Designer laccases: a vogue for high-potential fungal enzymes?

Review

Designer laccases: a vogue for high-potential fungal enzymes? Caroline J. Rodgers1*, Christopher F. Blanford1*, Stephen R. Giddens2, Pari Skamnioti2, Fraser A. Armstrong1 and Sarah J. Gurr2 1 2

Department of Chemistry, South Parks Road, University of Oxford, Oxford OX1 3QR Department of Plant Sciences, South Parks Road, University of Oxford, Oxford OX1 3RB

Laccases are blue multicopper oxidases that catalyse the four-electron reduction of O2 to water coupled with the oxidation of small organic substrates. Secreted basidiomycete white-rot fungal laccases orchestrate this with high thermodynamic efficiency, making these enzymes excellent candidates for exploitation as industrial oxidants. However, these fungi are less tractable genetically than the ascomycetes, which predominantly produce lower-potential laccases. We address the state-ofplay regarding expression of high reduction potential laccases in heterologous hosts, and issues regarding enzyme glycosylation status. We describe the synergistic role of structural biology, particularly in unmasking structure–function relationships following genetic modification and their collective impact on laccase yields. Such recent research draws closer the prospect of industrial quantities of designer, fit-for-purpose laccases. Introduction Laccases (EC 1.10.3.2) are blue multi-copper oxidases that catalyse the four-electron reduction of O2 to water, coupled with the oxidation of small organic (generally aromatic) substrates. Their characteristic blue colour is caused by a copper atom that is thought to be the primary electron acceptor. The first laccase, from the Japanese lacquer tree, Rhus vernicifera, was described in 1883 [1]. Subsequently, laccases and laccase-like proteins have been described in plants, fungi (ascomycetes and basidiomycetes), arthropods, bacteria [2] and in bovine rumen microflora [3]. Laccases play diverse roles in nature: lignification, delignification, oxidative plant stress management, and fungal morphogenesis and virulence [2]. Collectively, laccases exploit a disparate range of natural substrates. Individually, substrate usage varies between species, as do their constituent laccases. Laccases are functionally diverse, thermostable and environmentally friendly catalysts: they occur naturally, use air and produce water as a by-product. Their importance is reflected in the broad spectrum of reported applications [4,5] (Supplementary Material). However, there exists a real need to produce prolific quantities of low-cost enzymes for distinct industrial applications [6]. The question is how? Of the multi-copper oxidases, the white-rot fungal laccases court considerable industrial interest. Their ability *

Corresponding author: Gurr, S.J. ([email protected]) Equal contribution..

Glossary ABTS: common abbreviation for 2,20 -azino-bis(3-ethylbenzthiazoline-6-sulphonic acid), a non-phenolic electron donor frequently used in spectrophotometric assays of laccase activity. Ascomycete: fungus of the phylum Ascomycota, in which sexual spores (ascospores) are formed within an ascus (bag). Basidiomycete: fungus of the phylum Basidiomycota, in which sexual spores (basidiospores) are carried on a basidium (club-shaped structure). Bioreactor: device for production of enzymes or chemicals from a microbial cell culture. Vessel can range in size and complexity, from batch cultures grown on agar to submerged cultures in computer-controlled, continuous-flow reactors on a cubic metre scale. Blue copper oxidase: a protein carrying at least one type 1 copper atom that catalyses a reduction or oxidation involving molecular oxygen; includes multicopper oxidases such as laccase and bilirubin oxidase. Directed evolution: genetic modification strategy employing mutagenesis, sometimes iterative, coupled with phenotypic selection to create novel, synthetic proteins. Glycan: an oligosaccharide. In fungal laccases, these consist of branched mannose groups, linked to two N-acetyl-D-glucosamine groups, N-linked to asparagine. Glycosylation: enzymatic site-specific process that link glycans to proteins or lipids; a form of co-translational and post-translational modification. Isoelectric point: pH at which a chemical species has no net electrical charge. Mediator: redox-active small molecules and complexes for shuttling electrons between an electrode and more slowly diffusing proteins. Protein Data Bank (PDB): repository for 3-D structural information, typically from X-ray crystallography or nuclear magnetic resonance (NMR) spectroscopy. Structures identified by a four-character alphanumeric code (e.g. 1kya). URL: http://www.pdb.org/ Pseudohalide: monovalent anionic groups such as azide and cyanide that possess sufficient electronegativity to resemble a halide. Reduction potential: measure of electrochemical driving force required to add electron(s) to chemical species. Higher values imply a greater affinity for electron(s). SI unit: Volt. Saturation mutagenesis: genetic modification strategy that generates all possible mutations at one or more pre-determined target site(s). Site-directed mutagenesis: creation of a mutation at a defined locus. Solid-state fermentor: fermentation with little or no free liquid present, orchestrated on an inert synthetic substrate or with a natural organic substrate support. Standard state: in electrochemistry, 1 bar pressure for gaseous species and unit activity for all dissolved species. (Activity is often taken as equivalent to concentration, so this implies pH  0.) Symbolised by degree sign. Standard hydrogen electrode (SHE): commonly employed reference electrode to facilitate comparison of reduction potentials. Defined as exactly 0 V at standard state at all temperatures. Often measurements are acquired using other reference electrodes, then converted to this scale. Also known as the normal hydrogen electrode. Potentials herein are all relative to SHE. Submerged broth fermentation: growth of a microbe in nutrient-rich liquid medium that is free of all other organisms (axenic) and perfused with oxygen. Trickle-bed reactor: a bioreactor that consists of a fixed-bed solid support, perfused by trickle feeding, that is semi-continuously harvested. Type 1/type 2/type 3: classification of copper atoms in some copper proteins based on spectroscopic properties. Type 1 copper (in its +2 oxidation state) gives strong blue colour to laccases (and other blue copper proteins) because of a charge transfer from copper to coordinating Cys (absorption maximum around 600–610 nm). Oxidised type 1 and type 2 coppers can be observed by electron spin resonance spectroscopy. Type 3 coppers always pair with coupled opposing electron spins rendering them invisible by electron spin resonance spectroscopy, but absorb light in the near UV around 330 nm.

