TABLE 2.

Sequence alignment of substrate binding loops in MCOs^a

Protein	Loop I	Loop II	Loop III	Loop IV
Laccases
Phanerocaete chrysosporium MCO1	LNAQYLSPSGPIGGSA	TLQQTDMFTYKLPGQNPD-N	FDNLPSGASRAYM	VLDLVLENNDNGD
Cryptococcus neoformans Lac	IIAALATPEGYKGNIA	T--SVA-LSCMFGAVSQE-G	-NVLGNTFQGYGF	---T-VI---D--
Cucumis cinereus Lac	-IPA---P-SIQG--A	AQPNKG-RNGLAGTFANGVN	--QLGFSGGRFTI	---AGVL---GGP
Trametes villosa Lcc3	-TPA------PL----	ANPNRANTTGFANGI----N	----TFNGSEFFI	---GGVT---GGP
Ceriporiopsis subvermispora Lcs-1	-AAA-----STLT--F	ANPNNG-NMGFANGI----N	----TFNGTNLFI	---GNII---AGP
Agaricus bisporus Lcc-1	-ILAPDATNEFFSSGI	A-PMTGGNPDRNPNLNISLT	---AQPNAPFFDI	---G------EGA
Thanatephorus cucumeris Lac4	-VLEKQMFSTNNTALL	A-PMTVAGAGTNANLDPTNV	-RSTVDGILRFTF	---HHR----GAD
Cryptococcus parasiticus Lac	-TAD--ELVVYTQSNA	VTFGGGGFCGKSNNPY---P	----STTTRKWTI	---ATGN---ALP
Pleurotus ostreatus Pox1	-VVA-------PQNAV	ADPNLG-STGFDGGI----N	--AFDVTNFELTI	---ALAV---GGP
Trametes versicolor Lac3	-VAA------NVGPAF	ANPNFG-NVGFTGGI----N	----NFNGTNFFI	---ATAA-APGAP
Trametes versicolor Lcc2	-TAA------RLGPRF	ANPNFG-TVGFAGGI----N	----NFNGTNFFI	---ATAL-APGAP
Nicotiana tabacum Lac	-TEAVINEAIKSGLA-	ASPFMDAPIAVDNVT----A	MPTVALLQAHFFG	---DTGIIAPEN-
Melanocarpus albomyces Lac	-RAA-DDLVHFTQNNA	VTFGGQAACGGSLNPH---P	-DLTGTPLFVWKV	---EGPF---SLP
Class	----------------	--------------------	-------------	--------XX---
All	----------------	--------------------	-------------	-------------
Ascorbate oxidases
Phanerochaete chrysosporium MCO1	LNAQYL-S-PSGPIGGSAG	TLQQTDMFTYKLPGQNPDN	FDNLPSGASRAY	VLDLVLENNDNGD
Cucurbita pepo LaoZ	QEVG-LSSKPIRWIGEPQ-	TRARHPNTPPGLTLLNYLP	LPPTPYLGAMKY	QNANMMKENLSET
Arabidopsis thaliana AOZ	QELA-LSSRPMRWIGEPQ-	VRGREPKTPQALTVINYVD	VPVTPYLGSIRY	QNANVLKGVISEI
Cucurbita melo AOZ	QEVG-LKSNPMRWIGELQ-	VRGRKPKTSPALLLFNYLP	LPSTPYLGAIKF	QNANALTNNTSEI
Medicago truncatula AOZ	QEVG-LSSAPMRWIGEPQ-	VRGRKPSTPQALTILNYKP	LPTTPYLGSIKF	QNANQLNGNGSEI
Nicotiana tabacum AOZ	QEVD-LSSNPLRWIGEPQ-	VRGREPKTPQGLTLLNYLP	LPTQLYLGSIRY	QNANALAKDVSEI
Class	XX---X-X-X-XXXXX-X-	-X-X-X-X---X---XX--	-X---XXX----	XXXX------XX-
All	-----O-O-O---OO----	-----------O---O---	------------	-------------
Ferroxidases
Phanerochaete chrysosporium MCO1	L-NAQYLSPSGPIGGSA	TLQQTDMFTYKLPGQNPDN	FDNLPSGASRAY	VLDLVLENNDNGD
Neurospora crassa Fet3	L-QPRFMSKYNP----T	ASMDTTLFDT-IPPGLNTN	MNNLASGANYAF	IVQIVLNNLDSGR
Arrula adeninivorans Fet3	LNKEEFLTLYNP----T	SIFDENLFDV-IPSDLGMN	MTNLDNGVNYAF	VVEIVVNNNDAGF
Saccharomyces cerevisiae Fet3	L-IPNFMSRFNP----T	QRVDDTMLDV-IPKDLELN	MDNLDDGVNYAF	VIEIVLNNLDTGK
Schizocaccharomyces pombe Fet3	LVPDEFKTWKNP----T	AYMDESLFDT-IPDNYNPN	FFTLGDGANYAE	VVDVIIDNHDTGK
Candida albicans Fet3	I-GPAFLTRFNP----T	NGVDTTMLDS-VPADLQVN	MNVLNDGINYAF	IVDIVLNNFDTGK
Saccharomyces cerevisiae Fet3	L-TKSFMSVYNP----T	QKFDDTMLDV-IPSDLQLN	MDNLKNGVNYAF	IVEIVLNNQDTGT
Class	-----X----XX----X	---X----X---X-----X	---X---X-XXX-	-------X-X-X-
All	-----------O-----	------------O-----O	---O--O---O-	-------O-O-O-

^aWithin each class of MCOs (laccases, ascorbate oxidases, and ferroxidases), a subset of sequences, excluding close protein homologues, was selected and aligned. Multiple alignments containing the selected sequences for each class were independently realigned with the MCO1 sequence. The MCO1 regions predicted to be in contact with the substrate ligand (Table 1) were mapped in these multiple alignments, which allowed identification of the corresponding regions in laccases, ascorbate oxidases, and ferroxidases. For each protein class, conserved identical residues within the group of selected sequences are indicated by X. Residues conserved in each group and in the MCO1 sequence are indicated by boldface type and by O in the alignment.