123 An evidence of laccases in archaea - Springer Link

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K. K. Sharma · R. C. Kuhad (). Lignocellulose Biotechnology Laboratory,. Department of Microbiology,. University of Delhi South Campus, Benito Juarez Road,.
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Indian J Microbiol (June 2009) 49:142–150

Indian J Microbiol (June 2009) 49:142–150

ORIGINAL ARTICLE

An evidence of laccases in archaea Krishna Kant Sharma · Ramesh Chander Kuhad

Received: 20 April 2009 / Accepted: 13 May 2009

Abstract Laccases (benzenediol:oxygen oxidoreductase, EC 1.10.3.2) are a diverse group of multicopper oxidases that catalyze the oxidation of a variety of aromatic compounds. Here we present evidence for distribution of laccases among archaea and their probable functions. Putative laccase genes have been found in different archaeal groups that might have branched off early during evolution, e.g. Haloarcula marismortui ATCC 43049, Natronomonas pharaonis DSM2160, Pyrobaculum aerophilum IM2, Candidatus Nitrosopumilus maritimus SCM1, Halorubrum lacusprofundi ATCC 49239. Most of the archaeal multicopper oxidases reported here are of Type 1 and Type 2 whereas type 3 copper-binding domain could be found in Pyrobaculum aerophilum IM2 and Halorubrum lacusprofundi ATCC49239. An analysis of the genome sequence database revealed the presence of novel types of two-domain laccases in archaea. Keywords Archaea · Multicopper oxidase · Laccase · Genome · Cluster of orthologus groups

K. K. Sharma · R. C. Kuhad () Lignocellulose Biotechnology Laboratory, Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi - 110 021, India E-mail: [email protected]

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Introduction Laccases are one of the best-known members of the multicopper protein family, also known as benzenediol:oxygen oxidoreductase, EC 1.10.3.2 [1]. They are the model enzymes of multicopper oxidases (MCOs) which participate in (1) cross-linking of monomers, (2) degradation of polymers, and (3) ring cleavage of aromatic compounds [2, 3, 4, 5]. Being the simplest enzyme that combines all three known organic Cu(II) magnetic types in a single molecule, laccase has been particularly well studied with respect to its intramolecular electron transfer reactions [6]. Phylogenetically, laccases are member of MCOs family including ascorbate oxidase (EC 1.10.3.3), cytochrome c oxidase (EC 1.9.3.1), and ceruloplasmin (sometimes referred to as ferroxidase; EC 1.16.3.1). Commonly, a three-domain multicopper laccases have been reported from eukaryotes, e.g., fungi, lichens, plants and insects [4, 7–9]. There are some evidences, however, for its existence in prokaryotes; a protein with typical features of multicopper oxidase enzyme family, which are mainly involved in cell pigmentation and metal oxidation [10, 11]. The first bacterial laccase was detected in the plant root-associated bacterium, Azospirillum lipoferum, where it was shown to be involved in melanin formation [12]. The most well-characterized bacterial laccase was isolated from Sinorhizobium meliloti, which has been described as a 45-kDa periplasmic protein with isoelectric point at pH 6.2 and ability to oxidize syringaldazine [13]. The completely sequenced archaeal genomes potentially encode many functionally uncharacterized genes for novel enzymes of biotechnological interest [14]. Current decade has witnessed the determination of the complete archaeal

-

-

Candidatus Nitrosopumilus maritimus SCM1

Halorubrum lacusprofundi ATCC 49239

PAE1888

Pyrobaculum aerophilum IM2

HlacDRAFT_ 1345

NmarDRAFT_ 0728

PAE1888

NP1598A

rrnAC1378

Pan 1a

nirK

rrnAC2853

nirK

Haloarcula marismortui ATCC 43049

Natronomonas pharaonis DSM2160

Locus tag

Gene

Multicopper oxidase, Type 3

Multicopper oxidase, Type 3

Multicopper oxidase

Nitrite reductase copper containing

Membrane protein Pan 1

Nitrite reductase coppercontaining protein

Product

771769..773064 (+) (1296 bp)

295350..2964405 (-) (1056 bp)

1114128..1115561

773076..774164 (+) (1089bp)

1227027..1228178 (-) (1152bp)

