Thacker2and Ursula Ku¨es1 1 Georg-August-University Go¨ttingen, Institute of Forest Botany, Go¨ttingen, Germany 2 Duke University, Department of Biology, Durham, NC, USA Multicopper oxid
Trang 1related multicopper oxidase protein sequences
Patrik J Hoegger1, Sreedhar Kilaru1, Timothy Y James2, Jason R Thacker2and Ursula Ku¨es1
1 Georg-August-University Go¨ttingen, Institute of Forest Botany, Go¨ttingen, Germany
2 Duke University, Department of Biology, Durham, NC, USA
Multicopper oxidases (MCOs) are a family of enzymes
comprising laccases (EC 1.10.3.2), ferroxidases
(EC 1.16.3.1), ascorbate oxidase (EC 1.10.3.3), and
ceruloplasmin This family in turn belongs to the
highly diverse group of blue copper proteins which
contain from one to six copper atoms per molecule
and about 100 to > 1000 amino acid residues in the
single peptide chain [1] MCOs have the ability to
cou-ple the oxidation of a substrate with a four-electron
reduction of molecular oxygen to water The electron
transfer steps in these redox reactions are coordinated
in two copper centres that usually contain four copper
atoms In a redox reaction catalyzed by an MCO,
elec-trons from the substrate are accepted in the mononu-clear centre (type 1 copper atom) and then transferred
to the trinuclear cluster (one type 2 and two type
3 copper atoms), which serves as the dioxygen binding site and reduces the molecular oxygen upon receipt of four electrons The type 1 copper is bound to the enzyme by two histidine and one cysteine residue in the T1 centre, whereas eight histidine residues in the T2⁄ T3 cluster serve as ligands for the type 2 and type 3 copper atoms [2–5] Based on the conservation
of the amino acid ligands, two consensus patterns (G-X-[FYW]-X-[LIVMFYW]-X-[CST]-X8-G-[LM]-X3 -[LIVMFYW] and H-C-H-X3-H-X3-[AG]-[LM]) were
Keywords
basidiomycetes; evolution; phylogeny; wood
decay; white rot
Correspondence
P J Hoegger, Georg-August-University
Go¨ttingen, Institute of Forest Botany,
Buesgenweg 2, 37077 Go¨ttingen, Germany
Fax: +49 551392705
Tel: +49 5513914086
E-mail: phoegge@gwdg.de
Website: http://wwwuser.gwdg.de/uffb/
mhb/
Database
Protein sequence alignments are available in
the EMBL-ALIGN database under the
acces-sion numbers ALIGN_000939 and
ALIGN_000940
(Received 24 October 2005, revised
17 March 2006, accepted 23 March 2006)
doi:10.1111/j.1742-4658.2006.05247.x
A phylogenetic analysis of more than 350 multicopper oxidases (MCOs) from fungi, insects, plants, and bacteria provided the basis for a refined classification of this enzyme family into laccases sensu stricto (basidiomyc-etous and ascomyc(basidiomyc-etous), insect laccases, fungal pigment MCOs, fungal ferroxidases, ascorbate oxidases, plant laccase-like MCOs, and bilirubin oxidases Within the largest group of enzymes, formed by the 125 basidi-omycetous laccases, the gene phylogeny does not strictly follow the species phylogeny The enzymes seem to group at least partially according to the lifestyle of the corresponding species Analyses of the completely sequenced fungal genomes showed that the composition of MCOs in the different spe-cies can be very variable Some spespe-cies seem to encode only ferroxidases, whereas others have proteins which are distributed over up to four differ-ent functional clusters in the phylogenetic tree
Abbreviations
ABTS, 2,2¢-azinobis(3-ethylbenzo-6-thiazolinesulfonic acid); DHN, 1,8-dihydroxynaphthalene; L -DOPA, 3,4-dihydroxyphenylalanine; LMCO, laccase-like multicopper oxidase; MCO, multicopper oxidase.
