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Originally published In Press as doi:10.1074/jbc.M702694200 on July 19, 2007

J. Biol. Chem., Vol. 282, Issue 37, 26963-26970, September 14, 2007
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A Novel ADP-forming Succinyl-CoA Synthetase in Thermococcus kodakaraensis Structurally Related to the Archaeal Nucleoside Diphosphate-forming Acetyl-CoA Synthetases*

Kenichi Shikata, Toshiaki Fukui, Haruyuki Atomi, and Tadayuki Imanaka1

From the Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan

Received for publication, March 29, 2007 , and in revised form, July 19, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
We have identified and characterized a structurally novel succinyl-CoA synthetase (SCS) from the hyperthermophilic Archaea Thermococcus kodakaraensis. The presence of an SCS completes the metabolic pathway from glutamate to succinate in Thermococcales, which had not been clarified because of the absence of classical SCS homologs on their genomes. The SCS from T. kodakaraensis (SCSTk) is a heteromeric enzyme ({alpha}2beta2) encoded by TK1880 ({alpha}-subunit) and TK0943 (beta-subunit). Although both SCSTk and classical SCSs harbor the five domains present in enzymes of the acyl-CoA synthetase (nucleoside diphosphate-forming) superfamily, the domain order and distribution among subunits in SCSTk ({alpha}-subunit, domains 1-2-5; beta-subunit, domains 3-4) are distinct from those of classical SCSs ({alpha}-subunit, domains 1-2; beta-subunit, domains 3-4-5) and instead resemble the acetyl-CoA synthetases from Pyrococcus furiosus (ACSs IPf and IIPf). Comparison of the four Thermococcales genomes revealed that each strain harbors five {alpha}- and two beta-subunit homologs. Sequence similarity suggests that the beta-subunit of SCSTk is also a component of the presumed ACS II from T. kodakaraensis (ACS IITk). We coexpressed the {alpha}/beta-genes of SCSTk (TK1880/TK0943) and of ACS IITk (TK0139/TK0943). ACS IITk recognizes a broad range of hydrophobic/aromatic acid compounds, as is the case with ACS IIPf, whereas SCSTk displays a distinct and relatively strict substrate specificity for several acids, including succinate. This indicates that the {alpha}-subunits are responsible for the distinct substrate specificities of SCSTk and ACS IITk.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Thermococcus and Pyrococcus species are hyperthermophilic Archaea that preferentially utilize peptides/amino acids for cell growth (1). It is presumed that amino acids are first deaminated to 2-oxo acids by aminotransferases, with 2-oxoglutarate as a key amino acceptor (2). The generated glutamate is converted back to 2-oxoglutarate by oxidative deamination catalyzed by glutamate dehydrogenase (3). The 2-oxo acids are then converted to CoA thioester compounds by oxidative decarboxylation. Ferredoxin-dependent oxidoreductases catalyze these reactions, and a number of enzymes with distinct substrate specificities have been identified in Pyrococcus furiosus (4–7). Closely related genes are also found on the genomes of Thermococcus kodakaraensis (8), Pyrococcus abyssi (9), and Pyrococcus horikoshii (10). The final step is the hydrolysis of the thioester bond, releasing a carboxylic acid and CoA accompanied by substrate level phosphorylation, an important energy conservation reaction in these hyperthermophiles. The ADP-forming acetyl-CoA synthetases I and II from P. furiosus (ACSs IPf2 and IIPf) have been identified to be involved in this step (11–14) and exhibit activity not only for acetyl-CoA but also for branched-chain acyl-CoAs (ACSs IPf and IIPf) and aryl-CoAs (ACS IIPf) (13). The substrate specificities of the two enzymes are consistent with the preferential consumption of Leu, Ile, and Phe by P. furiosus during growth on amino acids (15). The same tendencies are also observed in T. kodakaraensis (16). However, although to a smaller extent compared with hydrophobic/aromatic amino acids, consumption of other amino acids is consistently observed during growth of T. kodakaraensis. The enzyme(s) involved in the metabolism of these amino acids have not been identified.

In contrast to the wide distribution of AMP-forming ACSs, the presence of ADP-forming ACSs seems to be limited to protists such as Entamoeba histolytica (17) and Giardia lamblia (18) and the Archaea. Archaeal ADP-forming ACSs have been characterized from the hyperthermophilic P. furiosus (11–13), Archaeoglobus fulgidus (19, 20), Methanocaldococcus jannaschii (20), and Pyrobaculum aerophilum (21) and the halophilic Haloarcula marismortui (21, 22). The enzymes display a common structure and are composed of two {alpha}- and two beta-subunits ({alpha}2beta2) or are homodimers of {alpha}-beta fusion proteins. They have been proposed to be members of the nucleoside diphosphate (NDP)-forming acyl-CoA synthetase superfamily (18). ADP/GDP-forming succinyl-CoA synthetase (SCS) also belongs to this superfamily and catalyzes the reversible conversion of succinyl-CoA to succinate and CoA concomitant with substrate level phosphorylation of ADP/GDP (23, 24). Generally, SCS functions in the tricarboxylic acid cycle of aerobic organisms, and it has been suggested that the mammalian ATP-dependent enzyme serves a catabolic role, whereas the GTP-dependent enzyme is involved in succinyl-CoA synthesis (24). Many Archaea also possess closely related homologs of SCS on their genomes. Although the SCSs in aerobic Archaea are presumed to function in the tricarboxylic acid cycle, their roles in anaerobic Archaea have not been clarified. Interestingly, despite the presence of 2-oxoglutarate:ferredoxin oxidoreductase homologs, which produce succinyl-CoA from 2-oxoglutarate, genes encoding SCS are missing on the genomes of Thermococcus, Pyrococcus, and Methanosarcina.

