Pyrroloquinoline Quinone (PQQ) Biosynthesis

The overall objective of this proposal is to elucidate the biosynthesis pathway for pyrroloquinoline quinone (PQQ) formation. PQQ is an important redox-active cofactor used by a number of bacterial dehydrogenases. PQQ is also important for human health and its role as a vitamin in mammals has recently been suggested. Although much is known about the function of enzymes that use PQQ as cofactor, relatively little is known about the chemical steps and therefore the function of the enzymes involved in PQQ biosynthesis. Six gene products (PqqA-F) are required to derive PQQ from glutamate and tyrosine residues encoded in the precursor peptide PqqA. In previous work we successfully characterized the last step of PQQ biosynthesis and discovered that PqqC (EC 1.3.3.11) is a novel cofactor-less oxygenase. With the stabilization of intermediate structures we achieved critical milestones on the way to understand the mechanistically intriguing problem of how dioxygen is activated for catalysis in the cofactorless oxygenase PqqC. We were able to successfully elucidate crystal structures of several key active site mutants (H154N, R157S, Y175F, R179S and doublemutant Y175S/R179S) in complex with AHQQ (PNAS 2004; Biochemistry 2007, BMC-Biochemistry 2008, Proteins 2010 in press). These complex structures revealed several reaction intermediates that we now want to further characterize by neutron crystallography and NMR spectroscopy. In addition, we plan to extend our work on the remaining PQQ biosynthesis proteins and study their reactions using biophysical methods combined with mutagenesis of critical residues. We have already established proof of concept with obtaining the structure of PqqB and active protein for PqqE and PqqF.