0167-7799/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibtech.2009.11.001 Available online 4 December 2009

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Review to oxidise organic molecules, especially in the presence of synthetic laccase mediators such as 2,20 -azino-bis(3-ethylbenzthiazoline-6-sulphonic acid (ABTS), makes them attractive enzymes [7]. Despite this, there is no ideal laccase fit for all purposes, but there exists a real possibility of designing improved industrial enzymes. Laccases have been reviewed extensively from a broad [8,9] and focused perspective [4,6,10–18]. Here, we highlight the ‘‘potential’’ of high-potential laccases, and focus on methods of producing commercially-relevant laccases, rather than on industrial applications [5,6,19,20]. We provide an update on laccase structure, particularly on glycosylation status. In the context of desirable attributes of laccases as robust industrial enzymes, we review their physicochemical properties and draw attention to bottlenecks in the production of tailor-made enzymes. We evaluate recent developments aimed at enhancing the performance of fungal laccases, when expressed in native (self/homologous) and non-native (heterologous) hosts, and comment on the need for scale-up. Laccase glycosylation: a structural perspective Our current understanding of laccases derives largely from analysis of over 100 fungal enzymes [15,21]. The emerging picture is of predominantly monomeric proteins (typically 55–85 kDa), with most species producing several isozymes with an isoelectric point around pH 4. Not all are extracellular enzymes, but secreted laccases are glycosylated (up to 25%) [22], with typically 3–10 glycosylation sites (Table 1), predicted from their amino acid sequence (e.g. N-glycosylation sites found at Asn-X-Thr/Ser sites). The glycosylation pattern (Figure 1a) consists of branched, a-linked mannose chains connected to Asn through two N-acetylglucosamine molecules by three b-linkages [22]. Crystal structures give insight into substrate binding site properties [23] and alert investigators to the position of glycosylation sites. To date, X-ray crystal structures have been released for 10 laccases from nine fungal species that carry all four copper atoms (Table 1). Basidiomycete structures predominate; Melanocarpus albomyces Lac1 is the only resolved ascomycete structure, but release of structures of the thermophilic Thielavia arenaria and Myceliophthora thermophila laccases is anticipated [24,25]. Laccases pose certain difficulties for crystallographers, which arise from problems associated with separating out isozymes, their complex glycosylation status, or both [26]. Collectively, these give rise to disordered conformations [27,28]. To varying degrees, the glycans of certain laccases have been characterised by crystallographic techniques (Table 1). However, components more remote from the polypeptide are often too disordered to be resolved fully, and complementary techniques are clearly required to analyse them (see Recommendations). Suggested roles for laccase carbohydrate moieties include stabilising the copper centres, directing protein secretion, protecting against proteolysis, and enhancing thermostability [12,29], but their precise roles remain obscure [30]. Enzyme modification studies, discussed later, are beginning to shed light on their influence. 64

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Laccase: physicochemical properties Catalytic activity Laccases oxidise phenolic substrates, Mn2+ (in presence of chelators), and a number of xenobiotic compounds [9]. The mechanism is complex; although its main features are known [10,31], the complete pathway remains to be elucidated. The four copper ions essential for catalytic activity are buried within the protein. The substrate undergoes one-electron oxidation by electron transfer to the blue type 1 copper located near the substrate binding pocket (Figure 1b). This copper is rapidly re-oxidised by longrange intramolecular electron transfer, via a conserved His–Cys–His motif, to the trinuclear copper cluster (Figure 1c) [26,32]. O2 binds between the two type 3 coppers and is reduced to water without the release of active peroxide intermediates [2]. During the catalytic cycle, the type 1 copper must be oxidised and reduced four times. Laccase reduction potentials Laccases have been classified as low-, medium- and highpotential based on equilibrium potentiometric titrations of the type 1 site [33], often without regard to the influence of pH on this potential. These titrations are performed by recording the intensity of light absorption around 600 nm as the potential is varied. From voltametric studies, it appears that a high catalytic potential for the reduction of O2 to water correlates with a high reduction potential for the type 1 copper [42]. Here, we consider laccases with type 1 reduction potentials greater than 0.6 V versus the standard hydrogen electrode (SHE; Table S1, Supplementary Information). In the high-potential Trametes villosa laccase Lcc1 (ET1Cu = 0.79 V vs. SHE at pH 5.5), for example, the catalytic potential for O2 reduction is 200 mV lower than the O2/ 2H2O couple [33,34], which makes the enzyme more thermodynamically efficient than platinum catalysts used in fuel cells [20,35]. Fungal laccases have higher reduction potentials for the copper sites compared with those from other organisms, for example, R. vernicifera (ET1Cu = 0.43 V vs. SHE) [36]. However, the accuracy of gathered data depends upon whether the recorded reduction potentials reflect measurement of single or multiple laccase isozymes [21]. Isozymes arise by multiple processes (e.g. differential post-translational modification or allelic gene variants), which cause variation in the physicochemical properties between isozymes within a single species [21,37]. Although the potential for the type 1 copper appears to correlate with catalytic potential, an equilibrium reduction potential measured for the trinuclear-site coppers is less likely to be related. Unlike the type 1 copper that undergoes continuous outer-sphere redox cycling, the type 2/type 3 site reacts specifically with O2 by an inner-sphere reaction, which produces reactive and short-lived intermediate states [10]. These active, oxygenated catalytic states must be highly oxidising and probably feature spontaneous (towards higher potentials) intramolecular electron transfer. In contrast, published titrations considered the inactive resting state (outside the catalytic cycle) or denatured protein, thereby generating a wide-range of potentials [36,38], which are difficult to interpret in a catalytic context. We conject that the entire electron-transfer relay be

