Structure-dependent modulation of substrate binding and biodegradation activity of Pirin proteins towards plant flavonols.

Abstract

Pirin is a non-heme metalloprotein that occurs widely in human tissues and is highly\xa0conserved across\xa0all 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 their catalytic specificity remain poorly understood. In this work, by screening against a panel of plant flavonoids, we found that both Pirins catalyze the oxidative degradation of a\xa0wide\xa0spectrum of flavonol analogues, and release carbon monoxide (CO) in the process. This shows that Pirin acts on a broad range of substrates and could represent a novel dietary source of CO in vivo. Although the kinetic profiles differ substantially between two Pirins, the identified substrate structures all share a\xa02,3-double bond, 3-hydroxyl and\xa04-oxo\xa0groups on their flavonol\xa0backbone , which contribute to the specific enzyme-substrate interaction. While hPirin is iron-dependent, YhhW is identified as a novel nickel-containing dioxygenase member of the bicupin family. Besides the expanded Pirin activity, 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. In addition, YhhW features a flexible Ω-loop near the catalytic cavity, which may help stabilize the reaction intermediates via a Ni-flavonol complex. The structure-dependent modulation of substrate binding to the catalytic cavity adds to understanding the differential dispositions of natural flavonols by human and bacterial Pirins.