DNA Sequence-]. DNA Sequencing and Mapping, Vol. 2, pp. 41 5-41 8 Reprints available directly from the publisher Photocopying permitted by license only
0 1992 Harwood Academic Publishers GmbH Printed in the United Kingdom
SHORT COMMUNICATION
Nucleotide sequence of the Synechococcus sp. PCC7942 hernEgene encoding the homologue of mammalian uroporphyrinogen decar boxylase Mitochondrial DNA Downloaded from informahealthcare.com by McMaster University on 12/27/14 For personal use only.
J. A. K. W. KIEL, A. M. TEN BERGE and G. VENEMA Department o f Genetics, Centre of Biologicaf Sciences, Kerklaan 30, NL-975 7 NN Haren, The Netherlands EMBL Data Library Accession No. Z11705
We have determined the complete nucleotide sequence of a Synechococcus sp. PCC7942 gene encoding the homologue of mammalian uroporphyrinogen decarboxylase (UROD). The gene, designated hem€, encoded a polypeptide of 354 amino acids with a molecular weight of 39,283. The primary sequences of the polypeptide encoded by hem€ and human and rat UROD had 32.5% identical amino acid residues. No invariant cysteine residues were found, despite the fact that UROD isolated from different sources has been shown to be inhibited by sulfhydryl reagents. The knowledge of the primary structure of this cyanobacterial protein may be helpful in better understanding the structural alterations and functional abnormalities of UROD in patients suffering from Porphyria Cutanea Tarda (PCT).
rinogen. In humans, a dominantly inherited disorder, Porphyria Cutanea Tarda (PCT), characterized by increased excretion of uroporphyrin and some of its decarboxylation products, i s caused by a partial deficiency of UROD activity (for review see Sweeney, 1986). Partially purified UROD has been studied from many sources including rat liver (Mukerji and Pimstone, 1986), Euglena gracilis Z (Juknat et a/., 1989) and the prokaryote Rhodopseudornonas palustris (Koopmann and Batlle, 1987). Furthermore, the enzyme has been purified to homogeneity from human and chicken erythrocytes (de Verneuil etal., 1983; Kawanishi etal., 1983), from bovine liver (Straka and Kushner, 1983), as well as from Saccharomyces cerevisiae (Felix and Brouillet, 1990). In addition, cDNAs complementary to UROD mRNA obtained from human and rat spleen were cloned and the nucleotide sequences determined (Romeo et a/., 1984, 1986; Romana ef a/., 1987). The genes encoded proteins of approximately 41 kDa that were highly similar (9Oo/o identity o n the amino acid level). Recently, we cloned and characterized the glgB gene encoding the glycogen branching enzyme from the cyanobacterium Synechococcus sp. PCC7942 o n a 3.9 kb f s t l fragment in plasmid p K V N l (Kiel et a/., 1989). Sequence analysis of the 2.7 kb Pstl-EcoRV fragment of the chromosomal insert in p K V N l showed the presence of an incomplete open reading frame (ORF) of 79 amino acids that started 59 nucleotides downstream from the
KEY WORDS: amino acid sequence, Anacystis nidulans R2, glycogen branching enzyme, heme and chorophyll synthesis, Porphyria Cutanea Tarda, primary sequence comparison
EXPERIMENTAL PROCEDURE A N D DISCUSSION Uroporphyrinogen decarboxylase (UROD, EC 4.1.1.37) is a cytosolic enzyme involved in the biosynthesis of heme and chlorophyll (Granick and Beale, 1978). It catalyzes the sequential removal of the four carboxyl groups from the acetate side chains of uroporphyrinogen to yield coproporphy-
Address for correspondence: Dr I. A. K. W. Kiel, Dept. of Genetics, Centre of Biological Sciences, Kerklaan 30, NL-9751 N N Haren, The Netherlands. Tel. (050) 632092, Fax. (.050)632348.
