Multistep, Eight-Electron Oxidation Catalyzed by the Cofactorless Oxidase, PqqC: Identification of Chemical Intermediates and Their Dependence on Molecular Oxygen

Abstract

The final step of the biosynthesis of the prokaryotic cofactor, PQQ, is catalyzed by PqqC, a cofactorless oxidase that brings about a ring closure and overall eight-electron oxidation of its substrate. Time-dependent acid quenching and subsequent HPLC separation and mass spectrometric analyses of reaction mixtures were performed in order to correlate the structures of intermediates with previously observed UV/Vis signatures. The reaction is composed of four stepwise oxidations: three steps use O2 as the two-electron acceptor and the fourth uses hydrogen peroxide (H2O2). The chemical nature of the intermediates, the stoichiometry of the reaction and their dependence on the oxygen concentration indicate that the third oxidation uses the product H2O2 from the preceding step to produce water. The last oxidation step can also be studied separately and is a reaction between O2 and PQQH2 trapped in the active site. This oxidation is approximately 10 times slower than the reoxidation of PQQH2 in solution. From the order of the four oxidation steps and their sensitivity to O2 concentration, we propose a progressive closure of the active site as the enzyme proceeds through its catalytic cycle.

Demonstration That the Radical S-Adenosylmethionine (SAM) Enzyme PqqE Catalyzes de Novo Carbon-Carbon Cross-linking within a Peptide Substrate PqqA in the Presence of the Peptide Chaperone PqqD

“…1A) was identified more than 25 years ago (4,30), and more recent bioinformatics studies demonstrated a conservation of gene order that includes an occasional gene fusion between PqqD with either the C terminus of PqqC or the N terminus of PqqE (2). However, the description of function for each gene product from this operon has lagged considerably, with PqqC representing the only enzyme within the pathway successfully characterized with regard to structural, spectroscopic, and kinetic properties (5,6,31). Although PqqE had been shown to be a radical SAM enzyme (22), it had not been possible to demonstrate a reaction between the peptide substrate PqqA and PqqE, suggesting either that PqqA must be modified before it can interact with PqqE or that key components of the PqqE system were missing (7).…”

Recent progress in studies on the health benefits of pyrroloquinoline quinone

“…32,33) In all cases, pqqA encodes a small polypeptide, typically 20-30 amino acids in length, containing a conserved glutamate and tyrosine that serve as the backbone in PQQ biogenesis. 34,35) The glutamate and tyrosine undergo post-translational modifications to form the intermediate 3a-(2-amino-2-carboxyethyl)-4,5-dioxo-4,5,6,7,8,9-hexahydroquinoline-7,9-dicarboxylic acid (AHQQ). 32,34) PqqC is the most characterized and has been shown to catalyze the eight-electron oxidation and ring cyclization of AHQQ to form PQQ.…”

“…34,35) The glutamate and tyrosine undergo post-translational modifications to form the intermediate 3a-(2-amino-2-carboxyethyl)-4,5-dioxo-4,5,6,7,8,9-hexahydroquinoline-7,9-dicarboxylic acid (AHQQ). 32,34) PqqC is the most characterized and has been shown to catalyze the eight-electron oxidation and ring cyclization of AHQQ to form PQQ. 35) A large number of bacteria extracellularly excrete PQQ and is indicated as micrograms per mL of broth culture.…”

Intrigues and Intricacies of the Biosynthetic Pathways for the Enzymatic Quinocofactors: PQQ, TTQ, CTQ, TPQ, and LTQ

“…50 Moreover, a progressive closure from an observed open to a closed conformation plays a primordial role in the catalysis (Figure 6). 53 Second, PqqD, a small 10-kDa protein with no homology to any characterized protein, has an indispensable role in the biosynthesis of PQQ that is not yet elucidated. Third, a strictly anaerobic PqqE must function with an O 2 -dependent PqqC and likely, an O 2 -dependent non-heme iron oxygenase (PqqB).…”