Trang 2defined for the MCOs (PROSITE PDOC00076, http://
us.expasy.org/prosite/) Compared with other members
of the MCO family, ceruloplasmin, responsible for iron
homeostasis in vertebrates, is rather unusual, as it has
five to six copper atoms per molecule [6] Therefore,
this enzyme will not be further discussed in this paper
Laccases in the broader sense by far make up the
largest subgroup of MCOs, originating from bacteria,
fungi, plants, and insects Laccase was first discovered
in the sap of the Japanese lacquer tree Rhus vernicifera
[7], hence the name Subsequently, laccases were also
found in various basidiomycetous and ascomycetous
fungi and, until now, the fungal laccases account for
the most important group with respect to number and
extent of characterization
Laccases were found in almost all wood-rotting
fungi analyzed so far [8] It has become evident that
laccases can play an important role in lignin
degrada-tion [9] even though one of the strongest lignin
degra-ding species, Phanerochaete chrysosporium, does not
produce a typical laccase [10] The precise function of
the enzyme in this process, however, is still poorly
understood [9,11] Besides delignification, fungal
lac-cases have been associated with various organismal
interactions (intra- and interspecific) and
developmen-tal processes such as fruiting body formation [12,13],
pigment formation during asexual development [14,15],
pathogenesis [16–18], competitor interactions [19]
Lac-cases of saprophytic and mycorrhizal fungi have also
been implicated in soil organic matter cycling, e.g
deg-radation of soil litter polymers or formation of humic
compounds [20,21]
Several lines of evidence (capacity to oxidize lignin
precursors, localization in lignifying xylem cell walls,
higher expression in xylem compared to other tissues)
suggest the involvement of plant laccases in the
lignifi-cation process [22–25] However, given the complexity
of the laccase gene families in plant species, additional,
so far not specified functions unrelated to lignin
for-mation have been proposed [26] Due to the
ferroxi-dase activity of the MCO LAC2-2 from Liriodendron
tulipifera and expression studies of the Arabidopsis
thaliana laccase gene family, the term ‘laccase-like
multicopper oxidases’ or LMCOs was introduced in
order to account for their potential multiplicity of
functions [27,28] All 17 of the A thaliana LMCOs
were shown to be expressed and the expression
pat-terns suggested that LMCO function in A thaliana
probably extends well beyond lignification [28]
In insects, laccases seem to play an important role in
cuticular sclerotization [29,30] In Drosophila
melano-gaster, a role in the melanization pathway during the
insect’s immune response [31] and in Manduca sexta a
role in the oxidation of toxic compounds in the diet and⁄ or in the iron metabolism has been proposed [32] Laccases have only recently been discovered in bac-teria and their classification and function are still con-troversial The first report of a bacterial laccase was from the Gram-negative soil bacterium Azospirillum lipoferum [33] and the enzyme was suggested to be involved in melanization [34] The Bacillus subtilis endospore coat protein CotA is a laccase required for the formation of spore pigment [35] and was recently shown to have also bilirubin oxidase (EC 1.3.3.5) activity [36] Other bacterial MCOs like the copper efflux protein CueO from Escherichia coli and the cop-per resistance protein CopA from Pseudomonas syrin-gae and Xanthomonas campestris were considered pseudo-laccases due to the dependence of the 2,6-dimethoxyphenol oxidation on Cu2+addition [37] This plethora of functions of the various laccases implicates the capability of oxidizing a wide range of substrates, which by the use of mediators (oxidizable low-molecular-weight compounds) can even be greatly extended [38] Therefore, laccases are very interesting enzymes for various biotechnological applications Most of the proposed uses for laccases are based on the ability to produce a free radical from a suitable substrate The multifaceted consecutive secondary reac-tions of the radicals are responsible for the versatility
of possible applications [39]
A novel MCO with weak laccase and strong ferroxi-dase activity was identified in P chrysosporium [10] Ferroxidase activity was also detected in a heterolo-gously expressed laccase from Cryptococcus neoformans [40] The role of ferroxidase has been analyzed exten-sively in Saccharomyces cerevisiae The yeast ferroxi-dase Fet3p is a plasma membrane protein that, along with the iron permease Ftr1p, is part of a high affinity iron uptake system [41] Next to its function in iron metabolism, a protective role by suppressing copper and iron cytotoxicity has been suggested [42]
