X-ray and EPR Characterization of the Auxiliary Fe-S Clusters in the Radical SAM Enzyme PqqE

Ian Barr, Troy A. Stich, Anthony S. Gizzi, Tyler L. Grove, Jeffrey B. Bonanno, John A. Latham, Tyler Chung, Carrie M. Wilmot, R. David Britt, Steven C. Almo, Judith P. Klinman

Research output: Contribution to journalArticle

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Abstract

The Radical SAM (RS) enzyme PqqE catalyzes the first step in the biosynthesis of the bacterial cofactor pyrroloquinoline quinone, forming a new carbon-carbon bond between two side chains within the ribosomally synthesized peptide substrate PqqA. In addition to the active site RS 4Fe-4S cluster, PqqE is predicted to have two auxiliary Fe-S clusters, like the other members of the SPASM domain family. Here we identify these sites and examine their structure using a combination of X-ray crystallography and Mössbauer and electron paramagnetic resonance (EPR) spectroscopies. X-ray crystallography allows us to identify the ligands to each of the two auxiliary clusters at the C-terminal region of the protein. The auxiliary cluster nearest the RS site (AuxI) is in the form of a 2Fe-2S cluster ligated by four cysteines, an Fe-S center not seen previously in other SPASM domain proteins; this assignment is further supported by Mössbauer and EPR spectroscopies. The second, more remote cluster (AuxII) is a 4Fe-4S center that is ligated by three cysteine residues and one aspartate residue. In addition, we examined the roles these ligands play in catalysis by the RS and AuxII clusters using site-directed mutagenesis coupled with EPR spectroscopy. Lastly, we discuss the possible functional consequences that these unique AuxI and AuxII clusters may have in catalysis for PqqE and how these may extend to additional RS enzymes catalyzing the post-translational modification of ribosomally encoded peptides.

Original languageEnglish (US)
Pages (from-to)1306-1315
Number of pages10
JournalBiochemistry
Volume57
Issue number8
DOIs
StatePublished - Feb 27 2018

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Electron Spin Resonance Spectroscopy
Paramagnetic resonance
Spectrum Analysis
X ray crystallography
X Ray Crystallography
X-Rays
Spectroscopy
Catalysis
X rays
Cysteine
Enzymes
Carbon
PQQ Cofactor
Ligands
Mutagenesis
Peptides
Biosynthesis
Post Translational Protein Processing
Site-Directed Mutagenesis
Aspartic Acid

PubMed: MeSH publication types

  • Journal Article
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

Cite this

Barr, I., Stich, T. A., Gizzi, A. S., Grove, T. L., Bonanno, J. B., Latham, J. A., ... Klinman, J. P. (2018). X-ray and EPR Characterization of the Auxiliary Fe-S Clusters in the Radical SAM Enzyme PqqE. Biochemistry, 57(8), 1306-1315. https://doi.org/10.1021/acs.biochem.7b01097

X-ray and EPR Characterization of the Auxiliary Fe-S Clusters in the Radical SAM Enzyme PqqE. / Barr, Ian; Stich, Troy A.; Gizzi, Anthony S.; Grove, Tyler L.; Bonanno, Jeffrey B.; Latham, John A.; Chung, Tyler; Wilmot, Carrie M.; Britt, R. David; Almo, Steven C.; Klinman, Judith P.

In: Biochemistry, Vol. 57, No. 8, 27.02.2018, p. 1306-1315.

