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Cloning and characterization of the flavodoxin gene from Desulfovibrio desulfuricans
Biochimica et Biophysica Acta, 1089 (1991) 417-419 © 1991 Elsevier Science Publishers B.V. 0167-4781/91/$03.50 ADONIS 016747819100188U
417
BBAEXP 90252
Short Sequence-Paper
Cloning and characterization of the flavodoxin gene from Desul.fovibrio desulfuricans Larry R. Helms and Richard P. Swenson Department of Biochemistry, The Ohio State Unirersity, Columbus, OH (U.S~.) (Received 23 May 1991)
Key words: F'avodoxin; Structural gene sequence; Amino acid sequence; (Desulforibrio desulfuricans)
The gene coding for the flavodoxin protein from Desulfovibrio desulfuricans [Essex 6] (ATCC 29577) has been cloned and sequenced. The gene was identified on Southern blots of Hindlll-digested genomic DNA by hybridization to the coding region for the flavodoxin from Desu/fov/br/o vu/gar/s [HiMenhoroughl (Krey, G.D., Vanin, E.F. and Swenson, ILP. (1988) J. Biol. Chem. 263, 1~t36-15443). Ultimately, a 1.8 kb Taql fragment was cloned which contains an open reading frame of 447 nucleotides coding for an acidic protein of 148 amino acids and calculated molecular weight of 15 726. The derived amino acid sequence of this protein is 47% identical to the flavodoxin from D. mdgar/s. Regions of the polypeptide which form the flavin mononueleotide binding site are largely homologous; however, some perhaps significant differences are noted. The aromatic amino acid residues that flank the flavin isoalloxaziue ring in the D. vu/gar/s structure, Le., tryptophan-60 and tyrosine-98, are conserved in this flavodoxin. Flavodoxins are small, electron transfer proteins that contain one equivalent of noncovalently bound flavin mononucleotide as their only redox cofactor. The flavodoxins from a variety of sources have been identified and characterized to various extents, including, in some cases, the establishment of tertiary structures by X-ray crystallography [1-4]. Likewise, the flavodoxin proteins from several members of the Desulfocibrio genus have been purified and partially characterized [5]. Physical studies suggest that some members of this flavodoxin family may have distinctly different flavin binding sites; however, other evidence would seem to indicate significant similarities [6]. As an ongoing project, we have cloned and sequenced the flavodoxin genes from several of the Desulfovibrio family in order to establish the primary structure as well as other properties of these flavodoxins [2,7]. In this paper, we report the cloning and sequence analysis of th~ gene coding for the flavodoxin protein from D. desul~aricans.
The sequence data in this paper have beer~ submitted to the EMBL/GenBank Data Libraries and assigned the accession nmaber X59438. Correspondence: R.P. Swenson, Department of Biochemistry, 776 BioSciences Bldg., The Ohio State University, 484 West 12th Avenue, Columbus, OH 43210, U.S.A.
Cultures of Desulfot'ibrio desulfuricans [Essex 6 strain] (ATCC 29577, type strain) were obtained from American Type Culture Collection and propagated in modified Baar's media under anaerobic conditions [8]. Genomic DNA was isolated as previously described [2]. Restriction fragments of genomic DNA which contain the flavodoxin structural gene were identified by hybridization of Southern blots with radiolabeled probes under conditions similar to those described previously [7]. Two probes were utilized in this study - the coding region for the flavodoxin from D. rulgaris [Hiidenborough] (ATCC 29579) (as a 450 bp Hindlll fragment) [2] and that from D. gigas (ATCC 29494) (as an 800 bp Ncol-HindIIl fragment (unpublished results)). Both probes were found to hybridize consistently to a 4.0 kb fragment in Southern blots of HindIll-digested D. desulfuricans genomic DNA. A mini-library of sizeselected H/ndIII fragments (3.8 to 5.5 kb) was constructed in the Bluescript vector (Stratagene) and screened by colony hybridization with the 800 bp D. gigas probe using previously described methods [6]. Of the 900 recombinant clones, three putative positives were identified and subsequently found to contain a 4.0 kb Hindlll fragment which hybridized strongly. The region responsible for hybridization was confined by restriction mapping to a 1.6 kb HindlII-Taqi fragment at the 5' end of this fragment. Nucleotide sequence analysis of this fragment by the Sanger dideoxy
418
TaqI ~ I l l
s'---I
NcolNcoI T
~.I 3
T
...,
lO0bp
Fig. I. Partial reslriction map and sequencing strategy for the 1.8 kb
Taqi fragment containing the flavodoxin gene from D. desulfuricans. The black rectangle represents the coding region for the flavodoxin gene. The arrows represent the regions sequenced on each strand of the UNA.
