupp; Yu Cong Zheng; Mohd Farid Abdul-Halim; Hui Jie Pan; Jörg Kahnt; Georgia Angelidou; Nicole Paczia; Xile Hu; Kyle Costa; Seigo Shima

doi:10.1002/anie.202213239

The Function of Two Radical-SAM Enzymes, HcgA and HcgG, in the Biosynthesis of the [Fe]-Hydrogenase Cofactor

Francisco J. Arriaza-Gallardo, Sebastian Schaupp, Yu Cong Zheng, Mohd Farid Abdul-Halim, Hui Jie Pan, Jörg Kahnt, Georgia Angelidou, Nicole Paczia, Xile Hu, Kyle Costa, Seigo Shima

Plant and Microbial Biology

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6 Scopus citations

Abstract

In the biosynthesis of the iron-guanylylpyridinol (FeGP) cofactor, 6-carboxymethyl-5-methyl-4-hydroxy-2-pyridinol (1) is 3-methylated to form 2, then 4-guanylylated to form 3, and converted into the full cofactor. HcgA-G proteins catalyze the biosynthetic reactions. Herein, we report the function of two radical S-adenosyl methionine enzymes, HcgA and HcgG, as uncovered by in vitro complementation experiments and the use of purified enzymes. In vitro biosynthesis using the cell extract from the Methanococcus maripaludis ΔhcgA strain was complemented with HcgA or precursors 1, 2 or 3. The results suggested that HcgA catalyzes the biosynthetic reaction that forms 1. We demonstrated the formation of 1 by HcgA using the 3 kDa cell extract filtrate as the substrate. Biosynthesis in the ΔhcgG system was recovered by HcgG but not by 3, which indicated that HcgG catalyzes the reactions after the biosynthesis of 3. The data indicated that HcgG contributes to the formation of CO and completes biosynthesis of the FeGP cofactor.

Original language	English (US)
Article number	e202213239
Journal	Angewandte Chemie - International Edition
Volume	61
Issue number	50
DOIs	https://doi.org/10.1002/anie.202213239
State	Published - Dec 12 2022

Bibliographical note

Funding Information:
We thank Thomas Lie and John A. Leigh for providing us with the mutated strains. We thank Yasuhiro Takahashi for providing the pRKISC plasmid. F.J.A.‐G. was supported by International Max Planck Research School (IMPRS) in Marburg. We thank Wolfgang Buckel and Rudolf K. Thauer as IMPRS Thesis Advisory Committee members. This work was supported by Deutsche Forschungsgemeinschaft, Priority Program, Iron‐Sulfur for Life (SPP1927, SH87/1‐1 and SH87/1‐2) and by the Max Planck Society to S. Sh. and by the Swiss National Science Foundation (200021_181977) to X. H.. K.C. acknowledges support from the US Department of Energy, Office of Science, Basic Energy Sciences, under grant number DE‐SC0019148. Open Access funding enabled and organized by Projekt DEAL. M. maripaludis

Publisher Copyright:
© 2022 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.

Keywords

Acyl Ligands
Biosynthesis
FeGP Cofactor
Radical S-Adenosyl Methionine Enzymes
[Fe]-Hydrogenase

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10.1002/anie.202213239

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Arriaza-Gallardo, F. J., Schaupp, S., Zheng, Y. C., Abdul-Halim, M. F., Pan, H. J., Kahnt, J., Angelidou, G., Paczia, N., Hu, X., Costa, K., & Shima, S. (2022). The Function of Two Radical-SAM Enzymes, HcgA and HcgG, in the Biosynthesis of the [Fe]-Hydrogenase Cofactor. Angewandte Chemie - International Edition, 61(50), Article e202213239. https://doi.org/10.1002/anie.202213239

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title = "The Function of Two Radical-SAM Enzymes, HcgA and HcgG, in the Biosynthesis of the [Fe]-Hydrogenase Cofactor",

abstract = "In the biosynthesis of the iron-guanylylpyridinol (FeGP) cofactor, 6-carboxymethyl-5-methyl-4-hydroxy-2-pyridinol (1) is 3-methylated to form 2, then 4-guanylylated to form 3, and converted into the full cofactor. HcgA-G proteins catalyze the biosynthetic reactions. Herein, we report the function of two radical S-adenosyl methionine enzymes, HcgA and HcgG, as uncovered by in vitro complementation experiments and the use of purified enzymes. In vitro biosynthesis using the cell extract from the Methanococcus maripaludis ΔhcgA strain was complemented with HcgA or precursors 1, 2 or 3. The results suggested that HcgA catalyzes the biosynthetic reaction that forms 1. We demonstrated the formation of 1 by HcgA using the 3 kDa cell extract filtrate as the substrate. Biosynthesis in the ΔhcgG system was recovered by HcgG but not by 3, which indicated that HcgG catalyzes the reactions after the biosynthesis of 3. The data indicated that HcgG contributes to the formation of CO and completes biosynthesis of the FeGP cofactor.",

keywords = "Acyl Ligands, Biosynthesis, FeGP Cofactor, Radical S-Adenosyl Methionine Enzymes, [Fe]-Hydrogenase",