2531326..2532411 (-) (1086bp)

DNA coordinates

0.65

0.38

0.50

0.65

0.63

0.65

GC

Putative archaeal laccase compared with other novel laccases (http://img.jgi.doe.gov)

Genome

Table 1

ZP_02015243

ZP_02024104

AAL63794

YP_326457

AAV46307

AAV47603

Accession

431

351

477

362

383

361

AA

No

Yes

No

No

No

No

Transmembrane helices

Yes

Yes

No

No

No

Yes

Signal peptide

Blue (Type 1) copper subtype Multicopper oxidase, type 2 Multicopper oxidase, type 3

Copper-containing nitrite reductase, Multicopper oxidase, type 2 Multicopper oxidase, type 3

Copper ion binding Oxidoreductases activity Cupredoxin Multicopper oxidase, copper-binding site Multicopper oxidase, type 2 Multicopper oxidase, type 3 (preliminary) Laccase

Nitrite reductase, coppercontaining

Copper ion binding Electron carrier activity Oxidoreductase activity Twin-arginine translocation pathway signal; Cupredoxin Blue (type 1) copper subtype

Copper-containing nitrite reductase Cupredoxin Blue copper subtype Multicopper oxidase types

IMG term/families

Indian J Microbiol (June 2009) 49:142–150 143

123

123 YE0712

yacK

FET3.1

pco A

aniA

Yersinia enterocolitica enterocolitica 8081

Pichia stipitis CBS 6054

Escherichia coli APEC O1

Burkholderia pseudomallei 1710 b

BPUM_0542

cotA

FET5

Bacillus pumilus

Saccharomyces cerevisiae YFL041 W

Bmal 10_ 03000556

Burkholderia mallei 10399

BURPS1710b_ A0477

APECO1_ OIR119.2

PICST_89638

Locus tag

Gene

(Continued)

Genome

Table 1

Multicopper oxidase

Outer spore coat protein A

Hypothetical protein

Multicopper oxidase domain protein

Copper resistant protein PcoA

Multicopper oxidase

Hypothetical Protein

Product

49139..51007 (+) (1869bp)

577315..578844

176678..178141

659851..661314

135300..137123

131793..136959 (-) (5167 bp)

827646..829247 (+) (1602bp)

DNA coordinates

0.42

0.44

0.68

0.68

0.52

0.42

0.53

GC

NP_116612

YP_ 001485796

ZP_01346491

YP_335636

YP_ 001481473

XP_ 001385046

YP_ 001005057

Accession

622

509

487

487

607

626

533

AA

No

No

Yes

No

Transmembrane helices

Yes

Yes

Yes

No

Signal peptide

Copper ion binding Ferroxidase activity Iron ion binding Metal ion binding Oxidoreductase activity Protein binding

Copper ion binding Electron carrier activity Heme binding Iron ion binding Nitrite reductase Oxidoreductases activity

Twin-arginine translocation pathway signal Copper resistant protein-CopA family

Cupredoxin Multicopper oxidase, type 2 Multicopper oxidase, type 3

Multicopper oxidase, copperbinding site Twin-arginine translocation pathway signal Cupredoxin Multicopper oxidase, type 2 Multicopper oxidase, type 3

IMG term/families

144 Indian J Microbiol (June 2009) 49:142–150

CG32838

HEPH

Cp

Drosophila melanogaster

Homo sapiens

Ratus norvegicus GK/Ox Cp

RP13-238N7.1

Dmel_CG32838

Ceruloplasmin

Hephaestin

CG32838

Acidic Laccase

-

Cryptococcus neoformans var. neoformans JEC 21 CNM02420

Laccase precursor

NCU04528.1 NCU04528.1

Product

Neruospora crassa OR74A

Locus tag

Gene

(Continued)

Genome

Table 1

0.40

0.49

0.59

GC

97809765..97854827 0.37 (+) (3700 bp)

65730180..65834740 0.39 (+) (4279 bp)

1341942..1342517 (+) (576 bp)

727710..730327 (-) (2618bp)

78434..80350 (-) (1917bp)

