ISBN: 978-83-60043-10-3 - eurobic9

Eurobic9, 2-6 September, 2008, Wrocław, Poland
P73. A Near-atomic Resolution Crystal Structure of Melanocarpus
Albomyces Laccase
N. Hakulinen a , J. Kallio a , M. Andberg b , A. Koivula b , K. Kruus b , J. Rouvinen
a
Dept. of Chem. , University of Joensuu, Yliopistokatu 7, 80100, Joensuu, Finland
e-mail: nina.hakulinen@joensuu.fi
b
VTT Technical Research Centre of Finland, PO Box 1000, 02044 , Espoo, Finland
Laccases (E.C. 1.10.3.2, p-diphenol dioxygen oxidoreductases) are redox enzymes that use molecular oxygen to
oxidize various phenolic compounds. They share the arrangement of the catalytic sites with other blue multicopper
oxidases including ascorbate oxidase, ceruloplasmin, CueO, and Fet3p. For catalytic activity, four copper
atoms are needed: one type-1 (T1) copper forming a mononuclear site, one type-2 (T2) copper and two type-3
(T3 and T3´) coppers forming a trinuclear site. Reducing substrates are oxidized near the mononuclear site and
then electrons are transferred to the trinuclear site, where dioxygen is reduced to water.
Melanocarpus albomyces is an ascomycete fungus expressing a thermostable laccase with a neutral pH optimum.
The three-dimensional structure of M. albomyces laccase (MaL) at 2.4 Å was solved among first complete
laccase structures [1]. In MaL structure, dioxygen was observed at the first time inside the trinuclear site. At the
present, laccase structures show wide variety of trinuclear site geometries having one or two oxygen atoms. Our
recent studies have shown that the trinuclear site of laccase is sensitive to X-rays and the observed structure may
depend on the data collection strategy or the intensity of the beam [2].
We have recently solved the three-dimensional structure of recombinant M. albomyces laccase (rMaL) at 1.3 Å
resolution [3]. At the moment, this is the highest resolution that has been attained for any multicopper oxidase.
This structure confirmed our earlier proposal regarding the dynamic behaviour of the trinuclear site and it
allowed us to describe important solvent cavities of the enzyme. T2 and T3 solvent cavities, and a putative SDSgate,
formed by Ser142, Ser510 and the C-terminal Asp556 of rMaL, were found. We also observed a
2-oxohistidine, an oxidized histidine, possibly caused by a metal-catalysed oxidation by the trinuclear site
coppers. To our knowledge, this is the first time that 2-oxohistidine has been observed in a protein crystal
structure.
References:
[1] Hakulinen, N., Kiiskinen, L.-L., Kruus, K., Saloheimo, M., Paananen, A., Koivula, A. and Rouvinen J.
(2002) Nature Structural Biology 9, 601
[2] Hakulinen, N., Kruus, K., Koivula, A. and Rouvinen, J. (2006) Biochem. Biophys. Res. Comm. 350, 929
[3] Hakulinen, N., Andberg, M., Kallio, J., Kruus, K., Koivula, A. and Rouvinen, J. (2008) J. Struct. Biol.
162, 29
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