author = "Arriaza-Gallardo, \{Francisco J.\} and Sebastian Schaupp and Zheng, \{Yu Cong\} and Abdul-Halim, \{Mohd Farid\} and Pan, \{Hui Jie\} and J{\"o}rg Kahnt and Georgia Angelidou and Nicole Paczia and Xile Hu and Kyle Costa and Seigo Shima",

note = "Funding Information: We thank Thomas Lie and John A. Leigh for providing us with the mutated strains. We thank Yasuhiro Takahashi for providing the pRKISC plasmid. F.J.A.‐G. was supported by International Max Planck Research School (IMPRS) in Marburg. We thank Wolfgang Buckel and Rudolf K. Thauer as IMPRS Thesis Advisory Committee members. This work was supported by Deutsche Forschungsgemeinschaft, Priority Program, Iron‐Sulfur for Life (SPP1927, SH87/1‐1 and SH87/1‐2) and by the Max Planck Society to S. Sh. and by the Swiss National Science Foundation (200021\_181977) to X. H.. K.C. acknowledges support from the US Department of Energy, Office of Science, Basic Energy Sciences, under grant number DE‐SC0019148. Open Access funding enabled and organized by Projekt DEAL. M. maripaludis Publisher Copyright: {\textcopyright} 2022 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.",

year = "2022",

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doi = "10.1002/anie.202213239",

language = "English (US)",

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T1 - The Function of Two Radical-SAM Enzymes, HcgA and HcgG, in the Biosynthesis of the [Fe]-Hydrogenase Cofactor

AU - Arriaza-Gallardo, Francisco J.

AU - Schaupp, Sebastian

AU - Zheng, Yu Cong

AU - Abdul-Halim, Mohd Farid

AU - Pan, Hui Jie

AU - Kahnt, Jörg

AU - Angelidou, Georgia

AU - Paczia, Nicole

AU - Hu, Xile

AU - Costa, Kyle

AU - Shima, Seigo

N1 - Funding Information: We thank Thomas Lie and John A. Leigh for providing us with the mutated strains. We thank Yasuhiro Takahashi for providing the pRKISC plasmid. F.J.A.‐G. was supported by International Max Planck Research School (IMPRS) in Marburg. We thank Wolfgang Buckel and Rudolf K. Thauer as IMPRS Thesis Advisory Committee members. This work was supported by Deutsche Forschungsgemeinschaft, Priority Program, Iron‐Sulfur for Life (SPP1927, SH87/1‐1 and SH87/1‐2) and by the Max Planck Society to S. Sh. and by the Swiss National Science Foundation (200021_181977) to X. H.. K.C. acknowledges support from the US Department of Energy, Office of Science, Basic Energy Sciences, under grant number DE‐SC0019148. Open Access funding enabled and organized by Projekt DEAL. M. maripaludis Publisher Copyright: © 2022 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.

PY - 2022/12/12

Y1 - 2022/12/12

N2 - In the biosynthesis of the iron-guanylylpyridinol (FeGP) cofactor, 6-carboxymethyl-5-methyl-4-hydroxy-2-pyridinol (1) is 3-methylated to form 2, then 4-guanylylated to form 3, and converted into the full cofactor. HcgA-G proteins catalyze the biosynthetic reactions. Herein, we report the function of two radical S-adenosyl methionine enzymes, HcgA and HcgG, as uncovered by in vitro complementation experiments and the use of purified enzymes. In vitro biosynthesis using the cell extract from the Methanococcus maripaludis ΔhcgA strain was complemented with HcgA or precursors 1, 2 or 3. The results suggested that HcgA catalyzes the biosynthetic reaction that forms 1. We demonstrated the formation of 1 by HcgA using the 3 kDa cell extract filtrate as the substrate. Biosynthesis in the ΔhcgG system was recovered by HcgG but not by 3, which indicated that HcgG catalyzes the reactions after the biosynthesis of 3. The data indicated that HcgG contributes to the formation of CO and completes biosynthesis of the FeGP cofactor.

AB - In the biosynthesis of the iron-guanylylpyridinol (FeGP) cofactor, 6-carboxymethyl-5-methyl-4-hydroxy-2-pyridinol (1) is 3-methylated to form 2, then 4-guanylylated to form 3, and converted into the full cofactor. HcgA-G proteins catalyze the biosynthetic reactions. Herein, we report the function of two radical S-adenosyl methionine enzymes, HcgA and HcgG, as uncovered by in vitro complementation experiments and the use of purified enzymes. In vitro biosynthesis using the cell extract from the Methanococcus maripaludis ΔhcgA strain was complemented with HcgA or precursors 1, 2 or 3. The results suggested that HcgA catalyzes the biosynthetic reaction that forms 1. We demonstrated the formation of 1 by HcgA using the 3 kDa cell extract filtrate as the substrate. Biosynthesis in the ΔhcgG system was recovered by HcgG but not by 3, which indicated that HcgG catalyzes the reactions after the biosynthesis of 3. The data indicated that HcgG contributes to the formation of CO and completes biosynthesis of the FeGP cofactor.

KW - Acyl Ligands

KW - Biosynthesis

KW - FeGP Cofactor

KW - Radical S-Adenosyl Methionine Enzymes

KW - [Fe]-Hydrogenase

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SN - 1433-7851

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The Function of Two Radical-SAM Enzymes, HcgA and HcgG, in the Biosynthesis of the [Fe]-Hydrogenase Cofactor

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