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Nucleotide sequence of the Rhodobacter capsulatus hemH gene
Gene, 170 (1996) 149-150 ©1996 Elsevier Science B.V. All rights reserved. 0378-1119/96/$15.00
149
GENE 09547
Nucleotide sequence of the Rhodobacter capsulatus hemH gene (Ferrochelatase; heme; photosynthesis; tetrapyrrole)
Ekaterina Kanazireva and Alan J. Biel Department of Microbiology, Louisiana State University, Baton Rouge, LA 70808, USA Received by J. Wild: 18 October 1995; Accepted: 30 October 1995; Received at publishers: 27 November 1995
SUMMARY
The last step in heme synthesis is the insertion of iron into the ring of protoporphyrin IX. The enzyme which catalyzes this reaction, ferrochelatase (FC), is encoded by the hemH gene. A clone containing this gene from Rhodobacter capsulatus, a purple non-sulfur photosynthetic bacterium, has been sequenced. A single open reading frame was found which could encode a protein of 351 amino acids. This putative protein is very similar to other FC and contains the FC signature sequence.
Rhodobacter capsulatus (Rc), a purple non-sulfur photosynthetic bacterium, synthesizes four tetrapyrrole endproducts: bacteriochlorophyll, heme, vitamin B12 and siroheme. The photosynthetic bacteria are the only group of organisms that synthesize all four of these tetrapyrroles. Bacteriochlorophyll and heme, the quantitatively major tetrapyrroles, share a common biosynthetic pathway from aminolevulinate to protoporphyrin IX. The pathways leading to vitamin Blz and siroheme branch off from the common tetrapyrrole pathway at uroporphyrin III. Heme is synthesized by chelating iron into the protoporphyrin ring. This reaction is catalyzed by ferrochelatase (FC), the product of the hemH gene. The hemH genes from several organisms, including plants, animals and bacteria such as Escherichia coli (Ec) and Bacillus subtilis, have been cloned and sequenced (Nakahashi et al., 1990; Brenner and Frasier, 1991; Hansson et al., 1991; Miyamoto et al., 1991; 1994). The aim of this study was to determine the nt and deduced Correspondence to: Dr. A.J. Biel, Department of Microbiology, Louisiana State University, Baton Rouge, LA 70808, USA. Tel. (1-504) 388-2791; Fax (1-504) 388-2597; e-mail: [email protected] Abbreviations: aa, amino acid(s); B., Bacillus; bp, base pair(s); E., Escherichia; Ec, E. coli; FC, ferrochelatase(s); hemH, gene encoding FC; kb, kilobase(s) or 1000 bp; nt, nucleotide(s); R., Rhodobacter; Rc, R. capsulatus; X, any aa. SSDI 0378-1119(96)00845-4
aa sequences of the Rc hemH gene and its gene product, respectively. The Rc hemH gene was previously cloned by complementation of an Ec hemH deletion mutant (Kanazireva and Biel, 1995). Expression of the cloned hemH gene results in elevated FC activity in both Ec and Rc. The hemH gene was localized to a 1.7-kb BamHI fragment. Fig. 1 shows the nt sequence of the hemH region. A single open reading frame of 1056 nt was found which could code for a 351-aa protein of 46 210 Da. The protein possesses 41% identity and 59% similarity to the Ec FC and 21% identity and 44% similarity to the B. subtilis FC (Miyamoto et al., 1991; Hansson and Hederstedt, 1992). The Rc FC has a sequence, beginning at Va1214, that is almost identical to the consensus FC signature aa sequence [LIVMF] (3)-X-S-X-H-[GS]-[LIVM]-P-X (4,5)-[DENK]-X-G-D-X-Y (Bairoch and Bucher, 1994). The only difference between the Rc sequence and the consensus sequence is the substitution of Lys228 for Arg. This is the first reported sequence of a hemH gene from a photosynthetic bacterium.
ACKNOWLEDGEMENTS
This work was supported by grant MCB-9304999 from the National Science Foundation.
