A mixed-valent Fe(II)Fe(III) species converts cysteine to an oxazolone/thioamide pair in methanobactin biosynthesis

Yun Ji Park; Richard J. Jodts; Jeffrey W. Slater; Reyvin M. Reyes; Valerie J. Winton; Rana A. Montaser; Paul M. Thomas; William B. Dowdle; Anahi Ruiz; Neil L. Kelleher; J. Martin Bollinger; Carsten Krebs; Brian M. Hoffman; Amy C. Rosenzweig

doi:10.1073/pnas.2123566119

A mixed-valent Fe(II)Fe(III) species converts cysteine to an oxazolone/thioamide pair in methanobactin biosynthesis

Yun Ji Park, Richard J. Jodts, Jeffrey W. Slater, Reyvin M. Reyes, Valerie J. Winton, Rana A. Montaser, Paul M. Thomas, William B. Dowdle, Anahi Ruiz, Neil L. Kelleher, J. Martin Bollinger, Carsten Krebs, Brian M. Hoffman, Amy C. Rosenzweig^*

^*Corresponding author for this work

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

17 Scopus citations

Abstract

The iron-containing heterodimeric MbnBC enzyme complex plays a central role in the biosynthesis of methanobactins (Mbns), ribosomally synthesized, posttranslationally modified natural products that bind copper with high affinity. MbnBC catalyzes a four-electron oxidation of a cysteine residue in its precursor-peptide substrate, MbnA, to an oxazolone ring and an adjacent thioamide group. Initial studies of MbnBC indicated the presence of both diiron and triiron species, complicating identification of the catalytically active species. Here, we present evidence through activity assays combined with electron paramagnetic resonance (EPR) and M€ossbauer spectroscopic analysis that the active species is a mixed-valent, antiferromagnetically coupled Fe(II)Fe(III) center. Consistent with this assignment, heterologous expression of the MbnBC complex in culture medium containing less iron yielded purified protein with less bound iron but greater activity in vitro. The maximally activated MbnBC prepared in this manner could modify both cysteine residues in MbnA, in contrast to prior findings that only the first cysteine could be processed. Site-directed mutagenesis and multiple crystal structures clearly identify the two essential Fe ions in the active cluster as well as the location of the previously detected third Fe site. Moreover, structural modeling indicates a role for MbnC in recognition of the MbnA leader peptide. These results add a biosynthetic oxidative rearrangement reaction to the repertoire of nonheme diiron enzymes and provide a foundation for elucidating the MbnBC mechanism.

Original language	English (US)
Article number	e2123566119
Journal	Proceedings of the National Academy of Sciences of the United States of America
Volume	119
Issue number	13
DOIs	https://doi.org/10.1073/pnas.2123566119
State	Published - Mar 29 2022

