Lipoylation of Acyltransferase Components of α-Ketoacid Dehydrogenase Complexes

Lipoic acid is a prosthetic group of the acyltransferase components of the pyruvate, α-ketoglutarate, and branched chain α-ketoacid dehydrogenase complexes, protein X of the eukaryotic pyruvate dehydrogenase complex, and H-protein of the glycine cleavage system. We have purified lipoyl-AMP:Nε-lysine lipoyltransferase I and II from bovine liver mitochondria employing apoH-protein as an acceptor of lipoic acid (Fujiwara, K., Okamura-Ikeda, K., and Motokawa, Y. (1994) J. Biol. Chem. 269, 16605-16609). In this study, we demonstrated the lipoylation of the lipoyl domains of the mammalian pyruvate (LE2p), α-ketoglutarate (LE2k), and branched chain α-keto acid (LE2b) dehydrogenase complexes using the purified lipoyltransferase I and II. Lipoyltransferase I and II lipoylated LE2p and LE2k as efficiently as H-protein, but the lipoylation rate of LE2b was extremely low. Comparison of amino acid sequences surrounding the lipoylation site of these proteins shows that the conserved glutamic acid residue situated 3 residues to the N-terminal side of the lipoylation site is replaced by glutamine (Gln-41) in LE2b. When Gln-41 of LE2b was changed to Glu, the rate of lipoylation increased about 100-fold and became comparable to that of LE2p and LE2k. The replacement of the glutamic acid residue of LE2p (Glu-169) and LE2k (Glu-40) by glutamine resulted in decrease in the lipoylation rate more than 100-fold. These results suggest that the glutamic acid residue plays an important role in the lipoylation reaction possibly functioning as a recognition signal. Gly-27 and Gly-54 of LE2k are also well conserved among the lipoyl domains of the α-ketoacid dehydrogenase complexes and H-protein. The mutagenesis experiments of these residues indicated that the glycine residue situated 11 residues to the C-terminal side of the lipoylation site (Gly-54 of LE2k) is important for the folding of lipoyl domain, and that existence of a small residue such as Gly or Cys at the position is essential for the lipoylation of these proteins.

Five lipoate-containing proteins are known in vertebrate: acyltransferase components of the pyruvate, ␣-ketoglutarate, and branched chain ␣-ketoacid dehydrogenase complexes (E2p, E2k, and E2b, respectively), protein X of the pyruvate dehydrogenase complex, and H-protein of the glycine cleavage system. Lipoate attaches to the ⑀-amino group of the specific lysine residue of the proteins via an amide linkage. The lipoyllysine residue functions as a carrier of intermediates of the reactions and reducing equivalents interacting with the active sites of the components of the complexes (1)(2)(3)(4). Early studies of the lipoylation of apoE2p using crude enzyme preparations from Escherichia coli and Streptococcus faecalis showed that lipoyl-AMP is an activated intermediate of the reaction (5). We have purified lipoyl-AMP:N ⑀ -lysine lipoyltransferase (lipoyltransferase) from bovine liver mitochondria using apoHprotein as an acceptor of lipoate (6). Two isoforms, lipoyltransferase I and II, were separated by the chromatography on a hydroxylapatite column. They showed similar molecular mass (about 40 kDa), catalytic properties, and behavior on column chromatographies except on hydroxylapatite. They catalyze the transfer of the lipoyl moiety from lipoyl-AMP to apoH-protein, but have no ability to activate lipoate to lipoyl-AMP. Lipoylation of H-protein in bovine liver, therefore, requires lipoateactivating enzyme that has been partially purified from bovine liver (7). On the contrary, lipoylation of E2p in E. coli is catalyzed by a single enzyme, lipoate-protein ligase (8,9).
Our current interest is to determine whether the lipoyltransferases purified from bovine liver are involved in the lipoylation of E2 components of the ␣-ketoacid dehydrogenase complexes. To address the question, we used the apolipoyl domain of E2 components translated in vitro with a reticulocyte lysate system as a protein substrate. The results presented here show that lipoyltransferase I and II can lipoylate the lipoyl domain of E2 components as well as H-protein. Site-directed mutagenesis experiments indicated that the glutamic acid residue situated 3 residues on the N-terminal side of the lipoylation site is important for the lipoylation of E2 components and the glycine residue situated 11 residues on the C-terminal side of the lipoylation site is responsible for the folding of the lipoyl domain in accordance with the previous observations with H-protein (10).