150 1 CGGCAGTTTCCA~TGGTTTGGCTGACCGGCTGGAAACCG~TGAAAGCCAGCAAA~3%CC GCTGCGCCCGGGCTCGGCCGCCTGTTCCTTGACCGAAATT~GCAAAACGCCG~GGG GT~GGGCGCGCAATC~TGCGC~ATATTGTT~GCTTTT~TTGACCCCAAATG~ AAAGCTATACCTCCGGGACCGGACAGC~TCAGGACACGT~TGACGATTGCCAACCG~ M T I A N R I
60 120 180 240 7
TCCTTCCGCACGCCCCCGCCGACCATCCGCCGGTGCCGGTGCCGCGTGTCGGCGTGCTTT L P H A P A D H P P V P V P R W G V L L 2 7
300
TGGCC~TCTCGGCACGCCCGACGCCACCGACTACTGGTCGATGCGCCGCTATCTG~CG A N L G T P D A T D Y W S M R ~ Y L N E 4 7
360
~TTCCTGTCCGACCGGCGGGTGATCGACTATCCGCTCTGGAAATGGCAGCCGCTGTTGc F L S D R R V I D Y P L W K W Q P L L Q 6 7
420
AGCTGATCATCCTGTCG~GCGGCCCTTCACCTCGGGC~C~CTATCGCTCGATCTGGA
480
L
I
I
L
S
K
R
P
F
T
S
G
N
N
Y
R
S
I
W
N
8
7
ACGAGGAGCGCGACGAAAGCCCCTTGATGACGAT~CCCGCGAT~GGTGCGC~GCTGC E E R D E S P L M T I T R D Q V R K L R I 0 7
540
GCGCCGCGGTCGAGACCCGCTATGGCGCGGGC~TGTGGTGGTCGATTTCTGCATGCGCT A A V E T R Y G A G N V V V D F C M R Y I 2 7
600
ACGGC~CCCCTCCACCCGTGACGTGCTCGATGA~TGCTGGCGCAGGGCTGCG~CGCA G N P S T R D V L D D M L A Q G C E R I I 4 7
660
TCCTGTTCCTGCCGCTTTATCCGC~TATGCGGGCGC~CCTCGGC~CGGCG~CGACC L F L P L Y P Q Y A G A T S A T A N D Q I 6 7
720
AGTTCTTCCGCGCGCTGATG~GGTGAAACGC~GCCCGCCGCCCGCACCGTGCCGG~T F F R A L M Q V K R Q P A A R T V P E Y I 8 7
780
ATTTCGC~GGCCGAGCTATATCGAGGCGCTGGCCAGCTC~TCGAGCGGGTCTATGCGA F A R P S Y I E A L A S S V E R V Y A T 2 0 7
840
CGCTCGACACCCGGCCCGACGTGCTGGTGGCCTCTTATCAC~CATGCCCAAACGCTATC L D T R P D [ V L V A S Y H G M P K R Y H 2 2 7
900
ACCGCGAGGGCGACCCCTATCATTGCC~TGCCAGAAAAC~CGC~CTTTTGCGCG~C ~ E G D P Y ] H C Q C Q K T S R L L R E R 2 4 7
960
GGCTGGGCTGGGGGCCGGATTCGATCGACACCACCTTTCAATCCGTCTTCGG~CCGAGG L G W G P D S I D T T F Q S V F G T E E 2 6 7
1020
~TGGCTGCGCCCCTATACGGTCGAGCATGTGGTGCAG~GGCGG~GCGGGC~G~GA W L R P Y T V E H V V Q L A E A G K K N 2 8
1080 7
ACATCGCGGTGATTTCCCCCGCCTTTTCCGCCGATTG~TCGAGACGCTCGAGGAAAT~ I A V I S P A F S A D C I E T L E E I N 3 0 7
1140
ACGGCGAGATCCGCGAGGCGTTTG~CATGCGGGCGGCGAAAGCTT~CCTATGTGCCCT G E I R E A F E H A G G E S F T Y V P C 3 2 7
1200
GCCTG~CGACGACGACCTGCACATCGCCGCGCTTCTGG~GT~TCGAGGAAAATCTGG L N D D D L H I A A L L E V V E E N L A 3 4 7
1260
CGGGCTGGATCGACTGATCCGCCCGGCCGCGACCGGA~TGAAAAAGCCGCGGGCCCCCT G W I D *
1320
TTGGCTTGCCAAGGATCCGGGTCTCAGATCAG~CGACCT~GTGCCGACCTGCGC~GT TCG~GTTCCTCGACATGCTGGTTG~GGCCGATG~GCCGTTCGACGACTTGCGG CCGATCTTGCGGGTGTCGTGGGTGCCGTGGATGCGGT~TACTGC~GCTCAGATA~TC GCGCGCGTGCCCCAGCGGA~GTCCGGACCGGCTTCGACGAAAATCCGGCCCATTCCGGG
1380 1440 1500 1560
351
Fig. 1. Nucleotide sequence of the Rc hemH gene (OenBank accession No. U34391) and the putative aa sequence of its product, FC. The double underlined nt constitute the putative Shine-Dalgarno site. The region was sequenced by making a series of nested deletions of pCAP123 (Kanazireva and Biel, 1995) and using the Circum~nt Thermal Cycle Sequencing Kit (New England Biolabs, Beverly, MA, USA). The bracketed aa residues indicate the region that corresponds to the FC signature sequence.