In this study, we aimed to identify the missing SCS in T. kodakaraensis, which would contribute to the efficient breakdown of glutamate or glutamine. The presence of the enzyme was anticipated from the accumulation of succinate observed in the medium when T. kodakaraensis was grown on amino acids along with pyruvate. Our results reveal the presence of a structurally novel type of SCS in T. kodakaraensis, which corresponds to an uncharacterized isoform of ACSs of the Archaea.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Strains, Plasmids, and Media—T. kodakaraensis KOD1 (25) cells were grown under anaerobic conditions at 85 °C in nutrient-rich growth medium containing 30.4 g/liter Marine Art SF-1 agent (Senju Pharmaceutical Co., Osaka, Japan), 5.0 g/liter yeast extract, and 5.0 g/liter Tryptone supplemented with 1.0 g/liter sulfur or 5.0 g/liter pyruvate (MA-YT-Pyr medium). Escherichia coli DH5{alpha} cells and plasmid pUC118 were used for general DNA manipulation and sequencing. E. coli BL21-CodonPlus(DE3)-RIL cells (Stratagene, La Jolla, CA) and pET21a(+) (Novagen, Madison, WI) were used for heterologous gene expression. E. coli strains were cultivated in LB medium (10 g/liter Tryptone, 10 g/liter yeast extract, and 5 g/liter NaCl (pH 7.0)) at 37 °C. Ampicillin was added to the medium at a concentration of 100 µg/ml when needed.

Partial Purification of SCS from T. kodakaraensis (SCSTk)—T. kodakaraensis cells grown in MA-YT-Pyr medium (4.8 liters) were harvested and lysed in buffer A (50 mM Tris-HCl buffer (pH 7.5)) containing 0.1% (w/v) Triton X-100. The cell-free extract was subjected to sequential chromatography with a Resource Q anion exchange column (6 ml), a Mono Q HR 5/5 anion exchange column, a HiLoad 26/60 Superdex 200 pg gel filtration column, a Resource ISO hydrophobic column (6 ml), and a Superdex 200 HR 10/30 gel filtration column (GE Healthcare, Little Chalfont, UK). The fractions displaying SCS activity were collected and combined. In the cases of anion exchange and hydrophobic chromatography, the proteins were eluted with a linear gradient of 0–1.0 M NaCl and 1.5 to 0 M ammonium sulfate in buffer A, respectively. The protein samples were dialyzed against the initial buffers prior to application. For gel filtration, protein samples concentrated by ultrafiltration with Centricon YM-30 (Millipore Corp., Bedford, MA) were applied to the columns equilibrated with buffer A containing 150 mM NaCl. The molecular mass was calibrated with standard proteins of thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), aldolase (158 kDa), albumin (67 kDa), ovalbumin (43 kDa), chymotrypsinogen A (25 kDa), and RNase A (13.7 kDa). Protein concentration was determined with a Bio-Rad protein assay kit using bovine serum albumin as a standard. The N-terminal amino acid sequences of protein bands separated by SDS-PAGE were determined by using a Model 491 cLC protein sequencer (Applied Biosystems, Foster City, CA) after electroblotting the proteins onto a polyvinylidene difluoride membrane (Millipore Corp.).

Overexpression of SCSTk and ACS IITk Genes in E. coli—The expression plasmids for the SCSTk and ACS IITk genes were constructed as follows. First, the TK1880 and TK0139 genes encoding {alpha}-subunits and the TK0943 gene encoding the common beta-subunit were independently amplified from T. kodakaraensis genomic DNA with the following primer sets: TK1880, 5'-CCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGGAGACCCCGAACCTGGACTTCCTG-3' (sense) and 5'-TTAGGATCCTCAAAGCCTCCCGTTCTCCCTC-3' (antisense); TK0139, 5'-ATACATATGACGTTCGACTACTTCTTCAAGCC-3' (sense) and 5'-TTAGGATCCTCACTCCTCCCTCAGCAAGCCAACG-3' (antisense); and TK0943, 5'-CCCTCTAGAATTCAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGAGCGCCAAAGAAGAGGCCC-3' (sense) and 5'-CTTGTCGACTCACTCTTTCTTTTCTGGAGCTTTC-3' (antisense). The underlined sequences indicate restriction sites for XbaI, BamHI, NdeI, BamHI, EcoRI, and SalI, respectively, and the italic sequences indicate ribosome-binding sequences aimed to promote efficient translation in E. coli. The amplified fragments were subcloned into pUC118 and then digested with the appropriate restriction enzymes. The fragments for the TK1880 and TK0139 genes were individually inserted into pET21a(+) at the corresponding sites, and the fragment for TK0943 was further inserted into each of the resulting plasmids so that the TK0943 gene was properly oriented downstream of the {alpha}-subunit gene. The plasmids obtained (pET-scs and pET-acs2) were sequenced to confirm the absence of unintended mutations and used to transform E. coli BL21-CodonPlus(DE3)-RIL cells. Gene expression was induced at the mid-exponential growth phase by the addition of 0.1 mM isopropyl beta-D-thiogalactopyranoside, followed by additional incubation for 4 h at 37 °C. After expression, cells were harvested, suspended in buffer A, and disrupted by sonication. The soluble protein fraction after centrifugation (15,000 x g, 15 min) was heat-treated at 80 °C for 10 min and centrifuged to remove heat-labile proteins from the host. The supernatant was subjected to sequential chromatography using Resource Q, Resource ISO, and Superdex 200 HR 10/30 columns according to the methods described above with a slight modification in the linear gradient for the hydrophobic column (1.8 to 0 M ammonium sulfate in buffer A). The final protein sample was dialyzed against buffer A and subjected to enzyme assay.