termination method [9] established that although the flavodoxin structural gene was present, a portion coding for approx. 20 amino acids at the amino-terminal was missing. Knowing that the orientation of the gene was from the Hindlll to the Taril site in the 5' to 3' direction, Southern blots of genomic DNA digested with Taql plus several other restriction enzymes were screened to identify a larger fragment containing the entire coding region. A 1.8 kb Taql fragment was identified using the radiolabeled 1.6 kb Hindlll-Taql fragment as a probe. Again, a mini-library of sizeselected Taql fragments was constructed by ligation into the C/al site of the Bluescript (Stratagene) vector. One clone out of 600 recombinants was found to contain the plasmid carrying the 1.8 kb Taql fragment.
The nucleotide sequencing scheme as well as the partial restriction map of the 1.8 kb Taql fragment containing the flavodoxin gene from D. desulfuricans is shown in Fig. 1. The nucleotide sequence spanning the structural gene is shown in Fig. 2. An open reading frame of 447 nucleotides which begins with an ATG initiation codon at nucleotide 77 is observed. A possible Shine-Dalgarno sequence (underlined) was found six nucleotides upstream from the initiation codon. The promoter region is probably not present in this Taql fragment. The open reading frame codes for an acidic protein (calculated isoelectric point of 4.0) of 148 amino acids with a calculated molecular weight of 15 726. The derived amino acid sequence (included in Fig. 2) is homologous to other flavodoxins from this family, sharing a sequence identity of 47% and 48% with the flavodoxins from D. r,ulgaris [2] and D. salexigens [7], respectively. The regions that form the flavin mononucleotide binding site are largely homologous as are the hydroxyamino acids that hydrogen bond to the ribityl side chain and phosphate moiety. Just as with other flavodoxins isolated from the De.~ulfovibrio family [2,7] this flavodoxin contains the same two aromatic residues (tryptophan-60 and tyrosine-98) that fank the isoalloxazine ring in the D. vulgaris flavodoxin crystal structure [1]. However, this represents the only trypto-
5' CAAGCTTATCGAACATCCCTATCCATTTGtUU~ATGAATTTCAATTTT/O,A~T~GACGC~k~AG~TC ATG AGT AAG GTA CTG ATT GTT T'rT GGT TCC &GC &CC GGC bAT ACG GAA AGC ATC GCC GAG MET SER LYS VAL LEU l i e VAL PHE GLY SER SER THR GLY ASN THR GLU SL~ ILE MA GIJ4 10 20 I AAG CTT GAA GAA CTG ATT GCC GCT GGC GGG CAC GAb. GTC ACG CTG CTT AAC GCG GCG GAC LYS LEU GLU GLU LEU I I E ~ AIA GLY GLY HIS GLU VAL THR LEU I.KU ASN MA AIA ASP 30 40 GCC TCG GCA GAA AAT CTG GCT GAC GOT TAT GAT GCA GTG CTG T~T GGC TGC TCG GCC TGG Pti~ GLY CYS SER MA TRP AIA SFR MA GLU ASN LZU ALA ASP GL¥ TYR ASP MA VkL ~ 50 60 GGC ATG GAA OAT CTG G/dk ATG CAG GAC GAC TTT TTA TCC CTG TTT GAG GAA 'Fir AAC CGC GLY MET GLU ASP LEO GLU MET GIN ASP ASP PHE LEU SER LEU PIlE GLU GLU PHE ASN kRG 70 80 ATC GGG CTG GCT GGC CGC tAG GTA GCC GCC TTT GCA TCC GGC