Laccases of prokaryotic origin: enzymes at the interface of protein science and protein technology

Abstract

The ubiquitous members of the multicopper oxidase family of enzymes oxidize a range of aromatic substrates such as polyphenols, methoxy-substituted phenols, amines and inorganic compounds, concomitantly with the reduction of molecular dioxygen to water. This family of enzymes can be broadly divided into two functional classes: metalloxidases and laccases. Several prokaryotic metalloxidases have been described in the last decade showing a robust activity towards metals, such as Cu(I), Fe(II) or Mn(II) and have been implicated in the metal metabolism of the corresponding microorganisms. Many laccases, with a superior efficiency for oxidation of organic compounds when compared with metals, have also been identified and characterized from prokaryotes, playing roles that more closely conform to those of intermediary metabolism. This review aims to present an update of current knowledge on prokaryotic multicopper oxidases, with a special emphasis on laccases, anticipating their enormous potential for industrial and environmental applications.

Introduction

Laccases are enzymes belonging to the large family of multicopper oxidases (MCOs) that are encoded in the genomes of organisms in all three Domains of Life—Archaea, Bacteria and Eukarya. MCOs are unique among copper proteins in that they contain one of each of the “biological” copper sites, type 1 (T1), type 2 (T2) and the binuclear type 3 (T3). MCOs couple the one-electron oxidation of a range of aromatic phenols and amines, benzenethiols, and even some inorganic compounds such as low-valent metals to the reduction of dioxygen to water. Their redox reactions depend on the presence of two catalytic sites: the mononuclear T1 copper centre that is the primary acceptor of electrons and a trinuclear centre, comprising T2 and T3 copper sites, involved in dioxygen reduction to water [1]. Most MCOs are proteins that contain approximately 500 amino acid residues, and are composed of three Greek key β-barrel cupredoxin domains (domains 1, 2 and 3) that come together to form the three spectroscopically distinct types of Cu sites [1, 2]. The T1 copper site is in domain 3 and the T2/T3 trinuclear copper cluster is at the border between domains 1 and 3, possessing ligands from each domain. The catalytic mechanism of MCOs involves (1) reduction of the T1 Cu site by the oxidized substrate, (2) electron transfer from the T1 Cu site to the trinuclear centre and (3) O₂ reduction by the trinuclear cluster [1, 3–6].

MCOs have been recognized for a long time as “moonlighting” proteins, i.e. multifunctional enzymes capable of changing the functions in response to their cellular localization and/or differential expression or due to alterations in the concentration of substrates [7]. Even though all MCOs exhibit some oxidase activity towards aromatic compounds, in general they appear to form two functional classes, as observed by Stoj and Kosman [8]: enzymes that oxidize organic substrates with higher efficiency than metal ions, the laccases (benzenediol: oxygen oxidoreductases, EC 1.10.3.2), and those that oxidize metal ions, such as Fe(II), Cu(I) and/or Mn(II), with higher efficiency as compared with organic substrates (EC 1.16.3). The latter enzymes are thus designated as metallo-oxidases and the most prominent members of this group are human ceruloplasmin (hCp) and yeast ferroxidase Fet3p from the Eukarya domain, known to be critically involved in the metal homeostasis mechanisms of the respective organisms [9]. Metal homeostasis operates mainly in aerobic metabolism, where metals such as iron and copper though essential for cells, readily participate in reactions that result in the production of highly reactive oxygen species. It has been proposed that metalloxidases act as cellular protectors by shifting the Cu(II)/Cu(I) and Fe(III)/Fe(II) ratios towards the less toxic forms of copper and iron, Cu(II) and Fe(III). Laccase´s metabolic roles are closer to intermediary metabolism (lignification, delignification, oxidative plant stress management, morphogenesis or virulence, among others) and because of their wide range of aromatic substrates and lack of requirement for exogenous co-factors, they have found application in a large number of biotechnological applications.

