Biochhnica et Biophysica Acta, 1129(1991) 119-123 © 1991 Elsevier Science Publishers B.V. All rights reserved Ill67-4781/91/$03.511
BBAEXP 90289
119
Short Sequence-Paper
Lipoamide dehydrogenase of Staphylococcus aureus: nucieotide sequence and sequence analysis Harri Hemil~i hzstitute of Bioteclmolol,,y, Unit'ersityof Helsinki, Helsblki (Finland) (Received 14 August 1991)
Key words: Lipoamide dehydrogenase; Pyruvate dehydrogenase: Ribosome: Membrane transport: pdhD; (StaphyhJcoccu.~aureu.~)
A complex of four proteins was previously isolated from Staphylococcus aureus. The complex had a strong interaction with membrane bound ribosomes, which suggested that it may be involved in protein secretion. However, the complex was identified as pyruvate dehydrogenase (PDH), which disproved the direct role of the complex in protein secretion. Here we report the nucleotide sequence of the last gene of the S. aureus pyruvate dehydrogenase operon, pdhD, which encodes lipoamide dehydrogenase (LPD). The pdhD gene encodes a protein of 468 amino acids, with a molecular mass of 49.5 kDa. The protein is closely related to other lipoamide dehydrogenases from bacteria and eukaryotes. The possible role of membrane bound lipoamide dehydrogenase is briefly discussed.
Lipoamide dehydrogenase (LPD; EC 1.8.1.4) is a ubiquitous flavoprotein, which catalyzes the NAD+-de pendent oxidation of dihydrolipoamide in a number of multienzyme complexes (for a review see Ref. 1). It serves as the E3-subunit for three 2-oxo acid dehydrogenase multienzyme complexes: pyruvate dehydrogenase (PDH), 2-oxoglutarate dehydrogenase ( O G D H ) and branched chain 2-oxo acid dehydrogenase (BCDH). Also, it functions as the L-subunit for the glycine cleavage system (GCS). So far, two isoenzymes of LPD have been found in Escherichia coil [2,3] and mammals [4], while three have been identified in Pseudomonas putida [5-7]. Although the role of the different isoenzymes has not been finally clarified, one isoenzyme in E. coil, encoded by the Ipd gene, functions in PDH, O G D H and GCS [2,8]. In P. putida, Glc-isoenzyme is used in PDH, O G D H and GCS [7], while the Val-isoenzyme is used in B C D H [5]. Previously, a complex of four proteins was isolated from S. aureus [9,10]. This complex was supposed to
participate in protein secretion, since it was attached to membrane bound ribosomes. A homologous complex was isolated from the closely related species Bacillus subtilis [11,12]. However, the complex was identified as PDH, which undermined the original hypothesis of its direct participation in protein secretion [I3,14]. In this paper, we report the nucleotide sequence and deduced amino acid sequence for the last gene of the S. aureus pyruvate dehydrogenase operon, pdhD, which encodes LPD. S. aureus pdhD gene was sequenced P om plasmid pMBR1 [15], which encodes the entire pdh operon (Fig. 1). The nucleotide sequence of pdhD and its deduced amino acid sequence are shown in Fig. 2. The
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The sequence data in this paper have been submitted to the EMBL/Genbank Data Libraries under the accession number X58434. Abbreviations: LPD, lipoamide dehydrogenase; PDH, pyruvate dehydrogenase; OGDH, 2-oxoglutarate dehydrogenase: BCDH. branched chain 2-oxo acid dehydrogenase; GCS, glycine cleavage system. Correspondence: H. Hemil~i,Institute of Biotechnology, Valimotie 7, 00380 Helsinki, Finland.
Fig. 1. Physical map of the S. aureus pdh operon. The bold line represents the region sequenced so far in this report and in I14]. The genes pdlu4, pdhB. pdhC and pdhD encode the PDH subunits Ela. El/3. E2 and E3. respectively. The sizes of the Ela and EI~ proteins have been determining using SDS-PAGE [9]. and their order was determined by deletion studies [15]. PI. P2 and P3 refer to the putative promoters implied by northern analysis of the pdh operon [15]. The restriction enzyme sites for EcoRI (E). Hindill (H). Psti (P), $tyl (S). Accl (A) and Xbal (X) are indicated. The sites outside of the sequenced region were determined by digestions of the pMBRI [15].
