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 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.
Wecksler
Stephen R
SR
Department of Chemistry and Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA.
Stoll
Stefan
S
Tran
Ha
H
Magnusson
Olafur T
OT
Wu
Shu-Pao
SP
King
David
D
Britt
R David
RD
Klinman
Judith P
JP
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D019976:Cloning, Organism; D003839:Deoxyadenosines / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D007711:Klebsiella pneumoniae / Q000201:enzymology* / Q000378:metabolism*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D045542:PQQ Cofactor / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D012436:S-Adenosylmethionine / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D001426:Bacterial Proteins; D003839:Deoxyadenosines; D045542:PQQ Cofactor; D012436:S-Adenosylmethionine