Funding

ACKNOWLEDGMENTS. We acknowledge staff and instrumentation support from the Structural Biology Facility at Northwestern University, the Robert H. Lurie Comprehensive Cancer Center of Northwestern University, and National Cancer Institute Cancer Center Support Grant P30 CA060553. We thank Zdzislaw Wawrzak for assistance with crystallographic data analysis and Dr. Grace Kenney for preparation of the MbnBC D208S and H210S variant plasmids. This work was supported by NIH grants GM118035 (A.C.R.), F32 GM131665 (Y.J.P.), T32GM008382 (R.J.J.), F32 GM136156 (J.W.S.), T32GM008449 (R.M.R.), GM127079 (C.K.), GM111097 (B.M.H.), and P41 GM108569 (N.L.K.) and NSF grants MCB1908587 (B.M.H.) and CHE2108583 (C.K. and J.M.B.). ICP-MS analysis was performed at the Northwestern University Quantitative Bio-element Imaging Center generously supported by National Aeronautics and Space Administration Ames Research Center Grant NNA04CC36G. This work utilized the LS-CAT beam-lines of the Advanced Photon Source, which is a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract DE-AC02-06CH11356. Use of LS-CAT Sector 21 was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor (Grant 085P1000817). We acknowledge staff and instrumentation support from the Structural Biology Facility at Northwestern University, the Robert H. Lurie Comprehensive Cancer Center of Northwestern University, and National Cancer Institute Cancer Center Support Grant P30 CA060553. We thank Zdzislaw Wawrzak for assistance with crystallographic data analysis and Dr. Grace Kenney for preparation of the MbnBC D208S and H210S variant plasmids. This work was supported by NIH grants GM118035 (A.C.R.), F32 GM131665 (Y.J.P.), T32GM008382 (R.J.J.), F32 GM136156 (J.W.S.), T32GM008449 (R.M.R.), GM127079 (C.K.), GM111097 (B.M.H.), and P41 GM108569 (N.L.K.) and NSF grants MCB1908587 (B.M.H.) and CHE2108583 (C.K. and J.M.B.). ICP-MS analysis was performed at the Northwestern University Quantitative Bio-element Imaging Center generously supported by National Aeronautics and Space Administration Ames Research Center Grant NNA04CC36G. This work utilized the LS-CAT beam-lines of the Advanced Photon Source, which is a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract DE-AC02-06CH11356. Use of LS-CAT Sector 21 was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor (Grant 085P1000817).

Keywords

MbnBC
methanobactin
natural products biosynthesis
nonheme iron

ASJC Scopus subject areas

General

Access to Document

10.1073/pnas.2123566119

Cite this

Park, Y. J., Jodts, R. J., Slater, J. W., Reyes, R. M., Winton, V. J., Montaser, R. A., Thomas, P. M., Dowdle, W. B., Ruiz, A., Kelleher, N. L., Bollinger, J. M., Krebs, C., Hoffman, B. M., & Rosenzweig, A. C. (2022). A mixed-valent Fe(II)Fe(III) species converts cysteine to an oxazolone/thioamide pair in methanobactin biosynthesis. Proceedings of the National Academy of Sciences of the United States of America, 119(13), Article e2123566119. https://doi.org/10.1073/pnas.2123566119

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title = "A mixed-valent Fe(II)Fe(III) species converts cysteine to an oxazolone/thioamide pair in methanobactin biosynthesis",

abstract = "The iron-containing heterodimeric MbnBC enzyme complex plays a central role in the biosynthesis of methanobactins (Mbns), ribosomally synthesized, posttranslationally modified natural products that bind copper with high affinity. MbnBC catalyzes a four-electron oxidation of a cysteine residue in its precursor-peptide substrate, MbnA, to an oxazolone ring and an adjacent thioamide group. Initial studies of MbnBC indicated the presence of both diiron and triiron species, complicating identification of the catalytically active species. Here, we present evidence through activity assays combined with electron paramagnetic resonance (EPR) and M€ossbauer spectroscopic analysis that the active species is a mixed-valent, antiferromagnetically coupled Fe(II)Fe(III) center. Consistent with this assignment, heterologous expression of the MbnBC complex in culture medium containing less iron yielded purified protein with less bound iron but greater activity in vitro. The maximally activated MbnBC prepared in this manner could modify both cysteine residues in MbnA, in contrast to prior findings that only the first cysteine could be processed. Site-directed mutagenesis and multiple crystal structures clearly identify the two essential Fe ions in the active cluster as well as the location of the previously detected third Fe site. Moreover, structural modeling indicates a role for MbnC in recognition of the MbnA leader peptide. These results add a biosynthetic oxidative rearrangement reaction to the repertoire of nonheme diiron enzymes and provide a foundation for elucidating the MbnBC mechanism.",

keywords = "MbnBC, methanobactin, natural products biosynthesis, nonheme iron",

author = "Park, {Yun Ji} and Jodts, {Richard J.} and Slater, {Jeffrey W.} and Reyes, {Reyvin M.} and Winton, {Valerie J.} and Montaser, {Rana A.} and Thomas, {Paul M.} and Dowdle, {William B.} and Anahi Ruiz and Kelleher, {Neil L.} and Bollinger, {J. Martin} and Carsten Krebs and Hoffman, {Brian M.} and Rosenzweig, {Amy C.}",