Prokaryotic multicopper oxidases

The first reports of MCO activity in prokaryotes came from (1) Azospirilium lipoferum, associated with pigment synthesis [10], (2) the marine bacterium Marinomonas mediterranea [11], later shown to arise from a peculiar enzyme showing laccase as well as tyrosinase activity [12], (3) Escherichia coli, related with metal resistance [13], and (4) Bacillus subtilis, originating from an outer spore coat enzyme involved in the synthesis of a brown spore pigment [14, 15]. These studies were followed by the identification and characterization of an exponentially increasing number of prokaryotic MCOs as predicted in the seminal bioinformatic work by Alexandre and Zhulin [16]. Importantly, two MCOs have been characterized from the microorganism of the Archaea domain: LccA, an extracellular salt-tolerant laccase with high activity for phenolic substrates, purified from the halophile archeon Haloferax volcanii [17] and the recombinant thermoactive and thermostable McoP from the hyperthermophilic archaeon Pyrobaculum aerophilum. The latter showed a remarkable Cu(I) and Fe(II) oxidation activity and ability to use nitrous oxide as well as dioxygen as electron acceptor [18].

Prokaryotic metalloxidases

There is a strong body of evidence for the link between prokaryotic multicopper oxidases and their involvement in either copper or iron homeostasis mechanisms, emerging from genetic, physiological and biochemical studies in a variety of strains such as Escherichia coli [13, 19–21], Pseudomonas aeruginosa [22], the soil bacteria Geobacter sulfurreducens [23, 24], Aquifex aeolicus [25], Myxococcus xanthus [26, 27], Pyrobaculum aerophilum [18], Xanthomonas campestris [28, 29], Rhodobacter capsulatus [30, 31], Rhodococcus erythropolis [32] and in the acidophilic biobleaching Acidithiobacillus thiooxidans [33], as well as in the pathogenic Staphylococcus aureus [34], Campylobacter jejuni [35], Legionella pneumophila [36], Salmonella enterica serovar thyphimurium [37] and Mycobacterium tuberculosis [38]. The vast majority of these enzymes harbour signal peptides indicating their export and putative role in the periplasmic space or at extracellular milieu [16, 39].

Prokaryotic metalloxidases from the MCO family have also been involved in bacterial Mn oxidation in terrestrial and aquatic environments as demonstrated for strains such as Leptothix sp. [40], Pseudomonas putida [41, 42], Pedomicrobium sp. [43], Bacillus sp. strain SG-1 [44, 45] and B. pumillus [46]. The physiological relevance of bacterial Mn(II) oxidation is at present unclear, but it is likely related to the energy or carbon metabolism of the microorganisms or cellular protection against reactive radical species or other environmental hazards [47]. The best characterized Mn oxidase from the MCO family is MnxG from Bacillus sp. strain PL-12 which seems to be involved in the two energetically distinct Mn(II) to Mn(IV) oxidation steps [48].

The first three-dimensional structure of a prokaryotic metalloxidase was reported for E. coli CueO [49, 50], followed by P. aerophilum McoP [51] and C. jejuni McoC [52]. A higher efficiency in the oxidation of Cu(I) and Fe(II) metals as compared with organic substrates was measured in these enzymes, and in A. aeolicus McoA, together with a Cu(II) dependent activation of enzymatic activity [18, 25, 52, 53]. A striking structural feature of these metalloxidases is (1) the occurrence of charged or polar residues in the substrate binding site, such as carboxylates present in the yeast ferroxidase Fet3p [8], and (2) a restricted or blocked access to the copper T1 centre, because of the presence of an additional loop region containing short α-helices.

