Besides the archetypical laccases discussed in Section 2.1.1, various seemingly "unconventional" laccases or analogs have been reported. Some of the isolated laccases (with sequences highly homologous to typical multi-Cu oxidases) appear as "yellow" or "white" when purified, indicating the lack of a canonical "blue" T1 Cu site. The site might be modified by oxidized substrates, as proposed for the Pleurotus ostreatus or Panus tigrinus "yellow" laccase [157, 158], or contain other metal ions (for example, Zn2+ or Fe3+), as proposed for the P. ostreatus "white" laccase . Often, a "yellow" or "white" laccase may oxidize non-phenolic compounds in the absence of mediator, or exhibit a more desirable pH or thermal profile than its "blue" counterpart.
Recently reported laccase analogs, classified as such based on their catalyzed reactions, include a dimeric (43 kDa subunit) Tricholoma giganteum (mushroom) oxidase which also shows an anti HIV-1 reverse transcriptase activity , a dimeric (40 kDa subunit) Tellima grandiflora (plant) oxidase , and an 85 kDa Nephotettix cincticeps (insect) oxidase . However, these oxidases have an N-terminus either unknown or not homologous to typical laccases. Thus more structural and enzymological information is needed to determine whether these oxidases belong to the laccase (or multi-Cu oxidases) family.
The discovery of novel laccases is propelled by the ever-increasing numbers of sequenced genomes and the revealed genetic information. The huge amount of data has sparked development of tools for computational analysis to link sequence with biological function. These tools have naturally also been employed to study the classical laccases. More than 100 plant and fungal laccase sequences were thus analyzed using sequence alignment  to identify signature sequences for laccases. A phylogenetic analysis including more than 350 multi-Cu oxidases was reported in another study . The largest group of enzymes in the latter study was indeed fungal laccases, which clearly separated in two clusters of sequences from basidiomycetes and sequences from ascomycetes. The sequences were selected for the study by the presence of four conserved Cu-oxidase consensus patterns and the study also included a number of bacterial protein sequences. These bacterial sequences clustered quite separately from all of the eukaryotic proteins. A couple of bacterial sequences were even excluded, as they could not be aligned without loss of resolution in the phylogenetic analysis. The example serves to illustrate the difficulty in classification of bacterial laccase sequences that have emerged amongst the multicopper oxidases in the last decade. The Pfam database is a collection of protein families and domains and it contains multiple protein alignments and profile-HMMs of these . In Pfam, the multicopper oxidases constitute a clan with seven Pfam members. The classical fungal lac-cases contain elements from no less than three members of this clan. Some of the new bacterial laccases differ from this archetype and have a completely different domain organization.
The expansion of the laccase enzyme class with prokaryotic members clearly represents both a great opportunity and a challenge as well to the field [164, 165, 165a]. Bacterial laccase-like enzymes have been discovered in Azospirillum, Bacillus, Escherichia, Marinomonas, Pseudomonas, Streptomyces, Thermus, and Xan-thomonas genera. Many bacterial laccase genes have a signal-like sequence, but the cellular localization and biological role of these enzymes is far from well-established. Some genes, such as the laccases from Streptomyces coelicolor and Thermus thermophilus, include a Tat-signal sequence, which labels the gene-product for translocation via the Tat-pathway [166, 167]. Indeed, S. coelicolor laccase activity has been detected in the media when overexpressed homologously. Lac-case activity has in other instances been located to the surface of spores, such as those of Bacillus subtilis , where it is involved in production of pigmentation offering protection against UV radiation.
Comparison of amino acid sequences of bacterial laccases reveals a large degree of diversity. This variation is reflected in several molecular parameters of the laccases. The molecular weight spans the range from 32 kDa for S. coelicolor laccase  to approximately 65, 70, or 75 kDa for B. subtilis, S. lavendulae, or S. cya-neus laccase [168-170]. The higher levels of structural organization also differ. Some bacterial laccases, such as the polyphenol oxidase from Marinomonas mediterranea, are active as monomers , while others, such as the homotrimeric EpoA from S. griseus, are active with quaternary structure . Based on sequence analysis, the small laccases from S. coelicolor and S. griseus likely lack the domain 2 (Pfam id: Cu-oxidase), which is present in all fungal laccases . The small laccases have a full complement of copper atoms and it is plausible that their multimeric organization somehow compensate for intradomain interactions otherwise present in laccases . Consequently, the bacterial laccases may have (reducing) substrate clefts different from those in fungal laccases, resulting in different specificity, reactivity, stability, or other properties. However, the current molecular models do not provide sufficiently reliable information about such intermolecular interactions, and the 3D structures of these small laccases are yet to be elucidated.
Some enzymatic properties of bacterial laccases are similar to those of fungal laccase (Tables 2.1 and 2.2). For instance, S. griseus laccase has a Topt of ~40 °C , T. thermophilus laccase has a pHopt of ~5 when oxidizing phenols , and B. halodurans laccase has a bell-shaped and monotonic pH profile for phenolic and non-phenolic substrates, respectively . However, other properties could be quite different. The E° (T1) differs significantly between the various bacterial laccases: S. coelicolor laccase has an E° of only 0.5 V , while the E° of M. mediterranea polyphenol oxidase is reported to be >0.9 V at pH 7 . The lac-cases are all reported to oxidize a variety of phenolic and non-phenolic substrates, such as syringaldazine, dimethoxyphenol, ABTS and K4Fe(CN)6. The laccase of S. griseus appears to be the exception as it oxidizes dimethoxyphenol but not syringaldazine .
A general trend of the bacterial laccases is their high degree of stability with the extremes being the laccase from T. thermophilus and S. coelicolor. The Topt for the T. thermophilus laccase is approximately 92 °C and its half-life of thermal in-activation is more than 14 h at 80 °C . S. coelicolor laccase can retain activity after boiling and treatment with SDS . Another striking feature of the bacterial laccases is their good catalytic activity under alkaline conditions. The activity of S. coelicolor laccase on dimethoxyphenol is reported to peak at pH 9.4 . It appears that the catalytic efficiency above neutral pH is rate-limited by the non-catalytic interaction between the substrate and the T1 Cu site. It is therefore likely that bacterial laccases bind negatively charged substrates better than positively or uncharged molecules. Other bacterial or archaeon laccases also demonstrate "unusual" pH, thermal, or inactivation-resistance properties. B. subtilis endospore coat CotA laccase is active at high pH and temperature, likely related to the requirement of the spore survival under extreme conditions [17, 168]. The laccase has been applied in dye bleaching at pH 9 and 60 °C, compared with ~ pH 7 and 40 °C suitable for fungal laccases . S. lavendulae and S. cyaneus have a laccase quite stable at ~70-75 °C [169, 170]. A 56 kDa laccase from B. halodurans can be stimulated by NaCl, in contrast to the halide inhibition of fungal laccases .
In summary, the bacterial laccases constitute a highly diverse subgroup within the laccase enzyme class. The diversity is apparent at the amino acid sequence level, which is naturally reflected in various physicochemical parameters of the enzymes (for example, molecular weight, pi, E°). The bacterial laccases differ from their fungal counterparts by enhanced temperature stability. Furthermore, they generally exhibit good activity above neutral pH. Their substrate specificity resembles that of the fungal laccases with a preference for aromatic phenolic substrates. Genome sequencing will undoubtedly lead to the discovery of even more bacterial laccases with properties different from the fungal laccases. The increase in diversity makes these laccases most interesting targets for protein engineering, either by site-directed mutagenesis  or gene shuffling (see Section 2.4.1).
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