Recent insights into oxidative metabolism of quercetin: catabolic profiles, degradation pathways, catalyzing metalloenzymes and molecular mechanisms.

Quercetin is the most abundant polyphenolic flavonoid (flavonol subclass) in vegetal foods and medicinal plants. This dietary chemopreventive agent has drawn significant interest for its multiple beneficial health effects ("polypharmacology") largely associated with the well-documented antioxidant properties. However, controversies exist in the literature due to its dual anti-/pro-oxidant character, poor stability/bioavailability but multifaceted bioactivities, leaving much confusion as to its exact roles in vivo. Increasing evidence indicates that a prior oxidation of quercetin to generate an array of chemical diverse products with redox-active/electrophilic moieties is emerging as a new linkage to its versatile actions. The present review aims to provide a comprehensive overview of the oxidative conversion of quercetin by systematically analyzing the current quercetin-related knowledge, with a particular focus on the complete spectrum of metabolite products, the enzymes involved in the catabolism and the underlying molecular mechanisms. Herein we review and compare the oxidation pathways, protein structures and catalytic patterns of the related metalloenzymes (phenol oxidases, heme enzymes and specially quercetinases), aiming for a deeper mechanistic understanding of the unusual biotransformation behaviors of quercetin and its seemingly controversial biological functions.

An artificial metalloprotein with metal-adaptive coordination sites and Ni-dependent quercetinase activity.

Engineering non-native metal active sites into proteins using canonical amino acids offers many advantages but is hampered by significant challenges. The TIM barrel protein, imidazole glycerol phosphate synthase from the hyperthermophilic organism Thermotoga maritima (tHisF), is well-suited for the construction of artificial metalloenzymes by this approach. To this end, we have generated a tHisF variant (tHisFEHH) with a Glu/His/His motif for metal ion coordination. Crystal structures of ZnII:tHisFEHH and NiII:tHisFEHH reveal that both metal ions bind to the engineered histidines. However, the two metals bind at distinct sites with different geometries, demonstrating the adaptability of tHisF. Only ZnII additionally ligates the Glu residue and adopts a tetrahedral geometry. The pseudo-octahedral NiII site comprises the two His and a native Ser residue. NiII:tHisFEHH catalyzes the oxidative cleavage of the flavanols quercetin and myricetin, providing an unprecedented example of an artificial metalloprotein with quercetinase activity.

Beauveria bassiana: Quercetinase production and genetic diversity.

Beauveria bassiana genetic diversity and ability to synthesize quercetin 2,3-dioxygenase (quercetinase) were analyzed. B. bassiana isolates, obtained from Brazilian soil samples, produced quercetinase after induction using 0.5 g/L quercetin. B. bassiana ATCC 7159 (29.6 nmol/mL/min) and isolate IP 11 (27.5 nmol/ml/min) showed the best performances and IP 3a (9.5 nmol/mL/min) presented the lowest level of quercetinase activity in the culture supernatant. A high level of polymorphism was detected by random amplified polymorphic DNA (RAPD) analysis. The use of internal-transcribed-spacer ribosomal region restriction fragment length polymorphism (ITS-RFLP) did not reveal characteristic markers to differentiate isolates. However, the ITS1-5.8S-ITS2 region sequence analysis provided more information on polymorphism among the isolates, allowing them to be clustered by relative similarity into three large groups. Correlation was tested according to the Person's correlation. Data of our studies showed, that lower associations among groups, level of quercetinase production, or geographical origin could be observed. This study presents the production of a novel biocatalyst by B. bassiana and suggests the possible industrial application of this fungal species in large-scale biotechnological manufacture of quercetinase.

A cupin domain-containing protein with a quercetinase activity (VdQase) regulates Verticillium dahliae's pathogenicity and contributes to counteracting host defenses.

We previously identified rutin as part of potato root responses to its pathogen Verticillium dahliae. Rutin was directly toxic to the pathogen at doses greater than 160 µM, a threshold below which many V. dahliae pathogenicity-related genes were up-regulated. We identified and characterized a cupin domain-containing protein (VdQase) with a dioxygenase activity and a potential role in V. dahliae-potato interactions. The pathogenicity of VdQase knock-out mutants generated through Agrobacterium tumefasciens-mediated transformation was significantly reduced on susceptible potato cultivar Kennebec compared to wild type isolates. Fluorescence microscopy revealed a higher accumulation of flavonols in the stems of infected potatoes and a higher concentration of rutin in the leaves in response to the VdQase mutants as compared to wild type isolates. This, along with the HPLC characterization of high residual and non-utilized quercetin in presence of the knockout mutants, indicates the involvement of VdQase in the catabolism of quercetin and possibly other flavonols in planta. Quantification of Salicylic and Jasmonic Acids (SA, JA) in response to the mutants vs. wild type isolates revealed involvement of VdQase in the interference with signaling, suggesting a role in pathogenicity. It is hypothesized that the by-product of dioxygenation 2-protocatechuoylphloroglucinolcarboxylic acid, after dissociating into phloroglucinol and protocatechuoyl moieties, becomes a starting point for benzoic acid and SA, thereby interfering with the JA pathway and affecting the interaction outcome. These events may be key factors for V. dahliae in countering potato defenses and becoming notorious in the rhizosphere.

A pirin-like protein from Pseudomonas stutzeri and its quercetinase activity.

A pirin-like protein from a marine denitrifying bacterium, Pseudomonas stutzeri Zobell has been heterologously expressed in E. coli and purified to homogeneity with metal-affinity and gel filtration chromatographies. The recombinant pirin-like protein has exhibited quercetinase activities upon the incorporation of a divalent metal ion, while its biological role remains unclear. In the case of Cu2+ the holo-protein demonstrated the highest activities and spectroscopic properties typical of type II Cu protein. A 3D-structual model constructed using the crystal structure of human pirin as temperate indicated that the metal biding site is constructed with 3His1Glu located in the consensus sequences in the N-terminal domain.

BEAUVERIA BASSIANA: quercetinase production and genetic diversity

Beauveria bassiana genetic diversity and ability to synthesize quercetin 2,3-dioxygenase (quercetinase) were analyzed. B. bassiana isolates, obtained from Brazilian soil samples, produced quercetinase after induction using 0.5 g/L quercetin. B. bassiana ATCC 7159 (29.6 nmol/mL/min) and isolate IP 11 (27.5 nmol/ml/min) showed the best performances and IP 3a (9.5 nmol/mL/min) presented the lowest level of quercetinase activity in the culture supernatant. A high level of polymorphism was detected by random amplified polymorphic DNA (RAPD) analysis. The use of internal-transcribed-spacer ribosomal region restriction fragment length polymorphism (ITS-RFLP) did not reveal characteristic markers to differentiate isolates. However, the ITS1-5.8S-ITS2 region sequence analysis provided more information on polymorphism among the isolates, allowing them to be clustered by relative similarity into three large groups. Correlation was tested according to the Person's correlation. Data of our studies showed, that lower associations among groups, level of quercetinase production, or geographical origin could be observed. This study presents the production of a novel biocatalyst by B. bassiana and suggests the possible industrial application of this fungal species in large-scale biotechnological manufacture of quercetinase.