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pKVN51, a BamHI-Bglll deletion derivative of pKVN21 (Kiel etal., 1989) (Fig. 2). Comparison of the primary sequence of the ORF with the sequences in the Swiss Protein database (release 20, November 1991) using FASTA (Pearson and Lipman, 1988) revealed similarity only to mammalian UROD. An alignment of the primary sequences is shown in Fig. 3. It appears that 32.5% of the amino acid residues are identical i n all proteins. Therefore, we conclude that p K V N l contains a gene encoding a cyanobacterial homologue of mammalian UROD. We propose to designate this gene hem€ in accordance to its designation in Escherichia coli and Bacillus subtilis A~CAGIIC~CGGMCCCACTGGCG~GCTCCTCTIICTCTGT~-~TCG (Sasarman et a/., 1975; Miczak et a/., 1976). RBS I I V A 1 3 As observed previously, only 59 nucleotides sepCGTCGTCTTCGCTCCCCCGAClTTlGCGCGClGCCCGmGAGGTTCTCGATCGCCCAC S S S L P R L L R A A R G E V L D R P P 23 arate the hem€ gene from the glg5 gene (Kiel et al., CCGTGTGGATGATGCGCCMGCTGGTCGETACATGMGGKTATCGCGATC~~~ V W ~ ~ R Q A G R Y I K V Y R D L R D K 1990). Furthermore, n o recognizable -35 and -1 0 43 A G T A C C C T G G C T T C C G T G A G C G T T C ~ ~ C G G A A C m ~ T C ~ ~ G C l Gpromoter C sequences could be found upstream of Y P G P R E R S E T P E L A I E I S L Q 63 Synechococcus sp. hem€. It would appear that a MCCGTTCCGCGCTTTCAAGCCCGACffiCGTCATCCTGTTCTCGGATATCCTCACGCCCT P F R A F K P D G V I L P S D I L T P L terminator-like structure is also absent downstream 03 TGCCGGGCATGGGCA'ITCCCTTCGACATCATKAGAGCAAACGCCCGATTCTGGAGCCGC hem€ (Fig. 1). These data suggest that both from P G W G I P P D I I E S K G P I L E P P 103 glg5 and hem€ are part of an operon-like structure. C G A T C C G C A C G G C T G ~ A A G ~ A G ~ C A ~ A T C T C ~ T C C ~ ~ ~ C ~
421
CTTTCATTCGTCCAA~CCCTTCGTCAGGAAGTCGGUACGAAGCCGCCGlGC
481
F I R P I L E T L R Q E V G N E A A V L 143 TAGGGTTTGCCGGTGCGCCATGGACTCTCGCGCXCTAlGCGATCGAAGGTAhAPsGCTCGA G P A G A P W T L A A Y A I E G K S S X 163
541
MACCTACGCCAACATCACTTGCCTTPKGGAACCGACGATCCTGCATGAGTTGT
Mitochondrial DNA Downloaded from informahealthcare.com by McMaster University on 12/27/14 For personal use only.
coding region of glgB (Kiel et a/., 1990). This ORF had similarity to mammalian UROD. In this report we present the complete nucleotide sequence of this gene, which appears to encode the Synechococcus sp. homologue of mammalian UROD. The nucleotide sequence of the region of plasmid pKVN1 downstream from Synechococcus sp. glg5 is presented in Fig. 1. Analysis of the sequence showed the presence of an O R F of 354 amino acids encoding a polypeptide with a molecular weight of 39,283. A protein of similar size was synthesized in an in vitro system from plasmid
1 61 121 181 241 301
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T Y A N I K H L A F S E P T I L H E L L 183 TGGGCAAGCTAGCCGACAACATTGCCATCTACCTCTGCCACCMAT"GAC%?CGTGCGC G X L A D N I A I Y L C H Q I D C G A Q 203 AGGTTGTTCAGCTGTTCGATTCTTGXCCGGTCAGCTGAGCCCGATCGATTACGACACCT V V Q L P D S W A G Q L S P I D Y D T P 223 TCGCTCTGCCTTATCAGCAGCGTGTCTTCCAGCAAGTGAAGGC~CACCCTGMGTGC A L P Y Q Q R V P Q Q V K A K H P E V P 243 CCTTMTTCTCTACATCAGCGGTAG%CTGGCGTAC%GA!XffiATCGGCCAGTCGGGCT L 1 L Y : S G S A G V L E R I I G Q S G C 263 GCGATATCGTCAGCGTCGATTGGACGGTTGA~TCGA%CTCGCCGCCGCTTGGGTC D I V S V D W T V D L L D A R R R L G P 283 CGGATATCGGCCTGCAAGGCAACATCGATCCGGGTGTGCTGTTTGGCTCTCAAGATTTCA D I G L Q G N I D P G V L P G S Q D F I 303 T C C G C G A T C G C A T T T T G G A C A C G G T G C G T M A G C M T T R D R I L D T V R K A G N Q R H I L N L 323
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X N L D Q L L A A S H ' 354 TGAGTAAGCGCGTCTTCATCACTGGGGCCAGCGGCTGTGTCGGCCACTATCTGACGGAAA
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Figure 1 Nucleotide sequence of the Synechococcus sp. hem€ gene. The sequence was determined for both D N A strands according to Tabor and Richardson (1987) using the T7 polymerase system (Pharmacia, Uppsala, Sweden) and denatured pKVNl DNA. Oligonucleotides were synthesized using an Applied Biosystems 381A DNA Synthesizer (Applied Biosystems, Inc., Foster City, Calif.). The nucleotide sequence has been deposited at EMBL and was assigned the accession number Z11705 RBS, putative ribosome binding site.
Figure 2 Analysis of in vitro synthesized proteins encoded by the Synechococcus sp. hem€ gene. ['5SJmethionine-labeled plasmid-encoded proteins were synthesized in vitro using an Escherichia coli cell-free coupled transcription-translation system (Arnersham International plc., UK). Protein products were SDS-10% polyacrylamide gel separated using 0.1% electrophoresis. Gels were fixed in 10% methanol-1 0% acetic acid, dried and exposed to Kodak Omat Films (XR-1). Lanes ( 1 ) no DNA; (2) pUC9; (3) pKVN51; (M) ["CJniethylated molecular weight markers (carbonic anhydrase, 30 kDa; ovalbumin 46 kDa and bovine serum albumin, 69 kDa; obtained from Amersham, Buckingharnshire (UK)). Plasmid pKVN51, a deletion derivative of pKVN21, carries a 1.6 kb Bglll-Pstl fragment of pKVNl containing the hem€ gene under the control of the Bacillus subtilis bacteriophage SP02 promoter (Kiel etal., 1989).
