Regulation of Coenzyme A Biosynthesis in the HyperthermophilicBacterium Thermotoga maritima

Takahiro Shimosaka,a Hiroya Tomita,a Haruyuki Atomia,b

Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto, Japana; JST, CREST, Tokyo, Japanb

ABSTRACT

Regulation of coenzyme A (CoA) biosynthesis in bacteria and eukaryotes occurs through feedback inhibition targeting type I andtype II pantothenate kinase (PanK), respectively. In contrast, the activity of type III PanK is not affected by CoA. As the hyper-thermophilic bacterium Thermotoga maritima harbors only a single type III PanK (Tm-PanK), here we examined the mecha-nisms that regulate CoA biosynthesis in this organism. We first examined the enzyme responsible for the ketopantoate reductase(KPR) reaction, which is the target of feedback inhibition in archaea. A classical KPR homolog was not present on the T. mari-tima genome, but we found a homolog (TM0550) of the ketol-acid reductoisomerase (KARI) from Corynebacterium glutami-cum, which exhibits KPR activity. The purified TM0550 protein displayed both KPR and KARI activities and was designated Tm-KPR/KARI. When T. maritima cell extract was subjected to anion-exchange chromatography, the fractions containing highlevels of KPR activity also displayed positive signals in a Western blot analysis using polyclonal anti-TM0550 protein antisera,strongly suggesting that Tm-KPR/KARI was the major source of KPR activity in the organism. The KPR activity of Tm-KPR/KARI was not inhibited in the presence of CoA. We thus examined the properties of Tm-PanK and the pantothenate synthetase(Tm-PS) of this organism. Tm-PS was not affected by CoA. Surprisingly however, Tm-PanK was inhibited by CoA, with almostcomplete inhibition in the presence of 400 �M CoA. Our results suggest that CoA biosynthesis in T. maritima is regulated byfeedback inhibition targeting PanK, although Tm-PanK is a type III enzyme.

IMPORTANCE

Bacteria and eukaryotes regulate the biosynthesis of coenzyme A (CoA) by feedback inhibition targeting type I or type II panto-thenate kinase (PanK). The hyperthermophilic bacterium Thermotoga maritima harbors a single type III PanK (Tm-PanK), pre-viously considered to be unaffected by CoA. By examining the properties of three enzymes involved in CoA biosynthesis in thisorganism, we found that Tm-PanK, although a type III enzyme, is inhibited by CoA. The results provide a feasible explanation ofhow CoA biosynthesis is regulated in T. maritima, which may also apply for other bacteria that harbor only type III PanK en-zymes.

Coenzyme A (CoA) is an essential cofactor found in all threedomains of life and is involved in numerous metabolic pathways

(1–3). In bacteria and eukaryotes, CoA is synthesized from pantothe-nate via five reactions catalyzed by pantothenate kinase (PanK),phosphopantothenoylcysteine synthetase (PPCS), phosphopanto-thenoylcysteine decarboxylase (PPCDC), phosphopantetheineadenylyltransferase (PPAT), and dephospho-CoA kinase (DPCK)(Fig. 1). In microorganisms and plants, pantothenate can be syn-thesized de novo from ketoisovalerate and �-alanine by ketopan-toate hydroxymethyltransferase (KPHMT), ketopantoate reduc-tase (KPR), and pantothenate synthetase (PS). In bacteria, �-alanine is synthesized from aspartate by aspartate 1-decarboxylase(ADC) (4, 5). Animals and some pathogenic bacteria do not har-bor the route from ketoisovalerate to pantothenate and thus relyon exogenous pantothenate for CoA synthesis.

Some bacteria do not harbor a classical KPR, and atypical pro-teins that display KPR activity have been reported. Ketol-acid re-ductoisomerase (KARI), encoded by the ilvC gene, catalyzes theKPR reaction (6–8). KARI is involved in the biosynthesis ofbranched-chain amino acids (Val, Leu, and Ile) and catalyzes theisomerization and reduction of acetohydroxybutyrate (for Ile)and acetolactate (for Val and Leu) (9). Disruption of the KARIgene in Corynebacterium glutamicum results in pantothenate aux-otrophy, indicating that KARI is responsible for the KPR reactionin this organism (7). Another atypical KPR (PanG) was recently

characterized in Francisella tularensis subsp. tularensis Schu S4(10). PanG is conserved in all sequenced Francisella species, andhomologs are also found in several pathogenic bacteria.

