J. Dohnálek1, T. Kovaľ1, L. Švecová1, 2, L. H. Østergaard3, T. Skálová1, J. Dušková1, J. Hašek1, P. Kolenko1,2, J. Stránský1, M. Trundová1
Covalent modifications of amino acids convey functionality, stability or interaction potential of a given protein. Many modifications are relatively well understood, such as glycosylation, disulphide bond formation, phosphorylation, methylation, co-factor covalent binding and other. Here we present a modification, relatively recently discovered and so far unique in proteins. The active site of bilirubin oxidase from the ascomycete plant pathogen Myrothecium verrucaria contains a covalent link between the side chains of tryptophan and histidine [1, 2]. Its presence was confirmed independently in several crystal structures and by mass spectrometry analysis [2, 3]. The role of this post-translational modification in substrate binding and oxidation is not sufficiently understood.
Protein bilirubin oxidase is used in medicine for determination of bilirubin level. Apart from bilirubin, this enzyme reacts strongly also with other organic and inorganic substrates, preferring different pH optima. The enzyme is also exploited in the textile industry, wood-processing industry, and, recently, also in nanotechnologies, especially in the construction of bio-fuel cells and biosensors.
The Trp396–His398 adduct is located at the T1 copper site of this multicopper oxidase and participates in oxidation of various types of substrates. The enzyme further transfers electrons from the T1 copper site to the trinuclear copper cluster in the protein interior, where reduction of O2 to two H2O molecules is catalysed.
The first structure of bilirubin oxidase in complex with one of its products, ferricyanide ion, shows interaction with the modified tryptophan side chain, Arg356, and with the active site-forming loop 393-398 [3]. Our structural and mutational studies also confirm that the adduct modifies T1 copper coordination and is important for the substrate binding and oxidation site [3]. The effects of the presence of the adduct vary with the type of the substrate being oxidized. The results imply that structurally and chemically distinct types of substrates, including bilirubin, utilize the Trp–His adduct mainly for binding and to a smaller extent for electron transfer [3].
The results have implications for technological applications of bilirubin oxidase and related enzymes.
This work was supported by the European Regional Development Fund (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), institutional support of the Institute of Biotechnology of the Czech Academy of Sciences, v. v. i. (RVO: 86652036), by the Ministry of Education, Youth, and Sports of the Czech Republic (LM2015043, support of Biocev-CMS).