Identification of Amino Acid Residues Responsible for C-H Activation in Type-III Copper Enzymes by Generating Tyrosinase Activity in a Catechol Oxidase.

Tyrosinases (TYRs) catalyze the hydroxylation of phenols and the oxidation of the resulting o-diphenols to o-quinones, while catechol oxidases (COs) exhibit only the latter activity. Aurone synthase (AUS) is not able to react with classical tyrosinase substrates, such as tyramine and l-tyrosine, while it can hydroxylate its natural substrate isoliquiritigenin. The structural difference of TYRs, COs, and AUS at the heart of their divergent catalytic activities is still a puzzle. Therefore, a library of 39 mutants of AUS from Coreopsis grandiflora (CgAUS) was generated and the activity studies showed that the reactivity of the three conserved histidines (HisA2 , HisB1 , and HisB2 ) is tuned by their adjacent residues (HisB1 +1, HisB2 +1, and waterkeeper residue) either to react as stronger bases or / and to stabilize a position permissive for substrate proton shuffling. This provides the understanding for C-H activation based on the type-III copper center to be used in future biotechnological processes.

In situ formation of the first proteinogenically functionalized [TeW6O24O2(Glu)]7- structure reveals unprecedented chemical and geometrical features of the Anderson-type cluster.

The chemistry of polyoxometalates (POMs) in a protein environment is an almost unexplored but highly relevant research field as important biological and pharmacological attributes of certain POMs are based on their interactions with proteins. We report on the A-type Anderson-Evans polyoxotungstate, [TeW6O24]6- (TEW), mediated crystallization of Coreopsis grandiflora aurone synthase (cgAUS1) using ∼0.24 mM protein and 1.0 mM TEW. The 1.78 Å crystal structure reveals the covalent binding of TEW to the protein under the formation of an unprecedented polyoxotungstate cluster, [TeW6O24O2(Glu)]7- (GluTEW). The polyoxotungstate-protein complex exhibits the first covalent bond between a protein and the A-type Anderson-Evans cluster, an archetype where up to now no hybrid structures exist. The polyoxotungstate is modified at two of its six addenda tungsten atoms, which covalently bind to the carboxylic oxygen atoms of glutamic acid (Glu157), leading to W-O distances of ∼2.35 Å. This ligand substitution reaction is accompanied by a reduction of the coordination number of two µ3 polyoxotungstate oxygen atoms. This is so far unique since all known hybridizations of the Anderson-Evans POM with organic units have been obtained via the functionalization of the B-type Anderson-Evans structure through its bridging oxygen atoms. The structure reported here proves the reactivity of this POM archetype's addenda atoms as it has been administered into the protein solution as a pre-assembled cluster. Moreover, the novel cluster [TeW6O24O2(Glu)]7- displays the great versatility of the Anderson-Evans POM class.

Aurone synthase is a catechol oxidase with hydroxylase activity and provides insights into the mechanism of plant polyphenol oxidases.

Tyrosinases and catechol oxidases belong to the family of polyphenol oxidases (PPOs). Tyrosinases catalyze theo-hydroxylation and oxidation of phenolic compounds, whereas catechol oxidases were so far defined to lack the hydroxylation activity and catalyze solely the oxidation of o-diphenolic compounds. Aurone synthase from Coreopsis grandiflora (AUS1) is a specialized plant PPO involved in the anabolic pathway of aurones. We present, to our knowledge, the first crystal structures of a latent plant PPO, its mature active and inactive form, caused by a sulfation of a copper binding histidine. Analysis of the latent proenzyme's interface between the shielding C-terminal domain and the main core provides insights into its activation mechanisms. As AUS1 did not accept common tyrosinase substrates (tyrosine and tyramine), the enzyme is classified as a catechol oxidase. However, AUS1 showed hydroxylase activity toward its natural substrate (isoliquiritigenin), revealing that the hydroxylase activity is not correlated with the acceptance of common tyrosinase substrates. Therefore, we propose that the hydroxylase reaction is a general functionality of PPOs. Molecular dynamics simulations of docked substrate-enzyme complexes were performed, and a key residue was identified that influences the plant PPO's acceptance or rejection of tyramine. Based on the evidenced hydroxylase activity and the interactions of specific residues with the substrates during the molecular dynamics simulations, a novel catalytic reaction mechanism for plant PPOs is proposed. The presented results strongly suggest that the physiological role of plant catechol oxidases were previously underestimated, as they might hydroxylate their--so far unknown--natural substrates in vivo.

Crystallization and preliminary crystallographic analysis of latent, active and recombinantly expressed aurone synthase, a polyphenol oxidase, from Coreopsis grandiflora.