EXPERIMENTAL PROCEDURES
Materials-T4 DNA ligase and TaKaRa Ex Taq DNA polymerase were purchased from Takara Shuzo (Shiga, Japan). Restriction endonucleases were from Toyobo (Tokyo, Japan). L-[ 35 S]Methionine (1000 Ci/mmol) and a rabbit reticulocyte lysate system were obtained from Amersham Corp. [1-14 C]Octanoic acid (0.055 Ci/mmol) was purchased from DuPont NEN. Lipoyl-AMP was prepared as described previously (6). [ 14 C]Octanoyl-AMP was synthesized essentially as described (6) using 250 Ci of [ 14 C]octanoic acid and isolated by high performance liquid chromatography on an ODS-120T column (4.5 ϫ 250 mm; Tosoh, Tokyo, Japan) with a linear gradient of 0.05 M sodium phosphate buffer, pH 5.5 (buffer A), and acetonitrile (buffer B). The gradient was developed from 10 to 45% B, and the elution of the product was monitored at 259 nm. Octanoyl-AMP was eluted at an acetonitrile concentration of 29%. Oligonucleotides were synthesized on an Applied Biosystems 392 DNA/RNA Synthesizer. cDNAs for rat E2p (11) and E2k (12) subcloned into pUC18 were kindly provided by Dr. Sadayuki Matuda (Department of Biology, Kanoya National Institute of Fitness and Sports, Kanoya, Japan). Lipoyltransferases were purified as described previously (6). The final preparations of lipoyltransferase I and II showed specific activities of 135 and 144 units/mg of protein, respectively. The activity of both enzyme preparations was adjusted to 1.67 ϫ 10 Ϫ3 unit/l. Activity of lipoyltransferase was assayed as described previously (6).
One unit of lipoyltransferase activity is defined as 1 nmol of H-protein lipoylated per min. cDNA for mature bovine H-protein subcloned into pTZ18U was prepared as described (13).
Cloning of cDNA for Bovine E2b-Standard molecular biology techniques were carried out essentially as described by Sambrook et al. (14). A bovine cDNA library (Clontech) was screened as described previously (15) using oligonucleotides synthesized according to the sequence reported by Griffin et al. (16). An EcoRI-XbaI fragment encoding the N-terminal 63 amino acid residues of E2b (16) and an XbaI-EcoRI fragment encoding the C-terminal 358 amino acid residues of E2b (16) were obtained. They were ligated at the XbaI site and subcloned into pTZ18U.
Construction of Plasmids Containing cDNA for the Lipoyl Domain of E2p, E2k, or E2b-The lipoyl domain and a part of the consecutive linker region of E2p, E2k, or E2b was generated by polymerase chain reaction. The oligonucleotides used for primers are listed below. The underlined nucleotides represent restriction sites which were introduced for cloning purposes. The nucleotides shown in boldface letters are initiator methionine codons introduced at the start site of the target sequences in 5Ј-primers and antisense stop codons in 3Ј-primers. If necessary, additional methionine codons were introduced in 3Ј-primers at the end of the target sequences to facilitate the labeling with . Amplification was carried out in a reaction mixture of 50 l containing 20 ng of linearized template plasmid, 50 pmol of each primer, 2.5 mM dNTP, 1 ϫ Ex Taq Buffer (Takara Shuzo), and 1.25 units of TaKaRa Ex Taq for 30 cycles at 94°C for 1 min, 52°C for 2 min, and 72°C for 3 min. The products were separated by electrophoresis on 1% agarose gels. Fragments of interest were extracted with QIAEX (Qiagen), digested with restriction enzymes, and subcloned into pTZ18U to obtain pLE2p, pLE2k, and pLE2b. The sequence of all amplified fragments was confirmed by DNA sequencing employing a 373A DNA sequencing system (Applied Biosystems).
In Vitro Transcription and Translation-Plasmids containing cDNA for the lipoyl domain or H-protein were linearized with SalI and purified by phenol/chloroform extraction and ethanol precipitation. mRNAs were synthesized in vitro as described previously (10). In vitro translation was carried out in the reaction mixture of 12.5 l containing 200 ng of mRNA, 1 l of [ 35 S]methionine, 1 l of translation mixture, 0.5 l of 2.5 M potassium acetate, 0.25 l of 25 mM magnesium acetate, and 5 l of rabbit reticulocyte lysate, and the mixture was incubated for 60 min at 30°C. In the case of LE2p, translation was carried out using 0.5 l of 2.5 M KCl instead of potassium acetate.