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Table 1. Crystal structures of fungal laccases carrying full complement of copper atomsa Fungus b Cerrena maxima

PDB code (resolution) [gene] c 3div (1.76 A˚) [—]f 2h5u (1.9 A˚) [—] f

N-glycosylation Available sites and glycans detectede sites d 3div: 9 3div: Asn51; Man-a-1,3-Man-a-1,6-[Man-]gManNAG2-Asn54; Asn208; Mang-NAG2Asn217; Asn292; NAG-Asn333h; Asn377; Asn416; NAG-NAG-Asn436g,h 2h5u: 10 2h5u: Asn51; Man-a-1,3-Man-a-1,6-ManNAG2-Asn54; Asn208; ManNAG2-Asn217; Asn292; Asn301; NAG-Asn333; Asn416; NAG-Asn436

Coriolopsis gallica

2vdz (1.7 A˚) [—]i 2vds (2.3 A˚) [—] i

5

2vdz: Asn51; NAG2-Asn54; Asn207; Asn335; NAG2-Asn433 2vds NAG2-Asn54; Asn207; Asn334; NAG2-Asn432

Lentinus tigrinus

2qt6 (1.5 A˚) [LCC1]j,k

6

2qt6:

Melanocarpus 2q9o (1.3 A˚) [LAC1]j,l,m,n,o 9 albomyces 2ih8 (2.0 A˚) [LAC1]j,l,m 2ih9 (2.0 A˚) [LAC1]j,l,m 1gw0 (2.4 A˚) [LAC1]j,l

3dkh (2.4 A˚) [LAC1]j,l,o,p 3fu7 (1.67 A˚) [LAC1]j,l,m,n,q 3fu8 (1.8 A˚) [LAC1]j,l,m,n,o,q 3fu9 (2.0 A˚) [LAC1]j,l,m,q

Man-a-1,6-Man-a-1,6-[Man-b(?)-1,3-]ManNAG2-Asn54 (plus nearby unlinked Man); Asn208; Asn292; Asn315; NAG-Asn376; NAG2-Asn435

2q9o: NAG-Asn39; ManNAG2-Asn88; Man-a-1,2-Man-a-1,3-ManNAG2-Asn201; NAG2Asn216; Asn231; NAG-Asn244; ManNAG2-Asn289; NAG2-Asn376; NAG-Asn396 2ih9: NAG-Asn39; Man-a-1,6-[Man-a-1,3-]ManNAG2-Asn88; NAG-Asn201; NAG2Asn216; Asn231; Asn244; ManNAG2-Asn289; NAG2-Asn376; NAG-Asn396 2ih9: NAG-Asn39; ManNAG2-Asn88; NAG-Asn201; ManNAG2-Asn216; Asn231; NDG-b-1-Asn244; ManNAG2-Asn289; NAG2-Asn376; NAG-Asn396 1gw0: NAG-Asn39; Man-b(?)-1,6-[Man-a-1,3-]Man-a-1,6-[Man-a-1,3-]ManNAG2Asn88; NAG-Asn201; ManNAG2-Asn216; Asn231; Asn244; Man-a-1,2-Man-a1,3-ManNAG2-Asn289; NAG2-Asn376; NAG2-Asn396 3dkh: NAG-Asn39; ManNAG2-Asn88; NAG-NAG-Asn201; ManNAG2-Asn216; Asn231; NAG-Asn244; ManNAG2-Asn289; NAG2-Asn376; NAG-Asn396 3fu7: NAG-Asn39; ManNAG2-Asn88; NAG-NAG-Asn201; NAG2-Asn216; Asn231; Asn244; ManNAG2-Asn289; NAG2-Asn376; NAG-Asn396 3fu8: NAG-Asn39; Man-a-1,6-ManNAG2-Asn88; NAG-NAG-Asn201; NAG2-Asn216; Asn231; Asn244; ManNAG2-Asn289; NAG2-Asn376; NAG-Asn396 3fu9: NAG-Asn39; NAG2-Asn88; NAG-Asn201; NAG-Asn216; Asn231; Asn244; NAG2-Asn289; NAG-Asn376; Asn396

Rigidoporus microporus r

1v10 (1.70 A˚) [—] s

5

1v10: None assigned in crystal structure. Asn337 and Asn435: 0–2 mannose + pentasaccharide core. No post-translational modification to Asn90, Asn184, Asn267. t

Trametes hirsuta u

3fpx (1.8 A˚) [LAC]

8

3fpx:

Trametes ochracea v

2hzh (2.6 A˚) [—]

6

2hzh: Asn51; NDG-NAG-Asn54h; Asn208; NDG-Asn333h; NAG-Man-Asn436h; Asn478

Trametes trogii

2hrg (1.58 A˚) [LCC1]w 2hrh (2.6 A˚) [LCC1] 1gyc (1.9 A˚) [LCC2]x

3

2hrg: Asn51; Man-a-1,6-ManNAG2-Asn54; NAG2-Asn433 2hrh: Asn51; NAG2-Asn54; NAG2-Asn433

1gyc: 8

1kya (2.4 A˚) [LAC1] y

1kya: 6 z

1gyc: NAG2-Asn54; Asn141; Asn208; NAG2-Asn217; NAG-Asn251; NAG-Asn333; NAG-Asn341; NAG2-Asn436 1kya: Asn51; NAG2-Asn54; Asn208; NAG-Asn217; NAG2-Asn333; NAG2-Asn436