Mitochondrial DNA Downloaded from informahealthcare.com by McMaster University on 12/27/14 For personal use only.
S YNECHOCOCCUS sp. hem€ GENE
In cyanobacteria, glycogen synthesis is a lightdependent process (Lehman and Wober, 1976). Furthermore, in Synechococcus sp. the hem€ gene product (UROD) i s presumably mainly involved in the synthesis of chlorophyll. Thus, the two genes may be coordinately expressed from a light-regulated promoter. Biochemical studies on partially purified and homogenous preparations of UROD from different sources have revealed that the enzyme is sensitive to sulfhydryl modification (de Verneuil eta/., 1983; Straka and Kushner, 1983; Kawanishi ef a/., 1983; Mukerji and Pimstone, 1986; Koopmann and Batlle, 1987; Felix and Brouillet, 1990). This indicates the presence of cysteine residue(s) at or near the active site of the enzyme. Surprisingly, no invariant cysteine residues can be found in Fig. 3, despite the presence of three cysteine residues in the cyanobacterial protein. We hypothesize that the cysteine residue in UROD susceptible to sulfhydryl modification, is close to the active site of the enzyme, but is not directly part of it. This implies that it is not essential for enzyme activity. Sulfhydryl modification of this residue may, however, result in an unfavourable conformation at the
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MEIVIGLGPQGFPELKNDTFLRRRWCEETDYTPVWCnRQAGRYLPEFRET~-QDFFSTC ---NGLGLQNFPELKNDTFLRAAWGEETDYTPVWCMRaMRDFFSTC MIASS----SLPRL-----LRARRGEVLDRPPYWMMRQAGR~DLRDKYPGFRERS 1
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ACKNOWLEDGEMENTS We thank Henk Mulder for the photography. This work was part of a project supported by a grant from the Netherlands Ministry of Economic Affairs in the framework of the "lntegraal Structuurplan Noorden des Lands". Addit,ional funding for the project was provided by AVEBE, Veendam, The Netherlands.
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REFERENCES
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active site, and thus inhibit enzyme activity. It is tempting to speculate that residue 35 i s the susceptible cysteine in mammalian UROD. This residue, which is substituted by methionine in the cyanobacterial enzyme, is part of a highly conserved region (Fig. 3). Other inhibition studies on UROD suggested that in addition to a cysteine residue also histidine residues and possibly a lysine group are required for enzyme activity (Kawanishi ef a/., 1983; Koopmann and Batlle, 1987). Figure 3 shows that t w o histidines (residues 189 and 339 in mammalian UROD) and three lysines (residues 104, 178 and 245 in mammalian UROD) are conserved also in the cyanobacterial protein. However, additional biochemical and genetic studies on both eukaryotic and prokaryotic enzymes are needed to provide a better understanding of the residues constituting the active site of UROD.
ALRDPEWASELGWFQAITLTRQR~GRVPLIGFAG~~~T~E~SS~Q~R RLRDPRRVASELGWFQAITLTRQQ~GRVPLIGFAG~~~~EffiSFK~Q~R A V H D L D P E E A T - P F I R P I L E T L R Q E V G N ~ ~ F A G A I K H ..I
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Figure 3 Alignment of the primary sequences of the Synechococcus sp. hem€ gene product and human and rat UROD. The primary sequences of the protein encoded by Synechococcus sp. hem€ and human and rat UROD (Romeo ef a/., 1986; Rornana et a/., 1987) were aligned using the CLUSTAL program of Higgins and Sharp (1989). Numbers to the left of the sequences indicate the position of the first amino acid shown relative to the N-terminal residue of each enzyme. For the primary sequence of rat UROD the numbering of the human primary sequence has been used, because the first codons of the gene encoding the rat enzyme were lacking on the cDNA. Dashes represent gaps introduced to maximize the similarity. *, Amino acid residue identical in all three cases; 0 ,conservative amino acid replacement. Syn, Synechococcus sp.
