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Nucleotide sequence of the narB gene encoding assimilatory nitrate reductase from the cyanobacterium Oscillatoria chalybea
BB ELSEVIER
Biochi~ic~a et Biophysica A~ta Biochimica et Biophysica Acta 1305 (1996) 19-24
Short sequence-paper
Nucleotide sequence of the narB gene encoding assimilatory nitrate reductase from the cyanobacterium Oscillatoria chalybea 1 Monika Unthan a, Werner Klipp b, Georg H. Schmid a,, a Biologie VIII: Zellphysiologie, Universitiit Bielefeld, Postfach 100131, D-33501 Bielefeld, Germany b Biologie VI: Genetik, Universit~t Bielefeld, Bielefeld, Germany Received 29 August 1995; revised 13 October 1995; accepted 17 October 1995
Abstract
The nucleotide sequence of the structural gene of nitrate reductase (narB) has been determined from the filamentous, non-heterocystous cyanobacterium Oscillatoria chalybea. The narB gene encodes a protein of 737 amino acid residues, which shows 61% identity to nitrate reductase of the unicellular cyanobacterium Synechococcus sp. PCC 7942 and only weak homologies to different bacterial molybdoenzymes, such as nitrate reductases or formate dehydrogenases. Keywords: Nitrate reductase; Molybdoenzymes; narB gene; Cyanobacterium; (O. chalybea)
Cyanobacterial nitrate reductases consist of single polypeptides of about 80-85 kDa containing one molybdenum cofactor and one [4Fe-4S] or two [2Fe-2S] cluster(s). Reduced ferredoxin was shown to be the physiological electron donor for the enzyme in a variety of cyanobacteria, such as Plectonema boryanum [1] or Synechococcus sp. PCC 7942, also known as Anacystis nidulans R2 [2]. In contrast to Synechococcus sp. PCC 7942, transcription of narB, the structural gene of nitrate reductase, occurred in OsciUatoria chalybea not only in the presence of nitrate but also in media containing arginine as nitrogen source [3,4]. Therefore, regulation of nitrate reductase in the two cyanobacteria is different. In order to study the structure of narB in O. chalybea the corresponding gene was cloned and sequenced. The following procedure was used to obtain chromosomal DNA from O. chalybea: after harvesting cultures of O. chalybea, the pelleted cells were resuspended in a saturated solution of sodium iodide (NaI) and incubated at 37°C for 60 min to remove exopolysaccharides. The NaI containing supernatant was removed after centrifugation, and sucrose buffer (50 mM Tris/50 mM NaCI/5 mM
* Corresponding author. Fax: +49 521 1065626. J The nucleotide sequence data reported in this paper have been submitted to the EMBL/GenBank Data Libraries under the accession number X89445. 0167-4781/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved SSDI 0 1 6 7 - 4 7 8 1 ( 9 5 ) 0 0 2 1 0 - 3
EDTA/20% sucrose/pH 8.0) containing 5 m g / m l lysozyme was added to the precipitate. Sucrose/lysozyme treatment was carded out at 37°C for 90 min. O. chalybea spheroplasts were collected by centrifugation and incubated with 2% SDS in sucrose buffer (37°C, 90 min). After repeated freezing ( - 8 0 ° C , 60 min) and thawing (37°C, 60 min) lysis of speroplasts occurred. Chromosomal DNA was obtained by phenol/chloroform extraction and ethanol precipitation employing standard DNA purification techniques [5]. The narB gene of O. chalybea was identified from size fractionated gene banks by hybridization with two oligonucleotides (marked in Fig. 1). These oligonucleotides were designed on the basis of cyanobacterial codon preference [6] according to two amino acid sequences highly conserved between nitrate reductase of Klebsiella pneumoniae (NasA) and related sequences of other organisms [7] including NarB of Synechococcus sp. PCC 7942 (EMBL database, accession No. X74597). Two DNA fragments have been cloned containing overlapping parts of the narB gene of O. chalybea. The plasmid pOCll containing a 3.7 kb HindIII fragment includes the 3' region of narB. The plasmid pOC21 carrying a 3.8 kb EcoRI fragment spans the 5' region of narB. Both DNA strands of a 3027 bp fragment from the narB gene region were sequenced using fluorescent dideoxynucleotides and an automatic laser fluorescent DNA sequencer. The complete nucleotide sequence of this DNA
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M. Unthan et al. / Biochimica et Biophysica Acta 1305 (1996) 19-24
region is presented in Fig. 1 and narB was shown to be located between positions 347 and 2560. The 3' end of an open reading frame (ORF1) is located immediately upstream of narB. The stop codon of narB is separated by only 14 bps from the ATG start codon of an additional open reading frame (ORF2). The narB gene encodes a protein of 737 amino acid residues with a calculated molecular weight of 81 648. As shown in Fig. 2 the deduced amino acid sequence of NarB from O. chalybea shows significant homology to NarB of