Enzyme Assays—Succinyl-CoA-forming activity in T. kodakaraensis extracts and in fractions during the purification procedure was measured by the hydroxamate method (26). The reaction was performed in a mixture (500 µl) composed of 1 mM CoASH, 5 mM ATP or GTP, 50 mM hydroxylamine, and 2.5 mM MgCl2 in 25 mM Tris succinate buffer at 70 °C for the appropriate periods of time. After the reaction, 350 µl of 20% trichloroacetic acid and 150 µl of 1 M FeCl3 were added to the mixture, and formation of the iron·succinohydroxymate complex derived from succinyl-CoA was determined spectrophotometrically at 520 nm. Authentic succinyl-CoA (Sigma) was used as a standard for calibration.

A continuous spectrophotometric assay monitoring CoA thioester formation from acids was applied for examination of the substrate specificities and kinetic properties of recombinant SCSTk and ACS IITk (21). The reaction mixture (1000 µl) was composed of 10 mM carboxylic acid, 1 mM ATP, 1.5 mM CoA, 5 mM MgCl2, 1.5 mM phosphoenolpyruvate, and 1.5 mM NADH in 50 mM MES-NaOH buffer (pH 6.5) together with a mixture of pyruvate kinase/lactate dehydrogenase from rabbit muscle (Sigma) as coupling enzymes. The ADP formation accompanied by CoA thioester formation at 55 °C was continuously monitored as a decrease in the absorbance of NADH at 340 nm. We confirmed that the stability and activity levels of the coupled enzymes were sufficient during the assay at 55 °C.

When measuring activity in the direction of CoA thioester cleavage, the formation of free CoASH was determined using 5,5'-dithiobis(2-nitrobenzoic acid) (13). The reaction was carried out in a mixture (1000 µl) containing 0.5 mM CoA thioester (isovaleryl-CoA or succinyl-CoA), 0.1 mM 5,5'-dithiobis(2-nitrobenzoic acid), 0.4 mM ADP, and 10 mM MgCl2 in 50 mM MES-NaOH buffer (pH 6.5) at 55 °C, and the increase in absorbance at 412 nm was continuously recorded.

Immunoprecipitation—Protein A-Sepharose Fast Flow (GE Healthcare) was used as the affinity resin for immunoprecipitation experiments. Five-hundred microliters of rabbit anti-serum (anti-TK0943 or anti-TK0465 serum) containing 1% bovine serum albumin was mixed with 100 µl of the resin suspension at 4 °C for 12 h. Antibody-bound resin was centrifuged, and the supernatant was removed. The pellet was washed five times with 1.5 ml of phosphate-buffered saline (136.9 mM NaCl, 8.1 mM Na2HPO4·12H2O, 2.68 mM KCl, and 1.47 mM KH2PO4), followed by the addition of 100 µl of cell extracts (2 mg/ml protein) of T. kodakaraensis grown in MA-YT-Pyr medium. After incubation at 4 °C for 1.5 h, the resin was washed three times with phosphate-buffered saline and then resuspended in 30 µl of the buffer. The suspension was heat-treated at 90 °C for 30 min and centrifuged to remove heavy and light chains of the antibodies, followed by SDS-PAGE analysis. The protein bands with a molecular mass of ~50 kDa (corresponding to the {alpha}-subunits) were sliced out and subjected to MALDI-TOF tandem mass spectrometry analysis at Hitachi High-Technologies Corp. (Hitachinaka, Japan).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of an SCS in Cell-free Extracts of T. kodakaraensis—We first examined the presence of SCS activity in T. kodakaraensis. The extract was prepared from cells grown in MA-YT-Pyr medium containing amino acids/peptides and pyruvate without elemental sulfur, and ATP-dependent succinyl-CoA formation from succinate and CoA was measured by the hydroxamate procedure. As a result, we observed 0.18 µmol of succinyl-CoA-forming activity/min/mg of total protein in the extract at 70 °C.


Figure 1
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  		FIGURE 1.
Purification of SCS from cell extracts of T. kodakaraensis. Arrowheads indicate the major proteins along with their respective N-terminal amino acid sequences and corresponding gene numbers on the T. kodakaraensis genome.

 
The protein responsible for the SCS activity was purified from the cell extracts. The crude extract was subjected to sequential column chromatography, and the enzyme was purified 221-fold based on the ATP-dependent activity (39.4 units/mg) (Table 1). SDS-PAGE analysis of the active fractions after the final purification step showed two major bands with molecular masses of 50 and 27 kDa, respectively (Fig. 1). The elution of a single peak with a molecular mass of ~160 kDa was observed by gel filtration chromatography, which supported a heterotetrameric conformation ({alpha}2beta2) of the enzyme composed of 50-kDa ({alpha}) and 27-kDa (beta) subunits. It should be noted that a lower GTP-dependent activity was always associated with the ATP-dependent activity through all purification steps, and neither ATP- nor GTP-dependent SCS activity could be detected in other fractions. These facts strongly suggested that a single SCS with broad nucleotide specificity was present in T. kodakaraensis cells. The final preparation displayed a specific activity of 14.3 units/mg in terms of GTP-dependent activity.