Trametes versicolor

Asn51; Man-a-1,3-Man-a-1,6-[Man-a-1,3-]ManNAG2-Asn54; Asn208; Man-a1,3-ManNAG2-Asn217; Asn292; NAG-Asn333; Asn377; NAG2-Asn436

a

The first laccase crystal structures reported, 1a65 and 1hfu (from C. cinereus), were depleted in their type 2 copper. A crystal structure has been described of an isozyme of Pycnoporous cinnarbarinus at 2.2 A˚ resolution, but no crystal structure has been deposited [80]. c A dash in the gene name means no match was found by BLAST analysis of the UniProt database. d Based on presence of Asn-X-Thr or Asn-X-Ser portions of the sequence. See Supplementary Information for Perl script used to automate their detection. e Bold text highlights N-glycosylation sites and the conserved positions. Italics denote glycans that appear in only one of the asymmetric units. Abbreviations: NAG = N-acetylD-glucosamine; Man = D-mannose; NDG = 2-acetylamino-2-deoxy-a-D-glucopyranose; ManNAG2 = Man-b-1,4-NAG-b-1,4-NAG-b-1; NAG-NAG/NAG2 = NAG-b-1,4-NAG-b-1; NAG = NAG-b-1. f Sequences for 2h5u and 3div are 91% and 94% identical, respectively, to Lac from T. hirsuta. They are reported as the same protein, but the assignment of 24 of the 499 residues differs. Tyr372 assigned meta nitration in 3div. g NAGs assigned as 2-(acetylamino)-1,5-anhydro-2-deoxy-D-glucitol (i.e., NAG without a 1-hydroxy). h Unlinked. i Shows  94% sequence identity with C. gallica Lac1; T. trogii Lcc1 and Trametes sp 30 Lac1. j Two molecules per asymmetric unit. k One of the proteins in the asymmetric unit shows elliptical electron density between the type 3 coppers that was assigned to peroxide. l Elliptical electron density between type 3 coppers assigned to a dioxygen species, except high-dose, decolourised 2ih9 structure where the reduced density is assigned to a mono-oxo species. Chloride assigned to the type 2 copper at the apex of the trinuclear cluster. The second NAG on Asn216 in 2ih9 is listed as 2-(acetylamino)-1,5-anhydro-2deoxy-D-glucitol. m Protein expressed in Trichoderma reesei. n His98 modified to an oxyhistidine, 3-(2-oxo-2H-imidazol-4-yl)-L-alanine. Chain A only in 3fu7. o Glycerol molecule(s) in organic substrate binding pocket. p L559A variant (final residue in the ‘‘C-terminal plug’’) [24] the end of which is 5–6 A˚ from one of the Type 3 copper atoms. Protein expressed in Saccharomyces cerevisiae. Ordered glycerol crystallised between the two proteins in the asymmetric unit. In chain B, no chloride was assigned near the apex of the type 2 copper and a third oxygen atom is located 3.1 A˚ from the O2 species assigned to the electron density between the type 3 coppers. q Soaked in 2,6-dimethoxyphenol, an organic substrate for laccase, for 4 s (3fu7), 10 s (3fu8), 20 min (3fu9). In 3fu7, some of the organic substrate had been oxidised and assigned as a para quinone and a C–O dimer; in 3fu9, the quinone was assigned to the electron density in the binding pocket. r Also known as Rigidoporus lignosus. s From hemihedrally twinned crystals; only 63% identity with sequence for Lcc; UniProt ID Q6H9H7. Asymmetric coordination of the type 3 coppers. t Determined by AspN digest and MALDI-TOF mass spectrometry [81]. u Also known as Coriolus hirsutus. v Also known as Coriolus zonatus. w Co-crystallised with 4-methylbenzoic acid on the surface near type 1 copper, three surface calcium ions, and three glycerol molecules. x Determined at room temperature, protein also known as LccI. y Four molecules per asymmetric unit, complexed with 2,5-xylidine near type 1 copper site. z Lac1 has seven possible sites, but the FASTA sequence for 1kya gives only six because Thr342 is displayed as Ala. b

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reduction potential have been identified following sitedirected mutagenesis [33] and comparison of crystal structures [26]. Raising the potential of the type 1 copper through sitedirected mutagenesis is complex. One aim is to duplicate this high potential in a high-yielding bacterial or yeast expression system. Thus, the inner coordination sphere of the type 1 copper in the low-potential laccase CotA from Bacillus subtilis was modified with Met!Phe/Leu mutations; the Leu variant produced the bigger increase in ET1Cu (about 0.1 V), but still 200 mV short of highpotential fungal laccases [43]. Residues that line the substrate binding pocket outside this coordination sphere also influence the reduction potential [44]. Here, a Glu!Thr mutation slightly raised the type 1 potential in the heterologously expressed low-potential laccase from M. albomyces (see below) without a significant change in catalytic activity as measured by ABTS [44] (Box 1, Figure 2). Other factors that influence reduction potential at the type 1 site remain to be discovered.