Synechococcus sp. PCC 7942 (61% identity). Identical amino acid residues are equally distributed over the entire length of the proteins. In contrast homologies to nitrate reductases of Alcaligenes eutrophus (NapA) [8], Klebsiella pneumoniae (NasA) [7] and Escherichia coli (NarG, NarZ) [9,10] are mainly restricted to nine domains and the overall homology is only about 30%. However, comparing the deduced amino acid sequences of nitrate reductases from A. eutrophus, K. pneumoniae and E. coli to each other, also
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Fig. 1. Nucleotide and deduced amino acid sequences of O. chalybea narB (nucleotides 347-2560) and partial sequences of ORF1 (nucleotides 1-327) and ORF2 (nucleotides 2575-3027). Two amino acid sequences highly conserved between nitrate reductases from a variety of organisms, which were used to design oligonucleotides for the isolation of O. chalybea narB DNA, are boxed. Three putative membrane-spanning helices of the ORF2 protein are underlined. The putative signal peptide in ORF2 is marked by shaded boxes (amino acid residues 1-48).
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M. Unthan et aL / Biochimica et Biophysica Acta 1305 (1996) 19-24
revealed only overall homologies in the range of approx. 30%. Although, the two cyanobacterial sequences exhibit significant homology to each other, they are as diverse from the other bacterial nitrate reductases as the others are from each other. As shown in Fig. 2, the domains conserved in nitrate reductases from different species are also present in other molybdoenzymes like formate dehydrogenases from Methanobacterium formicicum (FdhA) [11]. In addition, these domains were also found in formate dehydrogenases FdhF [12] and FdnG [13] from E. coli (data not shown). Four conserved domains (2, 4, 6, 8) marked in Fig. 2 have been identified not only in nitrate reductases and formate dehydrogenases but also in other molybdoenzymes like dimethylsulfoxide reductase DmsA [14] and biotin sulfoxide reductase BisC (SwissProt database, accession No. P20099) from E. coli. Therefore, these domains have been discussed to be involved in binding of the molybdenum-pterin cofactor [9]. A conserved cysteine motif (1) is located in the Nterminal part of bacterial nitrate reductases. Since the C-X2-C-X3-C-X39-C motif found in NarB of O. chalybea and Synechococcus sp. PCC 7942 resembles the C-X2-CX3-C-X30_36-Cmotif known to be involved in ligating one of the two [4Fe-4S] clusters in bacterial ferredoxins [15], it is likely that nitrate reductases and formate dehydrogenases harbour one [4Fe-4S] center. Domain 3 might be involved in catalytic activity since a conserved cysteine residue is present in all nitrate reductases and formate dehydrogenases sequenced so far. This cysteine residue is substituted by selenocysteine in formate dehydrogenase FdhF and FdnG from E. coli [12,13] an amino acid derivative known to be located in the active center of a variety of enzymes [16,17]. Three other conserved domains (5, 7 and 9) have been found in cyanobacterial nitrate reductases and in some other prokaryotic molybdoproteins. The functions of these regions remain unknown. In summary, the amino acid sequence comparisons presented in Fig. 2 indicate that nitrate reductases from cyanobacteria, which were shown to be closely related to each other, are clearly distinct from nitrate reductases from other bacterial species and homologies are limited to domains also found in a variety of other molybdoenzymes. Although the nitrate reductases from O. chalybea and Synechococcus sp. PCC 7942 seem to be regulated in different ways, no significant distinction in the enzyme
23
structures has been observed. The Synechococcus sp. PCC 7942 narB gene was shown to be part of the nirAnrtABCD-narB operon. The nirA gene encodes nitrite reductase [18] and nrtABCD are involved in nitrate uptake [19,20]. Partially sequenced genes located immediately upstream (ORF1) and downstream (ORF2) of narB in O. chalybea, respectively (Fig. 1), indicate that in this cyanobacterium narB is also part of an operon. ORF1 shows weak homology to nitrate transporter proteins from the filamentous fungus Aspergillus nidulans (CrnA) [21] and the green alga Chlamydomonas reinhardtii (Nar-3, Nar-4) [22]. However, no homology to NrtABCD nitrate transporter proteins in Synechococcus could be detected. ORF2 shows no homology to known genes or proteins. Hydropathy plots of the deduced amino acid sequence of ORF1 predicted three putative membrane-spanning helices (underlined in Fig. 1). The hydrophobic N-terminal part of ORF1 might be a secretory signal sequence. According to the - 3 / 1 rule (for review see [23]) ORF1 is predicted to be a secretory membrane protein which is cleaved between amino acid residues 48 and 49. This work was supported by the Deutsche Forschungsgemeinschaft within the research activity of the Forschergruppe Pu 28/14-3.