GAC CAG GtA TAT GAA CAT l i e GLY LEU MA GLY ARG LYS YAL MA MA PHE AIA SER GL¥ ASP GLIq GLU TYR GLU HIS 90 100 TTT TGC GGC GCG GTG CCT GCC &TT O ~ GAG CGC GCC tAG GAA CTG GGC GCG ACC ATC ArT PHZ CYS GLY AIA VAI, PRO tJ.A I I ~ GLU GLU ARG AIA LYS GLU LEU GLY MA THR ILZ ILK 110 120 GCC GAG GGG CTC /~G ATG G/~ GGC GAT GCC TCC AAC GAC CCC G/~ GCC GTG GC/. TCG TTT AIA GLU GL¥ LEU L¥S HEr GLU GL¥ ASP AIA SER ASN ASP PRO GLU /~A VAL MA SER PHE 130 140 GCC GAG GAT GTG CTC tAG CAG CTG T ~ tATTCT 3* MA GLU ASP VAL LEU LYS GIN LEU * * t Fig. 2. Nucleotide sequence of the flavodoxin gene from D. desulfuricans. The sequence shown i~ the non-transcribed strand. The translated amino acid sequence for the flavodoxin polypeptide is indicated below the open coding region of the gene. The termination codon is represented by three asterisks, the potential Shine-Dalgarno sequence is underlined.
419 phan residue in this flavodoxin, with a phenylalanine residue replacing a second tryptophan residue frequently observed within the carboxyl-terminal a-helix [2,7]. Other perhaps significant differences in the polypeptide loops forming the flavin binding site are noted. For example, a methionine residue replaces either an aspartate or glutamate at position 62, an acidic residue implicated in the stabilization of the electron transfer complex between flavodoxin and cytochrome c 3 [7,10]. Also, tyrosine-100 in the D. vulgaris [2] and D. salexigens [7] flavodoxins is replaced in the sequence by a histidine residue in this flavodoxin, This residue is within 6 ~ of the isoalloxazine ring in the D. vu/gar/s flavodoxin [1] and would represent the first example of a basic residue this close to the flavin in this family of flavodoxin, This research was supported by a grant from the National Institutes of Health (GM36490).
References I Mayhew, S.G. and Ludwig, M.L. (1975) in The Enzymes (Boyer, P.D, ed.), 3rd Edn., Vol. 12. pp, 57-118. 2 Krey, G.D., Vanin, E.F. and Swenson, R.P. (1988) J. Biol. Chem. 263, 15436-15443. 3 Smith, W,W., Partridge, K.A., Ludwig, M.L., Petsko, G.A., Tsernogiou. D., Tanaka, M. and Yasunobu, K.T. (1983) J, Mol. Biol. 165, 737-755. 4 Wakabayashi, S., Kimura, T., Fukuyama. K., Matsubara, H, and Rogers, L.J. (1989) Biochem. J. 263, 981-984. 5 Moura, I., Moura, JJ.G., Bruschi, M. and LcGall, J. (1980) Biochim. Biophys. Acta 591, 1-8. 6 Favaudon, V., LeGall, J. and Lhoste, J.M. (1980) in Flavins and Flavoproteins (Yagi, K. and Yamamo, eds.), pp. 373-386, Japan Scientific Societies Press, Tokyo. 7 Helms. LR., grey, G,D. and Swenson, R,P. (1990) Biochem, Biophys. Res. Commun. 168. 809-817. 8 Postgae, J.R. (1984) The Sulfate-Reducing Bacteria, 2nd Edn.. p. 32, Cambridge University Press, Cambridge. 9 Sanger, F., Nicklen. S. and Coulson, A.R. (1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467. 10 Stewart, D.E., LeGaU, J., Moura, 1., Moura, J.J.G., Peck, Jr, H,D., Xavier, A.V.0 Weiner, P.K. and Wampler, J,E. (1988) Biochemistry 27, 2444-2450.
