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Bilirubin oxidase

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Contents

Introduction

Bilirubin oxidase is an enzyme known to catalyze the oxidation of bilirubin to biliverdin by reducting O2 in water. It’s a multicopper oxidase which can exist under two different structures in the ascomycete Myrothecium verrucaria (cf 3abg). The 2xII form is a with 8 ligands NAG-NAG belonging to the family of oxidoreductases.

It was first isolated in 1981 by the scientists Sawao Murao and Noriaki Tanaka as they tried to find a microorganism able to decolore raw sewage and to use it as an analytical tool in clinical fields. Now, bilirubin oxidase may be used to determine free hemoglobin in icteric specimens and can be use as a treatment for neonatal jaundice.

Moreover, the bilirubin oxidase is an enzyme that is active in porphyrin and chlorophyll metabolism.

Structure

Multicopper oxidases family

Multicopper oxidases are enzymes which oxidise their substrate by accepting electrons at a mononuclear copper centre and transferring them to a trinuclear copper centre.[1]

Bilirubin oxidases are multicopper oxidases containing type 1, type 2, and type 3 coppers. Indeed, there is strong sequence homology between bilirubin oxidase and multicopper oxidases like laccase, ascorbate oxidase and even ceruloplasmin. Moreover, the sequence, characteristic of multicopper oxidase, is present in bilirubin oxidase. Copper is essential for the enzyme activity.[2]

Copper is classified into three types according to their optical and magnetic properties. Type 1 copper (or blue copper) shows many charge-transfer bands around 450 nm, 600 nm and 750 nm. The most peculiar band appears around 600 nm and represents the Cys to Cu(II) charge transfer. Type 2 copper (or nonblue copper) does not show any strong charge-transfer bands in the visible region. Type 3 coppers are not detectable by ESR because some are antiferromagnetically coupled. However, a hydroxide ion links them and so gives a strong absorption at 330 nm.[2] While type 2 and 3 coppers reduce dioxygen to two water molecules by forming a trinuclear center, type 1 copper transfers electrons from substrate to the trinuclear center. This is the peculiar sequence that forms an intramolecular electron-transfer pathway between the type 1 copper site and the trinuclear center composed of the type 2 and type 3 copper sites.[2]

Covalent bond between Trp396 and His398

Since the discovery of bilirubin oxidase, two cristal structures of native enzyme have been published independently. The structure shown here and described by Cracknell et al. reveals that the imidazole ring of —one of the T1Cu ligands — is abnormally close to the indole ring of , which raises the possibility that there is a . On the contrary in the structure described by Mizutani et al. (cf 3abg) the indole ring of turns away and does not form the covalent bond with the imidazole ring of [3].

Several studies proved that bilirubin oxidase has actually a post-translationally formed covalent bond between the imidazole ring of coordinated to type I copper and the indole ring of [3][4][5]. Indeed, the distance between and is 1.9 Å, which is shorter than the Van der Waals distance. Moreover, the torsion angle between the side chain rings of and is 65°, suggesting that the covalent link comprises a single bond [3].

Among the multicopper oxidases, bilirubin oxidase is the only one which forms this . It is due to the particular molecular environment of the residue, which is unique among the multicopper oxidases registered in the PDB. Indeed, the equivalent position is occupied by a glutamate side chain in multicopper oxidases from Thermus thermophilus (2xu9) and Campylobacter jejuni (3zx1), by aspartate in yeast Fet3p (1zpu), and by a main chain carbonyl oxygen atom in laccases from Antrodiella faginea (5ehf), Cerrena maxima (5h5u), Coriolus zonatus (2hzh), Lentinus sp. (3x1b), Lentinus tigrinus (2qt6), Steccherinum murashkinskyi (5mig), Trametes hirsuta (3fpx), and Trametes sp. AH28-2 (3kw7) [3].

The role of this covalent bond between the indole ring of and the imidazole ring of has been investigated using mutants of bilirubin oxidase (at the position 396). Their enzymatic activities have been compared with the one of wild type bilirubin oxidase. All the mutations have led to a significant decrease of the catalytic efficiency for bilirubin, compared to the wild type bilirubin oxidase. It proves that the has a major role in the enzymatic activity of bilirubin oxidase. However, it has no influence on the enzyme structure (except the replaced residue), since bilirubin can still bind to mutants not containing tryptophan at the position 396. Moreover, bilirubin is oxidized even in the absence of the , proving that its role in electron transfer is not crucial [5].

Function

The main function of this enzyme is to participate in the following chemical reaction: 2 bilirubin + O2 ↔ 2 biliverdin + 2 H2O

Applications

Diagnostic analysis and treatment

Bilirubin oxidase is used for diagnostic analysis of bilirubin in serum during medical examinations. It is useful in the field of clinical pathology for the diagnosis and treatment of jaundice and hyperbilirubinemia. It is used to determine two types of bilirubin: glucuronide-conjugated and unconjugated bilirubin separately.[6] Conjugated bilirubin can be quantitatively determined from a decrease in the absorbance at 450 nm caused by conjugated bilirubin oxidisation by bilirubin oxidase. The conjugated bilirubin values help for the evaluation of jaundice.[7] Finally, the addition of bilirubin to the cells has toxic effects, and the addition of bilirubin oxidase can reverse these effects. Thus, hyperbilirubinemia in newborn infants may be prevented by administering polyethylene glycol-conjugated bilirubin oxidase.[8]

