Pyrroloquinoline quinone biogenesis: Demonstration that PqqE from Klebsiella pneumoniae is a radical S-adenosyl-L-methionine enzyme

Stephen R. Wecksler, Stefan Stoll, Ha Tran, Olafur T. Magnusson, Shu-Pao Wu, David King, R. David Britt, Judith P. Klinman

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63 Scopus citations

Abstract

Biogenesis of pyrroloquinoline quinone (PQQ) in Klebsiella pneumoniae requires the expression of six genes (pqqA-F). One of these genes (pqqE) encodes a 43 kDa protein (PqqE) that plays a role in the initial steps in PQQ formation [Veletrop, J. S., et al. (1995) J. Bacteriol. 177, 5088-5098]. PqqE contains two highly conserved cysteine motifs at the N- and C-termini, with the N-terminal motif comprised of a CX 3 CX 2 C consensus sequence that is unique to a family of proteins known as radical S-adenosyl-L-methionine (SAM) enzymes [Sofia, H. J., et al. (2001) Nucleic Acids Res. 29, 1097-1106]. PqqE from K. pneumoniae was cloned into Escherichia coli and expressed as the native protein and with an N-terminal His 6 tag. Anaerobic expression and purification of the His 6 -tagged PqqE results in an enzyme with a brownish-red hue indicative of Fe-S cluster formation. Spectroscopic and physical analyses indicate that PqqE contains a mixture of Fe-S clusters, with the predominant form of the enzyme containing two [4Fe-4S] clusters. PqqE isolated anaerobically yields an active enzyme capable of cleaving SAM to methionine and 5′-deoxyadenosine in an uncoupled reaction (k obs =0.011±0.001 min -1 ). In this reaction, the 5′-deoxyadenosyl radical either abstracts a hydrogen atom from a solvent accessible position in the enzyme or obtains a proton and electron from buffer. The putative PQQ substrate PqqA has not yet been shown to be modified by PqqE, implying that PqqA must be modified before becoming the substrate for PqqE and/or that another protein in the biosynthetic pathway is critical for the initial steps in PQQ biogenesis.

Original languageEnglish
Pages (from-to)10151-10161
Number of pages11
JournalBiochemistry
Volume48
Issue number42
DOIs
StatePublished - 27 Oct 2009

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