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Fig, 2, Nucl¢otide and derived protein sequences of lipoamide dehydrogenas¢ from & auras VS. The nucleotide sequence of pdhD was d~t~rmined ~ m pMBRI [15] as previouslyd~scribcd [13,14]. Th~ ribosomal binding site in ~ n t of the initiation methionine is underlined. The inverted r~p~ut structures downstream of th~ stop codon ar~ marked by arrows under the sequence. Numbering of the sequence is consistent wilh th~ previously published I~lhC g~ne[14]. Both strands of the D N A were sequenced e~ept ~ r the 3' end starting at 2892 bp.
pdhD open reading frame of 1404 bp starts from the ATG-codon located at 1507 bp. A ribosomal binding site is located closely upstream from the initiation codon of pdhD. The pdhD gene encodes a protein of 468 amino acids, which has a molecular mass of 49.5 kDa (Fig. 2). Two inverted repeats are found after the stop codon (TAN at 2911 bp) of the open reading frame. The second of them may serve as the transcription terminator, since the energy of formation of the hairpin loop would be - 5 4 kJ/mol. In the closely related B. subtilis a single isoenzyme of LPD serves as the E3-subunit for both PDH and OGDH [16], and these two complexes appear to be independently regulated [17,18]. This suggests that the
pdhD gene of S. aureus may have its own promoter to express the gene in circumstances when the entire pdh operon is not transcribed. Furthermore, Northern blot analysis of S. aureus and B. subtilis has shown the presence of a short transcript from the pdh operon (1.6 and 1.7 kb, respectively) in addition to a large one covering the entire operon [13,15]. However, upstream from the S. aureus pdhD gene, we found no site closely resembling the known recognition sites of Gram-positive (r factors [19]. The sequences for B. subtilis and B. stearother. mophilus LPDs have been determined [13,20], and 73% of their amino acids are identical to the S. aureus enzyme (Fig. 3). The lipoamide dehydrogenases from
Fig. 3. Comparison of LPD sequences. Identical ( : ), and closely related amino acid~, t.), with a comparison value >_0.5 in the GAP mutation data matrix [27], are marked. The secondary structure assignments of the A. t'inelandii LPD [28] are shown below the alignment: a-helix (bold line}, O-sheet (thin line}. The conserved residues (* } and the residues with conservative substitutions (.) are indicated above the alignment. The residues which form hydrogen bond with FAD (F), and within the dimer interface (I) of A. rinelandii LPD [28] are indicated, as well as the active site cysteines (C). The sequences are numbered starting from the amino acid next to the initiation methionine.