DNA Coordinates

NP_036664

NP_620074

NP_724414

XP_568259

XP_956939

Accession

1059

1158

174

640

619

AA

No

No

No

Yes

No

Transmembrane helices

Yes

No

No

Yes

Yes

Signal peptide

Multicopper oxidase, type 1 Multicopper oxidase, type 2 Multicopper oxidase, type 3 Cupredoxin, copperbinding site

Iron ion binding, metal ion binding, oxidoreductases activity

Cupredoxin Multicopper oxidase, type 2

Multicopper oxidase, type 1 Multicopper oxidase, copperbinding site Cupredoxin Multicopper oxidase, type 2 Multicopper oxidase, type 3 (Preliminary) Laccase

IMG term/families

Indian J Microbiol (June 2009) 49:142–150 145

123

146

genome. A number of other genome projects are underway and it is no exaggeration to consider it the origin of a new science - a genome-based biology. The availability of complete archaeal genome and huge industrial applications of natural and modified archaeal enzymes has resulted in the exploration of biocatalysts from diverse sources. Surprisingly, till date, there are no reports of archaeal laccase gene.

Materials and methods To identify putative archaeal laccases, we applied in silico data mining, followed by exhaustive BLAST search of non-redundant (nr) protein sequence database (http: //www.ncbi.nlm.nih.gov/) and completed whole genome sequence provided by the Department of Energy- Joint Genome Institute (JGI) (http://img.jgi.doe.gov). The database of Cluster of Orthologus Groups of proteins (COGs) was used as a tool for phylogenetic classification of the proteins encoded in complete genomes of bacteria, archaea, and eukaryotes (http://img.jgi.doe.gov. and http: //www.ncbi.nlm.nih.gov/cog) [15]. Signal peptide sequence was predicted using SignalP (http://www.cbs.dtu.dk/services/signalp) and hydrophobicity analysis using the dense alignment surface algorithm (http://www.biokemi.su.se/~server/DAS/). Enzyme class (putative laccases) obtained from cluster of orthologous groups was predicted using ProtFun (http://www.cbs.dtu.dk/ servises/ArchaeaFun/). Three reconstruct methods, edit sequence, megalign, and tree view, were used to find the evolutionary trees or trees that best account for the observed variation in the group of protein sequences (DNASTAR, Inc., 3801 Reagent Street, Madison, WI 53705 USA). Each of these methods uses a different type of analysis and the reliability of the prediction was evaluated by the random re-sampling of the alignment (nucleotide substitution value).

Results and discussion Collection of 18 COGs from archaea, bacteria, and eukaryotes was compiled (http://img.jgi.doe.gov. and http: // www.ncbi.nlm.nih.gov/cog) (Table 1). The COGs were consistency of genome-specific best hit to the results of an exhaustive comparison of all protein sequences from the genomes. Genome-specific best hit resulted in very exhaustive genomic information of diverse multicopper oxidases. We also built trees grouping organisms based on the overall occurrence of molecular features, i.e. COG throughout the genomes of different archaea, bacteria, fungi (ascomyce-

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Indian J Microbiol (June 2009) 49:142–150