Figure 1. Structural insight into laccases from X-ray crystallography. (a) A stick model of a typical fungal glycan featuring a branched, a-linked mannose chain, blinked to two b-linked N-acetylglucosamines, b-linked to an Asn (from Asn54 in T. hirsuta Lac, PDB 3fpx). (b) A ribbon model of the X-ray crystal structure from T. versicolor Lac1 (PDB 1kya) with the coppers (orange circles) labelled by type and the organic substrate binding cleft highlighted in red. (c) A comparison of the architecture and sequence conservation between laccases from (top) T. versicolor Lcc1 (axial Phe in orange, PDB 1kya), (middle) Rigidoporus microporous laccase (axial Leu in orange, PDB 1v10), (bottom) B. subtilis CotA (axial Met in orange, PDB 1gsk). Coloured residue codes in the sequence alignment comprise the coordination spheres of the coppers; those around the type 1 copper are in italics.

considered a single entity rather than focusing on its components. Relationship between reduction potential and laccase structure In high-potential fungal laccases, such as in Trametes spp., the potential of the type 1 copper is typically 0.78–0.79 V vs SHE [39]. The unusual geometry around the type 1 copper is largely responsible for its high potential. Two His and one Cys are arranged roughly trigonally around the copper, and two non- (or weakly) coordinating residues sit within about 0.4 nm in the axial positions. One of the axial residues is invariably Ile; the other (Phe, Leu or Met, see Figure 1c) varies between laccases. There is a modest correlation between this second residue and the reduction potential of the type 1 copper, with Phe consistently producing high potentials [40]. A Phe!Met mutation lowers the reduction potential of the type 1 copper by as much as 110 mV [41]. Experiments with the closely-related bilirubin oxidase from the ascomycete Myrothecium verrucaria have shown that altering the type 1 site in a Met!Gln variant lowers the potential at which O2 is reduced (while also increasing its catalytic rate by 2.5–4-fold and removing pH dependence of the oxygen-reduction catalysis) [42]. Other residues important for modulating the type 1 copper 66

Laccase efficiency and enzyme inhibition Halides, pseudohalides, sulphides, carbonates and heavy metals decrease the catalytic efficiency of laccases [45]. Different modes of inhibition have been observed. For example, N-hydroxyglycine and fatty acids potentially block the phenolic substrate binding pocket [46–48]. Small anion inhibitors such as halides and pseudohalides probably block access to the trinuclear copper site, based on electron spin resonance spectroscopy, spectrophotometric assays and catalytic voltammetry [49–53]; the most effective of these, fluoride and azide, can lower activity by 50% at micromolar concentrations. X-ray structures corroborate spectroscopic assignments: azide sits between the type 3 coppers in one bacterial laccase structure [54] and structures of M. albomyces laccase assign a chlorine atom at the type 2 apex of the trinuclear cluster [55]. No corresponding structures for laccases from basidiomycetes exist yet, and structures incorporating a greater range of inhibitors must be resolved (see Recommendations in Box 2). Smaller and more electronegative halides inhibit laccases more effectively than those later in the periodic table (fluoride > chloride > bromide). There is much variation in the halide concentration required to inhibit a given laccase that does not correlate with its potential. With chloride, for example, the concentration required to halve the activity of laccase ranges from hundreds of micromolar to hundreds of millimolar between species [56]. To overcome this, we should exploit laccases from organisms such as salt-tolerant isolates of Flavodon flavus (see Recommendations, and Ref [57]); their enzymes might be better-suited for treatment of coloured effluents from the textile industry, which are typically alkaline and of high salinity [45,58]. Laccase modification: towards a designer molecule The biotechnological exploitation of laccases demands industrial-scale production of stable, active enzymes. This goal is unlikely to be realised with native, unmodified laccases in wild-type fungal strains: yields are too low and activity untailored to specific needs. Protein engineering offers a compelling pathway towards high-level expres-

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Box 1. Problems and pitfalls associated with laccase activity measurements  Catalytic competence of laccase in solution largely assessed spectophotometrically, using organic electron donors with characteristic redox potential and enzyme affinity. This electron source can be replaced by direct, controlled feed of electrons by adsorbing enzyme on an electrode. Figure 2a shows a typical profile of O2 reduction activity (measured as an electrical current) for immobilised laccase.  Solution assays for determining pH optima are problematic. Redox potential of some electron donors are independent of pH (e.g. ABTS–/ABTS2– is at 0.67 V, Figure 2b, green curve); for others (e.g. catechol or syringaldazine) electron and proton transfers are coupled, shifting reduction potential by approximately –60 mV per pH unit over a range of pH values (Figure 2b, magenta triangles).  Laccase activity profile versus pH is affected by pH dependence of enzyme catalytic potential and diffusion of substrates to enzyme. For four-electron, four-proton reduction of oxygen to water, onset of catalysis should shift by approximately 60 mV per pH unit. In reality, this might be lower, and below a certain pH value potential for catalysis may no longer increase with decreasing pH (Figure 2b, rainbow-coloured sigmoids).  Small-molecule diffusion can obscure true laccase activity. Limited aqueous solubility of O2 from air means maximum oxygen concentration in solution is about 0.27 mM: about the same as the

sion and efficient biocatalysis [16]. Herein lies the paradox: ascomycete fungi are considerably more amenable to genetic manipulation and industrial scale-up procedures, but the desirable high redox potential laccases lie predominantly within the less genetically tractable basidiomycete fungi. Hence, one goal is to combine attributes by creating high redox potential laccases in ascomycete fungi [18,26]. However, the expression of white-rot fungal laccases in heterologous systems has met with mixed success, when evaluated in terms of stability and boosted yield, as compared with the parent protein [6,13]. Below, we survey three different approaches to engineering a designer laccase, followed by a discussion on the rational alteration of glycosylation status. Site-directed mutagenesis Targeting the active site and substrate binding pocket. The growing library of fungal laccase structures, determined by X-ray crystallography (Table 1), helps inform rational choice of candidate residues for site-directed mutagenesis. One recurring set of target sequences are the residues that occupy the coordination sphere of the coppers, specifically to modify catalytic activity and potential. What emerges from this work is a catalogue of mutants with altered type 1 copper coordination [13]. Site-directed mutagenesis has met with variable success in improving the catalytic activity of laccases. For example, the basidiomycete Pycnoporus cinnabarinus laccase variants, produced with the dual purpose of identifying residues essential for catalysis and identifying strains with improved industrial performance, do not have superior laccase activity compared with that of wild-type strains [59]. More compellingly, an Asp!Asn modification to the hydrophobic substrate binding pocket in a Trametes versicolor laccase, expressed in Yarrowia lipolytica, was conjectured to lead to altered pH dependence, catalytic efficiency and permissivity of laccase towards xenobiotics that are not usually oxidised [60]. This single residue