REFERENCES Bairoch, A. and Bucher, P.: PROSITE: recent developments. Nucleic Acids Res. 22 (1994) 3583-3589. Brenner, D.A. and Frasier, F.: Cloning of murine ferrochelatase. Proc. Natl. Acad. Sci. USA 88 (1991) 849-853. Hansson, M. and Hederstedt, L.: Cloning and characterization of the Bacillus subtilis hemEH Y gene cluster, which encodes protoheme IX biosynthetic enzymes. J. Bacteriol. 174 (1992) 8081 8093. Hansson, M., Rutberg, L., Schr6der, I. and Hederstedt, L.: The Bacillus subtilis hem A X C D B L gene cluster, which encodes enzymes of the biosynthetic pathway from glutamate to uroporphyrinogen III. J. Bacteriol. 173 (1991) 2590-2599. Kanazireva, E. and Biel, A.J.: Cloning and overexpression of the Rhodobacter capsulatus hemH gene. J. Bacterioh 177 (1995) 6693-6694. Miyamoto, K., Nakahigashi, K., Nishimura, K. and Inokuchi, H.: Isolation and characterization of visible light-sensitive mutants of Escherichia coli K-12. J. Mol. Biol. 219 (1991) 393-398. Miyamoto, K., Tanaka, R., Teramoto, H., Masuda, T., Tsuji, H. and Inokuchi, H.: Nucleotide sequences of cDNA clones encoding ferrochelatase from barley and cucumber. Plant Physiol. 105 (1994) 769-770. Nakahashi, Y., Taketani, S., Okuda, M., Inoue, K. and Tokunaga, R.: Molecular cloning and sequence analysis of cDNA encoding human ferrochelatase. Biochem. Biophys. Res. Commun. 173 (1990) 748-755.
149
GENE 09547
Nucleotide sequence of the Rhodobacter capsulatus hemH gene (Ferrochelatase; heme; photosynthesis; tetrapyrrole)
Ekaterina Kanazireva and Alan J. Biel Department of Microbiology, Louisiana State University, Baton Rouge, LA 70808, USA Received by J. Wild: 18 October 1995; Accepted: 30 October 1995; Received at publishers: 27 November 1995
SUMMARY
The last step in heme synthesis is the insertion of iron into the ring of protoporphyrin IX. The enzyme which catalyzes this reaction, ferrochelatase (FC), is encoded by the hemH gene. A clone containing this gene from Rhodobacter capsulatus, a purple non-sulfur photosynthetic bacterium, has been sequenced. A single open reading frame was found which could encode a protein of 351 amino acids. This putative protein is very similar to other FC and contains the FC signature sequence.