Ascorbate oxidase catalyzes the oxidation of ascor-bic acid to monodehydroascorbate However, its spe-cificity is not as strict, as it was shown to oxidize also phenolic substrates typical for laccases [43] Despite extensive studies on structure, biochemistry, and expression of ascorbate oxidase in plant cells, the phy-siological roles remained uncertain [44] Ascorbate oxidase was suggested to modify the apoplastic redox state and thereby regulate growth and defence [44] De Tullio et al [45] proposed a function in dioxygen man-agement during photosynthesis, fruit ripening, and wound healing
With the availability of genomic sequences, a multi-tude of genes putatively coding for MCOs has been
Trang 3identified However, from only a small part of these
genes the product has been identified or even
charac-terized McCaig et al [28] proposed to categorize plant
LMCOs on the basis of sequence similarity and
phylo-genetic analysis until specific physiological functions
are defined They presented a classification of plant
LMCO sequences and, together with expression pro-files, provided strong evidence that most LMCOs from
A thaliana are not involved in lignification but may play a role in iron or other metal metabolisms In order to characterize plant and fungal laccases into distinct subgroups based on signature sequences,
basidiomycete laccases
ascomycete laccases
insect laccases
Cel NP 501502
fungal ferroxidases
Mgr Mco7
Sce AAB64948 Cgl XP 448078 Kla XP 452271
plant LMCOs
Pch AAO42609 MCO1 Pch AAS21669 MCO4 Pch AAS21659 MCO2 Mgr Mco1
Fgr Mco1 Uma Mco1 Ego NP 984335 Uma Mco3 Cne Mco5 Cne A36962 Cne Mco6 Cim Mco2
Fgr Mco10
CopA
Mtu CAA17652 Mbb NP 854527
Rca AAC16140 Bha BAB05801 Bha AAP57087 Lbh1 Ppu AAD24211 CumA Psy AAO54977 CumA Rsc NP 523089 Xfa NP 299954 Ret NP 660002 Mme AAF75831 PpoA
bilirubin oxidases 2
Cje CAB73936 Tth AAS81712 Bsu AAL63794 Aae AAC07157 SufI
CueO
99
99
69 97 99
96 97 99
60 61
97
70
60
83
99
99
97
98
64
57
83
90
75
92
0.1
plant and fungal ascorbate oxidases
fungal pigment MCOs (melanin DHN 1 )
laccases
sensu stricto
"ferroxidases/laccases"
Fig 1 Neighbour joining tree of multicopper oxidase amino acid sequences Sequences without accession number were derived from the genome sequences (see Experi-mental procedures) Bootstrap values are from 500 replications, only values ‡ 50% are shown ( 1 ) including enzymes involved in melanin synthesis by the 1,8-dihydroxy-naphtalene (DHN) pathway, and ( 2 ) including two sequences from ascomycetes.
Trang 4Kumar et al [46] analyzed over 100 laccase-like
sequences Here we present phylogenetic analyses and
a classification of over 350 MCO sequences, including
laccases, ascorbate oxidases, ferroxidases, and other,
not clearly assigned proteins from the animal, plant,
fungal, and bacterial kingdom
Results and discussion
MCO phylogenetic tree overview
After the different search and selection processes, a total
of 271 MCO amino acid sequences were obtained from
the NCBI GenBank Another 90 sequences were
retrieved from the publicly available genomic sequences
of basidiomycetous and ascomycetous fungi (see
Experi-mental procedures), resulting in a total number of 361
sequences The sequences cover various taxonomic
groups The 258 fungal sequences make up more than
two thirds of all sequences They were derived from 38
different basidiomycete, 30 ascomycete, and one
zyg-omycete species Further, a total of 62 plant sequences
(from one gymnosperm, 12 dicotyledon angiosperms,
and two monocotyledon angiosperms), 12 animal (from
one nematode and four insect species), and 29
prokary-otic sequences (from one archaea, 17 Gram-negative,
and six Gram-positive bacteria) were included in the
analysis In order to analyze the similarities among these
sequences, we used the neighbour joining method with
different distance estimation models (see Experimental
procedures) to construct phylogenetic trees based on the
manually adjusted ClustalX alignment Clades
consis-tent among trees were assigned and named according to
included sequences with known functions and⁄ or
enzy-matic characteristics (Fig 1, only tree based on the JTT
model shown) Based on the main clusters we propose
the following classification of MCOs (see below):
lac-cases sensu stricto (basidiomycetous and ascomycetous),
insect laccases, fungal pigment MCOs, fungal
ferrases, ascorbate oxidferrases, plant LMCOs, bilirubin
oxid-ases Nakamura and Go [47] recently presented a
comparison of blue copper proteins (including the
MCOs) and proposed an evolutionary scenario creating
the molecular diversity in this diverse assemblage of
proteins Focusing on the MCOs only, our analysis