The biosynthesis of CoA in bacteria and eukaryotes is regulatedby feedback inhibition. PanK is the main target of this regulation,and CoA, acetyl-CoA, and other CoA derivatives inhibit PanKactivity (2). In Escherichia coli, PPAT and KPHMT are also inhib-ited by CoA, but the effects are moderate in the case of PPAT (11),and concentrations of �1 mM CoA are necessary to inhibitKPHMT (12). PanK enzymes are classified into three types basedon their primary structure. Type I and type III PanKs are found ina wide range of bacteria, and the type II enzyme is mainly distrib-uted in eukaryotes. Type I and type II enzymes display the inhibi-

Received 23 January 2016 Accepted 4 May 2016

Accepted manuscript posted online 9 May 2016

Citation Shimosaka T, Tomita H, Atomi H. 2016. Regulation of coenzyme Abiosynthesis in the hyperthermophilic bacterium Thermotoga maritima. J Bacteriol198:1993–2000. doi:10.1128/JB.00077-16.

Editor: A. Becker, Philipps-Universität Marburg

Address correspondence to Haruyuki Atomi, atomi@sbchem.kyoto-u.ac.jp.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/JB.00077-16.

Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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tion by CoA and CoA derivatives described above. An exception isthe type II PanK from Staphylococcus aureus, which is not inhib-ited by CoA (13). On the other hand, type III PanK is not inhibitedby CoA. This has been experimentally demonstrated for the en-zymes from Helicobacter pylori and Bacillus subtilis (14).

In archaea, the mechanisms regulating CoA biosynthesis differfrom those of bacteria and eukaryotes (1, 15). In the hyperther-mophilic archaeon Thermococcus kodakarensis, the first four pro-teins of the CoA biosynthesis pathway and the protein necessaryfor �-alanine synthesis have been identified and characterized(16–20). Pantoate kinase (PoK) and phosphopantothenate syn-thetase (PPS) are unique to the archaea and replace PS and PanKin bacteria and eukaryotes in the conversion of pantoate to 4=-phosphopantothenate (Fig. 1) (20). The presence of PoK and PPShas also been demonstrated in Methanospirillum hungatei (21).The effects of CoA on KPHMT, KPR, PoK, and PPS from T. koda-karensis were examined (16–18, 20) and indicated that KPR is thetarget of feedback inhibition in this archaeon (17). The crystalstructure of KPR complexed with CoA and ketopantoate revealshow CoA inhibits the activity of KPR (22). PoK and PPS homologsare present on the majority of archaeal genomes, with exceptionslimited to Nanoarchaeum equitans, Korarchaeum cryptofilum, andmembers of the Thermoplasmatales, including Picrophilus torri-dus. A type I PanK from P. torridus was identified and character-ized, but the enzyme was not inhibited by CoA (23).

In this study, we examined the mechanisms regulating CoAbiosynthesis in the hyperthermophilic bacterium Thermotoga ma-ritima. The organism harbors a single type III PanK, suggestingthat regulation of CoA biosynthesis in T. maritima differs fromthat found in bacteria with type I enzymes and eukaryotes withtype II enzymes. We show that in T. maritima, a KARI homologfunctions as a KPR but is not affected by CoA. We further dem-onstrate that feedback inhibition targets the PanK reaction, al-though the enzyme is a type III PanK.

MATERIALS AND METHODSStrains and growth conditions. Thermotoga maritima 10099T was pur-chased from the Japan Collection of Microorganisms (JCM), RIKENBioResource Center (Japan). Cells were cultivated at 85°C in a nutrient-rich medium (artificial seawater-yeast extract-tryptone [ASW-YT]) un-der anaerobic conditions. ASW-YT medium consisted of 0.8� ASW (24),5.0 g liter�1 yeast extract, 5.0 g liter�1 tryptone, and 0.8 mg liter�1 resaz-urin. Prior to inoculation, Na2S was added to the medium until it becamecolorless. Escherichia coli strains DH5� and BL21-CodonPlus(DE3)-RILwere cultivated at 37°C in Luria-Bertani (LB) medium containing 100 mgliter�1 ampicillin. Unless mentioned otherwise, all chemicals were pur-chased from Wako Pure Chemicals (Osaka, Japan) or Nacalai Tesque(Kyoto, Japan).