121
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122 the Gram-positive bacteria are also aligned with the other known LPD sequences in Fig. 3. Recently, an alignment of four LPDs was published, but several of the gaps that were suggested [1] would be located within a-helix and/3-sheet regions, suggesting that the alignment is not correct [29], Fig. 3 contains 11 sequences so far known, which allows a more rigorous alignment analysis. Alignment of distantly related sequences may yield information of the most essential residues, which should be conserved in all of the sequences. Multiple sequence alignment complements crystallographic data, which do not indicate the limits for allowed amino acid exchanges. 77 amino acid residues (15%) are conserved in all of the sequences, and the lowest pairwise degree of similarity (38%) is between the enzymes of P. putida and S. ceret'isiae. The high degree of sequence similarity reflects the evolutionary constraints on LPD, which exists as a dimer and serves as a subunit in several enzyme complexes. The sequence for A. t'inelandii LPD is at the bottom of the alignment. The three-dimensional structure of the enzyme from A. vinelandii has recently been determined [28], and its secondary structural features are indicated in Fig. 3. Also, the residues which form hydrogen bonds with FAD, and within the dimer interface, are shown. Especially well conserved are the regions around the active site cysteines (Cys-48 and Cys-53 in the A. t'inelandii LPD), and the nucleotide binding folds. 31 conserved residues (40% of all conserved) are located in these three regions. Except for two /3-strands (amino acids 129-134 and 252-259) none of the gaps are located in the regions homologous to the a-helical or/3-sheet structures of A. cinelandii LPD~ His-450 of the A. cinelandii LPD apparently participates in catalysis [28], and the substitution of the corresponding His.0A4 in the E. coil enzyme by Gin nearly abolishes the activity [30]. The His residue is conserved in all sequences. A salt-bridge is formed between Lys-57 and Glu-194 of the A. cinelandii LPD [28] and these two residues are also conserved in all ~quences available. Glu-425 of the A. cinelandii LPD was suggested to play an important role in the dimer interface [28], yet it is substituted by Asp in the Grampositive LPD sequences (Fig. 3). The best characterized function of LPD is as the E.3-subunit of the 2-oxo acid dehydrogenase complexes (PDH, OGDH, BCDH). In mitochondria the PDH complex is tightly bound to complex I of the respiratory chain in the inner membrane suggesting a functional interaction between them [31]. There are also data that indicate that LPD may participate in the membranetransport of sugars and amino acids in E. coli. The import of ribose, galactose and maltose into E. coli appears to depend on lipoamide and LPD [32-35]. However, the deficiency of the major lipoamide dehy-
drogenase gene, lpd, did not decrease the sugar transport in E. coli [35]. This suggested that another isoenzyme may be involved in sugar transport, and subsequently the new isoenzyme was purified [3]. Furthermore, in E. coli LPD has been implicated to participate in the ubiquinone-mediated transport of amino acids through the plasma membrane [36]. However, in this case the protein affecting the transport was identified with an antiserum specific to the lpd isoenzyme of E. coli. The exact role of LPD in these transport processes is not clear. In S. aureus LPD (E3) was found to be tightly bound to the membrane-attached ribosomes [9,10], whereas in B. subtilis the E2 subunit of PDH was bound to membrane-attached ribosomes [1 I]. Recently, a mitochondrial protein of Neurospora crassa was cloned which was found to be attached to ribosomes and membranes, and it was considered