References [1] Mikami, B. and Ida, S. (1984) Biochim. Biophys. Acta 791,294-304. [2] Manzano, C., Candau, P., Gomez-Moreno, C., Relimpio, A.M. and Losada, M. (1976) Mol. Cell. Biochem. 10, 161-169. [3] Bednarz, J. and Schmid, G.H. (1991) Z. Naturforsch. 46c, 591-596. [4] Bednarz, J. and Schmid, G.H. (1992) Z. Naturforsch. 47c, 540-544. [5] Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning. A Laboratory Manual, 2nd Edn., Cold Spring Harbor Laboratory Press, Cold Spring Harbor. [6] Tandeau de Marsac, N. and Houmard, J. (1987) The cyanobacteria (Fay, P. and Van Baalen, C., eds.), pp. 251-302, Elsevier, Amsterdam. [7] Lin, J.T., Goldman, B.S. and Stewart, V. (1993) J. Bacteriol. 175, 2370-2378. [8] Siddiqui, R.A., Warnecke-Eberz, U., Hengsberger, A., Schneider, B., Kostka, S. and Friedrich, B. (1993) J. Bacteriol. 175, 5867-5876. [9] Blasco, F., Iobbi, C., Giordano, G., Chippaux, M. and Bonnefoy, V. (1989) Mol. Gen. Genet. 218, 249-256. [10] Blasco, F., Iobbi, C., Ratouchniak, J., Bonnefoy, V. and Chippaux, M. (1990)Mol. Gen. Genet. 222, 104-111. [11] Shuber, A.P., On-, E.C., Recny, M.A., Schendel, P.F., May, H.D., Schauer, N.L. and Ferry, J.G. (1986) J. Biol. Chem. 261, 1294212947.
Fig. 2. Alignment of predicted amino acid sequences of NarB from O. chalybea (O) and Synechococcus sp. PCC 7942 (S) with nitrate reductase from Alcaligenes eutrophus (NapA), domains of nitrate reductases from Klebsiella pneumoniae (NasA) and Escherichia coli (NarG), and formate dehydrogenase from Methanobacterium formicicum (FdhA). Amino acid residues are indicated in the standard single-letter code. Identical amino acid residues in the two cyanobacterial NarB proteins are boxed. Identical amino acid residues in the six aligned sequences are marked in boldface letters. Amino acid residues in FdhA or NapA also found in one or both of the cyanobacterial NarB sequences are indicated by vertical lines. Protein domains conserved in these sequences are underlined and numbered (for detailed discussion see text). A conserved cysteine residue in domain 3 which was shown to be selenocysteine in E. coli formate dehydrogenases FdhF and FdnG is marked by an asterisk.