note = "Funding Information: ACKNOWLEDGMENTS. We acknowledge staff and instrumentation support from the Structural Biology Facility at Northwestern University, the Robert H. Lurie Comprehensive Cancer Center of Northwestern University, and National Cancer Institute Cancer Center Support Grant P30 CA060553. We thank Zdzislaw Wawrzak for assistance with crystallographic data analysis and Dr. Grace Kenney for preparation of the MbnBC D208S and H210S variant plasmids. This work was supported by NIH grants GM118035 (A.C.R.), F32 GM131665 (Y.J.P.), T32GM008382 (R.J.J.), F32 GM136156 (J.W.S.), T32GM008449 (R.M.R.), GM127079 (C.K.), GM111097 (B.M.H.), and P41 GM108569 (N.L.K.) and NSF grants MCB1908587 (B.M.H.) and CHE2108583 (C.K. and J.M.B.). ICP-MS analysis was performed at the Northwestern University Quantitative Bio-element Imaging Center generously supported by National Aeronautics and Space Administration Ames Research Center Grant NNA04CC36G. This work utilized the LS-CAT beam-lines of the Advanced Photon Source, which is a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract DE-AC02-06CH11356. Use of LS-CAT Sector 21 was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor (Grant 085P1000817). Funding Information: We acknowledge staff and instrumentation support from the Structural Biology Facility at Northwestern University, the Robert H. Lurie Comprehensive Cancer Center of Northwestern University, and National Cancer Institute Cancer Center Support Grant P30 CA060553. We thank Zdzislaw Wawrzak for assistance with crystallographic data analysis and Dr. Grace Kenney for preparation of the MbnBC D208S and H210S variant plasmids. This work was supported by NIH grants GM118035 (A.C.R.), F32 GM131665 (Y.J.P.), T32GM008382 (R.J.J.), F32 GM136156 (J.W.S.), T32GM008449 (R.M.R.), GM127079 (C.K.), GM111097 (B.M.H.), and P41 GM108569 (N.L.K.) and NSF grants MCB1908587 (B.M.H.) and CHE2108583 (C.K. and J.M.B.). ICP-MS analysis was performed at the Northwestern University Quantitative Bio-element Imaging Center generously supported by National Aeronautics and Space Administration Ames Research Center Grant NNA04CC36G. This work utilized the LS-CAT beam-lines of the Advanced Photon Source, which is a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract DE-AC02-06CH11356. Use of LS-CAT Sector 21 was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor (Grant 085P1000817). Publisher Copyright: {\textcopyright} 2022 the Author(s).",

year = "2022",

month = mar,

day = "29",

doi = "10.1073/pnas.2123566119",

language = "English (US)",

volume = "119",

journal = "Proceedings of the National Academy of Sciences of the United States of America",

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Park, YJ, Jodts, RJ, Slater, JW, Reyes, RM, Winton, VJ, Montaser, RA, Thomas, PM, Dowdle, WB, Ruiz, A, Kelleher, NL, Bollinger, JM, Krebs, C, Hoffman, BM & Rosenzweig, AC 2022, 'A mixed-valent Fe(II)Fe(III) species converts cysteine to an oxazolone/thioamide pair in methanobactin biosynthesis', Proceedings of the National Academy of Sciences of the United States of America, vol. 119, no. 13, e2123566119. https://doi.org/10.1073/pnas.2123566119

A mixed-valent Fe(II)Fe(III) species converts cysteine to an oxazolone/thioamide pair in methanobactin biosynthesis. / Park, Yun Ji; Jodts, Richard J.; Slater, Jeffrey W. et al.
In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 119, No. 13, e2123566119, 29.03.2022.