Lipoylation and Octanoylation Studies-Lipoylation of apolipoyl domains was analyzed by nondenaturing polyacrylamide gel electrophoresis. Lipoylated lipoyl domains of E2p, E2k, and E2b were well separated from apolipoyl domains as previously demonstrated with H-protein (4,6). The lipoylation reaction was carried out in a mixture of 8 l containing 0.25-0.5 l of translation products labeled with [ 35 S]methionine including the lipoyl domain of about 7000 dpm, 40 mM potassium phosphate buffer, pH 7.8, 0.2 mg/ml bovine serum albumin, 10 M MnCl 2 , 50 M p-amidinophenylmethanesulfonyl fluoride, 0.1 mM lipoyl-AMP and lipoyltransferase. After incubation at 37°C for 60 min, 4 l of 3 ϫ sample buffer (1 ϫ sample buffer is 62.5 mM Tris-HCl, pH 6.8, 10% (v/v) glycerol, 0.002% (w/v) bromphenol blue) was added to the mixture, and 10 l of the mixture was subjected to 20% polyacrylamide gel electrophoresis (4). The proteins in the gel were electrotransferred onto Immobilon-P membrane (Millipore) in a semi-dry Sartoblot apparatus (Sartorius), and autoradiographed at room temperature. A percent lipoylation was determined by counting radioactivities of the apo-and hololipoyl domains on the membrane with a Fujix BAS 2000 Bio-Imaging analyzer (Fuji Photo Film, Tokyo, Japan).
Octanoylation reaction was performed as described for lipoylation but with some modifications due to the low specific radioactivity of [ 14 C]octanoyl-AMP. The reaction was conducted in a mixture of 16 l with 14-fold more amounts than lipoylation reaction of translation products prepared with non-radiolabeled methionine, 5 l of lipoyltransferase, and 50 M [ 14 -C]octanoyl-AMP. The other ingredients were the same as above. The reaction products were separated by 20% polyacrylamide gel electrophoresis and electrotransferred onto Immobilon-P membrane. After exposure of the membrane to an Imaging Plate, a detector of radioenergy, for 12 days, the radioactivities were analyzed and recorded with BAS 2000.

RESULTS AND DISCUSSION
Lipoylation of Lipoyl Domains of E2 Components-In order to test whether lipoyltransferase I and II that catalyze lipoylation of apoH-protein can lipoylate E2 components of the pyruvate, ␣-ketoglutarate, and branched chain ␣-ketoacid dehydrogenase complexes, we constructed the plasmids containing cDNA for the lipoyl domain of each E2 component as described under "Experimental Procedures." The plasmids were transcribed and translated in vitro. The translated products named LE2p, LE2k, and LE2b, respectively, were subjected to lipoylation with the purified lipoyltransferases. We have previously demonstrated that H-protein when lipoylated with lipoyltransferase moves faster than apoH-protein on nondenaturing polyacrylamide gel electrophoresis, since the apoprotein has an additional positive charge on the unmodified lysine residue (4,6). Similarly, the lipoyl-AMP-and lipoyltransferase-dependent alterations of mobility of lipoyl domains were observed in this experiment (Fig. 1), indicating that apolipoyl domains of acyltransferases are lipoylated as well with the purified lipoyltransferases that lipoylate H-protein of the glycine cleavage system. The fact that the faster migrating lipoyl domain on nondenaturing polyacrylamide gel electrophoresis is the lipoylated form has been demonstrated with the overexpressed human E2p (18). Fully lipoylated proteins were obtained with apolipoyl domains of E2p and E2k and apoH-protein as substrates when 6.7 ϫ 10 Ϫ4 unit of either lipoyltransferase I or II was employed (Fig. 1, A, B, and D), whereas much more amounts of lipoyltransferase were required to lipoylate the apolipoyl domain of E2b (Fig. 1C). Again lipoate plus ATP could not replace lipoyl-AMP in the lipoylation of apolipoyl domains (results not shown) as reported previously with apoH-protein (6). A minor component was detected when the in vitro translation products of pLE2p were subjected to nondenaturing polyacrylamide gel electrophoresis (Fig. 1A). pLE2p encodes the inner lipoyl domain of rat E2p spanning from Ser-127 to Pro-253 (11). The peptide has three methionine residues (Met-132, Met-143, and Met-145) in addition to the inserted initiator methionine. Either the codon for Met-143 or Met-145 is probably functional as an alternate initiator to produce the minor component, since the nucleotide sequences around the ATG codons conform the consensus sequence of Kozak (19). The minor component receives lipoate, since the mobility changed when incubated with lipoyl-AMP and lipoyltransferase (Fig.  1A).