417
BBAEXP 90252
Short Sequence-Paper
Cloning and characterization of the flavodoxin gene from Desul.fovibrio desulfuricans Larry R. Helms and Richard P. Swenson Department of Biochemistry, The Ohio State Unirersity, Columbus, OH (U.S~.) (Received 23 May 1991)
Key words: F'avodoxin; Structural gene sequence; Amino acid sequence; (Desulforibrio desulfuricans)
The gene coding for the flavodoxin protein from Desulfovibrio desulfuricans [Essex 6] (ATCC 29577) has been cloned and sequenced. The gene was identified on Southern blots of Hindlll-digested genomic DNA by hybridization to the coding region for the flavodoxin from Desu/fov/br/o vu/gar/s [HiMenhoroughl (Krey, G.D., Vanin, E.F. and Swenson, ILP. (1988) J. Biol. Chem. 263, 1~t36-15443). Ultimately, a 1.8 kb Taql fragment was cloned which contains an open reading frame of 447 nucleotides coding for an acidic protein of 148 amino acids and calculated molecular weight of 15 726. The derived amino acid sequence of this protein is 47% identical to the flavodoxin from D. mdgar/s. Regions of the polypeptide which form the flavin mononueleotide binding site are largely homologous; however, some perhaps significant differences are noted. The aromatic amino acid residues that flank the flavin isoalloxaziue ring in the D. vu/gar/s structure, Le., tryptophan-60 and tyrosine-98, are conserved in this flavodoxin. Flavodoxins are small, electron transfer proteins that contain one equivalent of noncovalently bound flavin mononucleotide as their only redox cofactor. The flavodoxins from a variety of sources have been identified and characterized to various extents, including, in some cases, the establishment of tertiary structures by X-ray crystallography [1-4]. Likewise, the flavodoxin proteins from several members of the Desulfocibrio genus have been purified and partially characterized [5]. Physical studies suggest that some members of this flavodoxin family may have distinctly different flavin binding sites; however, other evidence would seem to indicate significant similarities [6]. As an ongoing project, we have cloned and sequenced the flavodoxin genes from several of the Desulfovibrio family in order to establish the primary structure as well as other properties of these flavodoxins [2,7]. In this paper, we report the cloning and sequence analysis of th~ gene coding for the flavodoxin protein from D. desul~aricans.
The sequence data in this paper have beer~ submitted to the EMBL/GenBank Data Libraries and assigned the accession nmaber X59438. Correspondence: R.P. Swenson, Department of Biochemistry, 776 BioSciences Bldg., The Ohio State University, 484 West 12th Avenue, Columbus, OH 43210, U.S.A.
Cultures of Desulfot'ibrio desulfuricans [Essex 6 strain] (ATCC 29577, type strain) were obtained from American Type Culture Collection and propagated in modified Baar's media under anaerobic conditions [8]. Genomic DNA was isolated as previously described [2]. Restriction fragments of genomic DNA which contain the flavodoxin structural gene were identified by hybridization of Southern blots with radiolabeled probes under conditions similar to those described previously [7]. Two probes were utilized in this study - the coding region for the flavodoxin from D. rulgaris [Hiidenborough] (ATCC 29579) (as a 450 bp Hindlll fragment) [2] and that from D. gigas (ATCC 29494) (as an 800 bp Ncol-HindIIl fragment (unpublished results)). Both probes were found to hybridize consistently to a 4.0 kb fragment in Southern blots of HindIll-digested D. desulfuricans genomic DNA. A mini-library of sizeselected H/ndIII fragments (3.8 to 5.5 kb) was constructed in the Bluescript vector (Stratagene) and screened by colony hybridization with the 800 bp D. gigas probe using previously described methods [6]. Of the 900 recombinant clones, three putative positives were identified and subsequently found to contain a 4.0 kb Hindlll fragment which hybridized strongly. The region responsible for hybridization was confined by restriction mapping to a 1.6 kb HindlII-Taqi fragment at the 5' end of this fragment. Nucleotide sequence analysis of this fragment by the Sanger dideoxy
418
TaqI ~ I l l
s'---I
NcolNcoI T
~.I 3
T
...,
lO0bp
Fig. I. Partial reslriction map and sequencing strategy for the 1.8 kb
Taqi fragment containing the flavodoxin gene from D. desulfuricans. The black rectangle represents the coding region for the flavodoxin gene. The arrows represent the regions sequenced on each strand of the UNA.