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  		TABLE 1
Purification profile of SCSTk

 


Figure 2
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  		FIGURE 2.
Distinct domain order and distribution among subunits in selected members of the NDP-forming acyl-CoA synthetase superfamily. SCSEc represents the structure of classical SCSs.

 
The N-terminal amino acid sequences of the two proteins were determined to be XETPNL (X is an undefined residue) for the 50-kDa protein and SAXEEAL for the 27-kDa protein. A homology search against the T. kodakaraensis genome data base led to the identification of the corresponding genes, TK1880 and TK0943, for the 50- and 27-kDa subunits, respectively. TK1880 encodes a 469-amino acid protein (50.9 kDa) with METPNL as the N-terminal sequence, whereas the TK0943 product is a 243-amino acid protein (27.1 kDa) initiating with the sequence MSAKEEAL. The deduced molecular masses were in good agreement with those of the two proteins purified from T. kodakaraensis cells.

SCSTk Belongs to the Acyl-CoA Synthetase (NDP-forming) Superfamily—The deduced amino acid sequence of TK1880 displays high similarity (47% identical) to the {alpha}-subunits of the ADP-forming ACSs I and II from the closely related Archaea P. furiosus (PF1540 and PF0532, respectively). In addition, TK0943 displays 55% identity to the beta-subunit of ACS IPf (PF1787) and much higher identity (89%) to that of ACS IIPf (PF1837). The TK1880 and TK0943 proteins also displayed similarity to other members of the NDP-forming acyl-CoA synthetase superfamily, although to a lesser extent.

Bacterial and eukaryotic SCSs are the most studied enzymes in this superfamily. The detailed three-dimensional structure of SCS from E. coli (SCSEc), which consists of two small {alpha}-subunits (SucD) and two large beta-subunits (SucC), indicates the presence of five (sub)domains in the protein, domains 1-2 provided by the {alpha}-subunit and domains 3-4-5 composing the beta-subunit, as shown in Fig. 2 (23, 27). Based on the detailed structure of SCSEc and information provided in the Molecular Modeling Database structure summary (Protein Data Bank code 2SCU), the five domains of SCSEc are designated here as domain 1 ({alpha}-subunit residues 1–122), domain 2 ({alpha}-subunit residues 123–288), domain 3 (beta-subunit residues 19–102), domain 4 (beta-subunit residues 1–18 and 103–229), and domain 5(beta-subunit residues 230–388). The {alpha}-subunit is responsible for binding with the CoA substrate, and the active-site histidine (His246) resides in domain 2. Domains 3-4 of the beta-subunit harbor the ATP-grasp fold, and domain 5 harbors one of the three nucleotide-binding motifs found in the {alpha}beta-dimer. It has been pointed out that other members in this superfamily also commonly harbor these five domains, although the order and distribution of the domains between the two subunits display variation (18). Although all previously characterized classical SCSs share the same subunit and domain structure (domain order: {alpha}-subunit, 1-2; and beta-subunit, 3-4-5) (23, 24, 27, 28), the SCS identified here from T. kodakaraensis exhibits a distinct structure: domain 5 is fused to the {alpha}-subunit ({alpha}-subunit, domains 1-2-5; and beta-subunit, domains 3-4) (Fig. 2). The structure is therefore closer to those of the ACSs of Thermococcales (11–14) and the single polypeptide ACSs in G. lamblia (18). It should also be noted that the individual domains of SCSTk exhibit higher similarity to the domains of the Thermococcales and G. lamblia ACSs (>38% identical) than to those of the classical SCSs (<28% identical). These facts explain why functional prediction of TK1880 and TK0943 as SCSs was not possible based on the primary structure in T. kodakaraensis.


Figure 3
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  		FIGURE 3.
Diagram illustrating the paralog sets of {alpha}- and beta-subunits of NDP-forming acyl-CoA synthetase in Thermococcales. Gene products from T. kodakaraensis (TK), P. abyssi (PAB), P. furiosus (PF), and P. horikoshii (PH) that are >80% identical in amino acid sequence to one another are grouped together below each subunit. Combinations that have been experimentally verified to result in enzyme activity are connected.

 
Overexpression and Purification of Recombinant SCSTk and ACS IITk—The T. kodakaraensis genome harbors four genes (TK0139, TK0665, TK0944, and TK2127) paralogous to the {alpha}-subunit of SCSTk (TK1880) and one gene (TK0465) paralogous to the beta-subunit (TK0943). Members of the five- and two-paralog groups are similar in length (440–474 and 212–243 amino acids) and display overall similarity (41–50 and 55% identical) to the other members of the respective groups, indicating that their domain arrangements are common. Equivalent paralogous gene sets are also present on the genomes of P. abyssi (9), P. furiosus (29), and P. horikoshii (10), and each member exhibits particularly high identity (>80% at the amino acid levels) to only one member in the other strains (Fig. 3). The larger number of {alpha}-subunit genes implies that each beta-subunit may accommodate more than one type of {alpha}-subunit. Interestingly, the beta-subunit of SCSTk identified here (TK0943) corresponds to the beta-subunit of ACS IIPf (PF1837), suggesting that SCS and ACS II share the same beta-subunit in these hyperthermophiles.