The Role of the Glutamic Acid Residue Situated 3 Residues on the N-terminal Side of the Lipoyllysine Residue-Comparison of amino acid sequences around the lipoate attachment site of H-protein and acyltransferases of the ␣-ketoacid dehydrogenase complexes from various sources indicated that Gly-43, Glu-56, and Gly-70 of bovine H-protein are highly conserved among these proteins (10). Inspection of the sequences around the lipoylation site of E2 components indicates that among these conserved amino acid residues the glutamic acid residue corresponding to Glu-56 of H-protein is replaced by glutamine in E2b (Fig. 2). Intramitochondrial lipoylation of H-protein was greatly reduced when Glu-56 was changed to glutamine by site-directed mutagenesis (10). It is plausible that the presence of the glutamine residue at position 41 of E2b is the cause of the reduced rate of lipoylation observed above in the lipoylation with the purified lipoyltransferase. To test the possibility, LE2b-Q41E, a mutant lipoyl domain of E2b in which Gln-41 is replaced by Glu was generated in an in vitro translation reaction and subjected to lipoylation. As shown in Fig. 3 and Table  I, the lipoylation of LE2b-Q41E was greatly improved when compared with the wild-type LE2b. Initial velocity studies of lipoylation of the wild-type and the mutant LE2b indicated that the alteration of Gln-41 to Glu increased the rate about 100-fold, since LE2b-Q41E required 100-fold less amounts of lipoyltransferase I or II than the wild-type LE2b to obtain nearly the same initial velocity.
To investigate further the critical role of the glutamic acid residue, Glu-169 of LE2p and Glu-40 of LE2k were replaced by glutamine. The lipoylation rate of mutants LE2p-E169Q and LE2k-E40Q decreased dramatically when compared with the wild type (Table I and Fig. 4). These results together with the results obtained from the intramitochondrial lipoylation of Hprotein (10) indicate that the glutamic acid residue situated 3 residues to the N-terminal side of the lipoyllysine residue is important for the lipoylation with lipoyltransferase possibly functioning as a recognition signal for lipoyltransferase. The anionic charge appears to have a role in the recognition by lipoyltransferase. The side chain length of the glutamic acid residue appears to be also responsible, since replacement of Glu-56 of H-protein with Asp reduced the rate of lipoylation significantly (10). Griffin et al. (20) reported that the bovine mature E2b overexpressed in E. coli cells grown in a medium containing [2-3 H]lipoate received no radioactivity, whereas the inner lipoyl domain of human E2p was lipoylated in E. coli (18). These observations suggest that the mechanism of the interaction of the substrate and lipoyltransferase is similar in animals and bacteria. In the three-dimensional structure of lipoyl domain of E2p from E. coli (21), the glutamic acid residue is  1, 2, and 4) or absence (lanes 3 and 5) of lipoyl-AMP as described under "Experimental Procedures." The enzyme preparation of 0.4 l was used for LE2p, LE2k, and BH and 2 l was used for LE2b. The products were separated by nondenaturing polyacrylamide gel electrophoresis, electrotransferred onto Immobilon-P, and autoradiographed. Apo and lip indicate apoform and lipoylated form, respectively.

FIG. 2.
Comparison of the amino acid sequences surrounding the lipoic acid attachment site. The sequences of rat acetyltransferase (E2p) (11), rat succinyltransferase (E2k) (12), bovine acyltransferase of the branched chain ␣-ketoacid dehydrogenase complex (E2b) (16), and bovine H-protein (15) are compared. The lysine residue involved in lipoic acid attachment is marked with a star. The shadowed amino acid residues show the conserved residues which were chosen for mutagenesis. The numbers on the right and left refer to the positions of the amino acids of the mature proteins.  (lanes 2 and 6), 0.08 l (lanes 3 and 7), 0.4 l (lanes 4 and 8), and 2 l (lanes 5 and 9) was used. The autoradiographs were obtained as described in the legend to Fig. 1. The percent lipoylation was determined by quantitating radioactivities of lipoyl domains in panel A and panel B (lanes 1 and  6 -9) with a Bio-Imaging analyzer (C). E, LE2b; q, LE2b-Q41E. Apo and lip indicate apoform and lipoylated form, respectively. situated at the end of a ␤-strand preceding the tight ␤-turn in which the lysine residue to be lipoylated is located. No amino acid residues responsible for recognition for lipoyltransferase other than the glutamic acid residue have been reported. The conserved aspartic acid and alanine residues of E2 domain flanking the lipoyllysine residue, on the N-and C-terminal sides, appear to have no role for the recognition (22).
