Interaction of PqqE and PqqD in the pyrroloquinoline quinone (PQQ) biosynthetic pathway links PqqD to the radical SAM superfamilywz

PQQ is the essential cofactor for glucose and methanol dehydrogenases found predominantly in gram-negative bacteria. The biosynthetic operon for this prokaryotic cofactor is found in many pathogenic organisms, making the pathway an attractive target for new antibiotics. PQQ has also been found in high concentrations in human breast milk and is necessary for proper growth and development in mice, though its participation as a cofactor for mammalian enzymes remains to be proven. The biosynthetic pathway for this important cofactor is still poorly understood. It is known that PQQ is formed from the fusion of glutamate (Glu) and tyrosine (Tyr) (Scheme 1). The genes in the pqq operon from many organisms have been described and compared. In Klebsiella pneumoniae, there are six such genes designated pqqA-F. PqqA is a peptide with conserved Glu and Tyr that has been implicated as the substrate for biosynthesis. PqqB shares homology to the b-lactamase family, and PqqF is likely to function as a protease. PqqC catalyzes the ring closure and 8-electron oxidation of 3a-(2-amino-2-carboxyethyl)-4,5,-dioxo4,5,6,7,8,9,-hexahydroquinoline-7,9-dicarboxylic acid (AHQQ) in the final step of biosynthesis. PqqE contains a conserved cysteine triad that is found in the radical S-adenosyl-L-methionine (SAM) family, and has recently been shown to cleave SAM to methionine and 50-deoxyadenosine in an uncoupled reaction. Efforts to link the activity of PqqE to the cross-linking of the conserved Glu and Tyr residues within the putative substrate PqqA have, thus far, been unsuccessful. One of the least understood proteins within the biosynthesis pathway is PqqD. A BLAST search of this protein in the NCBI database yielded little information, other than revealing homology to PqqD proteins within the pqq operon. Further complicating the assignment of PqqD is the fact that in approximately 4% of PQQ-producing organisms, the genes for pqqC and pqqD are fused together to encode a single polypeptide chain. Due to this fact, several groups have hypothesized that PqqD may function to dissociate tightly bound PQQ from PqqC. The recent crystal structure for PqqD from Xanthomonas campestris, in which a putative PQQ binding site was identified at the dimer interface, appeared to support this role. Worth noting is that the PQQ binding site was identified by a docking study, and attempts to crystallize PqqD with PQQ have not been successful. In fact, there is little experimental evidence to support the role of PqqD as a ‘‘dissociase’’ or PQQ carrier protein, and Toyama et al. have shown that removal of the pqqD gene from the pqqC/D construct does not alter the function of PqqC. Furthermore, a pqqD knockout study demonstrated that this gene is critical for PQQ formation, arguing for a more prominent role in the biosynthetic pathway. Recently, we performed a refined BLAST search of PqqD uncovering a possible link between PqqE and PqqD. In a search for a ‘‘conserved domain’’ architecture of PqqD, a series of unknown proteins arose that contain a radical SAM domain fused to a PqqD domain (Fig. 1). While the open-reading frame from Dehalococcoides sp. CBDB1 (entry (6) in Fig. 1) has no known function, albA (entries (4) and (5) in Fig. 1) is involved in the biosynthesis of subtilosin A, with a possible role in the cross-linking of amino acid side chains. In this paper, we present the first evidence for a direct interaction of PqqE with PqqD. These data redirect the investigation of the role for PqqD within the PQQ biosynthetic pathway, and likely have important functional implications for the sub-family of radical SAM/PqqD domain containing enzymes. The DNA for pqqD from K. pneumoniae was cloned into E. coli and expressed as a native protein in high yield (see ESIz for details on cloning, purification, and characterization). N-terminal sequencing and high-resolution mass spectrometry confirmed the identity of the protein. The enzyme has no Scheme 1 Fusion of Glu and Tyr to make PQQ.