yielded a more resolved phylogeny of the MCO
sequences, providing the base for the (putative)
func-tional assignment of sequences
One of the most obvious features of the tree was
that the laccase sensu stricto sequences clustered
according to the taxonomical association of the
corresponding species The fungal laccases were clearly
separated in two clusters containing either exclusively
homobasidiomycete or filamentous ascomycete sequences, respectively (Fig 1) The former cluster included all the well characterized basidiomycete lac-cases (e.g from Coprinopsis cinerea, Pleurotus ostrea-tus, Pycnoporus cinnabarinus, Rhizoctonia solani, Trametes sp., Fig 2A, for references see Table 1) referred to as bona fide laccases [48] The latter contained most of the reported ascomycete laccases (from Aspergillus terreus [49], Botrytis cinerea [50], Cryphonectria parasitica [18], Gaeumanomyces graminis [51], Melanocarpus albomyces [52], Neurospora crassa [53], and Podospora anserina [54], as well as several previously undescribed sequences we deduced from whole genome sequences (Fig 2B) Similarly, all insect sequences grouped together (Fig 2C) Although the enzymatic activity-sequence link has been established for none of these animal sequences yet, expression data suggest that some of the enzymes included here are involved in cuticular sclerotization [32]
The fungal pigment MCO cluster included sequences from filamentous ascomycetes, ascomycetous yeasts and from basidiomycetes (Fig 2D) It contained the enzymes YA from Aspergillus nidulans and Abr2p from
A fumigatus, both of which are required in conidial pigment biosynthesis [14,15] More specifically, Abr2p was suggested to be involved in a DHN-melanin (named for the pathway intermediate 1,8-dihydroxy-naphthalene) biosynthesis pathway [15] YA has been named a laccase because of its ability to oxidize typical laccase substrates such as p-phenylenediamines, pyro-gallol, and gallic acid, however, no data on enzyme kinetics are available [14]
The fungal ferroxidase cluster comprised sequences from ascomycetous yeasts, filamentous ascomycetes and basidiomycetes (Fig 2E) It included the charac-terized Fet3 ferroxidases from the yeasts Arxula adeni-nivorans, Candida albicans, and S cerevisiae [55–57] and the sequence from gene abr1 neighbouring the putative laccase gene abr2 in a gene cluster for conidial pigment synthesis in Aspergillus fumigatus [15] In the neighbour joining tree based on p-distances, the ferr-oxidase cluster included three additional sequences (Ego_NP_984335, Fgr_Mco1, Mgr_Mco1) compared
to the PAM and JTT trees (not shown) These three sequences belong to a grade of sequences whose group-ing was not consistently supported between the differ-ent trees We marked them ‘ferroxidases⁄ laccases’ (in quotes to differentiate this grade from clusters⁄ clades) due to the presence of Mco1 from P chrysosporium [10] and a laccase from C neoformans, shown to polymerize 3,4-dihydroxyphenylalanine (l-DOPA) in melanin synthesis [17,58] These two enzymes were shown to have both strong ferroxidase and weak
Trang 5laccase activities and are thus not typical laccases
[10,40] This grade also included sequences from
fila-mentous ascomycetes (Fig 1)
Plant and fungal ascorbate oxidase sequences
grouped together separate from the laccase or
ferroxi-dase clusters (Fig 1) These sequences were further
divided into three closely related subclusters: one with
characterized and predicted plant ascorbate oxidases
[4,59,60], the second with predicted sequences from the
zygomycete Rhizopus oryzae, and the third with the so
far sole reported fungal ascorbate oxidase Asom from
Acremonium sp HI-25 [61] Further sequences in the
latter subcluster originated from other filamentous
ascomycetes and from the basidiomycete Ustilago
may-dis(Fig 2F)
The cluster with the sequences of characterized
lac-cases or LMCOs from the plants Acer pseudoplatanus,
L tulipifera, and Populus trichocarpa [23,62,63]
inclu-ded exclusively plant sequences (Fig 2G)
The bacterial sequences grouped clearly separate
from almost all eukaryotic proteins Two clusters were
obvious among the Eubacteria sequences, consisting of
copper resistance proteins (CopA, Fig 2H) and
cop-per efflux proteins (CueO, Fig 2J), respectively [64]
Only one Archaea and two fungal sequences were
among the eubacterial sequences: the undescribed MCO from the hyperthermophilic Pyrobaculum aero-philum, the bilirubin oxidase from the ascomycete Myrothecium verrucaria [65], and the closely related phenol oxidase from the ascomycete Acremonium murorum [66] The two fungal sequences belong to the third cluster among the bacterial sequences assigned