to be a ribosomal protein [37]. Interestingly, the protein was subsequently identified as subunit E2 of PDH [38]. This result as well as the results with Gram-positive bacteria [9-11], suggest some functional relationship between PDH, ribosomes and membranes, and warrants further investigations. This work was supported by grants from the Foundation for Biotechnicai and Industrial Fermentation Research, and the Emil Aaltonen Foundation. The author is grateful to Dr. L. Paulin for the synthesis of oligonucleotides, and to Dr. P. Russo for help in the preparation of the manuscript. References I Carothers, DJ,, Pons, G. and Patel, M.S. (1989) Arch. Biochem. Biophys. 268, 409-425. 2 Stephens, P.E., Lewis, H.M., Darlison, M.G. and Guest, J.R. (1983) Eur. J. Biochem. 135, 519-527. 3 Richarme, G. (1989) J. Bacteriol. 171, 6580-6585. 4 Carothers, D.J., Raefsky-Estrin, C., Pons, G. and Patel, M.S. (1987) Arch. Biochem. Biophys. 256, 597-605. 5 Burns, G., Brown, T., Hatter, K. and Sokatch, J.R. (1989) Eur. J. Biochem. 179, 61-69. 6 Burns, G, Sykes, P.J., Hatter, K. and Sokatch, J.R. (1989) J. Bacteriol. 171,665-668. 7 Palmer, J.A., Hatter, K. and Sokatch, J.R. (1991) J. Bacteriol. 173, 3109-3116. 8 Steiert, P.S, Stauffer, L.T. and Stauffer, G.V. (1990) J. Bacteriol. 172, 6142-6144. 9 Adler, L.-A. and Arvidson, S. (1984) J. Gen. Microbiol. 130, 1673-1682. I0 Adler, L.-A. and Arvidson, S. (1987) J. Gen. Microbiol. 133, 803-813. II Caulfield, M.P., Horiuchi, S., Tai, P.C. and Davis, B.D. (1984) Proc. Natl Acad. Sci. USA 81, 7772-7776. 12 Adler, L.-A. and Arvidson Z. (1984) FEMS Microbiol. Left. 23, 17-20. 13 Hemil~i,H., Palva, A., Paulin, L., Arvidson, S. and Palva, I. (1990) J. Bacteriol. 172, 5052-5063. 14 Hemil~i, H, Palva, A., Paulin, L., Adler, L.-A., Arvidson, S. and Palva, I. (1991) Res. Microbiol. 142, in press
123 15 16 17 18 19
Adler, L.-,~. and Arvidson, S. (19881J. Bacteriol. 170, 5337-5343. Hoch, J.A. artd Coukoulis, H.J. (19781 J. Bacterioi. 133, 265-269. Freese, E. and Fortnagel, U. (19691 J. Bacteriol. 99, 745-756. Ohn6, M. (19751J. Bacteriol. 122, 224-234. Helmann, J.D. and Chamberlin, M.J. (19881Annu. Rev. Biochem. 57, 839-872. 2(1 Borges, A., Hawkins, C.F., Packman, L.C. and Perham, R.N. (19901 Eur. J. Bioehem. 194, 95-102. 21 Otulakowski, G. and Robinson, B.H. (19871 J. Biol. Chem. 262, 17313-17318. 22 Pons, G., Raefsky-Estrin, C., Carothers, D.J., Pepin, R.A., Javed, A.A., Jesse, B.W., Ganapathi, M.K., Samols, D. and Patel, M.S. (19881 Proc. Natl Acad. Sci. USA 85, 1422-1426. 23 Browning, K.S., Uhlinger, D.J. and Reed, L.J. (19881 Proc. Natl Aead. Sei. USA 85, ! 831 - 1834. 24 Ross, J., Reid, G.A. and Dawes, I.W. (19881 J. Gen. Microbiol. 134, 1131-1139. 25 Benen, J.A.E., Van Berkel, WJ., Van Dongen, W.M., Miiller, F. and De Kok, A. (19891J. Gen. Microbiol. 135, 1787-1797. 26 Westphal, A.H. and De Kok, A. (19881 Eur. J. Biochem. 172, 299-305.
27 Devereux, J., Haeberli, P. and Smithies, O. (1984) Nucleic Acids Res. 12, 387-395. 28 Mauevi, A., Schierbeek, A.J. and Hol, W.G. (1991)J. Mol. Biol. 220, 975-994. 29 Lesk, A.M., Levilt. M. and Chothia, C. (1986) Protein Engin. I, 77-78. 3') Russell, G.C., Allison, N.J.. Williams, C.H. anti Guest. J.R. (1989) Ann. NY Acad. Sci. 573, 429-431. 31 Sumegi, B. and Srere, P.A. (1984) J. Biol. Chem. 259, 1504015045. 32 Richarme, G. (19851 J. Bacteriol. 162, 286-293. 33 Rieharme, G. (1985) Biochim. Biophys. Acta 815, 37-43. 34 Richarme, G. and Heine, H.-G. (1986) Eur. J. Biochem. 156, 399-405. 35 Richarme, G. (1987) Biochim. Biophys. Acta 893, 373-377. 36 Owen, P., Kaback. H.R. and Graeme-Cook, K.A. (1980) FEMS Microbiol. Left. 7, 345-348. 37 Kreader, C.A., Langer, C.S. and tleckman. J.E. (1989) J. Biol. Chem. 264, 317-327. 38 Russell, G.('. and Guest, J.R. (lqgl) Biochim. Biophys. Acla 1076, 225-232.