Figure 4
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  		FIGURE 4.
Levels of acyl-CoA-forming activity (ADP-forming) of recombinant SCSTk and ACS IITk for various organic acid compounds. The activity levels of SCSTk are indicated by black bars and those of ACS IITk with gray bars. Each acid compound is related to the metabolism of a particular amino acid, which is indicated in parentheses.

 
To examine whether TK0943 can act as the beta-subunit for both SCS and an ACS, we expressed the gene together with the TK1880 gene (encoding the SCS {alpha}-subunit) or the TK0139 gene (the T. kodakaraensis counterpart of the {alpha}-subunit gene of ACS IIPf) in E. coli. Recombinant proteins of TK1880/TK0943 (SCSTk) and TK0139/TK0943 (ACS IITk) were recovered as soluble proteins after heat treatment at 80 °C for 10 min and subsequently purified. SCSTk and ACS IITk were determined to be {alpha}2beta2-heterotetramers by gel filtration chromatography, the same quaternary structures as those reported for native SCSTk and ACS IIPf. The formation of {alpha}2beta2-heterotetramers and their high thermostability (see below) strongly suggest that TK0943 can assemble with either TK1880 or TK0139 as the {alpha}-subunit.

Biochemical Characterization of Recombinant SCSTk and ACS IITk—Purified recombinant SCSTk and ACS IITk exhibited ATP-dependent succinyl-CoA- and acetyl-CoA-forming activities at high temperature, respectively. Both enzymes showed the highest activity at pH 6.5, and the optimum temperatures were 75–80 °C for SCSTk and 75 °C for ACS IITk. The enzymes were highly thermostable, with half-lives of 90 min (SCSTk) and 120 min (ACS IITk) at 95 °C. At 98 °C, both enzymes showed half-lives of 50 min. The high thermostability of the proteins suggests that the decrease in activity observed in both enzymes at temperatures above 80 °C is most likely due to the instability of CoA thioesters in our assay system at high temperatures.

We coupled the ATP-dependent CoA thioester-forming activity with pyruvate kinase and lactate dehydrogenase at 55 °C to quantify ADP formation. As expected, high rates of NADH oxidation were observed with succinate (SCSTk) and acetate (ACS IITk), confirming that SCSTk and ACS IITk are ADP-forming enzymes. Using this assay, we examined the substrate specificity of SCSTk and ACS IITk with various organic acids likely to be generated through amino acid metabolism. As shown in Fig. 4, SCSTk showed high activity for succinate (16.2 units/mg), but malonate was not recognized as a substrate. Isovalerate and 3-methyl thiopropionate were converted by SCSTk with activity levels of approximately two-thirds compared with the succinate-converting activity. Although not direct intermediates of amino acid catabolism, glutarate (121%), adipate (59%), and butyrate (48%) also served as good substrates for SCSTk, whereas propionate (10%) and oxalate (9%) did not (data not shown). Considering the structures of these compounds, SCSTk seems to prefer mono- or dicarboxylates with a backbone of four or more carbons. In contrast, ACS IITk exhibited higher levels of activity for isovalerate (36.1 units/mg) than for acetate (15.6 units/mg) and could accept various acids with diverse hydrophobic structures such as 3-methyl thiopropionate (94% to isovalerate), 2-methyl butyrate (57%), isobutyrate (27%), and aromatic acids (phenyl acetate (77%), 4-hydroxyphenyl acetate (59%), and 3-indole acetate (34%)). The broad substrate specificity of ACS IITk for hydrophobic and aromatic acids corresponds well with that of the previously reported ACS IIPf. Our results clearly demonstrate that, despite the common beta-subunit, swapping of {alpha}-subunits in SCSTk and ACS IITk results in entirely different substrate specificities.

We further examined the kinetic properties of the SCSTk-catalyzed reactions in both directions (Table 2). The CoA thioester-forming activity of SCSTk was determined by monitoring ADP formation at 55 °C as described above. Interestingly, SCSTk displayed strong negative cooperation in succinate binding, and nonlinear regression analysis of the velocity data gave a dissociation constant of 2.8 mM for the first succinate molecule (Ks1), whereas the dissociation constant for the second substrate molecule (Ks2) was much larger (29 mM). Such negative cooperation was also observed in the reactions with isovalerate and 3-methyl thiopropionate as substrates (Table 2). The kinetic parameters indicated that succinate was the preferred substrate in the acyl-CoA-forming reaction. Acetate, the major substrate for ACS, was not efficiently converted to acetyl-CoA by SCSTk. Increases in activity were still observed at concentrations as high as 600 mM, and reliable curve fitting could therefore not be performed. The velocity of the reverse reaction was determined by monitoring the release of free CoA at 55 °C. Kinetic analysis with succinyl-CoA could not be performed because of the labile nature of succinyl-CoA at high reaction temperatures. We found that isovaleryl-CoA was much more stable than succinyl-CoA at these temperatures, and kinetic examinations were carried out with this substrate. SCSTk showed typical Michaelis-Menten kinetics with Km and kcat values of 9.1 µM and 54 s-1 active site-1, respectively. The kcat/Km value for isovaleryl-CoA was >10-fold higher than that for acetyl-CoA, again suggesting that SCSTk is not an enzyme involved mainly in acetate/acetyl-CoA conversion. Furthermore, the much higher affinity and kcat/Km values of the enzyme for isovaleryl-CoA compared with isovalerate (Table 2) support an in vivo function of SCSTk in catalyzing CoA thioester breakdown resulting in ATP production rather than acyl-CoA synthesis.


