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Background: Pirin, a non-heme metalloprotein that occurs widely in human tissues, typically functions as nuclear transcription regulators involved in cancer metastasis. We first revealed that Pirin homologs have the activity of "flavonolase" (quercetinase-like), i.e. catalyzing the oxidative decomposition of plant flavonols with the release of carbon monoxide (CO). (Guo et al.: ACS Chem. Biol. 2019, 14, 2629). However, its metabolic pathway and catalytic mechanism are not very clear.
Methods: Using quercetin and kaempferol as representative substrates, the generation of CO was scanned, the key metabolic products were detected by HPLC, UV-Vis, MS, NMR, ESR, 18O labeling and ferrous cofactor oxidation. Several characteristic intermediates and final catabolites were prepared characterized. The enzyme kinetics parameters were analyzed by substrate consumption method and product formation method, and a panel of Pirin inhibitors were screened.
Results: Unlike quercetinase, Pirin has low CO yield, and the characteristic intermediate is a triketone or cyclic 2-monooxygenation product. Only Fe(II)-Pirin has catalytic activity, and the enzyme activity can be regulated through the conversion of Fe(II)/Fe(III) valence state (e.g., using potassium ferrous or L-ascorbic acid), and it is speculated that Pirin participates in regulating cellular oxidative stress. A newly established analytical method based on multi-reaction monitoring of multiple flavonol metabolites catalyzed by Pirin was used to investigate the initial velocity conditions for the enzymatic reactions. The kinetic parameters Km of quercetin and kaempferol were 10 μM and 4 μM, respectively.
Conclusions: Pirin as flavonolase is a non-classical quercetinase that reveals an unusual flavonol ring cleavage pathway. Its catalytic centre, the non-haem iron cofactor, may serve as an important switch for sensing and regulating intracellular redox signals.