de Verneuil H., Sassa S., and Kappas A. (1983). Purification and properties of uroporphyrinogen decarboxylase from human erythrocytes. 1. Biol. Cbern. 258, 2454-2460. Felix F., and Brouillet N. (1990). Purification and properties of uroporphyrinogen decarboxylase from Sacc haromyces c-erevisiae. Eur. J. Biochem. 188, 393-403. Granick S., and Beale S.L. (1978). Hemes, chlorophylls, and related compounds: biosynthesis and metabolic regulation. Adv. Enzyrnol. 46,33-203. Higgins D.G., and Sharp P.M. (1989). Fast and sensitive multiple sequence alignments on a mrcrocomputer. Cornput. Appl. Biosci. 5, 151-1 54. Juknat A.A., Seubert A., Seubert S., and lppen H . (1 989). Studies on uroporphyrinogen decarboxylase of etiolated Euglena gracilis Z. Eur. 1. Biochem. 179, 423-428. Kawanishi S., Seki Y., and Sano S. ( 1 9 8 3 ) . Uroporphyrinogen decarboxylase, purification, properties, and inhibition by polychlorinated biphenyl isomers. 1. Biol. Chem. 258, 4285-4292. Kiel J.A.K.W., Elgersma H.S.A., Beldman G., Vossen J.P.M.J., and Venema G. (1989). Cloning and expression of the
Mitochondrial DNA Downloaded from informahealthcare.com by McMaster University on 12/27/14 For personal use only.
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J. A. K.
w.
branching enzyme gene (glgB) from the cyanobacterium Synechococcus sp. PCC7942 in Escherichia coli. Gene 78, 9-1 7. Kiel J.A.K.W., Boels J.M., Beldman G., and Venema G . (1990). Nucleotide sequence of the Synechococcus sp. PCC7942 branching enzyme gene (glgB); expression in Bacillus subtilis. Gene 89,77-84. Koopmann C.E., and Batlle A.M. del C. (1987). Biosynthesis of porphyrins in Rhodopseudornonaspalustris. VI. The effect of metals, thiols and other reagents on the activity of uroporphyrinogen decarboxylase. Int. J. Biochem. 19,373-377. Lehmann M., and Wober G. (1976). Accumulation, mobilization and turn-over of glycogen in the blue-green bacterium Anacystis nidulans. Arch. Microbiol. 11 1, 93-97. Miczak A,, Berek I., and lvanovics G. (1976). Mapping the uroporphyrinogen decarboxylase, coproporphyrinogen oxidase and ferrochelatase loci in Bacillus subtilis. Mol. Gen. Genet. 146,85-87. Mukerji S.K., and Pimstone N.R. (1986). In vitro studies of the mechanism of inhibition of rat liver uroporphyrinogen decarboxylase activity by ferrous iron under anaerobic conditions. Arch. Biochem. Biophys. 244,619-629. Pearson W.R., and Lipman D.J. (1988). Improved tools for biological sequence comparison. Proc. Natl. Acad. Sci. USA 85, 2444-2448. Romeo P-H., Dubart A,, Crandchamp B., de Verneuil H., Rosa
KIEL
Er AL. I.,
Nordmann Y., and Coossens M. (1984). Isolation and identification of a cDNA clone coding for rat uroporphyrinogen decarboxylase. Proc. Natl. Acad. Sci. USA 81, 3346-3350. Romeo P-H., Raich N., Dubart A,, Beaupain D., Pryor M., Kushner J., Cohen-Solal M., and Goossens M. (1986). Molecular cloning and nucleotide sequence of a complete human uroporphyrinogen decarboxylase cDNA. J. Biol. Chem. 261, 9825-9831. Romana M., Le Boulch P., and Romeo P-H. (1987). Rat uroporphyrinogen decarboxylase cDNA: nucleotide sequence and comparisQn to human uroporphyrinogen decarboxylase. NucleicAcids Res. 15, 7211. Sasarman A,, Chartrand P., Proschek R., Desrochers M., Tardif D., and Lapointe C. (1975). Uroporphyrin-accumulating mutant of fscherichia coli K-12. J. Bacteriol. 124, 1205-1 21 2. Straka J.C., and Kushner J.P. (1983). Purification and characterization of bovine hepatic uroporphyrinogen decarboxylase. Biochemistry 22,4664-4672. Sweeney G.D. (1986). Porphyria cutanea tarda, or the uroporphyrinogen decarboxylase deficiency diseases. Clin. Biochem. 19, 3-15. Tabor S., and Richardson C.C. (1987). DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Proc. Natl. Acad. Sci. USA 84,4767-4771.
0 1992 Harwood Academic Publishers GmbH Printed in the United Kingdom
SHORT COMMUNICATION
Nucleotide sequence of the Synechococcus sp. PCC7942 hernEgene encoding the homologue of mammalian uroporphyrinogen decar boxylase Mitochondrial DNA Downloaded from informahealthcare.com by McMaster University on 12/27/14 For personal use only.
