Structure and function of Aspergillus niger laccase McoG

Abstract

Biocatalysis 2017; 3: 1-16 p-phenylenediamine sulphate. However, the activities of H253A and H253N with 2-amino-4-methylphenol and 2-amino-4-methoxyphenol are strongly reduced compared to that of wild type. The redox potentials and electron transfer rates (k s ) of wild type and variants were determined (McoG wt E°' is +453 mV), and especially the reduced k s values of H253A and H253N show strong correlation with their low activity on phenolic compounds. In summary, our results suggest that the His253 adaptation of McoG can be beneficial for the conversion of phenolic compounds.Keywords: Aspergillus niger; crystal structure; laccase; redox potential; metalloprotein; multicopper oxidase Data deposition: Coordinates have been deposited with the Protein Data Bank. Accession codes are 5LM8, 5LWW and 5LWX.Abbreviations: CV, cyclic voltammetry; DT, decane-1-thiol; MCO, multicopper oxidase; MaL, laccase from Melanocarpus albomyces; TaL, laccase from Thielavia arenaria; SAM, self-assembled monolayer; SCE, saturated calomel electrode; SHE, standard hydrogen electrode; DMPPDA, N,N-dimethyl-p-phenylenediamine; ABTS, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid). IntroductionMulticopper oxidases (MCOs) form a family of redox enzymes that catalyze the reduction of molecular oxygen into water by a four-electron transfer process. It includes laccases (EC 1.10.3.2), ascorbate oxidases (EC 1.10.3.3), bilirubin oxidases (EC 1.3.3.5) and ferroxidases (EC 1.16.3.1), which are key enzymes in many biological processes of prokaryotic and eukaryotic organisms [1,2]. Their ability to catalyze the oxidation of various aromatic substrates with the concomitant reduction of molecular oxygen to water as sole byproduct makes them interesting green biocatalysts. The MCO catalyzed redox process is mediated by two copper centers, which contain Abstract: The ascomycete Aspergillus niger produces several multicopper oxidases, but their biocatalytic properties remain largely unknown. Elucidation of the crystal structure of A. niger laccase McoG at 1.7 Å resolution revealed that the C-terminal tail of this glycoprotein blocks the T3 solvent channel and that a peroxide ion bridges the two T3 copper atoms. Remarkably, McoG contains a histidine (His253) instead of the common aspartate or glutamate expected to be involved in catalytic proton transfer with phenolic compounds. The crystal structure of H253D at 1.5 Å resolution resembles the wild type structure. McoG and the H253D, H253A and H253N variants have similar activities with 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid or N,N-dimethyl-

A comparative structural analysis of the surface properties of asco-laccases

“…Based on the redox-potential-sequence-correlation described above, this could suggest that An L has a higher redox potential. However, the redox potential determined for An L is in the same range as for the other asco-laccases: An L 0.45 V [ 28 ]; Mt L 0.47 V [ 52 ]; Ma L 0.48 V [ 8 ] and Ta L 0.51 V [ 25 ]. On the other hand, a remarkably high redox potential of 0.72 V was reported for Ba L [ 26 ].…”

“…The asco-laccase fold is stabilized by two conserved disulphide bridges (Cys114-Cys540 and Cys298-332, Mt L numbering). A third disulphide bridge (Cys4-Cys12) located in the N-terminal extension is conserved in all the structures, except for An L which has a disordered N-terminal extension that could not be modelled in the crystal structure [ 28 ]. Ba L displays a fourth disulphide bridge between Cys201-Cys209 that seems to stabilize an extended loop (see Fig 5 ).…”

“…In An L Glu235 is replaced by a histidine (His253), and in both Ba L and An L an additional polar residue (Glu497 in Ba L) is found at the bottom of the pocket ( Fig 5B, 5F and 5H ); the other three asco-laccases contain a glycine in this position. Mutagenesis studies have shown that His253 in An L, similarly to Glu235, is essential for the oxidation of phenolic substrates [ 28 ]. The unique character of the T1-pockets in Ba L and An L demonstrates a previously ignored structural diversity within the asco-subgroup of laccases, that is reflected in the nature of the in vivo substrates.…”

“…With the aim of elucidating the structure and function of asco-laccases, we have undertaken the structure determination of the asco-laccase from the thermophilic fungus Myceliophthora thermophila ( Mt L) [ 22 ], The structure is known of four other asco-laccases (AA1_3): Ma L from Melanocarpus albomyces [ 23 , 24 ], Ta L from Thielavia arenaria [ 25 ], Ba L from Botrytis aclada [ 26 , 27 ] and An L from Aspergillus niger [ 28 ] ( S1 Table ). A significant evolutionary distance between the different asco-laccases is reflected in significant structural differences.…”

Making glycoproteins a little bit sweeter withPDB-REDO

“…Carbohydrate-renaming events.An example of incorrect modelling of N-glycosylation in the structure of the laccase McoG (PDB entry 5lm8;Ferraroni et al, 2017). The first NAG residue is attached to the O atom (OD1 in PDB nomenclature) of Asn103, rather than to the N atom.…”