24
M. Unthan et al. / Biochimica et Biophysica Acta 1305 (1996) 19-24
[12] Zinoni, F., Birkmann, A., Stadtman, T.C. and BiSck, A. (1986) Proc. Natl. Acad. Sci. USA 83, 4650-4654. [13] Berg, B.L., Li, J., Heider, J. and Stewart, V. (1991) J. Biol. Chem. 266, 22380-22385. [14] Bilous, P.T., Cole, S.T., Anderson, W.F. and Weiner, J.H. (1988) Mol. Microbiol. 2, 785-795. [15] Bruschi, M. and Guerlesquin, F. (1988) FEMS Microbiol. Rev. 54, 155-176. [16] Cone, J.E., Del Rio, R.M., Davis, J.N. and Stadtman, T.C. (1976) Proc. Natl. Acad. Sci. USA 73, 2659-2663 [17] B~Sck,A., Forchhammer, K., Heider, J., Leinfelder, W., Sawers, G., Veprek, B. and Zinoni, F. (1991) Mol. Microbiol. 5, 515-520
[18] Suzuki, I., Sugiyama, T. and Omata, T. (1993) Plant Cell Physiol. 34, 1311-1320. [19] Omata, T. (1991) Plant Cell Physiol. 32, 151-157. [20] Omata, T., Andriesse, X. and Hirano, A. (1993) Mol. Gen. Genet. 236, 193-202. [21] Unkles, S.E., Hawker, K,L., Grieve, C., Campbell, E.I., Montague, P. and Kinghorn, J.R. (1991) Proc. Natl. Acad. Sci. USA 88, 204-208. [22] Quesada, A., Galv~n, A. and Fernandez, E. (1994) Plant J. 5, 407-419. [23] Von Heijne, G. (1986) Nucleic Acids Res. 14, 4683-4690.
Biochi~ic~a et Biophysica A~ta Biochimica et Biophysica Acta 1305 (1996) 19-24
Short sequence-paper
Nucleotide sequence of the narB gene encoding assimilatory nitrate reductase from the cyanobacterium Oscillatoria chalybea 1 Monika Unthan a, Werner Klipp b, Georg H. Schmid a,, a Biologie VIII: Zellphysiologie, Universitiit Bielefeld, Postfach 100131, D-33501 Bielefeld, Germany b Biologie VI: Genetik, Universit~t Bielefeld, Bielefeld, Germany Received 29 August 1995; revised 13 October 1995; accepted 17 October 1995
Abstract
The nucleotide sequence of the structural gene of nitrate reductase (narB) has been determined from the filamentous, non-heterocystous cyanobacterium Oscillatoria chalybea. The narB gene encodes a protein of 737 amino acid residues, which shows 61% identity to nitrate reductase of the unicellular cyanobacterium Synechococcus sp. PCC 7942 and only weak homologies to different bacterial molybdoenzymes, such as nitrate reductases or formate dehydrogenases. Keywords: Nitrate reductase; Molybdoenzymes; narB gene; Cyanobacterium; (O. chalybea)
Cyanobacterial nitrate reductases consist of single polypeptides of about 80-85 kDa containing one molybdenum cofactor and one [4Fe-4S] or two [2Fe-2S] cluster(s). Reduced ferredoxin was shown to be the physiological electron donor for the enzyme in a variety of cyanobacteria, such as Plectonema boryanum [1] or Synechococcus sp. PCC 7942, also known as Anacystis nidulans R2 [2]. In contrast to Synechococcus sp. PCC 7942, transcription of narB, the structural gene of nitrate reductase, occurred in OsciUatoria chalybea not only in the presence of nitrate but also in media containing arginine as nitrogen source [3,4]. Therefore, regulation of nitrate reductase in the two cyanobacteria is different. In order to study the structure of narB in O. chalybea the corresponding gene was cloned and sequenced. The following procedure was used to obtain chromosomal DNA from O. chalybea: after harvesting cultures of O. chalybea, the pelleted cells were resuspended in a saturated solution of sodium iodide (NaI) and incubated at 37°C for 60 min to remove exopolysaccharides. The NaI containing supernatant was removed after centrifugation, and sucrose buffer (50 mM Tris/50 mM NaCI/5 mM
* Corresponding author. Fax: +49 521 1065626. J The nucleotide sequence data reported in this paper have been submitted to the EMBL/GenBank Data Libraries under the accession number X89445. 0167-4781/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved SSDI 0 1 6 7 - 4 7 8 1 ( 9 5 ) 0 0 2 1 0 - 3