Rhodobacter capsulatus (Rc), a purple non-sulfur photosynthetic bacterium, synthesizes four tetrapyrrole endproducts: bacteriochlorophyll, heme, vitamin B12 and siroheme. The photosynthetic bacteria are the only group of organisms that synthesize all four of these tetrapyrroles. Bacteriochlorophyll and heme, the quantitatively major tetrapyrroles, share a common biosynthetic pathway from aminolevulinate to protoporphyrin IX. The pathways leading to vitamin Blz and siroheme branch off from the common tetrapyrrole pathway at uroporphyrin III. Heme is synthesized by chelating iron into the protoporphyrin ring. This reaction is catalyzed by ferrochelatase (FC), the product of the hemH gene. The hemH genes from several organisms, including plants, animals and bacteria such as Escherichia coli (Ec) and Bacillus subtilis, have been cloned and sequenced (Nakahashi et al., 1990; Brenner and Frasier, 1991; Hansson et al., 1991; Miyamoto et al., 1991; 1994). The aim of this study was to determine the nt and deduced Correspondence to: Dr. A.J. Biel, Department of Microbiology, Louisiana State University, Baton Rouge, LA 70808, USA. Tel. (1-504) 388-2791; Fax (1-504) 388-2597; e-mail: [email protected] Abbreviations: aa, amino acid(s); B., Bacillus; bp, base pair(s); E., Escherichia; Ec, E. coli; FC, ferrochelatase(s); hemH, gene encoding FC; kb, kilobase(s) or 1000 bp; nt, nucleotide(s); R., Rhodobacter; Rc, R. capsulatus; X, any aa. SSDI 0378-1119(96)00845-4
aa sequences of the Rc hemH gene and its gene product, respectively. The Rc hemH gene was previously cloned by complementation of an Ec hemH deletion mutant (Kanazireva and Biel, 1995). Expression of the cloned hemH gene results in elevated FC activity in both Ec and Rc. The hemH gene was localized to a 1.7-kb BamHI fragment. Fig. 1 shows the nt sequence of the hemH region. A single open reading frame of 1056 nt was found which could code for a 351-aa protein of 46 210 Da. The protein possesses 41% identity and 59% similarity to the Ec FC and 21% identity and 44% similarity to the B. subtilis FC (Miyamoto et al., 1991; Hansson and Hederstedt, 1992). The Rc FC has a sequence, beginning at Va1214, that is almost identical to the consensus FC signature aa sequence [LIVMF] (3)-X-S-X-H-[GS]-[LIVM]-P-X (4,5)-[DENK]-X-G-D-X-Y (Bairoch and Bucher, 1994). The only difference between the Rc sequence and the consensus sequence is the substitution of Lys228 for Arg. This is the first reported sequence of a hemH gene from a photosynthetic bacterium.
ACKNOWLEDGEMENTS
This work was supported by grant MCB-9304999 from the National Science Foundation.
150 1 CGGCAGTTTCCA~TGGTTTGGCTGACCGGCTGGAAACCG~TGAAAGCCAGCAAA~3%CC GCTGCGCCCGGGCTCGGCCGCCTGTTCCTTGACCGAAATT~GCAAAACGCCG~GGG GT~GGGCGCGCAATC~TGCGC~ATATTGTT~GCTTTT~TTGACCCCAAATG~ AAAGCTATACCTCCGGGACCGGACAGC~TCAGGACACGT~TGACGATTGCCAACCG~ M T I A N R I
60 120 180 240 7
TCCTTCCGCACGCCCCCGCCGACCATCCGCCGGTGCCGGTGCCGCGTGTCGGCGTGCTTT L P H A P A D H P P V P V P R W G V L L 2 7
300
TGGCC~TCTCGGCACGCCCGACGCCACCGACTACTGGTCGATGCGCCGCTATCTG~CG A N L G T P D A T D Y W S M R ~ Y L N E 4 7
360
~TTCCTGTCCGACCGGCGGGTGATCGACTATCCGCTCTGGAAATGGCAGCCGCTGTTGc F L S D R R V I D Y P L W K W Q P L L Q 6 7
420
AGCTGATCATCCTGTCG~GCGGCCCTTCACCTCGGGC~C~CTATCGCTCGATCTGGA
480
L
I
I
L
S
K
R
P
F
T
S
G
N
N
Y
R
S
I
W
N
8
7
ACGAGGAGCGCGACGAAAGCCCCTTGATGACGAT~CCCGCGAT~GGTGCGC~GCTGC E E R D E S P L M T I T R D Q V R K L R I 0 7
540
