Alignment of individual peroxidase and LOX domains of the fusion protein

III. Results

3. Lipoxygenase pathway of selected cyanobacterial strains

3.3 Characterisation of a peroxidase-LOX fusion protein from Nostoc sp

3.3.1 Alignment of individual peroxidase and LOX domains of the fusion protein

A comparison of the deduced amino acid sequence alignment of the peroxidase coding domain with other peroxidase-like sequences from O. sativa, the coral P. homomalla and the bacterium Sulfitobacter sp. EE-36 showed that certain amino acids are conserved which could either be involved in heme binding or present catalytic residues of the active site of a peroxidase. The crystal structure of the AOS domain in the coral (Oldham et al.

2005) revealed, that three residues are important for the distal face of the heme group: Tyr-66, His-67 and Asn-137; the proximal-side residues are Arg-349 and Tyr-353. In the peroxidase domain of the cyanobacterial fusion protein the residues for the distal face of the heme group could be identified as His-41, Tyr-42 and Asn-118. (Fig. 25A, asterisks).

In contrast, the residues on the proximal face of the heme group differ from that of the coral. Instead of an arginine (Arg-349) in the coral a glycine (Gly-323) is situated at this position in Nostoc. sp. and at the position of the tyrosine in the coral (Tyr-353) a histidine (His-327) was found.

The alignment of the deduced amino acid sequence of the NspFP LOX-domain with known LOX sequences shows, that the residues involved in iron binding within the active site are highly conserved and identical to those from plant LOXs: 170, 175, His-342 and Asn-346. Although the fifth ligand did not align properly to the other sequences, it is represented by an isoleucine (Ile-429) as for other plant LOXs (Fig. 2B, li). At the position of the three determinants that may be involved in regiospecificity, NspFP contains different amino acids in comparison to the other LOX sequences from flowering plants (Sloane et al. 1995; Borngräber et al. 1996b; Hornung et al. 1999a): At the site described first by Sloane, a serine (Ser-229) is found instead of a phenylalanine in both plant LOXs (LOX2 At2 and PpLOX1; Fig. 2B, sl). These amino acids differ remarkably from the reported determinants for positional specificity of plant LOXs (Hornung et al. 1999a), precluding accurate prediction of positional specificity of the LOX-domain. In addition, an Arg residue reported to determine inverse substrate orientation in plant LOXs is missing as well from the NspFP (Fig. 2B, ho)(Hornung et al. 1999a). However, the amino acid residue determining the stereospecificity of LOXs (Coffa site, Fig. 2B, cof) is an alanine residue, suggesting that this enzyme is a S-specific LOX (Coffa and Brash 2004).

88 A)

Nostoc 1 ---MDL--NTYLKLLNLLDSESQKIMLELQA Plexaura 1 ---MTWKNFGFEIFGEKYGQEELEKRIKDE--HTPPPDSPVFGGLKLKLKKEKFK Sulfitobacter 1 ---MTDKKQTPPTTTDAGIRVQSDEHSLTVGPDGPIVLNDHY Oryza 1741 GSLLLPGCVLQENVILGALSVAPENAVLRRGGVYVGSQSPAMVKNTLLDEDERIEEMDQA

Nostoc 27 MFSAAGLALRG---RGTHTDGIIVKGNLTVLHS-SDVPSHSLFTPGKKYDVIF Plexaura 51 TLFTLGTTLKGF---RRATHTVGTGGIGEITIVND-PKFPEHEFFTAGRTFPARL Sulfitobacter 40 LLEQMANFNRE---RIPERQPHAKGSGAFGTFETTQDVSKYTMANIFQPGAKCDVVM Oryza 1801 YKKIVGNLAANLAITTMNVKSRYFHRIGVSGRGVLRMYEEIPSFPRHKIFASGKSFPVIV

Nostoc 76 RHANIVGG-AKDDALINGRGSAIRIGNIGDDLSKPRLLDLVLNTGEVFGLPTARLYHQFF Plexaura 102 RHANLK---YPDDAGADARSFSIKFADSDSDGP----LDIVMNTGEANIFWNSPSLEDFV Sulfitobacter 94 RFSTVAGERGSPDTWRDPRGFSIKM--YTDEG----IFDMVGNNTPIFFVRDPIKFQQFI Oryza 1861 RHSNSLS--ADDDARLDARGAAVRI--LSDNDGEAPLLDLTLKSGKAFYARTIADFATWL