J. A. K. W. KIEL, A. M. TEN BERGE and G. VENEMA Department o f Genetics, Centre of Biologicaf Sciences, Kerklaan 30, NL-975 7 NN Haren, The Netherlands EMBL Data Library Accession No. Z11705
We have determined the complete nucleotide sequence of a Synechococcus sp. PCC7942 gene encoding the homologue of mammalian uroporphyrinogen decarboxylase (UROD). The gene, designated hem€, encoded a polypeptide of 354 amino acids with a molecular weight of 39,283. The primary sequences of the polypeptide encoded by hem€ and human and rat UROD had 32.5% identical amino acid residues. No invariant cysteine residues were found, despite the fact that UROD isolated from different sources has been shown to be inhibited by sulfhydryl reagents. The knowledge of the primary structure of this cyanobacterial protein may be helpful in better understanding the structural alterations and functional abnormalities of UROD in patients suffering from Porphyria Cutanea Tarda (PCT).
rinogen. In humans, a dominantly inherited disorder, Porphyria Cutanea Tarda (PCT), characterized by increased excretion of uroporphyrin and some of its decarboxylation products, i s caused by a partial deficiency of UROD activity (for review see Sweeney, 1986). Partially purified UROD has been studied from many sources including rat liver (Mukerji and Pimstone, 1986), Euglena gracilis Z (Juknat et a/., 1989) and the prokaryote Rhodopseudornonas palustris (Koopmann and Batlle, 1987). Furthermore, the enzyme has been purified to homogeneity from human and chicken erythrocytes (de Verneuil etal., 1983; Kawanishi etal., 1983), from bovine liver (Straka and Kushner, 1983), as well as from Saccharomyces cerevisiae (Felix and Brouillet, 1990). In addition, cDNAs complementary to UROD mRNA obtained from human and rat spleen were cloned and the nucleotide sequences determined (Romeo et a/., 1984, 1986; Romana ef a/., 1987). The genes encoded proteins of approximately 41 kDa that were highly similar (9Oo/o identity o n the amino acid level). Recently, we cloned and characterized the glgB gene encoding the glycogen branching enzyme from the cyanobacterium Synechococcus sp. PCC7942 o n a 3.9 kb f s t l fragment in plasmid p K V N l (Kiel et a/., 1989). Sequence analysis of the 2.7 kb Pstl-EcoRV fragment of the chromosomal insert in p K V N l showed the presence of an incomplete open reading frame (ORF) of 79 amino acids that started 59 nucleotides downstream from the
KEY WORDS: amino acid sequence, Anacystis nidulans R2, glycogen branching enzyme, heme and chorophyll synthesis, Porphyria Cutanea Tarda, primary sequence comparison
EXPERIMENTAL PROCEDURE A N D DISCUSSION Uroporphyrinogen decarboxylase (UROD, EC 4.1.1.37) is a cytosolic enzyme involved in the biosynthesis of heme and chlorophyll (Granick and Beale, 1978). It catalyzes the sequential removal of the four carboxyl groups from the acetate side chains of uroporphyrinogen to yield coproporphy-
Address for correspondence: Dr I. A. K. W. Kiel, Dept. of Genetics, Centre of Biological Sciences, Kerklaan 30, NL-9751 N N Haren, The Netherlands. Tel. (050) 632092, Fax. (.050)632348.
41 5
41 6
J. A. K. W. KlEL €7 A t .
361
pKVN51, a BamHI-Bglll deletion derivative of pKVN21 (Kiel etal., 1989) (Fig. 2). Comparison of the primary sequence of the ORF with the sequences in the Swiss Protein database (release 20, November 1991) using FASTA (Pearson and Lipman, 1988) revealed similarity only to mammalian UROD. An alignment of the primary sequences is shown in Fig. 3. It appears that 32.5% of the amino acid residues are identical i n all proteins. Therefore, we conclude that p K V N l contains a gene encoding a cyanobacterial homologue of mammalian UROD. We propose to designate this gene hem€ in accordance to its designation in Escherichia coli and Bacillus subtilis A~CAGIIC~CGGMCCCACTGGCG~GCTCCTCTIICTCTGT~-~TCG (Sasarman et a/., 1975; Miczak et a/., 1976). RBS I I V A 1 3 As observed previously, only 59 nucleotides sepCGTCGTCTTCGCTCCCCCGAClTTlGCGCGClGCCCGmGAGGTTCTCGATCGCCCAC S S S L P R L L R A A R G E V L D R P P 23 arate the hem€ gene from the glg5 gene (Kiel et al., CCGTGTGGATGATGCGCCMGCTGGTCGETACATGMGGKTATCGCGATC~~~ V W ~ ~ R Q A G R Y I K V Y R D L R D K 1990). Furthermore, n o recognizable -35 and -1 0 43 A G T A C C C T G G C T T C C G T G A G C G T T C ~ ~ C G G A A C m ~ T C ~ ~ G C l Gpromoter C sequences could be found upstream of Y P G P R E R S E T P E L A I E I S L Q 63 Synechococcus sp. hem€. It would appear that a MCCGTTCCGCGCTTTCAAGCCCGACffiCGTCATCCTGTTCTCGGATATCCTCACGCCCT P F R A F K P D G V I L P S D I L T P L terminator-like structure is also absent downstream 03 TGCCGGGCATGGGCA'ITCCCTTCGACATCATKAGAGCAAACGCCCGATTCTGGAGCCGC hem€ (Fig. 1). These data suggest that both from P G W G I P P D I I E S K G P I L E P P 103 glg5 and hem€ are part of an operon-like structure. C G A T C C G C A C G G C T G ~ A A G ~ A G ~ C A ~ A T C T C ~ T C C ~ ~ ~ C ~
421
CTTTCATTCGTCCAA~CCCTTCGTCAGGAAGTCGGUACGAAGCCGCCGlGC
481
F I R P I L E T L R Q E V G N E A A V L 143 TAGGGTTTGCCGGTGCGCCATGGACTCTCGCGCXCTAlGCGATCGAAGGTAhAPsGCTCGA G P A G A P W T L A A Y A I E G K S S X 163
541
MACCTACGCCAACATCACTTGCCTTPKGGAACCGACGATCCTGCATGAGTTGT
Mitochondrial DNA Downloaded from informahealthcare.com by McMaster University on 12/27/14 For personal use only.