In the crystal structures of CueO and McoC, and in the model structure of McoA, an occlusion of the T1 Cu site by a methionine-rich secondary structure was observed (Fig. 1a). These Met-rich segments are similar to regions found in numerous proteins involved in copper homeostasis with a role in copper binding and activation [54, 55]. Indeed, X-ray studies of CueO allowed the identification of three additional copper-binding sites in the methionine-rich segment in positions that are putatively involved in an electron-transfer pathway which connects the substrate binding sites to the T1 copper centre (Fig. 1a, [50, 56, 57]). The deletion of the Met-rich segment in either E. coli CueO (14 Met residues in a 45 amino acid segment) or A. aeolicus McoA (12 Met residues in a 42 amino acid segment) resulted in a severe decrease in catalytic activity and a loss in the enzyme activation by exogenous Cu(II), providing evidence for the key role of this region in the modulation of the catalytic activity [25, 58]. Moreover, decreased K _m values were found for the larger aromatic substrates suggesting that the Met-segment impairs the efficient binding of larger substrates to the binding site due to steric effects [25, 58]. Methionine-rich regions were thus considered as a “cuprous oxidase” motifs related to bacterial Cu resistance [4]. The primary structure of Thermus thermophilus MCO [59] also reveals a presence of a methionine-rich region (Met293–Met305) that restricts the access to the copper T1 centre in its crystal structure (PDB 2XU9), however, no metalloxidase activity data were reported for this thermophilic enzyme. Importantly, not all prokaryotic metalloxidases with demonstrated or predicted Cu(I) activity exhibit methionine segments in their sequence, e.g. P. aerophilum McoP (which however shows a preference for ferrous rather than for cuprous oxidation in contrast to CueO, McoC or McoA), M. tuberculosis MmcO, A. thiooxidans CueO or L. pneumophila McL [18, 33, 36, 38] demonstrating that the structural determinants of metal specificity within the MCO family remain to be fully elucidated. In yeast Fet3p ferroxidase, the carboxylate residues close to the T1 Cu centre was shown to provide a binding site for Fe(II) and form part of the electron-transfer pathway from this substrate to the protein’s T1 Cu centre whilst the enzyme reactivity for Cu(I) uses an alternative pathway for electron transfer [4, 60].

Fig. 1.

Overall three-dimensional structure of E. coli CueO (PDB:1N68) [50] (a), Bacillus subtilis CotA-laccase (PDB:1W6L) [6] (b) and the trimer Streptomyces coelicor SLAC (PDB: 3CG8) [118] (c) with each cupredoxin domain colored in a different color (domain 1 in blue, 2 in green and 3 in orange). The copper atoms are shown as brown spheres, with the fifth regulatory Cu ion identified in the CueO structure, near the T1 Cu site

Prokaryotic laccases

The majority of laccases isolated within the Bacteria domain, i.e. enzymes with a known preference for organic over metal ions, were isolated from the Bacillus and Streptomyces genera despite the report of laccase activity in some other bacterial species [39, 61–65]. Interestingly, the gene coding for the phenolic oxidizing laccase of Archaeal origin Haloferax volcanii LccA, seems to had been most likely acquired by horizontal gene transfer from bacteria of the phyla Firmicutes (e.g. Bacillus) [17]. The most extensively studied laccase both from the fundamental and applied point of view, is the recombinant CotA laccase from B. subtilis [6, 15, 66–87], but laccases from B. halodurans [88], B. licheniformis [89, 90], Bacillus sp. HR03 [91], B. pumilus [46, 92], B. vallismortis [93], B. amyloliquefaciens [94], B. tequilenses [95], B. sphaericus [96], B. sp ADR [97] as well as from B. clausii [98] have also been isolated and characterized. Most of the laccases identified in Bacillus are found to be a part of the outer coat that protects spores from a diverse range of stresses, playing roles in the biosynthesis of a brown melanin-like spore pigment and protection from UV light and hydrogen peroxide [14, 15]. The X-ray crystal structures of CotA-laccase show the typical copper centre geometries and overall fold of multicopper oxidases with three cupredoxin domains [6, 66, 67]. In contrast to the structures of prokaryotic metalloxidases, the binding pocket close to the T1 copper centre is mainly formed by apolar aminoacids and appears to be broad and able to accommodate a number of bulky substrates (Fig. 1b).