Michaelis constant (KM) for reaction of O2 with enzyme, so limiting it to about half its maximum catalytic rate. Up to four organic substrates must be oxidised per O2, therefore, even though electron-donor concentration is in excess of enzyme concentration, these diffusion steps mean that intrinsic activity will be higher than measured by solution assays.  Solution-based assays reflect convolution of intrinsic activity, substrate affinity and substrate potential (Figure 2c), but their predictions are useful. What constitutes optimum pH depends on which laccase is being used and its end use:  For organic materials, e.g. for lignin degradation or bioremediation, activity should be determined using a structurally and electrochemically similar substrate.  For unmediated degradation of lignin, small-molecule analogues are better predictors of activity than ABTS.  In mediated degradation, enzyme effect on mediator is the best predictor of activity.  For applications that exploit electrocatalytic reduction of dioxygen, enzyme must be assessed on an electrode.  Cross-comparisons with different organic substrates are not straightforward. Assays must be conducted at the same pH, temperature, oxygenation and with normalised protein concentrations.

change did affect catalytic activity significantly, raising pH optimum by 1.4 units (see Box 1), which suggests significant alterations to interactions between the reducing substrate and the binding pocket. Recent support for this came from site-directed mutagenesis of single residues in the substrate binding pocket of recombinant T. versicolor Lccb laccase [37] (Table S1). Lccb showed significantly reduced activity towards the oxidation of anthracene as a result of a single point mutation in the substrate binding pocket. Three other isozyme families were characterised, which demonstrated significant variation between individual isozymes [37]. Such meticulous research highlights the need for rigorous characterisation of native isozymes, as well as their recombinant and mutagenised forms, in order that laccases can be selected carefully to optimise enzyme efficiency (see Recommendations). The study of the binding pocket of the lower-potential M. albomyces Lac1 (ET1Cu = 0.43–0.47 V [24]) has been enhanced by resolution of the crystal structures of its native and recombinant forms (Table 1). Moreover, recombinant expression is simplified because this fungus lies phylogenetically close to Saccharomyces cerevisiae, and thus, is wellequipped for efficient signal processing and post-translational modification [61–63]. Using this system, the lining of the binding pocket was changed with Glu!Asp/Thr mutations [44]. The target was chosen based on the structural resolution of a phenolic substrate absorbed into the crystal oriented towards this residue. With this docking point removed in the Thr mutant, the efficiency of catalytic oxidation of the phenolic compound fell dramatically, while activity towards the non-phenolic ABTS stayed at a similar level. In the Asp variant, catalytic efficiency towards 2,6-dimethylphenylalanine dropped and the pH optimum shifted upwards, while the ABTS activity profile remained similar, which exemplifies the cautions regarding solutionbased assays (Box 1). This carboxylate residue, and presumably its docking function, is conserved in laccases from ascomycetes and basidiomycetes, which underlines the need 67

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for additional site-directed mutagenesis research centred on this binding pocket (Recommendations). C-terminal modification. Research on C-terminal laccase modifications has focused primarily on those from ascomycetes. The crystal structure of M. albomyces reveals that the final four amino acids reenter the structure to form a plug that terminates neatly at a type 3 copper [55]. The function of this plug has been investigated by deleting the final four residues entirely — the mutant was practically inactive catalytically — or by substituting the final Leu for a shorter Ala (L559A) [24]; the latter mutant showed more complex changes to catalytic activity, azide tolerance and thermostability. The mutant crystal structure (Table 1) revealed alterations between the terminal carboxylate and the trinuclear copper cluster. Earlier directed evolution work on the laccase from the ascomycete Myceliophthora thermophila, expressed in S. cerevisiae, showed widely variable activity that was attributed to truncations of and/or mutations around its C terminus [63]. There exist few reports on the influence of the C terminus on basidiomycete laccases. Published structures of laccases from basidiomycetes do not show an analogous C-terminal plug. Wild-type T. versicolor Lcc2 laccase (expressed in P. pastoris) has been compared with a mutant in which 11 residues at the C terminus were replaced with a Cys residue, attempting to enhance enzyme affinity for a gold electrode [64]. This modification lowered the catalytic potential, but enhanced the rate of electron transfer from electrode to enzyme. In an alternative C-terminus modification strategy, a Stachybotrys chartarum laccase (ascomycete) was linked covalently to peptides that bind to carotenoids (e.g. lycopene from tomato), to target the laccase to stained areas of fabric. This resulted in more efficient bleaching [65]. There remains much scope for modification of the C terminus to produce enzymes with improved activity, particularly in basidiomycete laccases.

