Laccase versus Laccase-Like Multi-Copper Oxidase: A Comparative Study of Similar Enzymes with Diverse Substrate Spectra

Renate Reiss,Eric Eichhorn,Boris Schilling,Julian Ihssen,Michael Richter,Linda Thöny-Meyer,Claudio M Soares

doi:10.1371/journal.pone.0065633

Abstract

Laccases (EC 1.10.3.2) are multi-copper oxidases that catalyse the one-electron oxidation of a broad range of compounds including substituted phenols, arylamines and aromatic thiols to the corresponding radicals. Owing to their broad substrate range, copper-containing laccases are versatile biocatalysts, capable of oxidizing numerous natural and non-natural industry-relevant compounds, with water as the sole by-product. In the present study, 10 of the 11 multi-copper oxidases, hitherto considered to be laccases, from fungi, plant and bacterial origin were compared. A substrate screen of 91 natural and non-natural compounds was recorded and revealed a fairly broad but distinctive substrate spectrum amongst the enzymes. Even though the enzymes share conserved active site residues we found that the substrate ranges of the individual enzymes varied considerably. The EC classification is based on the type of chemical reaction performed and the actual name of the enzyme often refers to the physiological substrate. However, for the enzymes studied in this work such classification is not feasible, even more so as their prime substrates or natural functions are mainly unknown. The classification of multi-copper oxidases assigned as laccases remains a challenge. For the sake of simplicity we propose to introduce the term “laccase-like multi-copper oxidase” (LMCO) in addition to the term laccase that we use exclusively for the enzyme originally identified from the sap of the lacquer tree Rhus vernicifera.

Highlights

Multi-copper oxidases (MCOs) belong to a protein superfamily of enzymes which oxidize the substrate at a mononuclear copper center T1
The predicted molecular weight and the amino acid length of the studied enzymes was in a similar range (51–66 kDa, 470–600 aa), with the exception of the Streptomyces like multi-copper oxidase’’ (LMCO) (32.6 kDa, 297 aa).The alignment of the primary structures shows that all proteins contain the four strictly conserved copper ligand motifs (Fig. 1A)
From the crystal structures of B. subtilis and T. versicolor LMCOs it is known that the methionine, which is axially coordinated to copper T1 in bacterial LMCOs, is replaced by the non-coordinating residues leucine or phenylalanine [4,24]

Summary

Introduction

Multi-copper oxidases (MCOs) belong to a protein superfamily of enzymes which oxidize the substrate at a mononuclear copper center T1. Electrons are transferred internally to the trinuclear copper center T2/T3 where dioxygen is reduced by four electrons, yielding two water molecules. At least four copper atoms are present in MCOs and shared between the T1 and T2/T3 sites [2]. The sequence homology among MCOs is low, amino acid alignments show that the overall structures and copperbinding motifs are highly conserved [3]. The T1 copper atom is bound by two histidine residues and one cysteine residue, which forms a metallo-organic bond. The T2 copper atom of the trinuclear center is coordinated by two histidine ligands, while the two T3 coppers have in total six histidine ligands. The copper ligands are organized in four subsequent, strictly conserved motifs within the primary amino acid sequence HXHG, HXH, HXXHXH and HCHXXXHXXXXM/L/F. From the structural point of view, MCOs comprise 2, 3 or 6 homologous domains, each resembling a cupredoxin-like fold arranged in an (eight-stranded) Greek-key beta-barrel consisting of two b-sheets, each containing four strands [3,5]

Objectives

Methods

Results

Conclusion