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  		TABLE 2
Kinetic parameters of SCSTk at 55 °C

 
Immunoprecipitation of SCSTk and ACS IITk—As our in vitro data clarified that the single beta-subunit TK0943 can form an active enzyme with two distinct {alpha}-subunits, we next examined whether this is the case in vivo. We performed an immunoprecipitation analysis using specific antisera prepared against the individual purified beta-subunits TK0943 and TK0465. The precipitates were heat-treated to denature and remove heavy and light chains of the antibodies, followed by SDS-PAGE analysis. Multiple proteins with a molecular mass of ~50 kDa coprecipitated with the individual beta-subunits, and MALDI-TOF tandem mass spectrometry analyses identified TK0944, TK0665, TK1880, and TK0139 associated with TK0943 and also TK0944, TK0665, and TK0139 with associated TK0465 (data not shown). Although cross-reaction of the antibodies with the other beta-subunit was not observed in Western blot analysis, there is a possibility that cross-reaction occurred in the immunoprecipitation experiments. However, the presence of more than two types of {alpha}-subunits in each precipitate indicates that the beta-subunits assemble with multiple {alpha}-subunits in vivo.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Paralog sets of five {alpha}-subunits and two beta-subunits of acyl-CoA synthetase (NDP-forming) genes are completely conserved in the Thermococcales genomes. Among the 10 combinations possible, only two had been identified until now, ACSs IPf and IIPf (11–14). In this study, we found that one of the {alpha}-subunits (TK1880) in T. kodakaraensis is a component of a novel ADP-forming SCS with a distinct domain order and distribution among subunits compared with the classical SCS enzymes.

Despite the different order and distribution of the five domains among the {alpha}- and beta-subunits, it is presumed that members of the acyl-CoA synthetase (NDP-forming) superfamily share a common catalytic mechanism (18). In the case of CoA thioester formation by SCSEc, the {gamma}-phosphate of ATP is first transferred to the catalytic His246 residue in the {alpha}-subunit, and the phosphorylated imidazole ring reacts with acid and CoA to form the corresponding acyl-CoA molecule. The ATP- and CoA-binding sites are located within the ATP-grasp fold domain in the beta-subunit (domains 3-4) and the CoA-binding domain in the {alpha}-subunit (domain 1), respectively (23, 27, 30, 31). The catalytic His residue (corresponding to His261 in the TK1880 {alpha}-subunit) and several important residues that function in binding with the ATP·Mg2+ complex and CoA are well conserved among this superfamily (18). In contrast, the substrate-binding site has yet to be clarified even in the well studied SCSEc. The alteration of substrate specificity between SCSTk and ACS IITk in this study indicates the presence of the substrate-binding site in the {alpha}-subunits. In particular, the CoA ligase domains (domains 2 and 5) attached to the N-terminal CoA-binding domain in the {alpha}-subunit can be considered to be candidates for the substrate-binding domain. In the classical ATP- and GTP-dependent SCSs from pigeon, distinct beta-subunits (domain order 3-4-5) are responsible for large differences in the apparent Km values for succinate, in addition to a change in nucleotide specificity probably caused by replacement of the ATP-grasp fold (domains 3-4) (32). Taking these facts into consideration, domain 5 in the {alpha}-subunit of SCSTk and ACSTk seems likely to play a role in substrate recognition and binding.

Although not published in the literature, the crystal structures of PH0766 and PH1788 are available in the Protein Data Bank and provide valuable information in understanding how the archaeal SCS and ACS and the classical SCSs exhibit similar activity with different domain orientations (Fig. 5). P. horikoshii PH0766 and PH1788 are the {alpha}- and beta-subunits that correspond to the subunits of ACS II and ACS II/SCS in T. kodakaraensis, respectively. As in the case of the {alpha}-subunit of SCSTk (TK1880), PH0766 consists of domains 1-2-5. The crystal structure reveals a dimeric structure of the protein, in which domains 1-2 of one monomer ({alpha}1-subunit) make contact with domain 5 of the opposite monomer ({alpha}2-subunit). The arrangement of the three domains ({alpha}1-subunit, domains 1-2; and {alpha}2-subunit, domain 5) is strikingly similar to that observed in SCSEc ({alpha}-subunit, domains 1-2; and beta-subunit, domain 5). Furthermore, the surface of the {alpha}1-subunit (domains 1-2)/{alpha}2-subunit (domain 5) structure of PH0766 corresponding to the surface that interacts with domains 3 and 4 in SCSEc seems to be exposed to the solvent, indicating that a domain 3-4 protein can make contact in an orientation similar to that observed in SCSEc. As anticipated from its primary structure, the fold of PH1788 consisting of domains 3-4 (beta-subunit) is an ATP-grasp fold. As TK1880 and TK0943 can be expected to display a structure similar to those of the Pyrococcus counterparts, this model explains how SCSTk and SCSEc, with differences in their domain order and subunit distribution, can assemble into functional units of basically the same topology.