tous and basidomycetous), different forms of yeast, insects and mammals. Broadly these characteristics could be orthologs, homologs or folds. We focused on orthologs, i.e. multicopper oxidase from diverse archaeal, bacterial and eucaryal sources. Since our interest lies in the archaeal laccases (multicopper oxidase), we included fungal and bacterial COGs for comparative characterization and evolutionary distances among well-established laccases with the putative laccases from archaea. Mammalian multicopper oxidase, a homolog, was observed as outgroup in the rooted phylogenetic tree (Fig. 1). Although, they form different gene products i.e. ceruloplasmin in Rattus norvegicus and hephaestin in Homo sapiens but they share a common clade (Table 1). Laccase (cotA) from Bacillus subtilis-168 and Bacillus pumilusSAFR-032 were found to share a common clade and close ancestry with multicopper oxidase from Pyrobaculum aerophilum, an archaea. Moreover, P. aerophilum was also found to be evolutionary related to Escherichia coli APEC O1 (laccase) and Yersinia pestis KIM (hypothetical protein). Well-known laccases from Trametes versicolor were found to be closely related to Neurospora crassa OR74A, Cryptococcus neoformans var. neoformans JEC21 and Drosoplila melanogaster, a common fruit fly. Multicopper oxidase from different yeast, i.e. FET3_Yeast, Pichia stipitis CBS6054 (FET3.1), and Saccharomyces cerevisiae (FET5) share a common phylogenetic position. An unusual evolutionary history was also established between pathogenic proteobacteria, i.e. Burkholderia mallei and Burkholderia pseudomallei and an archaeal species, i.e. Haloarcula marismortui ATCC 43049 and Natronomonas pharanis DSM2160 (Fig. 1). A hydrophobicity analysis using the dense alignment surface algorithm suggested that putative archaeal laccases lack transmembrane regions, which verify that they are soluble protein (Table 1). The putative archaeal laccases identified here include NirK (nitrite reductase) from H. marismortui and N. pharaonis; Pan1a (membrane protein) from H. marismortui; PAE1888 (multicopper oxidase) from P. aerophilum (Table 1). Similarities between these proteins and multicopper oxidases were recognized, however, the proteins have not been analysed for laccase activity. Also, we have found putative laccases in representatives of bacteria, fungus, blue-green algae, insect and mammals. Based on the protein sequence analysis and E value cut-off of 10–7, fixed earlier by Alexandre and Zulin [10]. We could find that most of the BLAST search represented laccases with E value within the cut-off limit (Table 2). Archaeal multicopper oxidase (putative laccase) sequence annotation using Pfam protein families database (http: //pfam.sanger.ac.uk/) resulted in high Bits score, which

Indian J Microbiol (June 2009) 49:142–150

147 Burkholderia mallei 10399 Burkholderia pseudomallei 1710b Haloarcula marismortui ATCC 43049 Natronomonas pharaonis DSM 2160 Nostoc sp. PCC 7120 Candidatus Nitrosopumilus maritimus SCM Burkholderia multivorans ATCC 17616 Flavobacterium johnsoniae UW101 Uncultured bacterium Haloarcula marismortui ATCC Pan A Halorubrum lacusprofundi ATCC 49239 Bacillus thuringiensis serovar israelen Nocardia farcinica IFM 10152 FET3_YEAST Pichia stipitis CBS 6054 Saccharomyces cerevisiae Fet5p Neurospora crassa OR74A hypothetical Cryptococcus neoformans var. neoformans Melanocarpus albomyces Neurospora crassa OR74A laccase precur Drosophila melanogaster Trametes versicolor Cryptococcus neoformans var. neoformans Escherichia coli APEC O1 Yersinia pestis KIM Pyrobaculum aerophilum str. IM2 Bacillus pumilus SAFR-032 Bacillus subtilis subsp. subtilis Cyanothece sp. CCY0110 Lyngbya sp. PCC 8106 Synechococcus sp. RS9917 Burkholderia cenocepacia MC0-3 Homo sapiens - hephaestin Rattus norvegicus

312.7 300

250 200 150 100 Nucleotide substitutions (x100)

50

0

Fig. 1 Dendrogram constructed using multicopper oxidase from diverse archaeal, bacterial and eucaryal sources (http:// img.jgi.doe.gov) Table 2 Archaeal multicopper oxidase (putative laccase) sequence annotation using Pfam protein families database (http:// pfam.sanger.ac.uk/) Archaea

PfamA

AA

Entry type

Bits score

E value

P. aerophilum

Cu-oxidase 3

110

Domain

126.1

9.9e-35

Cu-oxidase

147

Domain

-20.4

0.015

Cu-oxidase 2

145

Domain

76.4

9.4e-20

Cu-oxidase 3

112

Domain

64.1

4.7e-16

Cu-oxidase 2

117

Domain

5.7

0.00028

H. marismortui ATCC43049

Cu-oxidase 3

115

Domain

33.1

6.4e-10

H. marismortui ATCC43049 Pan1

Cu-oxidase 3

111

Domain

43.1

6.7e-09

Cu-oxidase 2

125

Domain

61.8

2.3e-15

Cu-oxidase 3

111

Domain

58.9

1.7e-14

Cu-oxidase 2

113

Domain

38.8

2e-08

Cu-oxidase 3

115

Domain

17.2

2.7e-08

C. Nitropumilus maritimus

H. lacusprofundi ATCC49239 N. pharaonis DSM2160

was comparable to fungal laccases (Table 2). Further, each input sequence the server predicts enzyme/non-enzyme and enzyme class. The score consists estimated probability and

class/category to which the sequences belong (Table 3). Most of putative laccase sequences were found to be enzymes belonging to oxidoreductases. Even, few of them