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

TY - JOUR

T1 - A mixed-valent Fe(II)Fe(III) species converts cysteine to an oxazolone/thioamide pair in methanobactin biosynthesis

AU - Park, Yun Ji

AU - Jodts, Richard J.

AU - Slater, Jeffrey W.

AU - Reyes, Reyvin M.

AU - Winton, Valerie J.

AU - Montaser, Rana A.

AU - Thomas, Paul M.

AU - Dowdle, William B.

AU - Ruiz, Anahi

AU - Kelleher, Neil L.

AU - Bollinger, J. Martin

AU - Krebs, Carsten

AU - Hoffman, Brian M.

AU - Rosenzweig, Amy C.

N1 - Funding Information: ACKNOWLEDGMENTS. We acknowledge staff and instrumentation support from the Structural Biology Facility at Northwestern University, the Robert H. Lurie Comprehensive Cancer Center of Northwestern University, and National Cancer Institute Cancer Center Support Grant P30 CA060553. We thank Zdzislaw Wawrzak for assistance with crystallographic data analysis and Dr. Grace Kenney for preparation of the MbnBC D208S and H210S variant plasmids. This work was supported by NIH grants GM118035 (A.C.R.), F32 GM131665 (Y.J.P.), T32GM008382 (R.J.J.), F32 GM136156 (J.W.S.), T32GM008449 (R.M.R.), GM127079 (C.K.), GM111097 (B.M.H.), and P41 GM108569 (N.L.K.) and NSF grants MCB1908587 (B.M.H.) and CHE2108583 (C.K. and J.M.B.). ICP-MS analysis was performed at the Northwestern University Quantitative Bio-element Imaging Center generously supported by National Aeronautics and Space Administration Ames Research Center Grant NNA04CC36G. This work utilized the LS-CAT beam-lines of the Advanced Photon Source, which is a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract DE-AC02-06CH11356. Use of LS-CAT Sector 21 was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor (Grant 085P1000817). Funding Information: We acknowledge staff and instrumentation support from the Structural Biology Facility at Northwestern University, the Robert H. Lurie Comprehensive Cancer Center of Northwestern University, and National Cancer Institute Cancer Center Support Grant P30 CA060553. We thank Zdzislaw Wawrzak for assistance with crystallographic data analysis and Dr. Grace Kenney for preparation of the MbnBC D208S and H210S variant plasmids. This work was supported by NIH grants GM118035 (A.C.R.), F32 GM131665 (Y.J.P.), T32GM008382 (R.J.J.), F32 GM136156 (J.W.S.), T32GM008449 (R.M.R.), GM127079 (C.K.), GM111097 (B.M.H.), and P41 GM108569 (N.L.K.) and NSF grants MCB1908587 (B.M.H.) and CHE2108583 (C.K. and J.M.B.). ICP-MS analysis was performed at the Northwestern University Quantitative Bio-element Imaging Center generously supported by National Aeronautics and Space Administration Ames Research Center Grant NNA04CC36G. This work utilized the LS-CAT beam-lines of the Advanced Photon Source, which is a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract DE-AC02-06CH11356. Use of LS-CAT Sector 21 was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor (Grant 085P1000817). Publisher Copyright: © 2022 the Author(s).

