Investigation of the structure and mechanism of a PQQ biosynthetic pathway component, PqqC, and a bioinformatics analysis of potential PQQ producing organisms.
- Author(s): RoseFigura, Jordan M.
- Advisor(s): Klinman, Judith
- et al.
PQQ is an exogenous, tricyclic, quino-cofactor for a number of bacterial dehydrogenase reaction. It has also been proposed to play a role as a bacterial vitamin. The following work has defined 144 bacteria species that contain the machinery to form PQQ. PQQ formation is based on a conserved operon, pqqABCDEF, in Klebsiella pneumoniae. The last enzymatic step in the PQQ biogenesis pathway is catalyzed by PqqC and involves a ring closure and eight electron oxidation of the substrate AHQQ. Wild type (WT) PqqC and various active site mutants have been studied. The asparagine and alanine mutations at the histidine 84 position have supported the published mechanism and shown a new role for H84 as an active site acid. This was shown by anaerobic reactions, where H84A was capable of proceeding to a quinol intermediate anaerobically, but the more conservative H84N mutation was not. Aerobically, both mutations were able to form PQQ. Recent X-ray investigations of PqqC variants Y175F (with PQQ bound), H154S (with PQQ bound) and a R179S/Y175S double mutant (with AHQQ bound) show that the enzyme is alternately in a closed, open and open conformation, respectively. Though R179S/Y175S does not form the characteristic closed conformation seen in the WT-PQQ structure, it is still able to inititate a ring closure with AHQQ. Using apo-glucose dehydrogenase to assay for PQQ production, none of the mutants are capable of product formation. Spectrophotometric assays give insight into the incomplete reactions being catalyzed by the mutants. Active site variants Y175F, H154N and R179S form a quinoid intermediate anaerobically. Y175S is capable of reaction further and forms a quinol intermediate anaerobically after forming a quinoid. Y175F, H154N and R179S require O2 to proceed to the quinol intermediate. None of the mutants preclude substrate/product binding. Indicating that in all cases, oxidative chemistry is impeded because no mutants can react fully to form quinone even in the presence of O2. The residues targeted are proposed to form a proteinaceous core O2 binding structure that also contributes to O2 activation.