Trp–His covalent adduct in bilirubin oxidase is crucial for effective bilirubin binding but has a minor role in electron transfer

Abstract

Unlike any protein studied so far, the active site of bilirubin oxidase from Myrothecium verrucaria contains a unique type of covalent link between tryptophan and histidine side chains. The role of this post-translational modification in substrate binding and oxidation is not sufficiently understood. Our structural and mutational studies provide evidence that this Trp396–His398 adduct modifies T1 copper coordination and is an important part of the substrate binding and oxidation site. The presence of the adduct is crucial for oxidation of substituted phenols and it substantially influences the rate of oxidation of bilirubin. Additionally, we bring the first structure of bilirubin oxidase in complex with one of its products, ferricyanide ion, interacting with the modified tryptophan side chain, Arg356 and the active site-forming loop 393-398. 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.

Paired refinement under the control ofPAIREF

“…The functionality and versatility of PAIREF have been thoroughly tested on a number of cases. Here, we selected six structures and data sets to demonstrate the broad application potential of the tool: simulated data for lysozyme from Gallus gallus (SIM) (Holton et al, 2014), and measured data for thermolysin from Bacillus thermoproteolyticus (TL) (Winter et al, 2018), a cysteine-bound complex of cysteine dioxygenase from Rattus norvegicus (CDO) (Karplus & Diederichs, 2012), endothiapepsin from Cryphonectria parasitica in complex with fragment B53 (EP) (Huschmann et al, 2016), interferon gamma from Paralichthys olivaceus (POLI) (Zahradník et al, 2018) and bilirubin oxidase from Myrothecium verrucaria (BO) (Koval' et al, 2019). All the results are available from http://doi.org/10.5281/zenodo.3687267.…”

“…To demonstrate this functionality, we have analyzed the crystal structure of bilirubin oxidase in complex with ferricyanide (BO) (PDB entry 6i3j). The structure was previously refined at 2.59 Å resolution with hI/(I)i equal to 2 in the highest resolution shell (Koval' et al, 2019) as shown in Fig. 3(i).…”

Emerging Trends of Bilirubin Oxidases at the Bioelectrochemical Interface: Paving the Way for Self-Powered Electrochemical Devices and Biosensors

“…In the mvBOD active site, tryptophan and histidine side chains combine to form a unique type of covalent bond. Koval et al 53 Trp−His adduct in mvBOD participates in various engagements. Specifically, Trp396-N ε1 establishes hydrogen bonds with both the main chain oxygen of Phe354 and the side chain oxygen of Asn391.…”

“…The immediate environment of Trp−His in mvBOD is shaped by the side chain of Arg356 and main-chain atoms of the 393− 395 loop (Gly393, Asn394, and Gly395). 53 Akter et al 57 studied the post-translational cross-link between His and Trp residues at a Cu site in wild-type BOD (WT BOD). This cross-link affects the properties of the T1Cu sites.…”

Modelling covalent linkages in CCP4

“…Challenges typically encountered when modelling covalent linkages include detecting the presence of a covalent linkage, identifying the correct chemistry and obtaining appropriate restraints for use in refinement (Kleywegt, 2007;Zheng et al, 2014;Koval' et al, 2019). General mechanisms for generating and applying restraints between covalently bound components have existed for decades.…”