Moreover, when bilirubin is present in too high concentrations the spectrophotometric determination of plasma free hemoglobin isn’t possible due to the overlapping absorption bands of haemoglobin and bilirubin. Bilirubin oxidase is used to remove bilirubin in icteric samples in order to resolve this issue. By converting bilirubin to biliverdin, the enzyme eliminates its absorption in the 400 nm region and so it is possible to determine free hemoglobin concentration.[9]


Biofuel cell

In the field of power generation, biofuel cells provide an alternative to fossil energy. So far biofuell cells that were developed required specific enzymes that degrade and transform substrates present in physiologic fluids such as glucose and O2. These biofuel cells were used for medical devices power supply.[10] Now a new generation of biofuel cells use hydrogenase. Indeed, H2/O2 biofuel cells are designed based on two thermostable enzymes: an hyperthermophilic O2-tolerant hydrogenase for H2 oxidation and bilirubin oxidase for O2 reduction. [11] Both enzymes are immobilized on carbon nanofibers. This biofuel cell can deliver electricity over a wide range of températures and pH. Indeed, the thermostability of the enzymes allows the biofuel cell to work under extreme conditions, from 30 to 80°C.[10] H2/O2 biofuel cells can be used as alternative power supply for small electronic devices in a sustainable manner. However, research is ongoing; one issue is the enzyme instability on long term.[11]


References

  1. ↑ Wikipedia, Multicopper oxidase [1]
  2. ↑ 2.0 2.1 2.2 Shimizu A, Kwon JH, Sasaki T, Satoh T, Sakurai N, Sakurai T, Yamaguchi S, Samejima T. Myrothecium verrucaria bilirubin oxidase and its mutants for potential copper ligands. Biochemistry. 1999 Mar 9;38(10):3034-42. doi: 10.1021/bi9819531. PMID:10074356 doi:http://dx.doi.org/10.1021/bi9819531
  3. ↑ 3.0 3.1 3.2 3.3 Akter M, Tokiwa T, Shoji M, Nishikawa K, Shigeta Y, Sakurai T, Higuchi Y, Kataoka K, Shibata N. Redox potential-dependent formation of an unusual His-Trp bond in bilirubin oxidase. Chemistry. 2018 Aug 29. doi: 10.1002/chem.201803798. PMID:30156345 doi:http://dx.doi.org/10.1002/chem.201803798
  4. ↑ Kataoka K, Ito T, Okuda Y, Sakai Y, Yamashita S, Sakurai T. Roles of the indole ring of Trp396 covalently bound with the imidazole ring of His398 coordinated to type I copper in bilirubin oxidase. Biochem Biophys Res Commun. 2020 Jan 15;521(3):620-624. doi:, 10.1016/j.bbrc.2019.10.159. Epub 2019 Oct 31. PMID:31679691 doi:http://dx.doi.org/10.1016/j.bbrc.2019.10.159
  5. ↑ 5.0 5.1 Koval T, Svecova L, Ostergaard LH, Skalova T, Duskova J, Hasek J, Kolenko P, Fejfarova K, Stransky J, Trundova M, Dohnalek J. Trp-His covalent adduct in bilirubin oxidase is crucial for effective bilirubin binding but has a minor role in electron transfer. Sci Rep. 2019 Sep 23;9(1):13700. doi: 10.1038/s41598-019-50105-3. PMID:31548583 doi:http://dx.doi.org/10.1038/s41598-019-50105-3
  6. ↑ MP Biomedicals, Bilirubin Oxidase [2]
  7. ↑ Morimoto Y, Ishihara T, Takayama M, Kaito M, Adachi Y. Novel assay for measuring serum conjugated bilirubin and its clinical relevance. J Clin Lab Anal. 2000;14(1):27-31. PMID:10645982
  8. ↑ Kimura M, Matsumura Y, Konno T, Miyauchi Y, Maeda H. Enzymatic removal of bilirubin toxicity by bilirubin oxidase in vitro and excretion of degradation products in vivo. Proc Soc Exp Biol Med. 1990 Oct;195(1):64-9. doi: 10.3181/00379727-195-43119. PMID:2399262 doi:http://dx.doi.org/10.3181/00379727-195-43119
  9. ↑ Wong SS, Schenkel OJ. Quantification of plasma hemoglobin in the presence of bilirubin with bilirubin oxidase. Ann Clin Lab Sci. 1995 May-Jun;25(3):247-51. PMID:7605107
  10. ↑ 10.0 10.1 A. de Poulpiquet, A. Ciaccafava, R. Gadiou, S. Gounel, M.T. Giudici-Orticoni, N. Mano, E. Lojou, Reprinted from Electrochemistry Communications, Volume 42 (2014). [3]
  11. ↑ 11.0 11.1 A. de Poulpiquet, A. Ciaccafava, R. Gadiou, S. Gounel, M.T. Giudici-Orticoni, N. Mano, E. Lojou, Design of a H2/O2 biofuel cell based on thermostable enzymes (2014) [ https://doi.org/10.1016/j.elecom.2014.02.012 ]
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