coding region of glgB (Kiel et a/., 1990). This ORF had similarity to mammalian UROD. In this report we present the complete nucleotide sequence of this gene, which appears to encode the Synechococcus sp. homologue of mammalian UROD. The nucleotide sequence of the region of plasmid pKVN1 downstream from Synechococcus sp. glg5 is presented in Fig. 1. Analysis of the sequence showed the presence of an O R F of 354 amino acids encoding a polypeptide with a molecular weight of 39,283. A protein of similar size was synthesized in an in vitro system from plasmid
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T Y A N I K H L A F S E P T I L H E L L 183 TGGGCAAGCTAGCCGACAACATTGCCATCTACCTCTGCCACCMAT"GAC%?CGTGCGC G X L A D N I A I Y L C H Q I D C G A Q 203 AGGTTGTTCAGCTGTTCGATTCTTGXCCGGTCAGCTGAGCCCGATCGATTACGACACCT V V Q L P D S W A G Q L S P I D Y D T P 223 TCGCTCTGCCTTATCAGCAGCGTGTCTTCCAGCAAGTGAAGGC~CACCCTGMGTGC A L P Y Q Q R V P Q Q V K A K H P E V P 243 CCTTMTTCTCTACATCAGCGGTAG%CTGGCGTAC%GA!XffiATCGGCCAGTCGGGCT L 1 L Y : S G S A G V L E R I I G Q S G C 263 GCGATATCGTCAGCGTCGATTGGACGGTTGA~TCGA%CTCGCCGCCGCTTGGGTC D I V S V D W T V D L L D A R R R L G P 283 CGGATATCGGCCTGCAAGGCAACATCGATCCGGGTGTGCTGTTTGGCTCTCAAGATTTCA D I G L Q G N I D P G V L P G S Q D F I 303 T C C G C G A T C G C A T T T T G G A C A C G G T G C G T M A G C M T T R D R I L D T V R K A G N Q R H I L N L 323
1021
TGGGCCACGGCATCCTGCCCGGGACTCCTGAGGATMTGCCCGTCACTTCrnAGACGG
1081
CCAAAAACCTCGATCAACTCTTGGCAGCGAGTCACTMCTTGCGAGAGGCAATCACTGX
1141
X N L D Q L L A A S H ' 354 TGAGTAAGCGCGTCTTCATCACTGGGGCCAGCGGCTGTGTCGGCCACTATCTGACGGAAA
1201
CGTTGCTGCTCMCACAGACTATGAGCTGTTTCTGCTCGTCCGCGATCGCCAGAAGCTGA
G
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1261
CGATTCATGTCMGCGTCGGCCCGGAGTGGAGGTGATCGAAGCCGATATGGCGGACATCG
1321
ATCGCTTCCACGAGCTACTGCAAACCATCGACATIGCTGTTTIGACGGC'IGCAG
Figure 1 Nucleotide sequence of the Synechococcus sp. hem€ gene. The sequence was determined for both D N A strands according to Tabor and Richardson (1987) using the T7 polymerase system (Pharmacia, Uppsala, Sweden) and denatured pKVNl DNA. Oligonucleotides were synthesized using an Applied Biosystems 381A DNA Synthesizer (Applied Biosystems, Inc., Foster City, Calif.). The nucleotide sequence has been deposited at EMBL and was assigned the accession number Z11705 RBS, putative ribosome binding site.
Figure 2 Analysis of in vitro synthesized proteins encoded by the Synechococcus sp. hem€ gene. ['5SJmethionine-labeled plasmid-encoded proteins were synthesized in vitro using an Escherichia coli cell-free coupled transcription-translation system (Arnersham International plc., UK). Protein products were SDS-10% polyacrylamide gel separated using 0.1% electrophoresis. Gels were fixed in 10% methanol-1 0% acetic acid, dried and exposed to Kodak Omat Films (XR-1). Lanes ( 1 ) no DNA; (2) pUC9; (3) pKVN51; (M) ["CJniethylated molecular weight markers (carbonic anhydrase, 30 kDa; ovalbumin 46 kDa and bovine serum albumin, 69 kDa; obtained from Amersham, Buckingharnshire (UK)). Plasmid pKVN51, a deletion derivative of pKVN21, carries a 1.6 kb Bglll-Pstl fragment of pKVNl containing the hem€ gene under the control of the Bacillus subtilis bacteriophage SP02 promoter (Kiel etal., 1989).