GCGCCGCGGTCGAGACCCGCTATGGCGCGGGC~TGTGGTGGTCGATTTCTGCATGCGCT A A V E T R Y G A G N V V V D F C M R Y I 2 7
600
ACGGC~CCCCTCCACCCGTGACGTGCTCGATGA~TGCTGGCGCAGGGCTGCG~CGCA G N P S T R D V L D D M L A Q G C E R I I 4 7
660
TCCTGTTCCTGCCGCTTTATCCGC~TATGCGGGCGC~CCTCGGC~CGGCG~CGACC L F L P L Y P Q Y A G A T S A T A N D Q I 6 7
720
AGTTCTTCCGCGCGCTGATG~GGTGAAACGC~GCCCGCCGCCCGCACCGTGCCGG~T F F R A L M Q V K R Q P A A R T V P E Y I 8 7
780
ATTTCGC~GGCCGAGCTATATCGAGGCGCTGGCCAGCTC~TCGAGCGGGTCTATGCGA F A R P S Y I E A L A S S V E R V Y A T 2 0 7
840
CGCTCGACACCCGGCCCGACGTGCTGGTGGCCTCTTATCAC~CATGCCCAAACGCTATC L D T R P D [ V L V A S Y H G M P K R Y H 2 2 7
900
ACCGCGAGGGCGACCCCTATCATTGCC~TGCCAGAAAAC~CGC~CTTTTGCGCG~C ~ E G D P Y ] H C Q C Q K T S R L L R E R 2 4 7
960
GGCTGGGCTGGGGGCCGGATTCGATCGACACCACCTTTCAATCCGTCTTCGG~CCGAGG L G W G P D S I D T T F Q S V F G T E E 2 6 7
1020
~TGGCTGCGCCCCTATACGGTCGAGCATGTGGTGCAG~GGCGG~GCGGGC~G~GA W L R P Y T V E H V V Q L A E A G K K N 2 8
1080 7
ACATCGCGGTGATTTCCCCCGCCTTTTCCGCCGATTG~TCGAGACGCTCGAGGAAAT~ I A V I S P A F S A D C I E T L E E I N 3 0 7
1140
ACGGCGAGATCCGCGAGGCGTTTG~CATGCGGGCGGCGAAAGCTT~CCTATGTGCCCT G E I R E A F E H A G G E S F T Y V P C 3 2 7
1200
GCCTG~CGACGACGACCTGCACATCGCCGCGCTTCTGG~GT~TCGAGGAAAATCTGG L N D D D L H I A A L L E V V E E N L A 3 4 7
1260
CGGGCTGGATCGACTGATCCGCCCGGCCGCGACCGGA~TGAAAAAGCCGCGGGCCCCCT G W I D *
1320
TTGGCTTGCCAAGGATCCGGGTCTCAGATCAG~CGACCT~GTGCCGACCTGCGC~GT TCG~GTTCCTCGACATGCTGGTTG~GGCCGATG~GCCGTTCGACGACTTGCGG CCGATCTTGCGGGTGTCGTGGGTGCCGTGGATGCGGT~TACTGC~GCTCAGATA~TC GCGCGCGTGCCCCAGCGGA~GTCCGGACCGGCTTCGACGAAAATCCGGCCCATTCCGGG
1380 1440 1500 1560
351
Fig. 1. Nucleotide sequence of the Rc hemH gene (OenBank accession No. U34391) and the putative aa sequence of its product, FC. The double underlined nt constitute the putative Shine-Dalgarno site. The region was sequenced by making a series of nested deletions of pCAP123 (Kanazireva and Biel, 1995) and using the Circum~nt Thermal Cycle Sequencing Kit (New England Biolabs, Beverly, MA, USA). The bracketed aa residues indicate the region that corresponds to the FC signature sequence.
REFERENCES Bairoch, A. and Bucher, P.: PROSITE: recent developments. Nucleic Acids Res. 22 (1994) 3583-3589. Brenner, D.A. and Frasier, F.: Cloning of murine ferrochelatase. Proc. Natl. Acad. Sci. USA 88 (1991) 849-853. Hansson, M. and Hederstedt, L.: Cloning and characterization of the Bacillus subtilis hemEH Y gene cluster, which encodes protoheme IX biosynthetic enzymes. J. Bacteriol. 174 (1992) 8081 8093. Hansson, M., Rutberg, L., Schr6der, I. and Hederstedt, L.: The Bacillus subtilis hem A X C D B L gene cluster, which encodes enzymes of the biosynthetic pathway from glutamate to uroporphyrinogen III. J. Bacteriol. 173 (1991) 2590-2599. Kanazireva, E. and Biel, A.J.: Cloning and overexpression of the Rhodobacter capsulatus hemH gene. J. Bacterioh 177 (1995) 6693-6694. Miyamoto, K., Nakahigashi, K., Nishimura, K. and Inokuchi, H.: Isolation and characterization of visible light-sensitive mutants of Escherichia coli K-12. J. Mol. Biol. 219 (1991) 393-398. Miyamoto, K., Tanaka, R., Teramoto, H., Masuda, T., Tsuji, H. and Inokuchi, H.: Nucleotide sequences of cDNA clones encoding ferrochelatase from barley and cucumber. Plant Physiol. 105 (1994) 769-770. Nakahashi, Y., Taketani, S., Okuda, M., Inoue, K. and Tokunaga, R.: Molecular cloning and sequence analysis of cDNA encoding human ferrochelatase. Biochem. Biophys. Res. Commun. 173 (1990) 748-755.