Figure 5
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  		FIGURE 5.
Model indicating the proposed topology of SCSTk that would allow the enzyme to exhibit a domain orientation similar to those of the classical SCS proteins. a, the crystal structure of one dimer unit ({alpha}beta) of the tetrameric ({alpha}2beta2) SCSEc (Protein Data Bank code 2SCU), which is the product of the sucC and sucD genes, is shown on the left. The five domains are numbered and colored ({alpha}-subunit, domain 1 (green) and domain 2 (cyan); and beta-subunit, domain 3 (yellow); domain 4 (orange), and domain 5, magenta). The crystal structures of dimeric PH0766 (Protein Data Bank code 2CSU) and monomeric PH1788 from P. horikoshii, which correspond to the {alpha}- and beta-subunits of ACS II and ACS II/SCS from T. kodakaraensis, respectively, are shown in the middle and on the right. The colors of the domains correspond to those used for SCSEc. The domains of the two {alpha}-subunits ({alpha}1 and {alpha}2) of PH0766 are distinguished by numbering ({alpha}1-subunit, domains 1, 2, and 5, and {alpha}2-subunit, domain 1', 2', and 5'). The domains in the {alpha}-subunit correspond to residues 1–126 (domain 1), 127–274 (domain 2), and 275–453 (domain 5), and those in the beta-subunit correspond to residues 36–117 (domain 3) and 1–35 and 118–237 (domain 4). The structures were visualized using the software PyMOL 0.99 (33). b, the model indicates the similar domain orientation that may be taken by PH0766/PH1788 based on the structures shown in a. Domain colors correspond to those described for a.

 
The physiological functions of ACSs I and II have been supposed to be in the breakdown of the acyl-CoA molecules coupled to ATP generation (11–14). Although ACS IIPf (13, 14) and ACS IITk (this study) exhibit broad substrate specificities for a variety of acids with hydrophobic side chains, SCSTk is relatively specific, displaying relevant levels of activity for only a few acids. The activity levels and kinetic parameters of SCSTk for succinate, isovalerate, and 3-methyl thiopropionate suggest the involvement of this enzyme in the catabolism of Glu, Leu, and Met. However, considering that ACS IITk also displays high levels of activity for the latter two substrates, the major physiological role of SCSTk is most likely in the generation of ATP from succinyl-CoA. Indeed, in addition to large amounts of acetate production (+35.6 mM), we observed accumulation of succinate (+1.03 mM) stoichiometric to Glu/Gln consumption (-1.04 mM) in the medium when T. kodakaraensis was grown in the presence of amino acids along with pyruvate.3 As shown in Fig. 6, the identification of SCSTk completes a metabolic route for energy generation from glutamate to succinate via 2-oxoglutarate and succinyl-CoA by the function of glutamate dehydrogenase, 2-oxoglutarate:ferredoxin oxidoreductase, and SCSTk.


Figure 6
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  		FIGURE 6.
Diagram illustrating the catabolism of glutamate in T. kodakaraensis. S0, elemental sulfur; AT, amino acid:2-oxoacid aminotransferase; GDH, glutamate dehydrogenase; AlaAT, alanine aminotransferase; POR, pyruvate:ferredoxin oxidoreductase; VOR, 2-oxoisovalerate:ferredoxin oxidoreductase; IOR, indolepyruvate:ferredoxin oxidoreductase; KGOR, 2-oxoglutarate:ferredoxin oxidoreductase; Fdox, oxidized ferredoxin; Fdred, reduced ferredoxin.

 

   FOOTNOTES
 
* This work was supported by Grant-in-aid for Scientific Research 14103011 (to T. I.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed. Tel.: 81-75-383-2777; Fax: 81-75-383-2778; E-mail: imanaka{at}sbchem.kyoto-u.ac.jp.

2 The abbreviations used are: ACS IPf, acetyl-CoA synthetase I from P. furiosus; NDP, nucleoside diphosphate; SCS, succinyl-CoA synthetase; SCSTk, succinyl-CoA synthetase from T. kodakaraensis; MES, 4-morpholineethanesulfonic acid; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; SCSEc, succinyl-CoA synthetase from E. coli; ACS IITK, acetyl-CoA synthetase II from T. kodakaraensis. Back

