title: Purification, crystallization and preliminary X-ray study of the fungal laccase from Cerrena maxima.
abstract: Laccases are members of the blue multi-copper oxidase family that oxidize substrate molecules by accepting electrons at a mononuclear copper centre and transferring them to a trinuclear centre. Dioxygen binds to the trinuclear centre and, following the transfer of four electrons, is reduced to two molecules of water. Crystals of the laccase from Cerrena maxima have been obtained and X-ray data were collected to 1.9 A resolution using synchrotron radiation. A preliminary analysis shows that the enzyme has the typical laccase structure and several carbohydrate sites have been identified. The carbohydrate chains appear to be involved in stabilization of the intermolecular contacts in the crystal structure, thus promoting the formation of well ordered crystals of the enzyme. Here, the results of an X-ray crystallographic study on the laccase from the fungus Cerrena maxima are reported. Crystals that diffract well to a resolution of at least 1.9 A (R factor = 18.953%; R(free) = 23.835; r.m.s.d. bond lengths, 0.06 A; r.m.s.d. bond angles, 1.07 degrees) have been obtained despite the presence of glycan moieties. The overall spatial organization of C. maxima laccase and the structure of its copper-containing active centre have been determined by the molecular-replacement method using the laccase from Trametes versicolor (Piontek et al., 2002) as a structural template. In addition, four glycan-binding sites were identified and the 1.9 A X-ray data were used to determine the previously unknown primary structure of this protein. The identity (calculated from sequence alignment) between the C. maxima laccase and the T. versicolor laccase is about 87%. Tyr196 and Tyr372 show significant extra density at the ortho positions and this has been interpreted in terms of NO(2) substituents.
authors.affiliation: A. V. Shubnikov Institute of Crystallography, RAS, Leninskiy Prospect 59, 119333 Moscow, Russia.
authors.forename: Andrey V
authors.initials: AV
authors.lastname: Lyashenko
authors.forename: Nadegda E
authors.initials: NE
authors.lastname: Zhukhlistova
authors.forename: Azat G
authors.initials: AG
authors.lastname: Gabdoulkhakov
authors.forename: Yuliya N
authors.initials: YN
authors.lastname: Zhukova
authors.forename: Wolfang
authors.initials: W
authors.lastname: Voelter
authors.forename: Viatcheslav N
authors.initials: VN
authors.lastname: Zaitsev
authors.forename: Isabel
authors.initials: I
authors.lastname: Bento
authors.forename: Elena V
authors.initials: EV
authors.lastname: Stepanova
authors.forename: Galina S
authors.initials: GS
authors.lastname: Kachalova
authors.forename: Ol'ga V
authors.initials: OV
authors.lastname: Koroleva
authors.forename: Evgeniy A
authors.initials: EA
authors.lastname: Cherkashyn
authors.forename: Vladimir I
authors.initials: VI
authors.lastname: Tishkov
authors.forename: Victor S
authors.initials: VS
authors.lastname: Lamzin
authors.forename: Katja
authors.initials: K
authors.lastname: Schirwitz
authors.forename: Ekaterina Yu
authors.initials: EY
authors.lastname: Morgunova
authors.forename: Christian
authors.initials: C
authors.lastname: Betzel
authors.forename: Peter F
authors.initials: PF
authors.lastname: Lindley
authors.forename: Al'bert M
authors.initials: AM
authors.lastname: Mikhailov
mesh_terms: D001487:Basidiomycota / Q000737:chemistry / Q000201:enzymology*; D003460:Crystallization; D018360:Crystallography, X-Ray; D042845:Laccase / Q000737:chemistry* / Q000302:isolation & purification; D011487:Protein Conformation
chemical_list: D042845:Laccase