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Indian J Microbiol (June 2009) 49:142–150

Table 3 Prediction of enzyme class (putative laccases) from archaeal sources obtained from cluster of orthologous groups (http://www.cbs.dtu.dk/servises/ArchaeaFun/) Archaea P. aerophilum

C. Nitropumilus maritimus

C. Nitropumilus maritimus

H. marismortui ATCC43049

H. marismortui ATCC43049 Pan1

H. lacusprofundi ATCC49239

N. pharaonis DSM2160

Enzyme class

Enzyme

Prob/odd

Prob/odd

Oxidoreductase

0.247

1.112

Transferase

0.190

0.855

Hydrolase

0.196

0.882

Oxidoreductase

0.211

0.950

Transferase

0.210

0.945

Hydrolase

0.271

1.220

Oxidoreductase

0.234

1.053

Transferase

0.176

0.792

Hydrolase

0.223

1.004

Oxidoreductase

0.178

0.801

Transferase

0.186

0.837

Hydrolase

0.150

0.675

Oxidoreductase

0.297

1.337

Transferase

0.182

0.819

Hydrolase

0.177

0.797

Oxidoreductase

0.228

1.026

Transferase

0.154

0.693

Hydrolase

0.235

1.058

Oxidoreductase

0.202

0.909

Transferase

0.154

0.693

Hydrolase

0.153

0.689

were also found to be either transferase or hydrolases with marginal probability, which signifies its evolutionary primitiveness. This method relies on predicted proteins features like co-translational and post-translational modifications, secondary structure and simple physicochemical properties [14]. Archaeal two-domain multicopper blue proteins have not been previously studied. The BLAST programme was used to search the non-redundant (nr) set of protein sequences provided at NCBI (NIH, Bethesda). Sequence alignments around the copper binding sites of the novel two-domain archaeal multicopper blue proteins were studied (Fig. 2). Since the monomeric two-domain multicopper blue proteins cannot form interdomain copper binding sites, these proteins are presumed to aggregate to form homotrimers, like the case of nitrite reductase [16]. Domain alignment in our studies establishes archaeal MCOs as laccases (Fig. 2). Contrary to the statement that the gene repertoire overlapped more with Euryarchaeota than with bacteria or eukarya [17], here we could interestingly find all the multicopper oxidases of phylum Euryarchaeota to be deeply rooted and in a separate clade whereas shallow rooted multicopper

123

Enzyme class 0.597

1.194

0.400

0.800

0.420

0.840

0.576

1.152

0.496

0.992

0.601

1.202

0.610

1.220

oxidase from Crenarchaeota. For example, P. aerophilum was found to be phylogenetically closer to eubacteria (Fig. 1). The exceptional archaeal features make the archaeal domain of life an interesting area of research for novel protein. Translated proteins of multicopper oxidase reported here is of very diverse properties, i.e. coppercontaining nitrite reductase, membrane protein (Pan1), cupredoxin, oxidoreductases activity, multicopper oxidase type 2 and multicopper oxidase type 3. Archaeal multicopper oxidase share the conspicuous copper-binding site pattern with membrane protein (Pan1a), prokaryotic azurins, multicopper oxidase type 3 and nitrite reductase (NirK) fit in perfectly as a common ancestral form of multicopper oxidase. Most of the multicopper oxidase reported here were of Type 1 and Type 2, whereas in P. aerophilum IM2 and Halorubrum lacusprofundi ATCC49239, the entire three copper-binding domain could be found. An analysis of the genome sequence database revealed novel types of two-domain laccases. These twodomain proteins have a conspicuous combination of bluecopper and interdomain trinuclear copper binding residues, which is common in nitrite reductase, ceruloplasmin and

Indian J Microbiol (June 2009) 49:142–150

149 1

1

1ZPU_D query gi 2842755 gi 74696548 gi 74698345 gi 1175947 gi 81535779 gi 81816329 gi 75538832 gi 81841900

341 232 366 523 361 367 444 384 369 212

2

3

THPFHLHGHAFQTIQRDRTY. DHPMHIHNHRFQVTHKDGGK. PHPFHLHGHTFSIVRTAGST. AHPIHLHGHDFAVLRQSSKN. FHPFHLHGHEFQVVERSPEY. RHPFHLHGHNFQIVQKSPGF. AHPIHLHGHFFELVNGQERQ. YHPIHLHGHTFQMIKADGSP. AHPMHLHGHVFQVIAVNGTR. NHPIHLHGHSFAVTCTDGGW.