BBAEXP 90289
119
Short Sequence-Paper
Lipoamide dehydrogenase of Staphylococcus aureus: nucieotide sequence and sequence analysis Harri Hemil~i hzstitute of Bioteclmolol,,y, Unit'ersityof Helsinki, Helsblki (Finland) (Received 14 August 1991)
Key words: Lipoamide dehydrogenase; Pyruvate dehydrogenase: Ribosome: Membrane transport: pdhD; (StaphyhJcoccu.~aureu.~)
A complex of four proteins was previously isolated from Staphylococcus aureus. The complex had a strong interaction with membrane bound ribosomes, which suggested that it may be involved in protein secretion. However, the complex was identified as pyruvate dehydrogenase (PDH), which disproved the direct role of the complex in protein secretion. Here we report the nucleotide sequence of the last gene of the S. aureus pyruvate dehydrogenase operon, pdhD, which encodes lipoamide dehydrogenase (LPD). The pdhD gene encodes a protein of 468 amino acids, with a molecular mass of 49.5 kDa. The protein is closely related to other lipoamide dehydrogenases from bacteria and eukaryotes. The possible role of membrane bound lipoamide dehydrogenase is briefly discussed.
Lipoamide dehydrogenase (LPD; EC 1.8.1.4) is a ubiquitous flavoprotein, which catalyzes the NAD+-de pendent oxidation of dihydrolipoamide in a number of multienzyme complexes (for a review see Ref. 1). It serves as the E3-subunit for three 2-oxo acid dehydrogenase multienzyme complexes: pyruvate dehydrogenase (PDH), 2-oxoglutarate dehydrogenase ( O G D H ) and branched chain 2-oxo acid dehydrogenase (BCDH). Also, it functions as the L-subunit for the glycine cleavage system (GCS). So far, two isoenzymes of LPD have been found in Escherichia coil [2,3] and mammals [4], while three have been identified in Pseudomonas putida [5-7]. Although the role of the different isoenzymes has not been finally clarified, one isoenzyme in E. coil, encoded by the Ipd gene, functions in PDH, O G D H and GCS [2,8]. In P. putida, Glc-isoenzyme is used in PDH, O G D H and GCS [7], while the Val-isoenzyme is used in B C D H [5]. Previously, a complex of four proteins was isolated from S. aureus [9,10]. This complex was supposed to
participate in protein secretion, since it was attached to membrane bound ribosomes. A homologous complex was isolated from the closely related species Bacillus subtilis [11,12]. However, the complex was identified as PDH, which undermined the original hypothesis of its direct participation in protein secretion [I3,14]. In this paper, we report the nucleotide sequence and deduced amino acid sequence for the last gene of the S. aureus pyruvate dehydrogenase operon, pdhD, which encodes LPD. S. aureus pdhD gene was sequenced P om plasmid pMBR1 [15], which encodes the entire pdh operon (Fig. 1). The nucleotide sequence of pdhD and its deduced amino acid sequence are shown in Fig. 2. The
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Fig. 1. Physical map of the S. aureus pdh operon. The bold line represents the region sequenced so far in this report and in I14]. The genes pdlu4, pdhB. pdhC and pdhD encode the PDH subunits Ela. El/3. E2 and E3. respectively. The sizes of the Ela and EI~ proteins have been determining using SDS-PAGE [9]. and their order was determined by deletion studies [15]. PI. P2 and P3 refer to the putative promoters implied by northern analysis of the pdh operon [15]. The restriction enzyme sites for EcoRI (E). Hindill (H). Psti (P), $tyl (S). Accl (A) and Xbal (X) are indicated. The sites outside of the sequenced region were determined by digestions of the pMBRI [15].
