PDBsum entry 3dkh

Oxidoreductase

PDB id: 3dkh

Contents

Protein chains

558 a.a.

Ligands

NAG-NAG-BMA-MAN

NAG-NAG ×4

NAG-NAG-BMA ×4

NAG ×6

SO4 ×3

OXY ×2

GOL

Metals

_CU ×8

_CL

Waters ×706

References listed in PDB file

Key reference

• Title: Essential role of the c-Terminus in melanocarpus albomyces laccase for enzyme production, Catalytic properties and structure.
• Authors: M.Andberg, N.Hakulinen, S.Auer, M.Saloheimo, A.Koivula, J.Rouvinen, K.Kruus.
• Ref.: Febs J, 2009, 276, 6285-6300.
• PubMed id: 19780817

Abstract

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.