termination method [9] established that although the flavodoxin structural gene was present, a portion coding for approx. 20 amino acids at the amino-terminal was missing. Knowing that the orientation of the gene was from the Hindlll to the Taril site in the 5' to 3' direction, Southern blots of genomic DNA digested with Taql plus several other restriction enzymes were screened to identify a larger fragment containing the entire coding region. A 1.8 kb Taql fragment was identified using the radiolabeled 1.6 kb Hindlll-Taql fragment as a probe. Again, a mini-library of sizeselected Taql fragments was constructed by ligation into the C/al site of the Bluescript (Stratagene) vector. One clone out of 600 recombinants was found to contain the plasmid carrying the 1.8 kb Taql fragment.
The nucleotide sequencing scheme as well as the partial restriction map of the 1.8 kb Taql fragment containing the flavodoxin gene from D. desulfuricans is shown in Fig. 1. The nucleotide sequence spanning the structural gene is shown in Fig. 2. An open reading frame of 447 nucleotides which begins with an ATG initiation codon at nucleotide 77 is observed. A possible Shine-Dalgarno sequence (underlined) was found six nucleotides upstream from the initiation codon. The promoter region is probably not present in this Taql fragment. The open reading frame codes for an acidic protein (calculated isoelectric point of 4.0) of 148 amino acids with a calculated molecular weight of 15 726. The derived amino acid sequence (included in Fig. 2) is homologous to other flavodoxins from this family, sharing a sequence identity of 47% and 48% with the flavodoxins from D. r,ulgaris [2] and D. salexigens [7], respectively. The regions that form the flavin mononucleotide binding site are largely homologous as are the hydroxyamino acids that hydrogen bond to the ribityl side chain and phosphate moiety. Just as with other flavodoxins isolated from the De.~ulfovibrio family [2,7] this flavodoxin contains the same two aromatic residues (tryptophan-60 and tyrosine-98) that fank the isoalloxazine ring in the D. vulgaris flavodoxin crystal structure [1]. However, this represents the only trypto-
5' CAAGCTTATCGAACATCCCTATCCATTTGtUU~ATGAATTTCAATTTT/O,A~T~GACGC~k~AG~TC ATG AGT AAG GTA CTG ATT GTT T'rT GGT TCC &GC &CC GGC bAT ACG GAA AGC ATC GCC GAG MET SER LYS VAL LEU l i e VAL PHE GLY SER SER THR GLY ASN THR GLU SL~ ILE MA GIJ4 10 20 I AAG CTT GAA GAA CTG ATT GCC GCT GGC GGG CAC GAb. GTC ACG CTG CTT AAC GCG GCG GAC LYS LEU GLU GLU LEU I I E ~ AIA GLY GLY HIS GLU VAL THR LEU I.KU ASN MA AIA ASP 30 40 GCC TCG GCA GAA AAT CTG GCT GAC GOT TAT GAT GCA GTG CTG T~T GGC TGC TCG GCC TGG Pti~ GLY CYS SER MA TRP AIA SFR MA GLU ASN LZU ALA ASP GL¥ TYR ASP MA VkL ~ 50 60 GGC ATG GAA OAT CTG G/dk ATG CAG GAC GAC TTT TTA TCC CTG TTT GAG GAA 'Fir AAC CGC GLY MET GLU ASP LEO GLU MET GIN ASP ASP PHE LEU SER LEU PIlE GLU GLU PHE ASN kRG 70 80 ATC GGG CTG GCT GGC CGC tAG GTA GCC GCC TTT GCA TCC GGC