Nostoc 135 GSDFHQKSDMLASGSLRRY--AVEAALRNPDSFTELYYHTQLCYEWVDSKKKSRYARFRL Plexaura 155 PVEEGDAAEEYVYKNPYYYYNLVEALRRAPDTFAHLYYYSQVTMPFKAKDGKVRYCRYRA Sulfitobacter 148 RSQKRRADNNMRDHDMQW--DFWTLSPESAHQVTYLMGDRGIPKNWREMNGYSSHTYSLV Oryza 1917 VCGLPAREEQVKRSPHIR--DAVWGSLRSTDSYTVLHYYSNICRLLRFDDGREMYAKFKL

Nostoc 193 L----NPNQSTEGGLLDDSVEIGPRLVLPRKRGDTREKNYLRNEFRQRLTDGN-I-VEYV Plexaura 215 LPGDVDIKEEDESGRLTEE-EQRKIWIFSRHENEKRPDDYLRKEYVERLQKGP-VNYRLQ Sulfitobacter 206 NAEGEKFWVKF-HFHTDQGDGNAYLSQDEADKLAGTNGDYHRADLFNNIRDGNYPSWTLK Oryza 1975 RPADPDVPEDS-GKVVPRGILPPETGAIPRDEDDTRPLLFLADDFRRRVGSPDGVRYVFQ

Nostoc 247 LQAQFRSIEDVA--V--DCSNIWDPNTYPWLDIAAIVLNQDESENDYYQEIAYNPGNTHY Plexaura 273 IQIHEASPDDTA--TIFHAGILWDKETHPWFDLAKVSIKTPLSP-DVLEKTAFNIANQPA Sulfitobacter 265 WQIMPYEDAKTYRINPFDLTKVWPHEDYPLIEVGKLTLNRN--PTDFHTEIEQAAF---E Oryza 2034 LQLREVPTDAAARDVALDCTRPWDEAEFPYIDVGEVSIGRNL-PTEETEKLEFNPFLRCP

Nostoc 303 DLKLPNSYSVDDFASLGVSGALVH---YFGSIVRAERTQYLYGSKDDLPGKP--- Plexaura 330 SLGLLEAKSPEDYNSIGELRVAVY---TWVQHLRKLKIGSLVPAGQN--- Sulfitobacter 320 PNNMVPGVGLSPDKML---LARGFSYADAHRARLGVNYKQIPVNK-PVSP Oryza 2093 EVDVIPATSCAQSASIDHGRSLVYEICQRLRNGEPLPASWRAFLEQSDTKIDLSGCPVAA Nostoc 1 ---MDL--NTYLKLLNLLDSESQKIMLELQA Plexaura 1 ---MTWKNFGFEIFGEKYGQEELEKRIKDE--HTPPPDSPVFGGLKLKLKKEKFK Sulfitobacter 1 ---MTDKKQTPPTTTDAGIRVQSDEHSLTVGPDGPIVLNDHY Oryza 1741 GSLLLPGCVLQENVILGALSVAPENAVLRRGGVYVGSQSPAMVKNTLLDEDERIEEMDQA

89 B)

NspFP 124 FVDKQNGVKLHSIKIDDHEIT--PCQEQWQYAKRTYLQAEFLSQELKLHLARCHFNIEQY PhAOS-LOX 719 EPGPENP---IWTPHEENE---HDWMMAKFWLGVAESNFHQLNTHLLRTHLTTESF LOX2 At2 532 PPTAESENKF--VYTHGHDAT---THWIWKLAKAHVCSNDAGVHQLVNHWLRTHASMEPY PpLOX1 549 PPKTVGEERITRVLTTRKDDQLWKVNWEWELAKAHVSNNDITAHQVFSHFSRCHAVTEAV

NspFP 182 VMAIKRRLAPTHPVRAFINPHLEGLIFINSSAVPKIIGSTGFIPIASMLTQGSIVDVMKN PhAOS-LOX 769 ALSTWRNLASAHPVFKLLQPHIYGVLAIDTIGRKELIGSGGIVDQSLSLGGGGHVTFMEK LOX2 At2 587 IIATNRQLSTMHPVYKLLHPHMRYTLEINARARKSLINGGGIIESCFTPGKYAMELSSAA PpLOX1 609 IICSNRNLSKLHPLMQLLAPHFKSTLEINRQARATLIAAGGSIETHFTTRAYSLEMAAVN

NspFP 242 ELSKLSYMWNPI-ADLPRD---IPGDLFTPAATAYWELLNNYVEQGL PhAOS-LOX 829 CFKEVNLQDYHLPNALKKRGVDDPSK---LPGFYYRDDGLALWEAIETFIGE-I LOX2 At2 647 YKSMWRFDMEGLPADLVRRGMAEEDSSAECGVRLVIDDYPYAADGLLIWKAIKDLVES-Y PpLOX1 669 YKDTWTFESQALPTDLVARGMAVPDPDSPHGVRLVVEDYPYAADGLELWGALKAWHKE-Y