as bilirubin oxidases (Fig 2I) due to the correspond-ing activities described for CotA from B subtilis [36] and bilirubin oxidase from M verrucaria [65] The lat-ter enzyme is a MCO oxidizing bilirubin to biliverdin, but also typical laccase substrates like ABTS [2,2¢-azinobis(3-ethylbenzo-6-thiazolinesulfonic acid)] or syringaldazine [67] It was found in a screen of micro-organisms for decolourization of urine and faeces (containing bilirubin) in raw sewage [68] The biologi-cal role of bilirubin oxidase activity, however, is not known Biliverdin is the chromophore of bacteriophyt-ochromes, homologues of which were found in fungi, and it is also a precursor molecule in chromophore synthesis of plant and cyanobacterial phytochromes [69,70] Due to the lack of experimental data, how-ever, any connection between the chromophores (syn-thesis or degradation) and bilirubin oxidase remains purely speculative
Fig 2 Details of clusters from Fig 1 Sequences without accession number were derived from the genome sequences (see Experimental procedures) Bootstrap values are from 500 replications, only values ‡ 50% are shown (A) Basidiomycete laccases, (B) ascomycete lac-cases, (C) insect laclac-cases, (D) fungal pigment MCOs (melanin DHN), (E) fungal ferroxidases, (F) fungal and plant ascorbate oxidases, (G) plant LMCOs, (H) CopA (copper resistance), (I) bilirubin oxidases, and (J) CueO (copper efflux) Asterisks in (E) mark the ferroxidases where the corresponding genes are arranged in a mirrored tandem with an iron permease homologue Note: Cgo_Mco3, Clu_Mco2, Ctr_Mco1, Ctr_Mco2, and Ctr_Mco3 with frame shifts in the genomic sequences Species codes: Aad, Arxula adeninivorans; Aae, Aquifex aeolicus; Aau, Auricularia auricula-judae; Abi, Agaricus bisporus; Afu, Aspergillus fumigatus; Aga, Anopheles gambiae; Amu, Acremonium murorum; Ani, Emericella nidulans; Apo, Auricularia polytricha; Aps, Acer pseudoplatanus; Asp-HI, Acremonium sp HI-25; Ate, Aspergillus terreus; Ath, Arabidopsis thaliana; Bci, Botryotinia fuckeliana; Bha, Bacillus halodurans; Bpe, Bordetella pertussis; Bsu, Bacillus subtilis; Cal, Candida albi-cans; Cci, Coprinopsis cinerea; Cco, Coprinellus congregatus; Ccr, Caulobacter crescentus; Ccv-EN, Cucurbita cv Ebisu Nankin; Cel, Caenor-habditis elegans; Cga, Coriolopsis gallica; Cgl, Candida glabrata; Cgo, Chaetomium globosum; Cgu, Candida guilliermondii; Cim, Coccidioides immitis; Cje, Campylobacter jejuni; Cla, Colletotrichum lagenarium; Clu, Candida lusitanae; Cma, Cucurbita maxima; Cme, Cucumis melo; Cne, Filobasidiella neoformans; Cpa, Cryphonectria parasitica; Csa, Cucumis sativus; Csu, Ceriporiopsis subvermispora; Ctr, Candida tropical-is; Dha, Debaryomyces hansenii; Dme, Drosophila melanogaster; Eco, Escherichia coli; Ego, Ashbya gossypii; Fgr, Gibberella zeae; Ftr, Funa-lia trogii; Fve, Flammulina velutipes; Gar, Gossypium arboreum; Ggg, Gaeumannomyces graminis var graminis; Ggt, Gaeumannomyces graminis var tritici; Glu, Ganoderma lucidum; Gma, Glycine max; Kla, Kluyveromyces lactis; Led, Lentinula edodes; Lpe, Lolium perenne; Ltu, Liriodendron tulipifera; Mal, Melanocarpus albomyces; Mbb, Mycobacterium bovis ssp bovis; Mgr, Magnaporthe grisea; Mme, Marino-monas mediterranea; Mse, Manduca sexta; Mtr, Medicago truncatula; Mtu, Mycobacterium tuberculosis; Mve, Myrothecium verrucaria; Ncr, Neurospora crassa; Nta, Nicotiana tabacum; Oih, Oceanobacillus iheyensis; Osa, Oryza sativa (japonica cultivar-group); Pae, Pyrobaculum aerophilum; Pan, Podospora anserina; Pbt, Populus balsamifera ssp trichocarpa; Pch, Phanerochaete chrysosporium; Pci, Pycnoporus cinna-barinus; Pcl, Polyporus ciliatus; Pco, Pycnoporus coccineus; Per, Pleurotus eryngii; Phy, Pimpla hypochondriaca; PM1, Basidiomycete PM1; Pos, Pleurotus ostreatus; Ppu, Pseudomonas putida; Pra, Phlebia radiata; Pru, Panus rudis; Psa, Pycnoporus sanguineus; Psc, Pleurotus sajor-caju; Psp, Pleurotus sapidus; Psy, Pseudomonas syringae; Pta, Pinus taeda; Rca, Rhodobacter capsulatus; Ret, Rhizobium etli; Rmi, Rigidoporus microporus; Ror, Rhizopus oryzae; Rsc, Ralstonia solanacearum; Rso, Thanatephorus cucumeris; Sce, Saccharomyces cerevisi-ae; Sco, Schizophyllum commune; Sla, Streptomyces lavendulcerevisi-ae; Spo, Schizosaccharomyces pombe; Stm, Salmonella typhimurium; Sty, Salmonella typhi; Thi, Trametes hirsuta; Tpu, Trametes pubescens; Tsp420, Trametes sp 420; Tsp-AH, Trametes sp AH28-2; Tsp-C30, Trametes sp C30; Tsp-I62, Trametes sp I-62; Tth, Thermus thermophilus; Tts, Trachyderma tsunodae; Tve, Trametes versicolor; Tvi, Trametes villosa; Uma, Ustilago maydis; Vvo, Volvariella volvacea; Xca, Xanthomonas campestris; Xfa, Xylella fastidiosa; Yli, Yarrowia lipolyti-ca; Ype, Yersinia pestis.