Streptomyces laccases are extracellular enzymes, identified and characterized from a variety of species such as S. cyaneus [99, 100], S. griseus [101, 102], S. lavendulae [103], S. coelicolor [104], S. psammoticus [105, 106], S. ipomoea [107–109], S. sviceus Ssl1 [110, 111] as well as in S. violaceusniger, S. lividans TK24, S. viridosporus T7A and Amycolatopsis sp 75ib2 [112, 113] with putative roles in morphogenesis, sporulation, pigmentation, ligninocellulose degradation, bacteria–bacteria interactions or antibiotic production. The most extensively characterized is the laccase from S. coelicolor [5, 104, 114–120]. Streptomyces laccases are 2-domain bacterial laccases (usually denominated SLACs (small laccases)) as they lack the second of the three domains of typical MCOs [104] leading to a substantially different enzyme architecture [111, 113, 118]. Still, SLACs contain the four copper ions and spectroscopic and kinetic properties similar to those of common laccases [102, 104, 111]. Their crystal structures reveal a trimeric quaternary arrangement of the two-domain protein chains, resembling more the structures of nitrite reductases or human ceruloplasmin than those of typical laccases (Fig. 1c; [111, 118]). The T1 Cu ions located in domain 1 are close to each other, near the surface at the central part, forming a trimeric substrate binding site. SLACs are thought to result from the duplication of single-domain proteins of the cupredoxin family of the blue copper proteins and to represent key evolutionary intermediates of the three domain MCOs [121]. A pair of domains from the initial cupredoxin domain was duplicated twice more to form a six-domain MCO, such as human ceruloplasmin. A three-domain MCO was formed by a single addition to the double-domain protein, after modifications in the cupredoxin domains such as the adjustment of the copper-binding sites and evolution of the substrate binding pockets [2, 121].

Distinctive features of prokaryotic MCOs

Enzyme heterologous production and engineering

Recombinant DNA technology provides means for achieving high levels of enzyme production as well as redesigning nature’s catalysts at the molecular level, adapting their functions for applied ends. E. coli is the most common host for heterologous production of prokaryotic proteins benefiting from a range of genomic tools available. However, production of multicopper oxidases in the cytoplasm of E. coli faces a major drawback since the operating homeostasis systems maintain a cellular copper quota within a narrow range of around 10 μM under aerobic conditions [21, 122] which results invariably in the production of a recombinant copper-depleted population of enzymes [123]. A full complementation of copper in laccases is however, pivotal not only for the achievement of full catalytic efficiency but also for their maximal kinetic and thermodynamic stability [68, 76, 123–128]. This limitation can be overcome by changing the O₂ level during cultivation of recombinant E. coli expressing multicopper oxidases (Fig. 2a, [123]). Hence, the production of a fully copper-loaded population of proteins is achievable upon E. coli growth in aerobic to anaerobic (or microaerobic) conditions that allow for an 80-fold higher intracellular accumulation of copper (Fig. 2b, [123] ), resulting in a full metal incorporation into recombinant proteins as reported for MCOs from B. pumilus, B. sp HR03, B. amyloliquefaciens, B. clausii, Aeromonas hydrophila, Campylobacter jejuni CGUG11284, P. aerophilum, and S. coelicolor SLAC [18, 52, 91, 92, 94, 98, 129].

Fig. 2.

a UV–visible spectra of the as-isolated CotA species produced in aerobic (thin line) and in microaerobic (thick line) conditions. b Intracellular Cu content of E. coli cells during growth under aerobic (filled squares) and microaerobic (open squares) conditions (adapted from [123])

Taking advantage of the available tools for the genetic manipulation of bacterial genes and strains, a few studies report the optimization of laccases using protein engineering tools. The combination of rational and directed evolution approaches resulted in the improved ABTS oxidation efficiency in B. subtilis CotA-laccase [82, 83], higher levels of expression and improved activity for phenolics in B. licheniformis laccase [90] and in S. coelicolor SLAC [130], as well as an increase of solubility and thermostability in a bacterial laccase screened from a marine metagenomic library [131]. The construction of chimeric proteins capable of catalyzing two or more reactions provides a smart strategy that decreases enzyme-related costs in industrial processes by reducing production and purification operations. CotA-laccase has been successfully fused to a bacterial xylanase [87] and β-1,3-1,4-glucanase [86] showing enhanced activity, stability and potential for ligninocellulosic degradation in biorefinery applications.

Thermo and hyper-thermophilic MCOs

Enzymes from extremophiles are promising for industrial applications due to their high intrinsic thermal stability, revealing the robustness required for specific industrial processes. Laccases and metalloxidases showing optimal temperatures close or superior to 80 °C have been isolated from T. thermophilus, A. aeolicus and P. aerophilum [18, 25, 59, 132]. These thermo- and hyper-thermophilic enzymes exhibited a half-life of inactivation at 80 °C between 5 and 14 h, values far beyond those exhibited by any laccase characterized from fungal sources.