EDTA/20% sucrose/pH 8.0) containing 5 m g / m l lysozyme was added to the precipitate. Sucrose/lysozyme treatment was carded out at 37°C for 90 min. O. chalybea spheroplasts were collected by centrifugation and incubated with 2% SDS in sucrose buffer (37°C, 90 min). After repeated freezing ( - 8 0 ° C , 60 min) and thawing (37°C, 60 min) lysis of speroplasts occurred. Chromosomal DNA was obtained by phenol/chloroform extraction and ethanol precipitation employing standard DNA purification techniques [5]. The narB gene of O. chalybea was identified from size fractionated gene banks by hybridization with two oligonucleotides (marked in Fig. 1). These oligonucleotides were designed on the basis of cyanobacterial codon preference [6] according to two amino acid sequences highly conserved between nitrate reductase of Klebsiella pneumoniae (NasA) and related sequences of other organisms [7] including NarB of Synechococcus sp. PCC 7942 (EMBL database, accession No. X74597). Two DNA fragments have been cloned containing overlapping parts of the narB gene of O. chalybea. The plasmid pOCll containing a 3.7 kb HindIII fragment includes the 3' region of narB. The plasmid pOC21 carrying a 3.8 kb EcoRI fragment spans the 5' region of narB. Both DNA strands of a 3027 bp fragment from the narB gene region were sequenced using fluorescent dideoxynucleotides and an automatic laser fluorescent DNA sequencer. The complete nucleotide sequence of this DNA
20
M. Unthan et al. / Biochimica et Biophysica Acta 1305 (1996) 19-24
region is presented in Fig. 1 and narB was shown to be located between positions 347 and 2560. The 3' end of an open reading frame (ORF1) is located immediately upstream of narB. The stop codon of narB is separated by only 14 bps from the ATG start codon of an additional open reading frame (ORF2). The narB gene encodes a protein of 737 amino acid residues with a calculated molecular weight of 81 648. As shown in Fig. 2 the deduced amino acid sequence of NarB from O. chalybea shows significant homology to NarB of
Synechococcus sp. PCC 7942 (61% identity). Identical amino acid residues are equally distributed over the entire length of the proteins. In contrast homologies to nitrate reductases of Alcaligenes eutrophus (NapA) [8], Klebsiella pneumoniae (NasA) [7] and Escherichia coli (NarG, NarZ) [9,10] are mainly restricted to nine domains and the overall homology is only about 30%. However, comparing the deduced amino acid sequences of nitrate reductases from A. eutrophus, K. pneumoniae and E. coli to each other, also
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G C A T T A C A G T A G A A G ~ T T G G A A A C C G C A G C ~ G A T A T T ~ C G G A A T C A G ~ C G A G T G C T A T c T T T 306 W S M G I N Q S S E G T A K A R T L I N L H L M T A Q I ~26~ATGGT~GATGGGTATT~CC~T~T~AAc~Ac~c~Tc~cA~TTT~TT~TTACATTT~TGACC~TCAAATAGGT~ 336 P G A G P F S L IT G O P N A ~ GI G R E A G G L S H L L P 1351 A C C G ~ A G C G G G G C C A T T T T C T C T ~ C C ~ C A A C C C ~ C ~ T G G G A G G ~ G A G ~ G C A G ~ C T T T C T C A T ~ A T T A C C G G ~ T A 366 R V V K N A R H R S E V E K F W G L P V G R I A P E R G 1441 T C G A G T A G T A A A A A A T G C C C ~ c A T C G A T C G G ~ G T A G A A A A A T T T T G G G G A T T A C C A G T A G ~ C G C A T T G ~ C C G G A A A G G G G T T T G A C 396 V W E M I L G L E M G K V G L V W I A A T N P A V S M P 1531 G G T A T G G G A A A T ~ T T C T C G G T T T G G A A A T G G G A A A A G T T G G C T T G G T T T G G A T C G C G G C ~ C ~ T C C A G C G G T ~ G T A T G C C A ~ T T T 426 E R T K K A L S R S P F T V Y Q D A Y Y P T E T A 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Fig. 1. Nucleotide and deduced amino acid sequences of O. chalybea narB (nucleotides 347-2560) and partial sequences of ORF1 (nucleotides 1-327) and ORF2 (nucleotides 2575-3027). Two amino acid sequences highly conserved between nitrate reductases from a variety of organisms, which were used to design oligonucleotides for the isolation of O. chalybea narB DNA, are boxed. Three putative membrane-spanning helices of the ORF2 protein are underlined. The putative signal peptide in ORF2 is marked by shaded boxes (amino acid residues 1-48).