Trang 6Fungal MCO multigene families
The composition of the MCO arsenal of different
fungal taxonomic groups seems to be quite variable
Considering only complete fungal mco gene families,
i.e where whole genome sequences are available, half
of the basidiomycete and filamentous ascomycete
sequences (41 out of 84 total sequences) belong to the
laccase sensu stricto clusters (Table 2) The other
sequences of both basidiomycetes and filamentous
ascomycetes are distributed over the fungal pigment MCOs, ferroxidases, and ascorbate oxidases clusters or belong to no cluster In contrast, MCOs from the asc-omycetous yeasts belong almost all to the ferroxidases According to their grouping in the tree, four of the five MCOs from the zygomycete R oryzae seem to be ascorbate oxidases
The ferroxidases are the best represented group, being present in 19 of the 22 fungal genomes analyzed here (Table 2) In S cerevisiae, the ferroxidase Fet3p
A Tve B35883 Tsp-AH AAW28933 lacA Thi Q02497 Tve A35883 laccase A Thi AAA33104 Tsp-I62 AAB63444 Pox2 Tsp-I62 AAQ12269 Pox2 Thi AAL89554 072-1 Tpu AAM18407 Lap2 Tve AAL93622 laccase III Tve CAA77015 Lcc2 Tve AAL07440 Lac1 Tvi Q99044 LCC1 Tve BAA22153 CVL3 Tve CAD90888 Tsp-I62 AAB63445 Pox3 Tsp-AH AAW28934 lacC Pci AAG13724 Lac1 Pco BAB69776 Lcc1 Pco BAB69775 Lcc1 Pci AAC39469 Lcc3-1 Tts BAA28668 Ftr CAC13040 Lcc1 PM1 CAA78144 Tsp-C30 AAF06967 LAC1 Pcl AAG09229 Lcc3-1 Glu AAR82934 Fve AAR82931 Tve Q12718 LCC2 Tve AAC49828 LccI Tvi Q99046 LCC2 Tve AAL00887 Lac1 Tve AAW29420 lcc1 Pci AAD49218 Lcc3-2 Psa AAR20864 Tsp-I62 AAQ12267 Pox1 Tsp-I62 AAQ12268 Pox1 lcc1A Tsp-I62 AAB63443 Pox1 Tvi Q99055 LCC4 Tve Q12719 LCC4 Tve BAA23284 CVLG1 Tve Q12717 LCC5 Tvi Q99056 LCC5 Tpu AAM18408 Lap1A Tsp-C30 AAR00925 Lac3 Tsp-420 AAW28939 lacD Tsp-C30 AAM66349 Lac2 Pcl AAG09230 Lcc3-2 Tsp-420 AAW28936 lacA Tvi JC5355 laccase 3 Tvi Q99049 LCC3 Cga AAF70119 Lcc1 Led BAC06819 LeLcc3 Led AAT99291 LAC3VT Led AAT99289 LAC1DVT Led BAB84355 Lcc2 Led BAB83132 LeLcc2 Led AAT99286 LAC1AVT Led AAT99287 LAC1BVT Led AAF13038 Lac1 Led AAF13037 Lac1
Rmi AAQ82021 Lcc Rmi AAO38869 Lcc Pra CAA36379 Lac Rmi CAE81289 lcc1 Pos CAC69853 Poxa3 Psc CAD45379 Lac3 Abi Q12542 LCC2 Abi Q12541 LCC1
64
64
51 53 54
52
69
71
65
67 89
99
94 99 73
64 80
62
56 99 77 95
88 99 69
99
99
99 89
Led BAB84355 Lcc2 Led BAB83132 LeLcc2 Led AAT99286 LAC1AVT Led AAT99287 LAC1BVT Led AAF13038 Lac1 Led AAF13037 Lac1
Rmi AAQ82021 Lcc Rmi AAO38869 Lcc Pra CAA36379 Lac Rmi CAE81289 lcc1 Pos CAC69853 Poxa3 Psc CAD45379 Lac3 Abi Q12542 LCC2 Abi Q12541 LCC1 Pru AAW28932 lacA