A few studies have shed light on the stability of prokaryotic MCOs; the analysis of thermal unfolding data of A. aeolicus McoA or P. aerophilum McoP indicates melting temperatures above 100 °C with values ~20 °C higher than CotA, which is also considered to be a thermo-active and -stable enzyme (Fig. 3a, [15, 18, 123, 132]). Thermal unfolding of both proteins is a complex process pointing to three independent transitions which apparently correlate with the structural organization of three cupredoxin-like domains [133, 134]. Thermodynamic stability studies show that CotA-laccase has a high chemical stability, superior to that exhibited by the hyper-termophilic A. aeolicus McoA (Fig. 3b). The latter enzyme shows a very low heat capacity, which was associated to a peculiar strategy to fold and function at high temperatures, shared with a small group of thermophilic enzymes [132]. Copper ions are known to play a key role in the stability of MCOs [135]; for example, the unfolding rate constants of the apo-forms of CotA or McoA are 50- to 100-fold higher as compared with the corresponding holo-forms, as a result of the copper stabilizing effect in the protein structures [76, 77, 132].

Fig. 3.

a Excess heat capacity obtained from DSC scans of CotA and McoA. The thick lines represent the experimental data whereas the dashed lines represent the fit of the thermal transitions. b Fraction of unfolded (f _U) CotA and McoA by GdnHCl at pH 7.6 as measured by tryptophan fluorescence at 25 °C. The solid line is the fit according to the equation f _U = e^(−ΔG/RT)/1 + e^(−ΔG/RT) which assumes an equilibrium F↔U induced by GdnHCl (adapted from [68, 123, 132])

Neutral to alkaline optimal pH for aromatic amines and phenols

In general bacterial laccases show a clear preference for the neutral to alkaline pH range while fungal laccases such as Trametes versicolor TvL show maximal rates for aromatic phenolic or amine substrates in the acidic pH range [79]. Phenolic compounds exhibit acid–base transitions (pKa) within the neutral to alkaline pH range and therefore fungal laccases oxidize substrates in the phenolic form while bacterial laccases oxidize them in the phenolate form, i.e. after deprotonation of the phenolic group and above the respective compounds’ pKa values. This has been attributed to observed structural differences in the vicinity of the substrate binding site cavity: all known fungal laccases have a conserved Asp (pKa 3.9) or Glu (pKa 4.1) residue at that site proposed to have a role in stabilizing the phenoxy radical formed during the catalytic reaction [136, 137], which is not present in any bacterial laccase identified so far. Therefore the efficiency of oxidation of phenolics by bacterial laccases which lack a negatively charged residue close to the T1 Cu centre relies mostly on the protonation/deprotonation state equilibria of the compounds themselves showing thus, maximal rates at pH values close or above their pKa, in the neutral to alkaline range of pH values [71, 72, 79, 138].

In phenolic-mediated laccase reactions, the key element seems to lie in the chemical nature of the phenolic compound, determining both the stability and reactivity of the phenoxy radicals involved in the mediated reaction [139] and not in the properties of the enzyme [79]. For example, neutral pH is the optimal for the conversion of non-phenolic compounds in laccase mediator systems when phenolic compounds are used as redox mediators, with either a fungal (pH 3–4 optimal for phenolics oxidation alone) or bacterial laccase (optimal at pH 8–9) [79]. The mediating role of a phenolic compound relates to the half-life of its oxidized form, i.e. its phenoxy radical [138, 140] and thus the higher rates of mediated conversion of substrates at neutral pH most likely reflects the enhanced stability of the phenoxy radicals in these conditions [140].

Application of bacterial laccases

Biocatalysis is considered to be a key component for the development of a sustainable bio-economy and the use of enzymes as biocatalysts has been constantly growing in a range of industries. Laccases that work in air and produce water as the only by-product are industrially relevant enzymes, as they can potentially be used in a number of applications in food technology processes, the delignification of lignocellulosics, bioremediation, biosensors for analytical applications and others.

