Oxidative Metabolism Of Flavonols Catalyzed By Pirin Homologous Proteins

Pirin is a non-heme metalloprotein that occurs widely in human tissues and is highly conserved across all taxa.Pirin proteins typically function as nuclear transcription regulators,but two Pirin orthologs,YhhW(from Escherichia coli)and hPirin(from humans)were revealed to possess enzymatic activity of degrading quercetin.The exact role of Pirin homologs and the catalytic nature remains poorly understood.Therefore,the in-depth studies of the catalytic kinetics and structure-function relationship of Pirins show important significance for exploring the catalytic mechanism and thus the function of Pirin homologues.The main research work of this paper is as follows:(1)Pirin homologous proteins were overexpressed and purified.At the same time,the activity of Pirin protein was tested by screening against a panel of plant flavonoids.We found that both Pirins catalyze the oxidative degradation of a wide spectrum of flavonol analogues and release carbon monoxide(CO)in the process.Besides,we present the crystal structures of the native Ni-YhhW and tag-free Fe-hPirin,revealing the distinctive differences occurring at the metal-binding site.(2)By using five flavonol substrates(quercetin,kaempferol,morin,fisetin and myricetin)and in vitro drug metabolism reaction system combing with scanning modes of mass spectrometry,the metabolite profiles were systematically compared and identified.The cleavage pathway of flavonol metabolites was also discussed,further confirming that hPirin has the quercetin dioxygenase activity.(3)The kinetic parameters of hPirin towards kaempferol,quercetin and fisetin were determined using the established liquid chromatography analysis method.The optimal conditions for reaction have been optimized in several aspects.Under the optimized conditions,the values of K_m were9.36±0.61μM,10.86±0.47μM and 12.52±0.58μM,respectively.Based on these values,the relative activities against substrates with different structures were also compared.