3 T. Kanai, H. Imanaka, T. Fukui, K. Uwamori, Y. Omori, and T. Imanaka, unpublished data. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Itoh, T. (2003) J. Biosci. Bioeng. 96, 203-212[Medline] [Order article via Infotrieve]
  2. Adams, M. W. W. (1994) FEMS Microbiol. Rev. 15, 261-277[CrossRef][Medline] [Order article via Infotrieve]
  3. Robb, F. T., Park, J.-B., and Adams, M. W. W. (1992) Biochim. Biophys. Acta 1120, 267-272[CrossRef][Medline] [Order article via Infotrieve]
  4. Ma, K., Hutchins, A., Sung, S.-J., and Adams, M. W. W. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 9608-9613[Abstract/Free Full Text]
  5. Heider, J., Mai, X., and Adams, M. W. W. (1996) J. Bacteriol. 178, 780-787[Abstract/Free Full Text]
  6. Mai, X., and Adams, M. W. W. (1996) J. Bacteriol. 178, 5890-5896[Abstract/Free Full Text]
  7. Mai, X., and Adams, M. W. W. (1994) J. Biol. Chem. 269, 16726-16732[Abstract/Free Full Text]
  8. Fukui, T., Atomi, H., Kanai, T., Matsumi, R., Fujiwara, S., and Imanaka, T. (2005) Genome Res. 15, 352-363[Abstract/Free Full Text]
  9. Cohen, G. N., Barbe, V., Flament, D., Galperin, M., Heilig, R., Lecompte, O., Poch, O., Prieur, D., Quérellou, J., Ripp, R., Thierry, J.-C., Van der Oost, J., Weissenbach, J., Zivanovic, Y., and Forterre, P. (2003) Mol. Microbiol. 47, 1495-1512[CrossRef][Medline] [Order article via Infotrieve]
  10. Kawarabayasi, Y., Sawada, M., Horikawa, H., Haikawa, Y., Hino, Y., Yamamoto, S., Sekine, M., Baba, S., Kosugi, H., Hosoyama, A., Nagai, Y., Sakai, M., Ogura, K., Otsuka, R., Nakazawa, H., Takamiya, M., Ohfuku, Y., Funahashi, T., Tanaka, T., Kudoh, Y., Yamazaki, J., Kushida, N., Oguchi, A., Aoki, K., and Kikuchi, H. (1998) DNA Res. 5, 55-76[Abstract]
  11. Schäfer, T., and Schönheit, P. (1991) Arch. Microbiol. 155, 366-377
  12. Schäfer, T., Selig, M., and Schönheit, P. (1993) Arch. Microbiol. 159, 72-83[CrossRef]
  13. Mai, X., and Adams, M. W. W. (1996) J. Bacteriol. 178, 5897-5903[Abstract/Free Full Text]
  14. Musfeldt, M., Selig, M., and Schönheit, P. (1999) J. Bacteriol. 181, 5885-5888[Abstract/Free Full Text]
  15. Krahe, M., Antranikian, G., and Märkl, H. (1996) FEMS Microbiol. Rev. 18, 271-285[CrossRef]
  16. Kanai, T., Imanaka, H., Nakajima, A., Uwamori, K., Omori, Y., Fukui, T., Atomi, H., and Imanaka, T. (2005) J. Biotechnol. 116, 271-282[CrossRef][Medline] [Order article via Infotrieve]
  17. Field, J., Rosenthal, B., and Samuelson, J. (2000) Mol. Microbiol. 38, 446-455[CrossRef][Medline] [Order article via Infotrieve]
  18. Sánchez, L. B., Galperin, M. Y., and Müller, M. (2000) J. Biol. Chem. 275, 5794-5803[Abstract/Free Full Text]
  19. Labes, A., and Schönheit, P. (2001) Arch. Microbiol. 176, 329-338[CrossRef][Medline] [Order article via Infotrieve]
  20. Musfeldt, M., and Schönheit, P. (2002) J. Bacteriol. 184, 636-644[Abstract/Free Full Text]
  21. Bräsen, C., and Schönheit, P. (2004) Arch. Microbiol. 182, 277-287[CrossRef][Medline] [Order article via Infotrieve]
  22. Bräsen, C., and Schönheit, P. (2004) FEMS Microbiol. Lett. 241, 21-26[CrossRef][Medline] [Order article via Infotrieve]
  23. Fraser, M. E., James, M. N. G., Bridger, W. A., and Wolodko, W. T. (1999) J. Mol. Biol. 285, 1633-1653[CrossRef][Medline] [Order article via Infotrieve]
  24. Fraser, M. E., Hayakawa, K., Hume, M. S., Ryan, D. G., and Brownie, E. R. (2006) J. Biol. Chem. 281, 11058-11065[Abstract/Free Full Text]
  25. Atomi, H., Fukui, T., Kanai, T., Morikawa, M., and Imanaka, T. (2004) Archaea 1, 263-267[Medline] [Order article via Infotrieve]
  26. Takeda, Y., Suzuki, F., and Inoue, H. (1969) Methods Enzymol. 13, 153-160
  27. Wolodko, W. T., Fraser, M. E., James, M. N. G., and Bridger, W. A. (1994) J. Biol. Chem. 269, 10883-10890[Abstract/Free Full Text]
  28. Fraser, M. E., James, M. N. G., Bridger, W. A., and Wolodko, W. T. (2000) J. Mol. Biol. 299, 1325-1339[CrossRef][Medline] [Order article via Infotrieve]
  29. Robb, F. T., Maeder, D. L., Brown, J. R., DiRuggiero, J., Stump, M. D., Yeh, R. K., Weiss, R. B., and Dunn, D. M. (2001) Methods Enzymol. 330, 134-157[Medline] [Order article via Infotrieve]
  30. Joyce, M. A., Fraser, M. E., James, M. N. G., Bridger, W. A., and Wolodko, W. T. (2000) Biochemistry 39, 17-25[CrossRef][Medline] [Order article via Infotrieve]
  31. Fraser, M. E., Joyce, M. A., Ryan, D. G., and Wolodko, W. T. (2002) Biochemistry 41, 537-546[CrossRef][Medline] [Order article via Infotrieve]
  32. Johnson, J. D., Muhonen, W. W., and Lambeth, D. O. (1998) J. Biol. Chem. 273, 27573-27579[Abstract/Free Full Text]
  33. DeLano, W. L. (2002) The PyMOL Molecular Graphics System, DeLano Scientific, Palo Alto, CA

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