PY - 2022/3/29

Y1 - 2022/3/29

N2 - The iron-containing heterodimeric MbnBC enzyme complex plays a central role in the biosynthesis of methanobactins (Mbns), ribosomally synthesized, posttranslationally modified natural products that bind copper with high affinity. MbnBC catalyzes a four-electron oxidation of a cysteine residue in its precursor-peptide substrate, MbnA, to an oxazolone ring and an adjacent thioamide group. Initial studies of MbnBC indicated the presence of both diiron and triiron species, complicating identification of the catalytically active species. Here, we present evidence through activity assays combined with electron paramagnetic resonance (EPR) and M€ossbauer spectroscopic analysis that the active species is a mixed-valent, antiferromagnetically coupled Fe(II)Fe(III) center. Consistent with this assignment, heterologous expression of the MbnBC complex in culture medium containing less iron yielded purified protein with less bound iron but greater activity in vitro. The maximally activated MbnBC prepared in this manner could modify both cysteine residues in MbnA, in contrast to prior findings that only the first cysteine could be processed. Site-directed mutagenesis and multiple crystal structures clearly identify the two essential Fe ions in the active cluster as well as the location of the previously detected third Fe site. Moreover, structural modeling indicates a role for MbnC in recognition of the MbnA leader peptide. These results add a biosynthetic oxidative rearrangement reaction to the repertoire of nonheme diiron enzymes and provide a foundation for elucidating the MbnBC mechanism.

AB - The iron-containing heterodimeric MbnBC enzyme complex plays a central role in the biosynthesis of methanobactins (Mbns), ribosomally synthesized, posttranslationally modified natural products that bind copper with high affinity. MbnBC catalyzes a four-electron oxidation of a cysteine residue in its precursor-peptide substrate, MbnA, to an oxazolone ring and an adjacent thioamide group. Initial studies of MbnBC indicated the presence of both diiron and triiron species, complicating identification of the catalytically active species. Here, we present evidence through activity assays combined with electron paramagnetic resonance (EPR) and M€ossbauer spectroscopic analysis that the active species is a mixed-valent, antiferromagnetically coupled Fe(II)Fe(III) center. Consistent with this assignment, heterologous expression of the MbnBC complex in culture medium containing less iron yielded purified protein with less bound iron but greater activity in vitro. The maximally activated MbnBC prepared in this manner could modify both cysteine residues in MbnA, in contrast to prior findings that only the first cysteine could be processed. Site-directed mutagenesis and multiple crystal structures clearly identify the two essential Fe ions in the active cluster as well as the location of the previously detected third Fe site. Moreover, structural modeling indicates a role for MbnC in recognition of the MbnA leader peptide. These results add a biosynthetic oxidative rearrangement reaction to the repertoire of nonheme diiron enzymes and provide a foundation for elucidating the MbnBC mechanism.

KW - MbnBC

KW - methanobactin

KW - natural products biosynthesis

KW - nonheme iron

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UR - http://www.scopus.com/inward/citedby.url?scp=85126834998&partnerID=8YFLogxK

U2 - 10.1073/pnas.2123566119

DO - 10.1073/pnas.2123566119

M3 - Article

C2 - 35320042

AN - SCOPUS:85126834998

SN - 0027-8424

VL - 119

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

IS - 13

M1 - e2123566119

ER -

A mixed-valent Fe(II)Fe(III) species converts cysteine to an oxazolone/thioamide pair in methanobactin biosynthesis

Abstract

Funding

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Methanobactin biosynthetic protein complex of MbnB and MbnC from Methylosinus trichosporium OB3b, H210S mutant

Methanobactin biosynthetic protein complex of MbnB and MbnC from Methylosinus trichosporium OB3b at 2.31 Angstrom resolution

Methanobactin biosynthetic protein complex of MbnB and MbnC from Methylosinus trichosporium OB3b at 2.62 Angstrom resolution

Cite this

A mixed-valent Fe(II)Fe(III) species converts cysteine to an oxazolone/thioamide pair in methanobactin biosynthesis

Abstract

Funding

Keywords

ASJC Scopus subject areas

Access to Document

Other files and links

Fingerprint

Datasets

Methanobactin biosynthetic protein complex of MbnB and MbnC from Methylosinus trichosporium OB3b, H210S mutant

Methanobactin biosynthetic protein complex of MbnB and MbnC from Methylosinus trichosporium OB3b at 2.31 Angstrom resolution

Methanobactin biosynthetic protein complex of MbnB and MbnC from Methylosinus trichosporium OB3b at 2.62 Angstrom resolution

Cite this