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M. Unthan et aL / Biochimica et Biophysica Acta 1305 (1996) 19-24
revealed only overall homologies in the range of approx. 30%. Although, the two cyanobacterial sequences exhibit significant homology to each other, they are as diverse from the other bacterial nitrate reductases as the others are from each other. As shown in Fig. 2, the domains conserved in nitrate reductases from different species are also present in other molybdoenzymes like formate dehydrogenases from Methanobacterium formicicum (FdhA) [11]. In addition, these domains were also found in formate dehydrogenases FdhF [12] and FdnG [13] from E. coli (data not shown). Four conserved domains (2, 4, 6, 8) marked in Fig. 2 have been identified not only in nitrate reductases and formate dehydrogenases but also in other molybdoenzymes like dimethylsulfoxide reductase DmsA [14] and biotin sulfoxide reductase BisC (SwissProt database, accession No. P20099) from E. coli. Therefore, these domains have been discussed to be involved in binding of the molybdenum-pterin cofactor [9]. A conserved cysteine motif (1) is located in the Nterminal part of bacterial nitrate reductases. Since the C-X2-C-X3-C-X39-C motif found in NarB of O. chalybea and Synechococcus sp. PCC 7942 resembles the C-X2-CX3-C-X30_36-Cmotif known to be involved in ligating one of the two [4Fe-4S] clusters in bacterial ferredoxins [15], it is likely that nitrate reductases and formate dehydrogenases harbour one [4Fe-4S] center. Domain 3 might be involved in catalytic activity since a conserved cysteine residue is present in all nitrate reductases and formate dehydrogenases sequenced so far. This cysteine residue is substituted by selenocysteine in formate dehydrogenase FdhF and FdnG from E. coli [12,13] an amino acid derivative known to be located in the active center of a variety of enzymes [16,17]. Three other conserved domains (5, 7 and 9) have been found in cyanobacterial nitrate reductases and in some other prokaryotic molybdoproteins. The functions of these regions remain unknown. In summary, the amino acid sequence comparisons presented in Fig. 2 indicate that nitrate reductases from cyanobacteria, which were shown to be closely related to each other, are clearly distinct from nitrate reductases from other bacterial species and homologies are limited to domains also found in a variety of other molybdoenzymes. Although the nitrate reductases from O. chalybea and Synechococcus sp. PCC 7942 seem to be regulated in different ways, no significant distinction in the enzyme
23
structures has been observed. The Synechococcus sp. PCC 7942 narB gene was shown to be part of the nirAnrtABCD-narB operon. The nirA gene encodes nitrite reductase [18] and nrtABCD are involved in nitrate uptake [19,20]. Partially sequenced genes located immediately upstream (ORF1) and downstream (ORF2) of narB in O. chalybea, respectively (Fig. 1), indicate that in this cyanobacterium narB is also part of an operon. ORF1 shows weak homology to nitrate transporter proteins from the filamentous fungus Aspergillus nidulans (CrnA) [21] and the green alga Chlamydomonas reinhardtii (Nar-3, Nar-4) [22]. However, no homology to NrtABCD nitrate transporter proteins in Synechococcus could be detected. ORF2 shows no homology to known genes or proteins. Hydropathy plots of the deduced amino acid sequence of ORF1 predicted three putative membrane-spanning helices (underlined in Fig. 1). The hydrophobic N-terminal part of ORF1 might be a secretory signal sequence. According to the - 3 / 1 rule (for review see [23]) ORF1 is predicted to be a secretory membrane protein which is cleaved between amino acid residues 48 and 49. This work was supported by the Deutsche Forschungsgemeinschaft within the research activity of the Forschergruppe Pu 28/14-3.