Overexpression of the TM0550, TM0883, and TM1077 genes andpurification of the recombinant proteins. The TM0550, TM0883, andTM1077 genes were amplified from the genomic DNA of T. maritima

FIG 1 Coenzyme A biosynthesis pathway. In bacteria and eukaryotes, PS and PanK are responsible for the conversion of pantoate to 4=-phosphopantothenate.PoK and PPS replace the PS/PanK system in most archaea. Three different enzymes, KPR, KARI, and PanG, are known to catalyze the KPR reaction. PanKenzymes are classified into three types (types I to III) based on their primary structure. This study suggests that T. maritima utilizes KARI for the KPR reactionand that the type III PanK is inhibited by CoA. Enzymes utilized in T. maritima are indicated with (Tm). THF, tetrahydrofolate.

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using the primer set TM0550F/TM0550R (5=-AAAGGATCCCATATGGCAGTGATTTATTACGACA-3=/5=-AAAGAATTCTCACTCCTCATCGACGTTCCTCT-3=) for TM0550, TM0883F/TM0883R (5=-AAAGGATCCCATATGTACCTCCTCGTGGACGTGGGTAA-3=/5=-AAAGAATTCTCAATCTCCGAAGCAGAAATG-3=) for TM0883, and TM1077F/TM1077R (5=-AAAGGATCCCATATGAGAATCATAGAGACTATC-3=/5=-AAAGAATTCTCACCCCAGGATCGTGTTATC-3=) for TM1077. With useof NdeI and EcoRI, whose recognition sites are underlined, the amplifiedfragments and pET21a(�) expression vector (Merck KGaA, Darmstadt, Ger-many) were digested and ligated. After confirmation of the absence of unin-tended mutations, the constructed plasmids were individually introducedinto E. coli BL21-CodonPlus(DE3)-RIL. The procedures for gene expres-sion and protein purification were the same for all three proteins. Trans-formants were inoculated into LB medium containing ampicillin and cul-tivated at 37°C until the optical densities at 660 nm reached �0.4.Isopropyl-�-D-1-thiogalactopyranoside was added to a final concentra-tion of 0.1 mM to induce expression, and cells were cultivated for a further4 h. Cells were harvested by centrifugation (4°C, 5,000 � g, 15 min) andsuspended in 50 mM Tris-HCl (pH 7.5) with 150 mM NaCl. After cen-trifugation (4°C, 5,000 � g, 15 min), cells were suspended in 50 mMTris-HCl buffer (pH 7.5), disrupted by sonication, and centrifuged again(4°C, 5,000 � g, 15 min). The soluble cell extract was incubated at 90°C for10 min. After centrifugation (4°C, 5,000 � g, 15 min), the supernatant wasfiltered with a 0.2-�m New Steradisc Sterilized filter (Kurabo, Osaka,Japan) and then applied to a Resource Q 6-ml anion-exchange chroma-tography column (GE Healthcare, Little Chalfont, Buckinghamshire,United Kingdom). Protein was eluted with a linear gradient of NaCl (0 to1.0 M) in 50 mM Tris-HCl (pH 7.5) at a flow rate of 2.0 ml min�1. Thefractions were concentrated with an Amicon Ultra-4 10K centrifugal filter(Millipore, Billerica, MA), filtered, and then applied to a Superdex 20010/300 GL gel filtration column (GE Healthcare). Protein was eluted with50 mM Tris-HCl (pH 7.5), including 150 mM NaCl at a flow rate of 0.4 mlmin�1. For examining molecular mass, RNase A (13.7 kDa), carbonicanhydrase (29 kDa), conalbumin (75 kDa), and ferritin (440 kDa) (GEHealthcare) were used as standard proteins. All chromatography proce-dures were performed using an Äkta explorer system (GE Healthcare).The protein concentration was determined with the protein assay system(Bio-Rad, Hercules, CA) using bovine serum albumin as a standard.

Examination of KPR and KARI activities of the TM0550 protein.KPR activity was measured by consecutively monitoring the rate of de-crease in absorption of NADPH at 340 nm at 80°C using a spectropho-tometer. The reaction mixture contained 50 mM Tris-HCl (pH 7.5), 0.2mM NADPH (Oriental Yeast, Tokyo, Japan), 10 mM ketopantoate, 10mM MgCl2, and 10 �g ml�1 TM0550 protein. Ketopantoate was preparedby hydrolysis of dihydro-4,4-dimethyl-2,3-furandione (Sigma-Aldrich,St. Louis, MO) in 0.4 M NaOH for 1 h at 90°C. After preincubation of thereaction mixture without NADPH, the reaction was initiated by the addi-tion of NADPH. The rate of decrease in a reaction mixture withoutTM0550 protein was subtracted from each result. Modifications of thismethod are described when applied. KARI activity was measured by thesame method, except that ketopantoate was replaced with acetolactate.Acetolactate was prepared by hydrolysis of methyl 2-hydroxy-2-methyl-3-oxobutyrate (Sigma-Aldrich) in 0.1 M NaOH.