Figure 2. Different assay methods present different pH optima. (a) Simulation of a voltammogram for O2 reduction catalysed by an immobilised high-potential laccase around pH 4 (black line). The electron-donating role of the organic substrate is replaced by a potentiostated electrode to provide a readout of catalytic activity for any driving force. Points towards bottom left of the plot have highest activity and highest driving force (lowest potential). At the onset of catalysis (top right), the rate of reaction is limited by the availability of electrons to the enzyme. The catalytic rate increases with driving force until the enzyme cannot turn over any faster or the substrate cannot be supplied fast enough to keep up with the enzyme or both. (b) Extension of the simulation shown in (a) to a range of pH values. Colours of the sigmoidal traces represent potential-dependent activity at different pH values, with red as highest pH and violet as lowest. The vertical green line represents electron donors whose potential is not affected by pH (e.g. ABTS). The intersection of this line with each sigmoid represents the predicted activity for each pH. The position of the magenta triangles represent the measured activity, at a given potential and pH, for a mediator whose potential shifts with pH (e.g. syringaldazine). The intrinsic activity is taken as the limiting current at high driving force [as in (a)]. (c) Predicted activity versus pH profiles for each method. Each method presents a different pH optimum that spans one pH unit.

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Directed evolution Attempts to engineer laccases by directed evolution for use in lignin degradation have been reviewed briefly [18]. In general, low-reduction-potential ascomycete and bacterial laccases have been chosen because of their genetic tractability. The aim is to convert them into enzymes of higher reduction potential to obviate the need for mediators and extend the range of laccase substrates [18]. The first attempt at directed evolution considered heterologous expression of M. thermophila laccase in S. cerevisiae. Following 10 iterative rounds of mutagenesis, the laccase yield was raised some 170-fold and the enzyme showed improved thermostability, as compared with native enzyme activity [63]. More recently, directed evolution has been exploited to improve laccase activity and stability. For example, an improved variant M. thermophila laccase (expressed in yeast) emerged following two rounds of directed evolution. The variant withstood exposure to organic solvent concentrations known to promote unfolding and loss of catalytic activity in the native enzyme [66,67]. A S. cerevisiae strain that heterologously expressed a Pleurotus ostreatus laccase produced variants with greater catalytic activity and

Review stability. Molecular dynamics simulations combined with a hybrid synthetic crystal structure from T. versicolor and M. albomyces laccases were used to rationalise the functional roles of the principal mutations [68]. Collectively, these results demonstrate the value of site-directed mutagenesis in aiding interpretation of directed evolution studies. Combinatorial saturation mutagenesis and semi-rational design One way ahead is through combinatorial saturation mutagenesis techniques that are focused on the environment of the laccase copper atoms. Such an approach has been used to improve simultaneously functional expression and catalytic activity of a bacterial laccase from Bacillus licheniformis [69]. Among fungal laccases, saturation mutagenesis has been applied to two specific residues of recombinant M. thermophila laccase: S510, which lies within the VSG tripeptide common to low/medium potential laccases; and L513, an axial ligand of the type 1 copper (Figure 1). This led to a mutant with two contiguous substitutions around S510, which gave threefold higher turnover rates than with the parental enzyme [70]. In further work on this laccase, two mutations were introduced into the C terminus (G614D and E615K). The mutant enzyme showed better ABTS oxidation kinetics and increased K M;O2 , suggesting disturbed copper environments. Crystal structure data is needed to confirm these hypotheses [25].

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Laccase glycosylation: an engineering perspective We know little about glycosylation in basidiomycete laccases, save for glycan distribution patterns inferred from crystal structures. Glycosylation is likely to influence native and recombinant enzyme activity, but only recently have researchers begun to explore these structure–function relationships [71]. Site-directed mutagenesis on mutant laccases from Trametes sp. 420, which differ by an Asn!Gln mutation at one of each of the six potential N-glycosylation sites, exhibit differing specific activities with respect to each other and to wild-type recombinant laccase [72]. However, interpretation of these data is challenging, because of the need to normalise protein assays and to understand better the effect of modifications upon glycosylation status (see Recommendations). Recently, the N-glycosylation status of Pycnoporus sanguineus CelBMD001 laccase was determined by enzymatic rather than by engineering techniques. Stability and ABTS oxidation activity were decreased in the N-deglycosylated protein especially at low temperatures, which suggests that these effects are the result of localised variations in the laccase structure caused by the absence of glycans [71]. This merits investigation by targeted mutagenesis. Given this potential influence on activity, hyperglycosylation is an important consideration when attempting to mass produce these enzymes. S. cerevisiae and P. pastoris yeast expression systems are known to cause hyperglycosylation of non-native laccases, which might interfere with

Figure 3. Designer laccases. Modified, mass-produced laccases with improved stability, activity, and specificity will emerge tailor-made for disparate industrial purposes.

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Review protein folding and function [73]. The importance of posttranslational glycosylation during heterologous expression of laccases in different yeasts is now being evaluated. Recently, Trametes trogii laccase was produced in a Kluyveromyces lactis Golgi mutant strain, defective in O- and N- glycosylation pathways for laccase expression. The recombinant, glycan-free enzyme had significantly lower activity than recombinant laccase expressed in an unaltered K. lactis strain [74]. This suggests that glycans influence laccase properties significantly. Unfortunately, the protein yields were not compared. We must understand better and optimise the expression of recombinant laccases with faithfully reproduced glycosylation status. Subsequently, the role of glycans should be explored through systematic modification of glycosylation sites, coupled with robust structural comparisons and activity analyses (see Boxes 1 and 2). Mass production of laccase for industrial purposes? Much effort has been spent trying to reduce production costs and boost yields of laccases from wild-type strains of filamentous fungi grown in bioreactors [75]. This has been achieved by optimising growth conditions: modifying culture media (providing nutrients once, continuously or via fed-batch); adjusting environmental parameters (e.g. temperature, oxygen concentration, pH); and by addition of inducer molecules (e.g. copper, glycerol, Tween 80). Growth has been submerged in broth (SB), with fungus immobilised on supports, or as free cells or pellets, and in a variety of vessels/bioreactors (e.g. stirred tank, air-lift) or in solid-state fermentors (SSFs) [75]. Several different issues have emerged from SB growth of wild-type strains, such as problems with viscosity, protease activity, differing yields, unequal access to oxygen and usage of vast amounts of cooling water. However, mostly the issues were high costs and moderate yields. Despite this, the greatest laccase yield recorded in SB has been with Trametes pubescens [76]. Here, fed-batch SB culture offers several advantages: regulated substrate availability tightly controls growth rate, thus delimiting the rate of reaction and fungal metabolism, and avoiding catabolite repression and proteolysis. SSFs offer a cheaper, more environmentally benign alternative to SB; being better matched to the natural growth conditions of fungi with simpler downstream processing [77]. However, humidity, pH, temperature and aeration are not well regulated in SSF cultures. For the potential of designer laccases to be realised, effective, low-cost, efficient and green production systems are required in which overexpression of laccase can be induced. The future is one which combines the attributes of SSF and SB, such as by exploiting trickle bed reactors [75]. This potential has yet to be realised on an industrial scale but early indicators suggest that modest yields of laccases can be obtained over several weeks in laboratoryscale assays with the white-rot fungus Pleurotus ostreatus grown on sugarcane bagasse [78]. Conclusions and recommendations Although some laccases are being employed successfully in industry [5,6,12], no natural laccase combines the desired attributes of being stable and active over a range of temperatures and pH values, with high reduction potential, 70

