Fine tuning of the multicopper oxidase activity through the precise mutation of its active site vicinity

T. Kovaľ¹, L. Švecová¹, T. Skálová¹, J. Dušková¹, L. H. Østergaard², J. Dohnálek¹

¹Institute of Biotechnology of the Czech Academy of Sciences, v.v.i., Průmyslová 595, Vestec, 252 50, Czech Republic

²Novozymes A/S, Brudelysvej 26, DK-2880 Bagsværd, Denmark

tomas.koval@ibt.cas.cz

Enzymes belonging to blue multicopper oxidase family use four copper ions localized in two spatially separated sites to oxidize many different natural and artificial substrates and transfer electrons to oxygen. Due to this ability, multicopper oxidases are intensively studied and also used for many applications in biotechnology. For example, laccases are utilized in food industry for color enhancement, cork modification or brewing [1].

Our research is focused on bilirubin oxidase (BOX) from Myrothecium verrucaria (EC: 1.3.3.5) which is already used in medical diagnostics (e.g. level of bilirubin in blood). For its high redox potential and its ability to, based on reaction conditions, selectively oxidize chemically distinct types of substrates BOX is in the focus of the development of biosensors [2] and biofuel cells [3]. 

In our previous study, we kinetically characterized BOX activity towards four different substrates (ferrocyanide, ABTS, bilirubin and 2,6-dimethoxyphenol). We obtained the structure of BOX in complex with ferricyanide which is one of its products (PDB ID: 6I3J) and explained the role of the unique tryptophan – histidine covalent adduct present in its active site [4]. Based on these results and also on two new structures of complexes with BOX inhibitors we designed, expressed and enzymatically characterized several functional mutants of BOX targeting the vicinity of the active site. Interestingly, different point mutations have diverse effects on the activity of BOX toward the aforementioned substrates with some of them leading to an increase of activity. These recent results present a solid base for further optimization of BOX activity through mutagenesis and its future possible utilization in biotechnology.

1. Osma, J.F., Toca-Herrera, J.L. & Rodríguez-Couto, S. Uses of laccases in the food industry. Enzyme Res. 2010, 918761 (2010).

2. Monteiro, T., Moreira, M., Gaspar, S.B.R. et al. Almeida MG. Bilirubin oxidase as a single enzymatic oxygen scavenger for the development of reductase-based biosensors in the open air and its application on a nitrite biosensor. Biosens Bioelectron. 217, 114720 (2022).

3. Franco, J.H., Minteer, S.D. & De Andrade, A.R. Ethanol Biofuel Cells: Hybrid Catalytic Cascades as a Tool for Biosensor Devices. Biosensors (Basel). 11, 41 (2021).

4. Kovaľ, T., Švecová, L., Østergaard, L.H. et al. Trp–His covalent adduct in bilirubin oxidase is crucial for effective bilirubin binding but has a minor role in electron transfer. Sci Rep 9, 13700 (2019).

The work was supported by the institutional support of IBT CAS, v.v.i. (RVO: 86652036), ERDF (CZ.02.1.01/0.0/0.0/15_003/0000447, CZ.02.1.01/0.0/0.0/16_013/0001776 and CZ.1.05/1.1.00/02.0109), MEYS CR (CZ.02.1.01/0.0/0.0/16_019/0000778 and LM2018127, support of Biocev-CMS – core facilities Biophysical Methods, Crystallization of Proteins and Nucleic Acids, and Structural Mass Spectrometry of CIISB, part of Instruct-ERIC).