Mitochondrial DNA Downloaded from informahealthcare.com by McMaster University on 12/27/14 For personal use only.
S YNECHOCOCCUS sp. hem€ GENE
In cyanobacteria, glycogen synthesis is a lightdependent process (Lehman and Wober, 1976). Furthermore, in Synechococcus sp. the hem€ gene product (UROD) i s presumably mainly involved in the synthesis of chlorophyll. Thus, the two genes may be coordinately expressed from a light-regulated promoter. Biochemical studies on partially purified and homogenous preparations of UROD from different sources have revealed that the enzyme is sensitive to sulfhydryl modification (de Verneuil eta/., 1983; Straka and Kushner, 1983; Kawanishi ef a/., 1983; Mukerji and Pimstone, 1986; Koopmann and Batlle, 1987; Felix and Brouillet, 1990). This indicates the presence of cysteine residue(s) at or near the active site of the enzyme. Surprisingly, no invariant cysteine residues can be found in Fig. 3, despite the presence of three cysteine residues in the cyanobacterial protein. We hypothesize that the cysteine residue in UROD susceptible to sulfhydryl modification, is close to the active site of the enzyme, but is not directly part of it. This implies that it is not essential for enzyme activity. Sulfhydryl modification of this residue may, however, result in an unfavourable conformation at the
1
human rat
4
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MEIVIGLGPQGFPELKNDTFLRRRWCEETDYTPVWCnRQAGRYLPEFRET~-QDFFSTC ---NGLGLQNFPELKNDTFLRAAWGEETDYTPVWCMRaMRDFFSTC MIASS----SLPRL-----LRARRGEVLDRPPYWMMRQAGR~DLRDKYPGFRERS 1
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RSPEACCELTLQPLRRFPLDRRIlfSDILWPQRLGME~PSKGPSFPEPLREEQDLE RSPEACCELTLEPVRRFPLDRRIIFSDILVVPQ~~GKGPSFPEPLREERDLE
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120
Syn
112
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VRKQVliaALREAG~PVPMIIFAKffinFRLEELAQRGYEWGLD~~~ECVGK~
Syn
231
VFQQYKRKHPE-----VPLILYISGSAG~ERHGQSGCDlVS~~L~D~RR~PDI
human rat
300 300
Syn
286
T L Q G N L D P C ~ Y A S E E E I G Q L V K Q M L D D F G P H R Y I A N L G K TLQGELDPCALYASEEEIGRLVQQMLNDFGPQRYI~LGHGLYPDMDPE~G~LDA~K GLQGNIDPGVLFGSQDFIRURILDTVR~GNQRHI~LGflGILPGTPEDN~HFFETAN
human rat syn
360 360
120
ACKNOWLEDGEMENTS We thank Henk Mulder for the photography. This work was part of a project supported by a grant from the Netherlands Ministry of Economic Affairs in the framework of the "lntegraal Structuurplan Noorden des Lands". Addit,ional funding for the project was provided by AVEBE, Veendam, The Netherlands.
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(Received 12th February 1992)
WLYQRPQASHOLLRILTDRLVPYLVGQWAGAQALQLFESHIIGH~PQLFNXF~PYIRU ~YQKPVASHKLLGILTHALVPYLIGQV~GAQALQLFESHIIG~SELFSKF~PYIRU LRFSEPTILHELLGK~NII\IYLCHQlDCGAQWQLFDSWAGQLSPIDYDTFffiPYQQR
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REFERENCES
VIV(RYKAGLQK*GLTRHPMIIFAKDG:~FALEELAQAGYEWGLD~~~EPVGK~
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346
active site, and thus inhibit enzyme activity. It is tempting to speculate that residue 35 i s the susceptible cysteine in mammalian UROD. This residue, which is substituted by methionine in the cyanobacterial enzyme, is part of a highly conserved region (Fig. 3). Other inhibition studies on UROD suggested that in addition to a cysteine residue also histidine residues and possibly a lysine group are required for enzyme activity (Kawanishi ef a/., 1983; Koopmann and Batlle, 1987). Figure 3 shows that t w o histidines (residues 189 and 339 in mammalian UROD) and three lysines (residues 104, 178 and 245 in mammalian UROD) are conserved also in the cyanobacterial protein. However, additional biochemical and genetic studies on both eukaryotic and prokaryotic enzymes are needed to provide a better understanding of the residues constituting the active site of UROD.