Thermostability and effects of pH and temperature on activity of theTM0550 protein. For examining thermostability, TM0550 protein (1 mgml�1) in 50 mM Tris-HCl (pH 7.5) was incubated for various periods oftime at 70°C, 80°C, or 90°C. After incubation, the mixture was cooled onice, and residual KPR activity was measured. For examining the effects oftemperature on activity, the KPR reaction was performed at various tem-peratures in 50 mM HEPES (pH 7.5). For examining the effects of pH, theKPR reaction was performed at 80°C at various pH values using the fol-lowing 50 mM buffers: 2-morpholineethanesulfonic acid (MES) (pH 5.5to 6.5), piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES) (pH 6.5 to 7.5),Tris (pH 7.5 to 8.0), N,N-bis(2-hydroxyethyl)glycine (Bicine) (pH 8.0 to

9.0), and N-cyclohexyl-2-aminoethanesulfonic acid (CHES) (pH 9.0 to10.0).

Substrate specificity of the TM0550 protein and kinetic examinationof the reaction. For examining the substrate specificity of the TM0550protein, various ketoacids and acetolactate were used as substrates at afinal concentration of 10 mM. For kinetic analysis, activity assays wereperformed with various concentrations of ketoacids and acetolactate(with 0.2 mM NADPH) or NAD(P)H (with 10 mM ketopantoate)(NADH; Oriental Yeast). Kinetic parameters were calculated with IGORPro v. 5.03 (Wave-Metrics, Lake Oswego, OR).

Ketopantoate reductase in the cell extract of T. maritima. T. mari-tima was cultivated in ASW-YT medium at 85°C for 12 h and harvested bycentrifugation (4°C, 5,000 � g, 15 min). Cells were suspended in 0.8�ASW, centrifuged, resuspended with 50 mM Tris-HCl (pH 7.5), and dis-rupted by sonication. After centrifugation (4°C, 5,000 � g, 15 min), thesupernatant was applied to an Amicon Ultra-0.5 3K centrifugal filter (Mil-lipore) and centrifuged to remove low-molecular-weight compounds.The sample retained above the membrane was fractionated with the sameprocedure that was applied for purifying recombinant TM0550 proteinusing a Resource Q 6-ml anion-exchange chromatography column. TheKPR activity in 100-�l aliquots from each fraction was measured. Sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was per-formed on a 12.5% gel with the fractions that showed high KPR activity.Gels were either stained with Coomassie brilliant blue or used for Westernblot analysis. In the latter case, proteins were electroblotted onto an Am-ersham Hybond polyvinylidene difluoride (PVDF) membrane (GEHealthcare). After blocking with blocking reagents (GE Healthcare),membranes were hybridized with anti-TM0550 protein antisera gener-ated with the purified, recombinant protein. The membrane was washed,hybridized with horseradish peroxidase (HRP)-conjugated recombinantprotein G (dilution, 1:100,000; Invitrogen, Carlsbad, CA), and washedagain. For signal detection, the ECL advance Western blotting detectionsystem (GE Healthcare) and an ImageQuant LAS 500 system (GE Health-care) were used.

Examination of PS activity of the TM1077 protein and PanK activityof the TM0883 protein. PS activity was measured by monitoring the gen-eration of AMP by high-performance liquid chromatography (HPLC).The reaction mixture contained 50 mM Tris-HCl (pH 7.5), 4 mM ATP, 10mM KCl, 10 mM MgCl2, 4 mM pantoate, 4 mM �-alanine (Sigma-Al-drich), and 10 �g ml�1 TM1077 protein. Pantoate was prepared by hy-drolysis of D-(�)-pantolactone (Sigma-Aldrich) in 0.4 M NaOH for 1 h at90°C. Reactions were carried out at 80°C. PanK activity was measured bymonitoring the generation of ADP by HPLC. The reaction mixture con-tained 50 mM Tris-HCl (pH 7.5), 6 mM ATP, 10 mM KCl, 10 mM MgCl2,0.4 mM calcium pantothenate, and 100 �g ml�1 TM0883 protein. Reac-tions were carried out at 75°C. PS or PanK proteins were removed aftertheir reactions by ultrafiltration using an Amicon Ultra-0.5 10K cen-trifugal filter (Millipore), and 10-�l aliquots were applied to a Cosmo-sil 5C18-PAQ 4.6-mm-inside-diameter (i.d.) by 250-mm column (Naca-lai Tesque). Compounds were separated with 20 mM sodium phosphate(pH 6.1) at a flow rate of 1.0 ml min�1 at 40°C. Absorbance at 210 nm wasmeasured. For use as a standard compound, 4=-phosphopantothenate wassynthesized enzymatically with PanK from E. coli as previously described(20).