The Role of Conserved Glycine Residues-Highly conserved glycine residues reside 16 residues to the N-terminal side and 11 residues to the C-terminal side of the lipoylation site in E2 components and H-protein (Fig. 2). To investigate the role of these glycine residues in lipoylation of E2 components, these residues of LE2k (Gly-27 and Gly-54) have been replaced by serine or asparagine. On nondenaturing polyacrylamide gel electrophoresis, the mobilities of apoforms of LE2k-G54S and LE2k-G54N were greatly reduced when compared with the wild type (Fig. 5, A, C, and E), suggesting that the substitution caused a conformational change. The Gly to Ser substitution affected the lipoylation moderately, but the Gly to Asn substitution abolished the lipoylation (Table I and Fig. 5, C and E). On the other hand, the replacements of Gly-27 by Ser or Asn did not show any effects on the mobility on the nondenaturing polyacrylamide gel electrophoresis or the rate of lipoylation (Table I and Fig. 5, B and D). To confirm these observations, the incorporation of the radiolabeled octanoyl moiety from [ 14 C]octanoyl-AMP to wild-type and mutant LE2ks was examined. We employed [ 14 C]octanoyl-AMP as an alternative substrate for lipoyltransferase since radiolabeled lipoic acid is not available commercially, and lipoyltransferase can octanoylate apoE2k (Fig. 6) as well as apoH-protein (6). As shown in Fig. 6, LE2k, LE2k-G27S, and LE2k-G27N are equally octanoylated by lipoyltransferase II. The octanoylated LE2k-G54S shows a broad band with lowered mobility as compared with the wildtype LE2k on nondenaturing polyacrylamide gels in accordance with the results shown in Fig. 5C. As expected, LE2k-E40Q and LE2k-G54N were not octanoylated (Fig. 6). The same results were obtained with lipoyltransferase I (not shown). These results are consistent with the above observations with lipoyl-AMP. The results presented here indicate that Gly-54 has some role for the folding of the lipoyl domain. The effects of Gly-54 mutations on lipoylation apparently depend on the substituting amino acid residue. Therefore, Gly-54 appears not to be involved in the recognition by lipoyltransferase. Indeed, the amino acid residue corresponding to Gly-54 of the outer lipoyl domain of rat E2p is cysteine (Fig. 2). We previously reported the similar results with H-protein when Gly-70 corresponding to Gly-54 of rat E2k was replaced by Ser or Asn (10). The mutations apparently destabilized H-protein and reduced the   (lanes 2 and 6), 0.08 l (lanes 3 and 7), 0.4 l (lanes 4 and 8), and 2 l (lanes 5 and 9) was used. The autoradiographs were obtained as described in the legend to Fig. 1. The percent lipoylation was determined by quantitating radioactivities of lipoyl domains in panel A and panel B (lanes 1 and  6 -9) with a Bio-Imaging analyzer (C). E, LE2k; q, LE2k-E40Q. Apo and lip indicate apoform and lipoylated form, respectively. rate of lipoylation. The Gly-54 is located at the opposite site of the lipoyllysine residue in the lipoyl domain (21). A bulky residue at the position may prevent the accession of the lysine residue to be lipoylated to the catalytic site of lipoyltransferase. Interestingly, a glycine residue corresponding to Gly-54 of E2k is also present in biotin enzymes. Mutations of the glycine residue of E. coli biotin carboxyl carrier protein of acetyl-CoA carboxylase and ␣-subunit of human propionyl-CoA carboxylase to serine caused a less efficient biotinylation (23,24). The biotinylation of the mutant biotin carboxyl carrier protein is temperature-sensitive and the defect is more severe at 42°C than at 30°C (23), suggesting the incomplete folding of the protein to be biotinylated.
Conclusion-We demonstrated that lipoyltransferase I and II purified from bovine liver can lipoylate not only H-protein of the glycine cleavage system but also lipoyl domains of acyltransferases of the pyruvate, ␣-ketoglutarate, and branched chain ␣-ketoacid dehydrogenase complexes. No difference in the substrate specificity was found with lipoyltransferase I and II. The lipoyl domain of bovine E2b is not a good substrate for the lipoyltransferases. It remains to be established whether the in vivo lipoylation of E2b is catalyzed by the lipoyltransferases already described or whether the lipoylation requires a novel, as yet unidentified lipoyltransferase. The study of lipoylation of mutant lipoyl domains indicated that the glutamic acid residue situated 3 residues to the N-terminal side of the lipoylation site appears to be essential, but not exclusive, for the recognition by lipoyltransferase, and that the glycine residue located 11 residues to the C-terminal side of the lipoyllysine residue appears to be important for the folding of the lipoyl domain of E2 components.