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Fig, 2, Nucl¢otide and derived protein sequences of lipoamide dehydrogenas¢ from & auras VS. The nucleotide sequence of pdhD was d~t~rmined ~ m pMBRI [15] as previouslyd~scribcd [13,14]. Th~ ribosomal binding site in ~ n t of the initiation methionine is underlined. The inverted r~p~ut structures downstream of th~ stop codon ar~ marked by arrows under the sequence. Numbering of the sequence is consistent wilh th~ previously published I~lhC g~ne[14]. Both strands of the D N A were sequenced e~ept ~ r the 3' end starting at 2892 bp.
pdhD open reading frame of 1404 bp starts from the ATG-codon located at 1507 bp. A ribosomal binding site is located closely upstream from the initiation codon of pdhD. The pdhD gene encodes a protein of 468 amino acids, which has a molecular mass of 49.5 kDa (Fig. 2). Two inverted repeats are found after the stop codon (TAN at 2911 bp) of the open reading frame. The second of them may serve as the transcription terminator, since the energy of formation of the hairpin loop would be - 5 4 kJ/mol. In the closely related B. subtilis a single isoenzyme of LPD serves as the E3-subunit for both PDH and OGDH [16], and these two complexes appear to be independently regulated [17,18]. This suggests that the
pdhD gene of S. aureus may have its own promoter to express the gene in circumstances when the entire pdh operon is not transcribed. Furthermore, Northern blot analysis of S. aureus and B. subtilis has shown the presence of a short transcript from the pdh operon (1.6 and 1.7 kb, respectively) in addition to a large one covering the entire operon [13,15]. However, upstream from the S. aureus pdhD gene, we found no site closely resembling the known recognition sites of Gram-positive (r factors [19]. The sequences for B. subtilis and B. stearother. mophilus LPDs have been determined [13,20], and 73% of their amino acids are identical to the S. aureus enzyme (Fig. 3). The lipoamide dehydrogenases from
Fig. 3. Comparison of LPD sequences. Identical ( : ), and closely related amino acid~, t.), with a comparison value >_0.5 in the GAP mutation data matrix [27], are marked. The secondary structure assignments of the A. t'inelandii LPD [28] are shown below the alignment: a-helix (bold line}, O-sheet (thin line}. The conserved residues (* } and the residues with conservative substitutions (.) are indicated above the alignment. The residues which form hydrogen bond with FAD (F), and within the dimer interface (I) of A. rinelandii LPD [28] are indicated, as well as the active site cysteines (C). The sequences are numbered starting from the amino acid next to the initiation methionine.
121
STE, K T G V V ~ J ~ ; ; ; ; ; S ~ J ~ ; ; C ~ G L E T V , ; ; R . . YN . . . . T:;;V;:;;~;;;;~L~;~HVAKV;EEAKALAE • . . H;;VF. GEPKT;;DK | RTU~EKV;NQ;;G;~AGI~A;GRKVKWN;L~KFTGANTEEV 120 !! ,. IIII " I I " I'- I I I II::I:I'"'II I I I " '"I'"' '"" . I . III'"'III'"'II",,,, :" ,, : I:: : ""I: :I "'I'"'I: l'l Ill" ,,. ,-,, ,,, . ,,,, . ,, WGOFPIETOT I Vl GAGPGGYVAA!P~qILGOKVT IVEK..GN . . . . . LGGVCLNVGCIPSKALLHASHRFVEAOHSEN...LGVIA.ESVSLMFOKVClEFKSSWNKLTGGVEGL£KGNI~/NIVKGEAYFVDIINSLRY 1:)8
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Bacillus
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Pseudomonas p u t i d a (Vat)
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pseudomonas fLuorescens (G|c)
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676