Effect of CoA and acetyl-CoA on KPR, PS, and PanK activities. KPR,PS, and PanK activity measurements were carried out in the presence ofvarious concentrations of CoA and acetyl-CoA. Experiments with KPRwere performed with different concentrations of NADPH or ketopan-toate. In the experiments with PS and PanK, CoA or acetyl-CoA wasadded to the standard reaction mixtures described above.

RESULTSGenes potentially related to CoA biosynthesis in T. maritima.We searched the T. maritima genome for genes potentially in-volved in CoA biosynthesis. We used the amino acid sequences of

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the enzymes involved in CoA biosynthesis in E. coli and per-formed a BLAST search against the protein sequences of T.maritima. The genes identified were TM1728 (45.6% identicalwith E. coli KPHMT), TM0939 (36.0%, ADC), TM1077 (46.4%,PS), TM1687 (37.8%, PPCS/PPCDC), TM0741 (47.8%, PPAT),and TM1387 (26.7%, DPCK). T. maritima does not possess a typeI PanK but harbors a type III PanK encoded by TM0883, which hasbeen biochemically and structurally characterized (25, 26). Therewere no genes that encoded proteins with notable similarity toKPR from E. coli or to PanG from F. tularensis. On the other hand,we found that TM0550 was 54.5% identical to the ketol-acid re-ductoisomerase (KARI) of C. glutamicum, which displays KPRactivity (7). As type III PanKs are considered to be unaffected byCoA, we first focused on the KPR reaction, as this is the target offeedback inhibition in archaeal CoA biosynthesis (17). We thusexamined whether the KARI homolog TM0550 corresponded toKPR in T. maritima and whether it was inhibited by CoA.

Expression and purification of the TM0550 protein. TheTM0550 gene was expressed in E. coli, and the recombinant pro-tein was purified by heat treatment, followed by anion-exchangechromatography and gel filtration chromatography. The samplewas subjected to SDS-PAGE and the gel was stained with Coomas-sie brilliant blue. Only one band corresponding to the molecularmass of TM0550 (38,046 Da) was observed (Fig. 2A), indicatingthat the protein was purified to apparent homogeneity. Judgingfrom gel filtration chromatography, the molecular mass of theprotein was greater than that of ferritin, suggesting that it wascomposed of more than 12 subunits.

Basic enzymatic properties of the TM0550 protein. The puri-fied, recombinant TM0550 protein was incubated with NADPHand acetolactate or ketopantoate. In both cases, oxidation of NA-DPH was observed, suggesting that the protein exhibited bothKARI and KPR activities. KPR activity (generation of pantoatefrom ketopantoate) was confirmed by adding the reaction mix-ture to the PS reaction described below (see Fig. S1 in the supple-mental material). We thus designated the T. maritima TM0550protein Tm-KPR/KARI.

The effects of temperature on KPR activity were examined atvarious temperatures between 60°C and 90°C (Fig. 3A). Activitylevels continued to increase up to 90°C. The activation energy ofthe KPR reaction, based on the Arrhenius plot of the data, wascalculated to be 76.2 kJ mol�1 (Fig. 3B). To examine the effect of

pH, KPR activity was performed at various pH values at 80°C.Tm-KPR/KARI showed its highest activity at pH 7.5 (Fig. 3C). Thethermostability of Tm-KPR/KARI was examined by incubatingthe protein at 70°C, 80°C, and 90°C for various periods of time.The half-life of the protein was calculated to be 250 min, 90 min,and 7 min at 70°C, 80°C, and 90°C, respectively (Fig. 3D).