Research output: Contribution to journalArticle

Barr, I, Stich, TA, Gizzi, AS, Grove, TL, Bonanno, JB, Latham, JA, Chung, T, Wilmot, CM, Britt, RD, Almo, SC & Klinman, JP 2018, 'X-ray and EPR Characterization of the Auxiliary Fe-S Clusters in the Radical SAM Enzyme PqqE', Biochemistry, vol. 57, no. 8, pp. 1306-1315. https://doi.org/10.1021/acs.biochem.7b01097
Barr I, Stich TA, Gizzi AS, Grove TL, Bonanno JB, Latham JA et al. X-ray and EPR Characterization of the Auxiliary Fe-S Clusters in the Radical SAM Enzyme PqqE. Biochemistry. 2018 Feb 27;57(8):1306-1315. https://doi.org/10.1021/acs.biochem.7b01097
Barr, Ian ; Stich, Troy A. ; Gizzi, Anthony S. ; Grove, Tyler L. ; Bonanno, Jeffrey B. ; Latham, John A. ; Chung, Tyler ; Wilmot, Carrie M. ; Britt, R. David ; Almo, Steven C. ; Klinman, Judith P. / X-ray and EPR Characterization of the Auxiliary Fe-S Clusters in the Radical SAM Enzyme PqqE. In: Biochemistry. 2018 ; Vol. 57, No. 8. pp. 1306-1315.
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abstract = "The Radical SAM (RS) enzyme PqqE catalyzes the first step in the biosynthesis of the bacterial cofactor pyrroloquinoline quinone, forming a new carbon-carbon bond between two side chains within the ribosomally synthesized peptide substrate PqqA. In addition to the active site RS 4Fe-4S cluster, PqqE is predicted to have two auxiliary Fe-S clusters, like the other members of the SPASM domain family. Here we identify these sites and examine their structure using a combination of X-ray crystallography and M{\"o}ssbauer and electron paramagnetic resonance (EPR) spectroscopies. X-ray crystallography allows us to identify the ligands to each of the two auxiliary clusters at the C-terminal region of the protein. The auxiliary cluster nearest the RS site (AuxI) is in the form of a 2Fe-2S cluster ligated by four cysteines, an Fe-S center not seen previously in other SPASM domain proteins; this assignment is further supported by M{\"o}ssbauer and EPR spectroscopies. The second, more remote cluster (AuxII) is a 4Fe-4S center that is ligated by three cysteine residues and one aspartate residue. In addition, we examined the roles these ligands play in catalysis by the RS and AuxII clusters using site-directed mutagenesis coupled with EPR spectroscopy. Lastly, we discuss the possible functional consequences that these unique AuxI and AuxII clusters may have in catalysis for PqqE and how these may extend to additional RS enzymes catalyzing the post-translational modification of ribosomally encoded peptides.",
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T1 - X-ray and EPR Characterization of the Auxiliary Fe-S Clusters in the Radical SAM Enzyme PqqE

AU - Barr, Ian

AU - Stich, Troy A.

AU - Gizzi, Anthony S.

AU - Grove, Tyler L.

AU - Bonanno, Jeffrey B.

AU - Latham, John A.

AU - Chung, Tyler

AU - Wilmot, Carrie M.

AU - Britt, R. David

AU - Almo, Steven C.

AU - Klinman, Judith P.

PY - 2018/2/27

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N2 - The Radical SAM (RS) enzyme PqqE catalyzes the first step in the biosynthesis of the bacterial cofactor pyrroloquinoline quinone, forming a new carbon-carbon bond between two side chains within the ribosomally synthesized peptide substrate PqqA. In addition to the active site RS 4Fe-4S cluster, PqqE is predicted to have two auxiliary Fe-S clusters, like the other members of the SPASM domain family. Here we identify these sites and examine their structure using a combination of X-ray crystallography and Mössbauer and electron paramagnetic resonance (EPR) spectroscopies. X-ray crystallography allows us to identify the ligands to each of the two auxiliary clusters at the C-terminal region of the protein. The auxiliary cluster nearest the RS site (AuxI) is in the form of a 2Fe-2S cluster ligated by four cysteines, an Fe-S center not seen previously in other SPASM domain proteins; this assignment is further supported by Mössbauer and EPR spectroscopies. The second, more remote cluster (AuxII) is a 4Fe-4S center that is ligated by three cysteine residues and one aspartate residue. In addition, we examined the roles these ligands play in catalysis by the RS and AuxII clusters using site-directed mutagenesis coupled with EPR spectroscopy. Lastly, we discuss the possible functional consequences that these unique AuxI and AuxII clusters may have in catalysis for PqqE and how these may extend to additional RS enzymes catalyzing the post-translational modification of ribosomally encoded peptides.

AB - The Radical SAM (RS) enzyme PqqE catalyzes the first step in the biosynthesis of the bacterial cofactor pyrroloquinoline quinone, forming a new carbon-carbon bond between two side chains within the ribosomally synthesized peptide substrate PqqA. In addition to the active site RS 4Fe-4S cluster, PqqE is predicted to have two auxiliary Fe-S clusters, like the other members of the SPASM domain family. Here we identify these sites and examine their structure using a combination of X-ray crystallography and Mössbauer and electron paramagnetic resonance (EPR) spectroscopies. X-ray crystallography allows us to identify the ligands to each of the two auxiliary clusters at the C-terminal region of the protein. The auxiliary cluster nearest the RS site (AuxI) is in the form of a 2Fe-2S cluster ligated by four cysteines, an Fe-S center not seen previously in other SPASM domain proteins; this assignment is further supported by Mössbauer and EPR spectroscopies. The second, more remote cluster (AuxII) is a 4Fe-4S center that is ligated by three cysteine residues and one aspartate residue. In addition, we examined the roles these ligands play in catalysis by the RS and AuxII clusters using site-directed mutagenesis coupled with EPR spectroscopy. Lastly, we discuss the possible functional consequences that these unique AuxI and AuxII clusters may have in catalysis for PqqE and how these may extend to additional RS enzymes catalyzing the post-translational modification of ribosomally encoded peptides.

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JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

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