Csu AAC97074 Lcs1 Csu AAO26040 Lcs-1 Tsp-420 AAW28938 lacC Tsp-420 AAW28937 lacB Psc CAD45378 Lac2 Psc CAD45381 Lac5 Psp CAH05069 lac1 Psc CAD45377 Lac1 Pos Q12729 POX1 Pos AAR82932 Per AAV85769 pel3 Pos BAA85185 Psc CAD45380 Lac4 Pos AAR21094 Pos Q12739 POX2 Vvo AAR03582 lac3 Led BAB83131 LeLcc1 Led AAT99290 LAC2VT Pos CAA06292 PoxA1b Sco BAA31217 Cci BK004118 Lcc8 Cci BK004122 Lcc12 Cci BK004123 Lcc13 Cco CAD62686 Lac2 Cco CAB69046 Clac2 Cci BK004112 Lcc2 Cci BK004124 Lcc14 Cci BK004113 Lcc3 Cci AAR01244 Lcc3 Cci AAD30966 Lcc3 Cci BK004117 Lcc7 Cci AAR01248 Lcc7 Cci BK004116 Lcc6 Cci BK004121 Lcc11 Cci BK004111 Lcc1 Cci AY464531 Lcc1 Cci BK004125 Lcc15 Cci BK004115 Lcc5 Cci AAR01246 Lcc5 Cci BK004119 Lcc9 Cci BK004114 Lcc4 Cci BK004120 Lcc10 Vvo AAO72981 Lac1 Vvo AAR03585 lac6 Vvo AAR03583 lac5 Vvo AAR03581 lac2 Vvo AAR03584 lac4 Cci BK004126 Lcc16 Cci BK004127 Lcc17 Rso S68120 laccase 4 Rso Q02081 LCC4 Rso S68118 laccase 2 Rso Q02075 LCC2 Rso Q02079 LCC3 Rso P56193 LCC1
88 99 69
99
99
99 89
99
99
77
62 87
99
94 99
99 69
97 64
99 62
99
57 51 77
84
99 62
99 99 99
88 95 83 93
0.05
Trang 7Ctr Mco1 Cal EAK92029 FET32 Cal EAK92051 FET31 Ctr Mco2
Clu Mco1 Dha XP 461767 Cgu Mco1 Cal CAA70509 Fet3 Cal BAC10629 CaFET96 Ego NP 984228 Kla XP 456256 Sce AAA64929 Fet3 Cgl BAB62813 Fet3
Yli XP 502524 Yli XP 502500 Sce BAA09199 Cgl XP 448770 Kla XP 453305 Ego NP 983177 Cgu Mco2 Dha XP 459860 Clu Mco2 Ctr Mco3 Cal EAK97856 FET397 Spo CAA91955
Aad CAB90817 Afet3 Mgr Mco5 Fgr Mco5 Ncr CAD21075 Fgr Mco6 Cgo Mco3 Afu AAF03353 Abr1 Ncr Mco7
Mgr Mco9 Cgo Mco6 Ror Mco1 Yli XP 500278 Pch Mco5 Apo AAT73204 lac1
Uma Mco2 Cne Mco3 Cne Mco1 Cne Mco2 Cne Mco4
96
61
84 80 96 95
71
91 64
63
79 77
73
99 91
77 54
0.1
Dha XP 457262 Clu Mco3
Cgu Mco3
Aau AAR21095 Uma Mco4
Fgr Mco11 Mgr Mco11
Fgr Mco12 Mgr Mco10 Uma Mco5 Ani CAC59820 TilA Ani EAA65930 Ani Mco6 Afu AAF03349 Abr2 Fgr Mco9 Ani KSASL1 laccase I Ani P17489 YA Ani EAA58164
98 64
57
99 54
99 96
99 70
54 70
0.1
Dme AAF57331 Dme AAF57332 Dme AAN16124 Mse AAN17507 MsLac2 PhyC AD20461 Lac1 Mse AAN17506 MsLac1 Aga AAN17505 AgLac1 Dme AAF52771 Dme AAL48945 Dme AAL49165 Dme AAF56527
70 99 92
99 55
95 76
81
0.05
Ate BAA08486 DHGO Ncr KSNCLO NcrP 06811 LACC Ncr EAA27703 Ncr P10574 LACC Pan P78722 LAC2 Mal CAE00180 lac1 Cpa Q03966 LAC-1 Ggt CAD10749 Lac3 Ncr Mco5
Mgr Mco2 Ncr Mco8 Cla BAB32575 LAC1 Ncr CAD70438 Ncr Mco3 Cgo Mco1 Ncr Mco2 Cgo Mco4 Mgr Mco6 Fgr Mco2 Cgo Mco5 Ncr Mco6 Fgr Mco7 Ncr Mco4 Cim Mco1 Ani Mco1 Ncr Mco1 Cgo Mco2 Ggg CAD24841 Lac1 Ggt CAD10747 Lac1 Bci AAK77953 Lcc2 Bci AAK77952 Lcc1 Fgr Mco13 Fgr Mco4 Ani Mco2 Ggg CAD24842 Lac2 Ggt CAD10748 Lac2 Mgr Mco3
Fgr Mco3 Mgr Mco4
69 99
55 59
99 82 90
76
79
99 54
99
85 51 50 68
0.05
B
D C
E
*
*
*
*
*
*
*
*
*
*
*
*
Fig 2 (Continued).