1ZPU_D query gi 74697330 gi 74697836 gi 74697757 gi 74697110 gi 81426059 gi 116921 gi 81501284 gi 75402594

341 130 368 396 381 405 466 489 486 688

.[28] .[33] .[25] .[25] .[25] .[33] .[30] .[30] .[30] .[30]

2 3

THPFHLHG.[34].PMRRDT LYVRP QSNFVIRFK PHTFHVHG.[ 8].GVPTTT.[1].QQVAP GEEYTYEID PHPFHLHG.[18].PPQRDT VSVGA.[1].GDNVTIRFR PHPFHLHG.[18].PVRRDT TLVAM KNQTTIRFV PHPFHLHG.[18].PVRRDV TSVAI GNQTTIRFV DHPFHLHG.[24].PLSRDT VEIPA NGWVRLRFI THPIHLHG.[12].QVRKHT ISLNP AQRISYRVS THPIHLHG.[12].RVRKHT IDMPP GSKRSYRVT THPMHLHG.[12].QVRRHT VPVQP AQRISFLVT RHPMHIHG.[12].DPLLHT IEVPP GATAVADFD

452 222 465 490 475 511 557 580 577 779

31

312 452 329 463 656 469 486 535 475 462 305

NPGVWFFHCHIE. DPGIYLAHCHKV. NPGPWFLHCHID. NPGAWILHCHIA. NPGAWFFHCHID. NPGVWYFHCHVD. EPGDWAFHCHML. NPGVWVMHCHNN. EAAPWMLHCHHM. NPGDWAFHCHKS.

483 361 494 687 500 517 566 503 490 336

Fet3p H. marismortui ATCC43049 Multicopper oxidase (nirK) Laccase-4 precursor Hypothetical protein Ferro-O2-oxidoreductase FET5 precursor Copper-binding protein Copper-binding protein Multi-copper oxidase Multi-copper oxidase

453 223 466 491 476 512 558 581 578 780

ADNPGVWFFHC.[20]. ANQPGTHFYHC.[20]. TDNPGPWFLHC.[20]. TDNAGPWFLHC.[20]. TDNPGPWFLHC.[ 7]. TYNPGAWTLHC.[20]. ADARGNWAYHC.[17]. ADALGRWAYHC.[17]. ADALGRWAWHC.[17]. TEASGQWFFHC.[17].

483 253 496 521 493 542 585 608 605 807

Multicopper Oxidase (Fet3p) H.lacusprofundi ATCC49239 Multicopper Oxidase Laccase 2 Laccase Laccase 2 Extracellular multicopper oxidase Copper resistance protein Copper resistance protein A precursor copper resistance transmembrane protein Putative copper efflux ATPase

(A) Domain 2: Halorubrum lacusprofundi ATCC49239

(A) Domain 2: Haloarcula marismortui ATCC43049 (nirK) 2 3 1PF3_A query gi 2829670 gi 2833199 gi 81501284 gi 81426059 gi 13632794 gi 75329673 gi 75333948 gi 81777459

3 90 150 160 148 133 122 120 114 102 115

23 84 85 73 63 52 50 42 30 43

TLHWHGLE.[4].VDGGPQG NIDLHAVR.[1].PGGGAEA SIHWHGLH.[9].ANGVTEC TIHWHGIR.[9].VNGITEC SIHWHGIL.[7].VPGLSFH SIHWHGIL.[7].VPGLSFP SIHWHGII.[7].VPGLSFM TIHWHGVR.[9].PEFVTSV SLHWHGIR.[9].PAYITQC TIHWHGIR.[7].VPFLVQP

