Characterisation of an unusual cysteine pair in the Rieske carnitine monooxygenase CntA catalytic site

Mussa Quareshy; Muralidharan Shanmugam; Alexander D. Cameron; Timothy D. H. Bugg; Yin Chen

doi:10.1111/febs.16722

Characterisation of an unusual cysteine pair in the Rieske carnitine monooxygenase CntA catalytic site

Mussa Quareshy^*, Muralidharan Shanmugam^*, Alexander D. Cameron, Timothy D. H. Bugg, Yin Chen

^*Corresponding author for this work

Biosciences

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Abstract

Rieske monooxygenases undertake complex catalysis integral to marine, terrestrial and human gut-ecosystems. Group-I to -IV Rieske monooxygenases accept aromatic substrates and have well-characterised catalytic mechanisms. Nascent to our understanding are Group-V members catalysing the oxidation/breakdown of quaternary ammonium substrates. Phylogenetic analysis of Group V highlights a cysteine residue-pair adjacent to the mononuclear Fe active site with no established role. Following our elucidation of the carnitine monooxygenase CntA structure, we probed the function of the cysteine pair Cys206/Cys209. Utilising biochemical and biophysical techniques, we found the cysteine residues do not play a structural role nor influence the electron transfer pathway, but rather are used in a nonstoichiometric role to ensure the catalytic iron centre remains in an Fe(II) state.

Original language	English
Pages (from-to)	2939-2953
Number of pages	15
Journal	The FEBS journal
Volume	290
Issue number	11
Early online date	8 Jan 2023
DOIs	https://doi.org/10.1111/febs.16722
Publication status	Published - Jun 2023

Bibliographical note

Acknowledgments:
This work was supported by a Leverhulme Trust research grant (RPG-2016-307). MS acknowledges The University of Manchester and the National EPR Facility for financial supports. We thank the National EPSRC EPR service and Facility (NS/A000055/1, EP/W014521/1) for the acquisition of various EPR measurements, which are included in this manuscript. The authors acknowledge the support of the University of Warwick, School of Life Sciences Media preparation team of Cerith Harries and Caroline Stewart for preparing various reagents and culture media.

Keywords

carnitine
cw-EPR
cysteine residues
electron transfer
mononuclear Fe
Rieske monooxygenase

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title = "Characterisation of an unusual cysteine pair in the Rieske carnitine monooxygenase CntA catalytic site",

abstract = "Rieske monooxygenases undertake complex catalysis integral to marine, terrestrial and human gut-ecosystems. Group-I to -IV Rieske monooxygenases accept aromatic substrates and have well-characterised catalytic mechanisms. Nascent to our understanding are Group-V members catalysing the oxidation/breakdown of quaternary ammonium substrates. Phylogenetic analysis of Group V highlights a cysteine residue-pair adjacent to the mononuclear Fe active site with no established role. Following our elucidation of the carnitine monooxygenase CntA structure, we probed the function of the cysteine pair Cys206/Cys209. Utilising biochemical and biophysical techniques, we found the cysteine residues do not play a structural role nor influence the electron transfer pathway, but rather are used in a nonstoichiometric role to ensure the catalytic iron centre remains in an Fe(II) state.",

keywords = "carnitine, cw-EPR, cysteine residues, electron transfer, mononuclear Fe, Rieske monooxygenase",

author = "Mussa Quareshy and Muralidharan Shanmugam and Cameron, \{Alexander D.\} and Bugg, \{Timothy D. H.\} and Yin Chen",

note = "Acknowledgments: This work was supported by a Leverhulme Trust research grant (RPG-2016-307). MS acknowledges The University of Manchester and the National EPR Facility for financial supports. We thank the National EPSRC EPR service and Facility (NS/A000055/1, EP/W014521/1) for the acquisition of various EPR measurements, which are included in this manuscript. The authors acknowledge the support of the University of Warwick, School of Life Sciences Media preparation team of Cerith Harries and Caroline Stewart for preparing various reagents and culture media.",

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AU - Quareshy, Mussa

AU - Shanmugam, Muralidharan

AU - Cameron, Alexander D.

AU - Bugg, Timothy D. H.

AU - Chen, Yin

N1 - Acknowledgments: This work was supported by a Leverhulme Trust research grant (RPG-2016-307). MS acknowledges The University of Manchester and the National EPR Facility for financial supports. We thank the National EPSRC EPR service and Facility (NS/A000055/1, EP/W014521/1) for the acquisition of various EPR measurements, which are included in this manuscript. The authors acknowledge the support of the University of Warwick, School of Life Sciences Media preparation team of Cerith Harries and Caroline Stewart for preparing various reagents and culture media.

PY - 2023/6

Y1 - 2023/6

N2 - Rieske monooxygenases undertake complex catalysis integral to marine, terrestrial and human gut-ecosystems. Group-I to -IV Rieske monooxygenases accept aromatic substrates and have well-characterised catalytic mechanisms. Nascent to our understanding are Group-V members catalysing the oxidation/breakdown of quaternary ammonium substrates. Phylogenetic analysis of Group V highlights a cysteine residue-pair adjacent to the mononuclear Fe active site with no established role. Following our elucidation of the carnitine monooxygenase CntA structure, we probed the function of the cysteine pair Cys206/Cys209. Utilising biochemical and biophysical techniques, we found the cysteine residues do not play a structural role nor influence the electron transfer pathway, but rather are used in a nonstoichiometric role to ensure the catalytic iron centre remains in an Fe(II) state.

AB - Rieske monooxygenases undertake complex catalysis integral to marine, terrestrial and human gut-ecosystems. Group-I to -IV Rieske monooxygenases accept aromatic substrates and have well-characterised catalytic mechanisms. Nascent to our understanding are Group-V members catalysing the oxidation/breakdown of quaternary ammonium substrates. Phylogenetic analysis of Group V highlights a cysteine residue-pair adjacent to the mononuclear Fe active site with no established role. Following our elucidation of the carnitine monooxygenase CntA structure, we probed the function of the cysteine pair Cys206/Cys209. Utilising biochemical and biophysical techniques, we found the cysteine residues do not play a structural role nor influence the electron transfer pathway, but rather are used in a nonstoichiometric role to ensure the catalytic iron centre remains in an Fe(II) state.

KW - carnitine

KW - cw-EPR

KW - cysteine residues

KW - electron transfer

KW - mononuclear Fe

KW - Rieske monooxygenase

UR - https://www.scopus.com/pages/publications/85147042452

U2 - 10.1111/febs.16722

DO - 10.1111/febs.16722

M3 - Article

SN - 1742-464X

VL - 290

SP - 2939

EP - 2953

JO - The FEBS journal

JF - The FEBS journal

IS - 11

ER -

Characterisation of an unusual cysteine pair in the Rieske carnitine monooxygenase CntA catalytic site

Abstract

Bibliographical note

Keywords

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