Structure and function of Aspergillus niger laccase McoG

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(3ethylbenzothiazoline-6-sulphonic acid or N,N-dimethylResearch Article Open Access © 2017 Marta Ferraroni et al., published by De Gruyter Open. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 License. Marta Ferraroni#, Adrie H. Westphal#, Marco Borsari#, Juan Antonio Tamayo-Ramos, Fabrizio Briganti†, Leo H. de Graaff†, Willem J. H. van Berkel* Structure and function of Aspergillus niger laccase McoG *Corresponding author: Willem J. H. van Berkel, Laboratory of Biochemistry, Wageningen University & Research, Stippeneng 4, Wageningen 6708 WE, The Netherlands, E-mail: [email protected] Marta Ferraroni, Fabrizio Briganti, Department of Chemistry ‘Ugo Schiff’, University of Florence, Via della Lastruccia 3, I-50019 Sesto Fiorentino, Italy Adrie H. Westphal, Laboratory of Biochemistry, Wageningen University & Research, Stippeneng 4, Wageningen 6708 WE, The Netherlands Marco Borsari, Department of Chemical and Geological Sciences, University of Modena and Reggio Emilia, Via Campi 103, I-41125 Modena, Italy Juan Antonio Tamayo-Ramos, Leo H. de Graaff, Microbial Systems Biology, Laboratory of Systems and Synthetic Biology, Wageningen University & Research, Stippeneng 4, Wageningen 6708 WE, The Netherlands Juan Antonio Tamayo-Ramos, International Research Center in Critical Raw Materials for Advanced Industrial Technologies (ICCRAM), University of Burgos, Plaza Misael Bañuelos s/n, 09001, Burgos, Spain #Authors contributed equally †Prof. Dr. Fabrizio Briganti passed away on 22 June 2012. We dedicate this article to Dr. Leo H. de Graaff, who passed away on 6 October 2016. Unauthenticated Download Date | 12/16/17 12:08 PM 2 M. Ferraroni, et al. in total four copper atoms, classified as T1 (one copper), T2 (one copper), and T3 (two coppers) according to their spectroscopic characteristics [3]. Laccases form the largest subgroup within the MCO family and have received most of the attention in biochemical and biotechnological studies [2, 4]. They are generally extracellular monomeric glycoproteins with molecular weights ranging from 60 to 80 kDa, and up to 30% of their molecular weight can be made up of carbohydrates [4]. These enzymes are widely distributed in nature and in fungi, laccase activities have been related to the degradation of lignocellulose material, the production of pigments, processes of morphogenesis, sporulation, the involvement in pathogenesis toward plants and animals [5] and the oxidation of antibiotics produced by microorganisms [6]. In addition, laccases can oxidize non-phenolic compounds through the inclusion of redox mediators, with which they are able to oxidize e.g. lignin, cellulose or starch [7]. This ability is used in various biotechnological processes, including the breakdown of colorants, bioremediation, biopulping and the synthesis of pharmaceutical products [8]. Laccases are present in various groups of fungi, including yeasts [9], filamentous ascomycetes [10] and white [11] and brown rot fungi [12], as well as mycorrhizal species [13]. In particular, basidiomycete laccases of several Trametes and Pleurotus species have been well characterized. Aspergillus MCOs, included in the ascomycete laccases cluster, have received little attention. A significant number of these enzymes, including A. nidulans LccA, LccB and LccC [14] and the MCO group from A. niger remain largely uncharacterized [15]. Interestingly, A. niger MCOs have a low similarity to laccases present in the basidiomycete laccases cluster (around 25% identical). Thus, to obtain insight into the biological role and possible biotechnological potential of this particular group of MCOs, more knowledge about their structure and function is required. Recently, ten of these A. niger laccases were homologously overexpressed, revealing remarkable biochemical differences [10]. Here, we address the structural and catalytic properties of one of these, McoG, which has an unusual histidine (His253) in the active site, and in addition, the properties of three variants (H253D, H253A and H253N). 2 Material and methods