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

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 CX3CX2C 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 His6 tag. Anaerobic expression and purification of the His6-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-4...