Purification, crystallization and preliminary X-ray study of the fungal laccase from Cerrena maxima.
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.
Lyashenko
Andrey V
AV
A. V. Shubnikov Institute of Crystallography, RAS, Leninskiy Prospect 59, 119333 Moscow, Russia.
Zhukhlistova
Nadegda E
NE
Gabdoulkhakov
Azat G
AG
Zhukova
Yuliya N
YN
Voelter
Wolfang
W
Zaitsev
Viatcheslav N
VN
Bento
Isabel
I
Stepanova
Elena V
EV
Kachalova
Galina S
GS
Koroleva
Ol'ga V
OV
Cherkashyn
Evgeniy A
EA
Tishkov
Vladimir I
VI
Lamzin
Victor S
VS
Schirwitz
Katja
K
Morgunova
Ekaterina Yu
EY
Betzel
Christian
C
Lindley
Peter F
PF
Mikhailov
Al'bert M
AM
D001487:Basidiomycota / Q000737:chemistry / Q000201:enzymology*; D003460:Crystallization; D018360:Crystallography, X-Ray; D042845:Laccase / Q000737:chemistry* / Q000302:isolation & purification; D011487:Protein Conformation
D042845:Laccase