NspFP 285 LQPFEDELRTEVNAIQVDELFAELKERS---LYSGDQPPKYDSSE-LKSLLMYIIYH PhAOS-LOX 879 IAIFYKNDDDVKRDNEIQSWIYDVHKNGWRVNPGHQDHGVPASFESREQLKEVLTSLVFT LOX2 At2 706 VKHFYSDSKSITSDLELQAWWDEIKNKG---HYDKKDEPWWPKLNTTQDLSQILTNMIWI PpLOX1 728 VDIYYKDDAAVLQDSELMTWWTEMREKA---HEDKKDSHGWPELNSKEALVDILTTVIWI

NspFP 338 SSFLHSWANFKQYDDAG-NPNHVSMGDYSQYDQQTQDKIRF----SQRSLTWVLSSIRYN PhAOS-LOX 939 FSCQHAAVNFSQKDHYGFTPNAPAVLRHPPPKKKGEATLQ---SILSTLPSKSQA LOX2 At2 763 ASGQHAAINFGQYPFGGYVPNRPTLLRKLIPQE-TDPDYEMFMRNPQYSFLGSLPTQLQA PpLOX1 785 PSCLHAAVNFGQYDFAGFMPHHPTLTRRLLPEHGNEKDKADFNKNPEKYYLTSISNIDST

NspFP 393 SVAVYGSDLLKQLIREKSSILE---PGLPLEDLMMSINI--- PhAOS-LOX 991 AKAIATVYILTKFSEDERYLGNYSA---TAWEDKDALDAINRFQDKLEDISKKIKQRNEN LOX2 At2 822 TKVMAVQETLSTHSPDEEYLIELREVQRHWFQDEQVVKYFNKFSEELVKIEKTINERNKD PpLOX1 845 TTAMSVYEVLSAHCPIEEYIGERRG---NWTNNEKVLAAFKGFKESVNEADAVMRARNAD

NspFP --- PhAOS-LOX 1048 LEV---PYIYLLPERIPNGTAI--- LOX2 At2 882 KKLKNRTGAGMPPYELLLPTSPHGVTGRGIPNSISI PpLOX1 902 PKLRNRGGPVKMPYQLLRPHSKPGVTSMGVPNSITI

bo li li

NspFP --- PhAOS-LOX 1048 LEV---PYIYLLPERIPNGTAI--- LOX2 At2 882 KKLKNRTGAGMPPYELLLPTSPHGVTGRGIPNSISI PpLOX1 902 PKLRNRGGPVKMPYQLLRPHSKPGVTSMGVPNSITI

bo li li

Figure 25. Partially deduced amino acid sequence alignment of peroxidase and LOX domain of N. sp. SAG 25.82 fusion protein. LOX2 At2 (Acc. no. AAF79461) from A.

thaliana, PpLOX1 (Acc. no CAE47464) from P. patens, 13-LOX (Acc. no. AAL85880) PhAOS-LOX (Acc. no. AAC47283) from the coral P. homomalla, peroxidase (Acc. no XP_472850) from O. sativa and peroxidase (Acc. no. ZP_00955421) from S. sp. EE-36.

A) stars: conserved amino acids which are involved in the active site of the AOS coding domain of the fusion protein of P. homomalla and which are also present in the peroxidase domain of the fusion protein N. sp. B) stars: the residues involved in iron ligation (three histidines, an asparagine and an isoleucine; li), and the three determinants for substrate and regiospecificity (bo, according to Borngraber et al. 1996, sl, according to Sloane et al.

1995, ho, according to Hornung et al. 1999).

90 To isolate the fusion protein and the separate domains, gene specific primers were used to amplify the full length fusion protein and the separate domains from genomic DNA of N.

sp SAG 25.82. The obtained fragment of the full length fusion protein had a complete open reading frame of 2322 bp, encoding a protein of 774 amino acids with a molecular weight of 70.9k. The analysis of the three obtained fragments via gel electrophoresis showed, that the peroxidase domain as well as the full length fusion protein were about 1300 bp longer than expected from the published genomic sequence of NspFP (data not shown). Sequence analysis of the two fragments in question revealed the presence of a large transposon insertion within the catalase coding domain causing a frame shift. This insertion had highest homology to an insertion sequence, named IS891N putative transposase gene, which was identified in Nostoc ellipsosporum (Acc. no. U48694). Consequently, the insertion was removed via PCR mutagenesis to allow expression of the catalase coding domain and the intact fusion protein. To investigate the catalytic activities of the fusion protein and the separate domains, we made three different constructs, which were overexpressed in E. coli (DE3) expression strains.

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