Bilirubin oxidase from plant pathogen Myrothecium verrucaria (BO, EC 126.96.36.199) is a blue monomeric multicopper oxidoreductase (MCO) catalyzing oxidation of substrates consisting of tetrapyrrole (especially bilirubin to biliverdin), diphenols and aryl diamines. BO consists of 534 amino acid residues with a molecular mass of 60 kDa. Similarly to other MCOs the substrate binding site of BO is comprised of one Cu ion (T1) coordinated by one cysteine, two histidines and one methionine and behaves as an acceptor of an electron from substrate. Four electrons are transferred from T1 to the trinuclear copper cluster (TNC) coordinated by eight histidines, where molecular oxygen is reduced to water. BO is capable to oxidize a great variety of organic compounds with many applications in industry (e.g. pulp bleaching, delignification, drug detection and degradation of herbicides [1, 2, 3]). Two structures of BO have been already published in PDB (2XLL , 3ABG ), but none of them with a ligand in the substrate binding site. The substrate binding mechanism and the way of the electron transport from substrate to T1 site are still to be determined.
We solved two structures of BO using X-ray crystallography - native structure at 2.3 Å resolution and a structure with a small ligand in the substrate binding site at 2.6 Å resolution. The phase problem was solved by molecular replacement using a previously reported structure of BO (PDB code 2XLL ) as a model. Both structures are in the same space group F222, which is different in comparison to previously deposited structures (P1 in 2XLL , P63 in 3ABG ). Two molecules were localized in asymmetric unit. On the basis of our results we suggest two possible electron transfer routes from substrate to the T1 center.
This work is supported by the project „BIOCEV – Biotechnology and Biomedicine Centre of the Academy of Sciences and Charles University (CZ.1.05/1.1.00/02.0109), from the European Regional Development Fund.“, by the Ministry of Education, Youth and Sports of the Czech Republic (grant No. EE2.3.30.0029 and No. LG14009), by the Grant Agency of the Czech Technical University in Prague, grant No. SGS13/219/OHK4/3T/14, by Grant Agency of the Czech Republic, grant No. 15-05228S.