Crystal Structure of a Four-Copper Laccase Complexed with an Arylamine:  Insights into Substrate Recognition and Correlation with Kinetics^,

Abstract

Laccases are multicopper oxidases that catalyze the oxidation of a wide range of phenols or arylamines, and their use in industrial oxidative processes is increasing. We purified from the white rot fungus Trametes versicolor a laccase that exists as five different isozymes, depending on glycosylation. The 2.4 A resolution structure of the most abundant isozyme of the glycosylated enzyme was solved. The four copper atoms are present, and it is the first crystal structure of a laccase in its active form. The crystallized enzyme binds 2,5-xylidine, which was used as a laccase inducer in the fungus culture. This arylamine is a very weak reducing substrate of the enzyme. The cavity enclosing 2,5-xylidine is rather wide, allowing the accommodation of substrates of various sizes. Several amino acid residues make hydrophobic interactions with the aromatic ring of the ligand. In addition, two charged or polar residues interact with its amino group. The first one is an histidine that also coordinates the copper that functions as the primary electron acceptor. The second is an aspartate conserved among fungal laccases. The purified enzyme can oxidize various hydroxylated compounds of the phenylurea family of herbicides that we synthesized. These phenolic substrates have better affinities at pH 5 than at pH 3, which could be related to the 2,5-xylidine binding by the aspartate. This is the first high-resolution structure of a multicopper oxidase complexed to a reducing substrate. It provides a model for engineering laccases that are either more efficient or with a wider substrate specificity.

A Labile Regulatory Copper Ion Lies Near the T1 Copper Site in the Multicopper Oxidase CueO

“…CueO can oxidize a range of substrates in vitro, including catechols, siderophores, and Fe(II) (10), and this activity is greatly enhanced in the presence of excess (ϳ100 M) copper(II) ions (10,15), suggesting that binding of a labile copper is important for enzymatic activity. We recently determined the crystal structure of CueO at 1.4-Å resolution (15) and showed that its structure is similar to that of other multicopper oxidases, such as ascorbate oxidase (16) and several laccases (17)(18)(19). As expected, CueO contains a T1 "blue copper" site, near the point at which the larger substrate is believed to bind, as well as a trinuclear copper center, where oxygen reduction occurs.…”

Structural insight into the oxidation of sinapic acid by CotA laccase

“…One methoxy group of SA forms a hydrogen bond with the surface-exposed histidine His419, one of the Cu1 ligands, by N-O hydrogen bond. ABTS is reported bonding to His497, the other Cu1 ligand, which is another binding mode also observed in other fungal laccases/substrates complexes (Bertrand et al, 2002;Kallio et al, 2009;Matera et al, 2008). In CotA laccase, Cu1 is coordinated by two histidines including His419 and His497, both surface-exposed and available for substrate binding.…”

“…To date, four crystal structures of laccase/ligand complex have been reported: laccase from Trametes versicolor (TvL)/2,3-xylidine complex (Bertrand et al, 2002), laccase from Trametes trogii (TtL)/p-toluate complex (Matera et al, 2008), laccase from Melanocarpus albomyces (MaL)/2,6-dimethoxyphenol (DMP) complex (Kallio et al, 2009) and CotA/ABTS complex from Bacillus subtilis (Enguita et al, 2004). Among these ligands, DMP (a phenolic compound) and ABTS (a non-phenolic compound) are efficient synthetic mediators for laccases.…”

“…CotA is a protein isolated from the spore coat of B. subtilis and is characterized as a typical laccase (Bertrand et al, 2002;Martins et al, 2002). This enzyme has dual advantages of both thermostability and thermoactivity.…”

Evaluation of microorganisms performance in hemicellulosic hydrolyzate detoxification and sugarcane bagasse lignin removal for ethanol production

“…MnP and LiP structures are very similar in that they are heme-containing glycoproteins which require hydrogen peroxide as an oxidant (POULOS et al, 1993;. Laccase, a copper-containing oxidase, utilizes molecular oxygen as oxidant to oxidize phenolic rings to phenoxyl radicals (BERTRAND et al, 2002).…”