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halide/hydroxide tolerance and suitability for cost-effective production. High-potential laccases represent a means to exploit some of these desirable characteristics in designer enzymes (Figure 3). The development of robust heterologous expression systems offers several potential benefits over purifying laccase directly from fungal cultures. Firstly, many species secrete several laccases; recombinant methods allow expression of a single protein of known sequence. Secondly, recombinant methods can result in high protein yields, particularly if proteins are secreted by a host, which itself produces few other extracellular proteins. Thirdly, laccase production by wild-type strains is inextricably linked with secondary metabolism and its associated problems (e.g. protease activity and

Box 2. Recommendations: the way ahead (a lac and lustrous future?)  Effects of glycosylation on catalysis (especially aberrant glycosylation during heterologous expression) merit systematic investigation, both in solution and in immobilised systems.  Single-mutation shortcomings realised. Directed mutagenesis at single, then multiple sites in a systematic manner, followed by robust determinations of recombinant enzyme activity, compared with wild-type strain. Areas for exploration/exploitation include alterations to the binding pocket in laccases from basidiomycetes and investigations into pH optima, halide tolerance and thermal stability.  Choice of heterologous or homologous expression system should consider: (i) equipping gene with a constitutive or inducible promoter; and (ii) expression in phylogenetically close host or in self, but with native laccase genes deleted.  Annotated genome sequence data of fungi of industrial interest will reveal number of LAC genes in a given genome.  When LACs form part of a multigene family, quantitative realtime transcript profiling will unmask highly expressed members and conditions that control gene expression.  Laccase transcript studies will aid in robust assessment of a number of laccase isozymes before purity of individual laccases is confirmed [37].  Structural biology of laccases has advanced with an explosion in X-ray structures; significant gaps remain to be filled:  Need for a thorough exploration of structure and role of glycans. NMR could illuminate these features more effectively than crystallography.  Structures of related blue multi-copper oxidases must be resolved to develop better structure–function relationships and select features for rational mutagenesis. These include the structure of the Rhus vernicifera laccase and of at least one bilirubin oxidase.  Need for structures of laccase complexed with organic substrates [24], putative surface stabilisers [53,79] and inhibitors to complement the work already done on the low-potential laccase CotA from B. subtilis [54].  Limitations of solution-based assays (Box 1) realised and complementary electrochemical assays of adsorbed proteins adopted more widely [79], so ending the near total reliance on equilibrium titrations as the sole qualifiers of (electro)catalytic ability of laccases and other blue multi-copper oxidases.  Development of spectroscopic or computational methods that allow assessment of reduction potentials in enzymes turning over substrate rather than at rest (equilibrium).  Development of a consensus method for determining enzyme activity for exploitation in high-throughput assays.  More collaborative research required between biologists, chemists and chemical engineers to quicken pace of strategic discovery and facilitate effective scale-up to fermentations in industrial bioreactors.

Review uncontrolled growth). Production of recombinant laccases should also facilitate protein purification. Several outstanding issues remain to be addressed, including the need: (i) to understand the role of glycan composition and glycan position on laccase function; (ii) for a flexible, controllable and resilient heterologous expression system; (iii) to improve enzyme stability in demanding environments; and (iv) for lost-cost, environmentallybenign scale up of laccase production in bioreactors. Given these significant hurdles, we outline a series of recommendations in Box 2. If we can realise these, we will have a truly cosmopolitan industrial enzyme. Note added in proof Very recently the additive effect of mutations at three sites in the blue (but not multi-) copper protein azurin (one at the coordinating Met; two in the secondary coordination sphere, altering hydrophobic and hydrogen-bonding interactions) tuned the potential between 0.0 to 0.7 V vs SHE [82]. Acknowledgements This work was funded by a Leverhulme Trust award to FAA and SJG to support SRG and CFB (now an EPSRC Career Acceleration Fellow), an EPSRC studentship to CJR, BBSRC monies and a Leverhulme Trust Early Career Research Fellowship to support PS. We thank Rachel Heath and Kylie Vincent (Department of Chemistry, University of Oxford) for their critical appraisal of the manuscript, Nina Hakulinen (Department of Chemistry, University of Joensuu) for valuable discussions on laccase structural biology, Max Crispin (Department of Biochemistry, University of Oxford) for discussions on glycosylation, and Victoria Vowles and Paul Peters (Chemical Abstracts Service) for their help with Supplementary Figure S1.

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