title: Mechanistic studies on the tyrosinase-catalyzed formation of the anachelin chromophore.
abstract: The complex secondary metabolite anachelin, isolated from the freshwater cyanobacterium Anabaena cylindrica, is believed to act as siderophore, facilitating iron uptake. Its structure is characterized by a fascinating blend of polyketide, peptide, and alkaloid fragments. In particular, the tetrahydroquinolinium-derived chromophore is unique among natural products, and its biosynthesis is unknown. We propose a hypothesis for the biogenesis of the anachelin chromophore starting from a C-terminally bound L-Tyr residue. It is proposed that this amino acid is reductively aminated, methylated, and hydroxylated. Oxidation of this catechol diamine substrate by a tyrosinase would lead to an o-quinone, which would react by intramolecular aza-annulation and tautomerization to give the anachelin chromophore. In order to evaluate this hypothesis, a model substrate related to the proposed biogenetic precursor was prepared. It was shown that the enzyme tyrosinase is able to transform this substrate into an anachelin chromophore derivative, which corroborates the biogenetic hypothesis. In order to gain further insight into the mechanism of this transformation, we performed spectrophotometric reaction monitoring, allowing the formation of the expected product to be observed. In addition, a rise in absorption at around 250 nm might be due to the presence of a spiro five-membered ring intermediate resulting from an alternative 1,4-addition to the o-quinone. Lastly, we were able to show that the action of tyrosinase on this substrate follows Michaelis-Menten kinetics (k(cat)=123 s(-1) and K(m)=8.66 mM). Interestingly, the catalytic efficiency is decreased only by a factor of 30 relative to the natural substrate L-DOPA.
authors.affiliation: Laboratorium für Organische Chemie der Eidgenössischen Technischen Hochschule Zürich, ETH Hönggerberg, 8093 Zürich, Switzerland. gademann@org.chem.ethz.ch
authors.forename: Karl
authors.initials: K
authors.lastname: Gademann
mesh_terms: D001372:Aza Compounds / Q000494:pharmacology; D002384:Catalysis; D007700:Kinetics; D008543:Melanins / Q000096:biosynthesis / Q000737:chemistry; D015394:Molecular Structure; D014442:Monophenol Monooxygenase / Q000378:metabolism*; D009842:Oligopeptides / Q000096:biosynthesis* / Q000737:chemistry* / Q000378:metabolism; D010084:Oxidation-Reduction; D019800:Phenol / Q000737:chemistry; D011806:Quinolinium Compounds / Q000737:chemistry* / Q000378:metabolism; D013053:Spectrophotometry; D013379:Substrate Specificity
chemical_list: D001372:Aza Compounds; D008543:Melanins; D009842:Oligopeptides; D011806:Quinolinium Compounds; C420972:anachelin; D019800:Phenol; D014442:Monophenol Monooxygenase