Trang 8Oih NP 692267 CotA
Sla BAC16804 Bsu 1GSK CotA
Amu CAB75422 PpoA Mve Q12737 bilirubin oxidase
50 91
99 0.1
Ascomycetes Zygomycetes
Plants
F
G
J I
H
Basidiomycete
Cma P24792 AAO Ccv-EN S11027 Csa P14133 Cme AAF35911 AO4 Cme AAF35910 AO1 Mtr CAA75577 Nta Q40588 AAO Cme CAA71275 AO3 Ath NP 680176 At5g21105 Ath AAO30070 At5g21100 Ath T05020
Ror Mco2 Ror Mco5 Ror Mco3 Ror Mco4 Uma Mco6 Asp-HI BAA24288 Asom Cgo Mco7 Fgr Mco8 Mgr Mco8 Ani Mco3 Ani Mco4
99
81 51
72
67 98
86 99
97
65 92 84 50
0.1 Ath NP 182180 At2g46570
Gma AAM54731 Pbt CAA74104 Lac90 Ath NP 196158 At5g05390 Ath NP 181568 At2g40370 Osa BAB68098 Pta AAK37826 LAC4 Pta AAK37824 LAC2 Ath NP 196330 Ath NP 180580 At2g30210 Osa BAB92845
Osa BAC84596 Lpe AAL73970 LAC5-4 Osa BAD81779 Osa BAD82646 Ath AAF14041 Ath NP 195725 At5g01050 Ath NP 195724 At5g01040 Lpe AAL73969 LAC2-1 Ath AAF97830
Osa BAD15631 Osa BAD61379 Aps AAB09228 Gar AAR83118 lac1 Ath AAO50685 At5g48100 Ath NP 196498 At5g09360 Ltu AAB17194 LAC2-4
Ltu AAB17192 LAC2-2 Ltu AAB17193 LAC2-3 Osa BAB86452 Ath NP 200810 At5g60020 Pbt CAA74105 Lac110 Pta AAK37825 LAC3 Pta AAK37827 LAC5 Pta AAK37828 LAC6 Ltu AAB17191 LAC2-1 Osa BAB86465 Ath NP 180477 At2g29130 Pta AAK37823 LAC1 Ath NP 195946 At5g03260 Osa BAB90733
Osa BAB86450 Lpe AAL73968 LAC5-6 Pbt CAC14719 GLac3 Pbt CAA74103 Lac3 Pta AAK37830 LAC8 Pta AAK37829 LAC7 Ath NP 565881 At2g38080 Ath T01240
Nta JC5229 Ath NP 195739 At5g01190
51 53 57
73 65
71 78
99
99
99 54
73 52
81 60
53
99
99
56
63
0.05
Xca A36868 CopA homolog Xca AAM39893 CopA Bpe CAE43580 CopA Eco S52253 PcoA
Psy P12374 CopA Rsc CAD17807 CopA Ccr AAK22948
73
91 70
52 0.05
Ype Q8ZBK0 CueO Eco P36649 CueO Sty Q8Z9E1 CueO Stm Q8ZRS2 CueO 98
94
0.02 Fig 2 (Continued).
Trang 9Redox potential
Km
kcat
Km
kcat
Km
kcat
Trametes pubescens
Km
kcat
Km
kcat
Km
kcat
Km
kcat
Km
kcat
Km
kcat
Km
kcat
Km
kcat
Km
kcat
Pycnoporus cinnabarinus
Pycnoporus cinnabarinus
Km
kcat
Km
kcat
Km
kcat
Km
kcat
Km
kcat
Km
kcat
Ceriporiopsis subvermispora
Km
Km
Km
Km
Km
Km
Km
Trang 10Redox potential
Km
kcat
Km
kcat
Km
kcat
Km
kcat
Km
kcat
Km
Km
Km
Km
kcat
Km
kcat
Km
kcat
Km
kcat
Km
kcat
Km
kcat
Thanatephorus cucumeris
a ABTS,
b No.
c Km
1 ,
kcat
1Æs
1