The catalytic rate-limiting step in laccases is considered to be the oxidation of substrate at the T1 site, most probably controlled by the redox potential difference between this site and the trinuclear site. The redox potentials exhibited by the T1 Cu sites of laccases span over a broad range of values, from 400 mV for plant laccases to 790 mV (vs. NHE) for some fungal laccases [1]. High redox potentials are expected to allow for oxidation of an extended range of substrates and improve the effectiveness and versatility of the enzymes. All known prokaryotic MCOs show redox potentials at the lower end of these values. The conserved coordinating amino acids for the T1 copper site are two histidines and a cysteine, and the natural variations occur in the so-called axial position with a single interaction from a Met being the most common arrangement in bacterial laccases. Fungal laccases have the non-coordinating Phe or Leu at this position and these may contribute for the high E ^o observed in these enzymes [1, 8]. However, the redox potential in laccases is determined not only by the coordination geometry of the T1 copper centre but also by the nature of the second coordination sphere residues influencing solvent accessibility, hydrogen bonding, and dielectric anisotropy around the site as indicated in studies of enzyme variants carrying point mutations in the vicinity of the T1 copper site [68, 69, 111, 128, 141–145]. Noteworthy it was shown that the ´low redox potential´ B. subtilis CotA-laccase (Eº = 525 mV, showing a coordinated Met at the T1 centre) oxidizes, at faster rates high redox compounds such as the azo dye reactive black five or aromatic amines than the fungal laccases (Eº ~ 780 mV) and in the absence of redox mediators [71, 84, 85, 146–149].

Bacterial laccases have been successfully used for synthetic dye degradation [71, 72, 78, 94, 100, 107], the biobleaching, aging of kraft pulps [99, 108, 150], micropollutant degradation from wastewater treatment plants [151], the oxidation of polycyclic aromatic hydrocarbons [152], the dimerization of phenolic acids [89], the synthesis of bio-dyes, phenoxazine and phenoxazinone compounds [84, 85], but also in biofuel cells [80] and droplet-based microfluidic systems [81] amongst other biotechnological applications. It is expected that the number of laccase-based industrial oxidation processes will increase significantly in the next few years considering, for example, the lignocellulose biorefinery which represents a promising alternative source of renewable chemicals, materials, energy and fuels. Lignin is the most abundant aromatic polymer in Nature, and an important source of bulk and fine chemicals. Laccase, a prominent ligninolytic enzyme, will certainly have key roles in overcoming the lignin recalcitrance to degradation and valorization. The recent screening of metagenomic libraries of diverse natural ecosystems from bovine rumen microflora [153], mangrove soils [154], marine sources [155] or the guts of giant panda [156], termite [157] and wood-feeding beetle [158] provided novel insights into largely unexplored bacterial ligninocellulosic communities, and led to the identification of new bacterial lignin-degrading enzymes, including laccases, with diverse and new enzymatic properties.

Concluding remarks

MCOs are multifunctional enzymes able to oxidize organic and inorganic substrates with varied efficiency. Within prokaryotes the physiological roles of MCOs with demonstrated or predicted higher reactivity towards low-valent metal ions—“cuproxidases” and ferroxidases, involved in Cu and Fe metabolism—are far better characterized than that of laccases, and, in some cases, the molecular basis of that contribution has been characterized. So far, the vast majority of prokaryotic laccases have been isolated from the Bacillus and Streptomyces genera but the characterization of new members to the MCO family from Bacteria or Archaea tends to increase. Laccases of prokaryotic origin have been shown to perform reactions analogous to their fungal counterparts, despite the lower redox potential, and further benefit from interesting properties from the fundamental and technological perspectives such as higher optimal temperature and pH, salt tolerance and increased thermal and chemical stability. Bacterial systems for which genetic and molecular biological tools are well established allow the achievement of higher yields of enzyme production and the generation of more efficient and stable biocatalysts required for handling the harsh industrial-process conditions through the use of protein engineering approaches. There are many open fundamental questions related to MCOs and the relative simplicity of recombinant prokaryotic systems also provide advantage in structure-function studies in relation e.g. to the tunnability of MCO’s redox potential, the molecular determinants of thermal and chemical stability as well as the mechanisms of multifunctionality within MCOs. These has several general implications in the understanding of molecular recognition to ligands for drug discovery programs, the evolution of protein function over time, and in the engineering of proteins in the realm of biotechnology, in areas as diverse as synthetic biology and metagenomics.