ALRDPEWASELGWFQAITLTRQR~GRVPLIGFAG~~~T~E~SS~Q~R RLRDPRRVASELGWFQAITLTRQQ~GRVPLIGFAG~~~~EffiSFK~Q~R A V H D L D P E E A T - P F I R P I L E T L R Q E V G N ~ ~ F A G A I K H ..I
171
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Figure 3 Alignment of the primary sequences of the Synechococcus sp. hem€ gene product and human and rat UROD. The primary sequences of the protein encoded by Synechococcus sp. hem€ and human and rat UROD (Romeo ef a/., 1986; Rornana et a/., 1987) were aligned using the CLUSTAL program of Higgins and Sharp (1989). Numbers to the left of the sequences indicate the position of the first amino acid shown relative to the N-terminal residue of each enzyme. For the primary sequence of rat UROD the numbering of the human primary sequence has been used, because the first codons of the gene encoding the rat enzyme were lacking on the cDNA. Dashes represent gaps introduced to maximize the similarity. *, Amino acid residue identical in all three cases; 0 ,conservative amino acid replacement. Syn, Synechococcus sp.
de Verneuil H., Sassa S., and Kappas A. (1983). Purification and properties of uroporphyrinogen decarboxylase from human erythrocytes. 1. Biol. Cbern. 258, 2454-2460. Felix F., and Brouillet N. (1990). Purification and properties of uroporphyrinogen decarboxylase from Sacc haromyces c-erevisiae. Eur. J. Biochem. 188, 393-403. Granick S., and Beale S.L. (1978). Hemes, chlorophylls, and related compounds: biosynthesis and metabolic regulation. Adv. Enzyrnol. 46,33-203. Higgins D.G., and Sharp P.M. (1989). Fast and sensitive multiple sequence alignments on a mrcrocomputer. Cornput. Appl. Biosci. 5, 151-1 54. Juknat A.A., Seubert A., Seubert S., and lppen H . (1 989). Studies on uroporphyrinogen decarboxylase of etiolated Euglena gracilis Z. Eur. 1. Biochem. 179, 423-428. Kawanishi S., Seki Y., and Sano S. ( 1 9 8 3 ) . Uroporphyrinogen decarboxylase, purification, properties, and inhibition by polychlorinated biphenyl isomers. 1. Biol. Chem. 258, 4285-4292. Kiel J.A.K.W., Elgersma H.S.A., Beldman G., Vossen J.P.M.J., and Venema G. (1989). Cloning and expression of the
Mitochondrial DNA Downloaded from informahealthcare.com by McMaster University on 12/27/14 For personal use only.
41 8
J. A. K.
w.
branching enzyme gene (glgB) from the cyanobacterium Synechococcus sp. PCC7942 in Escherichia coli. Gene 78, 9-1 7. Kiel J.A.K.W., Boels J.M., Beldman G., and Venema G . (1990). Nucleotide sequence of the Synechococcus sp. PCC7942 branching enzyme gene (glgB); expression in Bacillus subtilis. Gene 89,77-84. Koopmann C.E., and Batlle A.M. del C. (1987). Biosynthesis of porphyrins in Rhodopseudornonaspalustris. VI. The effect of metals, thiols and other reagents on the activity of uroporphyrinogen decarboxylase. Int. J. Biochem. 19,373-377. Lehmann M., and Wober G. (1976). Accumulation, mobilization and turn-over of glycogen in the blue-green bacterium Anacystis nidulans. Arch. Microbiol. 11 1, 93-97. Miczak A,, Berek I., and lvanovics G. (1976). Mapping the uroporphyrinogen decarboxylase, coproporphyrinogen oxidase and ferrochelatase loci in Bacillus subtilis. Mol. Gen. Genet. 146,85-87. Mukerji S.K., and Pimstone N.R. (1986). In vitro studies of the mechanism of inhibition of rat liver uroporphyrinogen decarboxylase activity by ferrous iron under anaerobic conditions. Arch. Biochem. Biophys. 244,619-629. Pearson W.R., and Lipman D.J. (1988). Improved tools for biological sequence comparison. Proc. Natl. Acad. Sci. USA 85, 2444-2448. Romeo P-H., Dubart A,, Crandchamp B., de Verneuil H., Rosa
KIEL
Er AL. I.,
Nordmann Y., and Coossens M. (1984). Isolation and identification of a cDNA clone coding for rat uroporphyrinogen decarboxylase. Proc. Natl. Acad. Sci. USA 81, 3346-3350. Romeo P-H., Raich N., Dubart A,, Beaupain D., Pryor M., Kushner J., Cohen-Solal M., and Goossens M. (1986). Molecular cloning and nucleotide sequence of a complete human uroporphyrinogen decarboxylase cDNA. J. Biol. Chem. 261, 9825-9831. Romana M., Le Boulch P., and Romeo P-H. (1987). Rat uroporphyrinogen decarboxylase cDNA: nucleotide sequence and comparisQn to human uroporphyrinogen decarboxylase. NucleicAcids Res. 15, 7211. Sasarman A,, Chartrand P., Proschek R., Desrochers M., Tardif D., and Lapointe C. (1975). Uroporphyrin-accumulating mutant of fscherichia coli K-12. J. Bacteriol. 124, 1205-1 21 2. Straka J.C., and Kushner J.P. (1983). Purification and characterization of bovine hepatic uroporphyrinogen decarboxylase. Biochemistry 22,4664-4672. Sweeney G.D. (1986). Porphyria cutanea tarda, or the uroporphyrinogen decarboxylase deficiency diseases. Clin. Biochem. 19, 3-15. Tabor S., and Richardson C.C. (1987). DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Proc. Natl. Acad. Sci. USA 84,4767-4771.