References [1] Mikami, B. and Ida, S. (1984) Biochim. Biophys. Acta 791,294-304. [2] Manzano, C., Candau, P., Gomez-Moreno, C., Relimpio, A.M. and Losada, M. (1976) Mol. Cell. Biochem. 10, 161-169. [3] Bednarz, J. and Schmid, G.H. (1991) Z. Naturforsch. 46c, 591-596. [4] Bednarz, J. and Schmid, G.H. (1992) Z. Naturforsch. 47c, 540-544. [5] Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning. A Laboratory Manual, 2nd Edn., Cold Spring Harbor Laboratory Press, Cold Spring Harbor. [6] Tandeau de Marsac, N. and Houmard, J. (1987) The cyanobacteria (Fay, P. and Van Baalen, C., eds.), pp. 251-302, Elsevier, Amsterdam. [7] Lin, J.T., Goldman, B.S. and Stewart, V. (1993) J. Bacteriol. 175, 2370-2378. [8] Siddiqui, R.A., Warnecke-Eberz, U., Hengsberger, A., Schneider, B., Kostka, S. and Friedrich, B. (1993) J. Bacteriol. 175, 5867-5876. [9] Blasco, F., Iobbi, C., Giordano, G., Chippaux, M. and Bonnefoy, V. (1989) Mol. Gen. Genet. 218, 249-256. [10] Blasco, F., Iobbi, C., Ratouchniak, J., Bonnefoy, V. and Chippaux, M. (1990)Mol. Gen. Genet. 222, 104-111. [11] Shuber, A.P., On-, E.C., Recny, M.A., Schendel, P.F., May, H.D., Schauer, N.L. and Ferry, J.G. (1986) J. Biol. Chem. 261, 1294212947.
Fig. 2. Alignment of predicted amino acid sequences of NarB from O. chalybea (O) and Synechococcus sp. PCC 7942 (S) with nitrate reductase from Alcaligenes eutrophus (NapA), domains of nitrate reductases from Klebsiella pneumoniae (NasA) and Escherichia coli (NarG), and formate dehydrogenase from Methanobacterium formicicum (FdhA). Amino acid residues are indicated in the standard single-letter code. Identical amino acid residues in the two cyanobacterial NarB proteins are boxed. Identical amino acid residues in the six aligned sequences are marked in boldface letters. Amino acid residues in FdhA or NapA also found in one or both of the cyanobacterial NarB sequences are indicated by vertical lines. Protein domains conserved in these sequences are underlined and numbered (for detailed discussion see text). A conserved cysteine residue in domain 3 which was shown to be selenocysteine in E. coli formate dehydrogenases FdhF and FdnG is marked by an asterisk.
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M. Unthan et al. / Biochimica et Biophysica Acta 1305 (1996) 19-24
[12] Zinoni, F., Birkmann, A., Stadtman, T.C. and BiSck, A. (1986) Proc. Natl. Acad. Sci. USA 83, 4650-4654. [13] Berg, B.L., Li, J., Heider, J. and Stewart, V. (1991) J. Biol. Chem. 266, 22380-22385. [14] Bilous, P.T., Cole, S.T., Anderson, W.F. and Weiner, J.H. (1988) Mol. Microbiol. 2, 785-795. [15] Bruschi, M. and Guerlesquin, F. (1988) FEMS Microbiol. Rev. 54, 155-176. [16] Cone, J.E., Del Rio, R.M., Davis, J.N. and Stadtman, T.C. (1976) Proc. Natl. Acad. Sci. USA 73, 2659-2663 [17] B~Sck,A., Forchhammer, K., Heider, J., Leinfelder, W., Sawers, G., Veprek, B. and Zinoni, F. (1991) Mol. Microbiol. 5, 515-520
[18] Suzuki, I., Sugiyama, T. and Omata, T. (1993) Plant Cell Physiol. 34, 1311-1320. [19] Omata, T. (1991) Plant Cell Physiol. 32, 151-157. [20] Omata, T., Andriesse, X. and Hirano, A. (1993) Mol. Gen. Genet. 236, 193-202. [21] Unkles, S.E., Hawker, K,L., Grieve, C., Campbell, E.I., Montague, P. and Kinghorn, J.R. (1991) Proc. Natl. Acad. Sci. USA 88, 204-208. [22] Quesada, A., Galv~n, A. and Fernandez, E. (1994) Plant J. 5, 407-419. [23] Von Heijne, G. (1986) Nucleic Acids Res. 14, 4683-4690.