GAC CAG GtA TAT GAA CAT l i e GLY LEU MA GLY ARG LYS YAL MA MA PHE AIA SER GL¥ ASP GLIq GLU TYR GLU HIS 90 100 TTT TGC GGC GCG GTG CCT GCC &TT O ~ GAG CGC GCC tAG GAA CTG GGC GCG ACC ATC ArT PHZ CYS GLY AIA VAI, PRO tJ.A I I ~ GLU GLU ARG AIA LYS GLU LEU GLY MA THR ILZ ILK 110 120 GCC GAG GGG CTC /~G ATG G/~ GGC GAT GCC TCC AAC GAC CCC G/~ GCC GTG GC/. TCG TTT AIA GLU GL¥ LEU L¥S HEr GLU GL¥ ASP AIA SER ASN ASP PRO GLU /~A VAL MA SER PHE 130 140 GCC GAG GAT GTG CTC tAG CAG CTG T ~ tATTCT 3* MA GLU ASP VAL LEU LYS GIN LEU * * t Fig. 2. Nucleotide sequence of the flavodoxin gene from D. desulfuricans. The sequence shown i~ the non-transcribed strand. The translated amino acid sequence for the flavodoxin polypeptide is indicated below the open coding region of the gene. The termination codon is represented by three asterisks, the potential Shine-Dalgarno sequence is underlined.
419 phan residue in this flavodoxin, with a phenylalanine residue replacing a second tryptophan residue frequently observed within the carboxyl-terminal a-helix [2,7]. Other perhaps significant differences in the polypeptide loops forming the flavin binding site are noted. For example, a methionine residue replaces either an aspartate or glutamate at position 62, an acidic residue implicated in the stabilization of the electron transfer complex between flavodoxin and cytochrome c 3 [7,10]. Also, tyrosine-100 in the D. vulgaris [2] and D. salexigens [7] flavodoxins is replaced in the sequence by a histidine residue in this flavodoxin, This residue is within 6 ~ of the isoalloxazine ring in the D. vu/gar/s flavodoxin [1] and would represent the first example of a basic residue this close to the flavin in this family of flavodoxin, This research was supported by a grant from the National Institutes of Health (GM36490).
References I Mayhew, S.G. and Ludwig, M.L. (1975) in The Enzymes (Boyer, P.D, ed.), 3rd Edn., Vol. 12. pp, 57-118. 2 Krey, G.D., Vanin, E.F. and Swenson, R.P. (1988) J. Biol. Chem. 263, 15436-15443. 3 Smith, W,W., Partridge, K.A., Ludwig, M.L., Petsko, G.A., Tsernogiou. D., Tanaka, M. and Yasunobu, K.T. (1983) J, Mol. Biol. 165, 737-755. 4 Wakabayashi, S., Kimura, T., Fukuyama. K., Matsubara, H, and Rogers, L.J. (1989) Biochem. J. 263, 981-984. 5 Moura, I., Moura, JJ.G., Bruschi, M. and LcGall, J. (1980) Biochim. Biophys. Acta 591, 1-8. 6 Favaudon, V., LeGall, J. and Lhoste, J.M. (1980) in Flavins and Flavoproteins (Yagi, K. and Yamamo, eds.), pp. 373-386, Japan Scientific Societies Press, Tokyo. 7 Helms. LR., grey, G,D. and Swenson, R,P. (1990) Biochem, Biophys. Res. Commun. 168. 809-817. 8 Postgae, J.R. (1984) The Sulfate-Reducing Bacteria, 2nd Edn.. p. 32, Cambridge University Press, Cambridge. 9 Sanger, F., Nicklen. S. and Coulson, A.R. (1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467. 10 Stewart, D.E., LeGaU, J., Moura, 1., Moura, J.J.G., Peck, Jr, H,D., Xavier, A.V.0 Weiner, P.K. and Wampler, J,E. (1988) Biochemistry 27, 2444-2450.