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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*E-mail: firstname.lastname@example.org. ORCID Troy A. Stich: 0000-0003-0710-1456 John A. Latham: 0000-0002-3034-1768 Carrie M. Wilmot: 0000-0002-8706-0000 R. David Britt: 0000-0003-0889-8436 Judith P. Klinman: 0000-0001-5734-2843 Present Addresses ◇I.B.: Department of Natural Sciences and Mathematics, Dominican University of California, San Rafael, California 94901, United States. †J.A.L.: Department of Chemistry and Biochemistry, University of Denver, Denver, Colorado 80222, United States. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Financial support was provided by the National Institutes of Health (NIH), GM118117 to J.P.K., GM66569 to C.M.W., P01GM118303-01 and U54GM093342 to J.A.G. and S.C.A., U54GM094662 to S.C.A., R21 AI133329 to T.L.G and S.C.A., and GM104543 to R.D.B., and the Price Family Foundation to S.C.A. Notes The authors declare no competing financial interest.
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