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122 the Gram-positive bacteria are also aligned with the other known LPD sequences in Fig. 3. Recently, an alignment of four LPDs was published, but several of the gaps that were suggested [1] would be located within a-helix and/3-sheet regions, suggesting that the alignment is not correct [29], Fig. 3 contains 11 sequences so far known, which allows a more rigorous alignment analysis. Alignment of distantly related sequences may yield information of the most essential residues, which should be conserved in all of the sequences. Multiple sequence alignment complements crystallographic data, which do not indicate the limits for allowed amino acid exchanges. 77 amino acid residues (15%) are conserved in all of the sequences, and the lowest pairwise degree of similarity (38%) is between the enzymes of P. putida and S. ceret'isiae. The high degree of sequence similarity reflects the evolutionary constraints on LPD, which exists as a dimer and serves as a subunit in several enzyme complexes. The sequence for A. t'inelandii LPD is at the bottom of the alignment. The three-dimensional structure of the enzyme from A. vinelandii has recently been determined [28], and its secondary structural features are indicated in Fig. 3. Also, the residues which form hydrogen bonds with FAD, and within the dimer interface, are shown. Especially well conserved are the regions around the active site cysteines (Cys-48 and Cys-53 in the A. t'inelandii LPD), and the nucleotide binding folds. 31 conserved residues (40% of all conserved) are located in these three regions. Except for two /3-strands (amino acids 129-134 and 252-259) none of the gaps are located in the regions homologous to the a-helical or/3-sheet structures of A. cinelandii LPD~ His-450 of the A. cinelandii LPD apparently participates in catalysis [28], and the substitution of the corresponding His.0A4 in the E. coil enzyme by Gin nearly abolishes the activity [30]. The His residue is conserved in all sequences. A salt-bridge is formed between Lys-57 and Glu-194 of the A. cinelandii LPD [28] and these two residues are also conserved in all ~quences available. Glu-425 of the A. cinelandii LPD was suggested to play an important role in the dimer interface [28], yet it is substituted by Asp in the Grampositive LPD sequences (Fig. 3). The best characterized function of LPD is as the E.3-subunit of the 2-oxo acid dehydrogenase complexes (PDH, OGDH, BCDH). In mitochondria the PDH complex is tightly bound to complex I of the respiratory chain in the inner membrane suggesting a functional interaction between them [31]. There are also data that indicate that LPD may participate in the membranetransport of sugars and amino acids in E. coli. The import of ribose, galactose and maltose into E. coli appears to depend on lipoamide and LPD [32-35]. However, the deficiency of the major lipoamide dehy-
drogenase gene, lpd, did not decrease the sugar transport in E. coli [35]. This suggested that another isoenzyme may be involved in sugar transport, and subsequently the new isoenzyme was purified [3]. Furthermore, in E. coli LPD has been implicated to participate in the ubiquinone-mediated transport of amino acids through the plasma membrane [36]. However, in this case the protein affecting the transport was identified with an antiserum specific to the lpd isoenzyme of E. coli. The exact role of LPD in these transport processes is not clear. In S. aureus LPD (E3) was found to be tightly bound to the membrane-attached ribosomes [9,10], whereas in B. subtilis the E2 subunit of PDH was bound to membrane-attached ribosomes [1 I]. Recently, a mitochondrial protein of Neurospora crassa was cloned which was found to be attached to ribosomes and membranes, and it was considered to be a ribosomal protein [37]. Interestingly, the protein was subsequently identified as subunit E2 of PDH [38]. This result as well as the results with Gram-positive bacteria [9-11], suggest some functional relationship between PDH, ribosomes and membranes, and warrants further investigations. This work was supported by grants from the Foundation for Biotechnicai and Industrial Fermentation Research, and the Emil Aaltonen Foundation. The author is grateful to Dr. L. Paulin for the synthesis of oligonucleotides, and to Dr. P. Russo for help in the preparation of the manuscript. References I Carothers, DJ,, Pons, G. and Patel, M.S. (1989) Arch. Biochem. Biophys. 268, 409-425. 2 Stephens, P.E., Lewis, H.M., Darlison, M.G. and Guest, J.R. (1983) Eur. J. Biochem. 135, 519-527. 3 Richarme, G. (1989) J. Bacteriol. 171, 6580-6585. 4 Carothers, D.J., Raefsky-Estrin, C., Pons, G. and Patel, M.S. (1987) Arch. Biochem. Biophys. 256, 597-605. 5 Burns, G., Brown, T., Hatter, K. and Sokatch, J.R. (1989) Eur. J. Biochem. 179, 61-69. 6 Burns, G, Sykes, P.J., Hatter, K. and Sokatch, J.R. (1989) J. Bacteriol. 171,665-668. 7 Palmer, J.A., Hatter, K. and Sokatch, J.R. (1991) J. Bacteriol. 173, 3109-3116. 8 Steiert, P.S, Stauffer, L.T. and Stauffer, G.V. (1990) J. Bacteriol. 172, 6142-6144. 9 Adler, L.-A. and Arvidson, S. (1984) J. Gen. Microbiol. 130, 1673-1682. I0 Adler, L.-A. and Arvidson, S. (1987) J. Gen. Microbiol. 133, 803-813. II Caulfield, M.P., Horiuchi, S., Tai, P.C. and Davis, B.D. (1984) Proc. Natl Acad. Sci. USA 81, 7772-7776. 12 Adler, L.-A. and Arvidson Z. (1984) FEMS Microbiol. Left. 23, 17-20. 13 Hemil~i,H., Palva, A., Paulin, L., Arvidson, S. and Palva, I. (1990) J. Bacteriol. 172, 5052-5063. 14 Hemil~i, H, Palva, A., Paulin, L., Adler, L.-A., Arvidson, S. and Palva, I. (1991) Res. Microbiol. 142, in press
123 15 16 17 18 19
Adler, L.-,~. and Arvidson, S. (19881J. Bacteriol. 170, 5337-5343. Hoch, J.A. artd Coukoulis, H.J. (19781 J. Bacterioi. 133, 265-269. Freese, E. and Fortnagel, U. (19691 J. Bacteriol. 99, 745-756. Ohn6, M. (19751J. Bacteriol. 122, 224-234. Helmann, J.D. and Chamberlin, M.J. (19881Annu. Rev. Biochem. 57, 839-872. 2(1 Borges, A., Hawkins, C.F., Packman, L.C. and Perham, R.N. (19901 Eur. J. Bioehem. 194, 95-102. 21 Otulakowski, G. and Robinson, B.H. (19871 J. Biol. Chem. 262, 17313-17318. 22 Pons, G., Raefsky-Estrin, C., Carothers, D.J., Pepin, R.A., Javed, A.A., Jesse, B.W., Ganapathi, M.K., Samols, D. and Patel, M.S. (19881 Proc. Natl Acad. Sci. USA 85, 1422-1426. 23 Browning, K.S., Uhlinger, D.J. and Reed, L.J. (19881 Proc. Natl Aead. Sei. USA 85, ! 831 - 1834. 24 Ross, J., Reid, G.A. and Dawes, I.W. (19881 J. Gen. Microbiol. 134, 1131-1139. 25 Benen, J.A.E., Van Berkel, WJ., Van Dongen, W.M., Miiller, F. and De Kok, A. (19891J. Gen. Microbiol. 135, 1787-1797. 26 Westphal, A.H. and De Kok, A. (19881 Eur. J. Biochem. 172, 299-305.
27 Devereux, J., Haeberli, P. and Smithies, O. (1984) Nucleic Acids Res. 12, 387-395. 28 Mauevi, A., Schierbeek, A.J. and Hol, W.G. (1991)J. Mol. Biol. 220, 975-994. 29 Lesk, A.M., Levilt. M. and Chothia, C. (1986) Protein Engin. I, 77-78. 3') Russell, G.C., Allison, N.J.. Williams, C.H. anti Guest. J.R. (1989) Ann. NY Acad. Sci. 573, 429-431. 31 Sumegi, B. and Srere, P.A. (1984) J. Biol. Chem. 259, 1504015045. 32 Richarme, G. (19851 J. Bacteriol. 162, 286-293. 33 Rieharme, G. (1985) Biochim. Biophys. Acta 815, 37-43. 34 Richarme, G. and Heine, H.-G. (1986) Eur. J. Biochem. 156, 399-405. 35 Richarme, G. (1987) Biochim. Biophys. Acta 893, 373-377. 36 Owen, P., Kaback. H.R. and Graeme-Cook, K.A. (1980) FEMS Microbiol. Left. 7, 345-348. 37 Kreader, C.A., Langer, C.S. and tleckman. J.E. (1989) J. Biol. Chem. 264, 317-327. 38 Russell, G.('. and Guest, J.R. (lqgl) Biochim. Biophys. Acla 1076, 225-232.