89 149 159 147 132 121 119 113 101 114

1PF3_A 23 TLHWHGL.[2].PGE VDGGPQGIIPPGGKR 97 query 13 TFHVHGL SKD.[5].VPTTTGQQVAPGEEY 217 gi 74697989 74 SIHWHGI.[2].LGS.[5].VPGVTQCPIAPGDTL 154 gi 74696385 76 GIHWHGL.[2].SGS.[5].VPGITQCAIAIGQSM 156 gi 74696548 154 SVHWHGF.[2].FES.[5].VNGITECPIAPGDTF 227 gi 74696653 27 AMHWHGF.[2].DDT.[5].APGVSQCPIVPGKSY 109 gi 74698514 25 SIHFHGI.[2].KNT.[5].VVGLSQWAIQPGQSY 104 gi 81325064 50 SIHWHGI.[2].PPN.[3].VPGLSFAGIEPDGMY 127 gi 81796717 56 SVHWHGL.[2].PAN.[3].VPGMSFDGIAPGQEY 134 gi 75541127 53 SIHWHGI.[2].PPE.[3].VPGISFPGIEPGATF 130

12

PHQHG.[7].MGLAGLVVIED 139 Multicopper Oxidase (Cueo) YH YH.[3].PNLDM.[3].SGMFGMILVEP 198 H.marismortui ATCC43049 (nirK) YH SHFSA.[3].NGVVGTIVVNG 205 Laccase-2 precursor YH SHFSA.[3].NGIVGAIQIDG 193 Laccase precursor YH SHSGF.[3].TGVYGGLIIDP 177 Copper resistance transmemb protein YH SHSGF.[3].TGLYGAIVIEP 166 Copper resistance protein YH SHSGL.[3].EGVYGAIIIDA 164 Copper resistance protein A AH AHSSW.[2].ATVYGALIIRP 158 Diphenol oxidase (Laccase) YH AHISW.[2].STVYGPLIILP 146 Laccase-17 precursor YH PHCNT.[5].HGLTGVIVVEN 161 Metallo-oxidoreductase

312 98 218 155 157 228 110 105 128 135 131

SVTLNVDQ.[2].ATCWFHPHQHG.[7]. TYEIDANQ.[1].GTHFYHCHYQT.[5]. TYKFQATQ.[1].GTTWYHSHFSL.[3]. TYRFKVSQ.[1].GSTWYHSHFSL.[3]. TYRFRAMQ.[1].GSAWYHSHYSL.[3]. TYEFNASL.[1].GTSWYHAHYSA.[3]. TYQWRADT.[1].GTYWYHAHDKA.[3]. VYKIHVKQ.[1].GTYWYHSHSGF.[3]. LYRFALRQ.[1].GTYWYHSHSMF.[3]. SYRFTIRQ.[1].GTYWFHSHSGG.[3].

139 256 191 193 264 146 141 164 171 167

Multicopper Oxidase (Cueo) H.lacusprofundi ATCC49239 Multicopper Oxidase Laccase-2 precursor Hypothetical protein Hypothetical protein Hypothetical protein Multicopper Oxidase Multicopper Oxidase Copper resistance protein A Multicopper oxidase type 1

(B) Domain 3: Haloarcula marismortui ATCC43049 ( nirK)

(B) Domain 3: Halorubrum lacusprofundi ATCC49239

Fig. 2 Sequence alignment around the copper-binding (A). Domain 2; (B). Domain 3, in multicopper blue proteins of Haloarcula marismortui ATCC43049 (nirK) and Halorubrum lacusprofundi ATCC49239. The numbers 1, 2 and 3 at the top of the alignments, indicate the consensus positions of the type 1, type 2 and type 3 copper-binding residues, respectively. The residue colored in brown is of BCB sites. The residues colored in green are type 2/3 IDCB sites.

ascorbate oxidase (Fig. 2). So it can be presumed that the archaeal laccases might be the plausible ancestral form of nitrite reductase, ceruloplasmin and ascorbate oxidase.

Conclusion Our analysis strongly suggests that laccases could be obtained from archaeal sources with robust biocatalytic functions. Moreover, it opens up broad opportunities for novel biocatalyst with broad biotechnological applications. New functions of the multicopper oxidase family may emerge from the novel proteins we have identified in diverse archaeal species. Moreover, the biological functions of these proteins should be revealed by future laboratory experiments.

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