Aurone synthase (AUS), a member of a novel group of plant polyphenol oxidases (PPOs), catalyzes the oxidative conversion of chalcones to aurones. Two active cgAUS1 (41.6 kDa) forms that differed in the level of phosphorylation or sulfation as well as the latent precursor form (58.9 kDa) were purified from the petals of Coreopsis grandiflora. The differing active cgAUS1 forms and the latent cgAUS1 as well as recombinantly expressed latent cgAUS1 were crystallized, resulting in six different crystal forms. The active forms crystallized in space groups P2(1)2(1)2(1) and P12(1)1 and diffracted to ∼ 1.65 Šresolution. Co-crystallization of active cgAUS1 with 1,4-resorcinol led to crystals belonging to space group P3(1)21. The crystals of latent cgAUS1 belonged to space group P12(1)1 and diffracted to 2.50 Šresolution. Co-crystallization of recombinantly expressed pro-AUS with the hexatungstotellurate(VI) salt Na6[TeW6O24] within the liquid-liquid phase separation zone significantly improved the quality of the crystals compared with crystals obtained without hexatungstotellurate(VI).

Site-directed mutagenesis around the CuA site of a polyphenol oxidase from Coreopsis grandiflora (cgAUS1).

Aurone synthase from Coreopsis grandiflora (cgAUS1), catalyzing conversion of butein to sulfuretin in a type-3 copper center, is a rare example of a polyphenol oxidase involved in anabolism. Site-directed mutagenesis around the CuA site of AUS1 was performed, and recombinant enzymes were analyzed by mass spectrometry. Replacement of the coordinating CuA histidines with alanine resulted in the presence of a single copper and loss of diphenolase activity. The thioether bridge-building cysteine and a phenylalanine over the CuA site, exchanged to alanine, have no influence on copper content but appear to play an important role in substrate binding.

Latent and active aurone synthase from petals of C. grandiflora: a polyphenol oxidase with unique characteristics.

MAIN CONCLUSION: Aurone synthase belongs to the novel group 2 polyphenol oxidases and the presented kinetic characterization suggests a differing aurone biosynthesis in Asteraceae species compared to snapdragon. Aurone synthases (AUS) are polyphenol oxidases (PPO) physiologically involved in the formation of yellow aurone pigments in petals of various Asteraceae species. They catalyze the oxidative conversion of chalcones into aurones. Latent (58.9 kDa) and active (41.6 kDa) aurone synthase from petals of C. grandiflora was purified by a quantitative removal of pigments using aqueous two-phase separation and several subsequent chromatographic steps. The purified enzymes were identified as cgAUS1 (A0A075DN54) and sequence analysis revealed that cgAUS1 is a member of a new group of plant PPOs. Mass determination experiments of intact cgAUS1 gave evidence that the C-terminal domain, usually shielding the active site of latent polyphenol oxidases, is linked to the main core by a disulfide bond. This is a novel and unique structural feature of plant PPOs. Proteolytic activation in vivo leads to active aurone synthase possessing a residual peptide of the C-terminal domain. Kinetic characterization of purified cgAUS1 strongly suggests a specific involvement in 4-deoxyaurone biosynthesis in Coreopsis grandiflora (Asteraceae) that differs in various aspects compared to the 4-hydroxyaurone formation in Antirrhinum majus (Plantaginaceae): cgAUS1 is predicted to be localized in the thylakoid lumen, it possesses exclusively diphenolase activity and the results suggest that aurone formation occurs at the level of chalcone aglycones. The latent enzyme exhibits allosteric activation which changes at a specific product concentration to a constant reaction rate. The presented novel structural and functional properties of aurone synthase provide further insights in the diversity and role of plant PPOs.

Cloning and functional expression in E. coli of a polyphenol oxidase transcript from Coreopsis grandiflora involved in aurone formation.

Polyphenol oxidases are involved in aurone biosynthesis but the gene responsible for 4-deoxyaurone formation in Asteraceae was so far unknown. Three novel full-length cDNA sequences were isolated from Coreopsis grandiflora with sizes of 1.80kb (cgAUS1) and 1.85kb (cgAUS2a, 2b), encoding for proteins of 68-69kDa, respectively. cgAUS1 is preferably expressed in young petals indicating a specific role in pigment formation. The 58.9kDa AUS1 holoproenzyme, was recombinantly expressed in E. coli and purified to homogeneity. The enzyme shows only diphenolase activity, catalyzing the conversion of chalcones to aurones and was characterized by SDS-PAGE and shot-gun type nanoUHPLC-ESI-MS/MS.