Essential role of the C-terminus in Melanocarpus albomyces laccase for enzyme production, catalytic properties and structure.
The C-terminus of the fungal laccase from Melanocarpus albomyces (MaL) is processed during secretion at a processing site conserved among the ascomycete laccases. The three-dimensional structure of MaL has been solved as one of the first complete laccase structures. According to the crystal structure of MaL, the four C-terminal amino acids of the mature protein penetrate into a tunnel leading towards the trinuclear site. The C-terminal carboxylate group forms a hydrogen bond with a side chain of His140, which also coordinates to the type 3 copper. In order to analyze the role of the processed C-terminus, site-directed mutagenesis of the MaL cDNA was performed, and the mutated proteins were expressed in Trichoderma reesei and Saccharomyces cerevisiae. Changes in the C-terminus of MaL caused major defects in protein production in both expression hosts. The deletion of the last four amino acids dramatically affected the activity of the enzyme, as the deletion mutant delDSGL(559) was practically inactive. Detailed characterization of the purified L559A mutant expressed in S. cerevisiae showed the importance of the C-terminal plug for laccase activity, stability, and kinetics. Moreover, the crystal structure of the L559A mutant expressed in S. cerevisiae showed that the C-terminal mutation had clearly affected the trinuclear site geometry. The results in this study clearly confirm the critical role of the last amino acids in the C-terminus of MaL.
VTT Technical Research Center of Finland, P.O. Box 1000, FIN-02044 VTT, Finland. martina.andberg@vtt.fi
Martina
M
Andberg
Nina
N
Hakulinen
Sanna
S
Auer
Markku
M
Saloheimo
Anu
A
Koivula
Juha
J
Rouvinen
Kristiina
K
Kruus
D000595:Amino Acid Sequence; D002384:Catalysis; D004795:Enzyme Stability; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D042845:Laccase / Q000737:chemistry* / Q000378:metabolism; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D012441:Saccharomyces cerevisiae / Q000235:genetics; D020037:Sordariales / Q000201:enzymology*; D013329:Structure-Activity Relationship; D014242:Trichoderma / Q000235:genetics
D042845:Laccase