Substrate specificity of Tm-KPR/KARI and kinetic examina-tion. Substrate specificity was examined using various ketoacids(10 mM) as substrates. Tm-KPR/KARI exhibited the highest ac-tivity with ketopantoate in addition to relatively high levels ofactivity with ketobutyrate, ketoisovalerate, and ketovalerate (Fig.4). Kinetic examinations were performed with these four ketoac-ids and acetolactate. Among the four ketoacids, the initial velocitysaturated at much lower substrate concentrations with ketopan-toate (Fig. 5A) than with the other three ketoacids (Fig. 5B), andthe kcat/Km values were more than a magnitude higher with keto-pantoate (Table 1). With acetolactate as a substrate, high levels ofactivity were observed at low substrate concentrations, and sub-strate inhibition was observed at higher concentrations (Fig. 5C).We applied a typical equation for substrate inhibition, v Vmax[S]/(Ks1 � [S] � 1/Ks2 [S]2), where v is the initial reactionvelocity, Vmax is the maximum velocity, [S] is the substrate con-centration, Ks1 is the dissociation constant between the enzymeand the first substrate, and Ks2 is the dissociation constant betweenthe enzyme-substrate complex and the second, inhibitory, sub-strate. This equation fit well to our data, and the kinetic param-eters are shown in Table 1. A rough estimate (kcat/Ks1 9.58s�1 mM�1) suggests that the catalytic efficiency of the TM0550protein for the KARI reaction is even higher than that for theKPR reaction. In addition, a kinetic examination was per-formed with NADH and NADPH (Fig. 5D). A comparison ofthe kcat/Km values indicates that Tm-KPR/KARI prefersNADPH over NADH (Table 1).

Ketopantoate reductase activity in the cell extract of T. ma-ritima. We next examined whether Tm-KPR/KARI was the mainsource of KPR activity in T. maritima. T. maritima was cultivatedin ASW-YT medium, and cell extracts were prepared. NADPH-dependent KPR activity was observed in the cell extracts. The cellextracts were fractionated with an anion-exchange column, andKPR activity was examined with each fraction. Although a de-crease in NADPH was observed in the activity measurements withmany fractions, strikingly high levels of KPR activity were ob-served in fractions B5 to B8 (Fig. 6A). These fractions also dis-played KARI activity. An SDS-PAGE analysis of fractions B3 to C2is shown in Fig. 6B. We performed a Western blot analysis on thesefractions using anti-TM0550 protein antisera generated with thepurified, recombinant protein (Fig. 6C). We found that the pres-ence of the TM0550 protein clearly coincided with the presence ofKPR activity. The results suggest that Tm-KPR/KARI is at leastresponsible for the majority of KPR activity in T. maritima cells.

Effects of CoA and acetyl-CoA on Tm-KPR/KARI activity. Asour results suggested that Tm-KPR/KARI was the major source ofKPR activity in T. maritima, we examined whether the enzymewas inhibited in the presence of CoA or acetyl-CoA. In a previousstudy on the KPR from the archaeon T. kodakarensis, we observeda decrease in activity of �50% when 0.1 mM CoA or acetyl-CoAwas added to the reaction mixture in the presence of 0.2 mMNADH (17). Here we added various concentrations of CoA oracetyl-CoA in the presence of 5 mM ketopantoate and 0.2 mMNADPH. Even at concentrations of 1 mM CoA or acetyl-CoA, the

FIG 2 SDS-PAGE analysis of the purified TM0550 (A), TM0883 (B), andTM1077 (C) proteins. Each lane contains 1 �g of protein, and the gels werestained with Coomassie brilliant blue.

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KPR activity of Tm-KPR/KARI was not affected (Fig. 7). The sameexperiments were carried out in the presence of lower concentra-tions of substrate (0.5 mM ketopantoate or 0.02 mM NADPH),but inhibition by CoA was not observed. The results indicate thatTm-KPR/KARI is not a target of feedback inhibition.

Examination of the T. maritima type III PanK. As KPR activ-

ity of Tm-KPR/KARI was not affected by CoA, we next examinedthe type III PanK of T. maritima (Tm-PanK) encoded by TM0883.The recombinant protein was produced in E. coli and subjected toheat treatment, followed by anion-exchange chromatography andgel filtration chromatography. The sample was applied to SDS-PAGE, and the gel was stained with Coomassie brilliant blue.Only one band corresponding to the molecular mass of theTM0883 protein (27,154 Da) was observed (Fig. 2B). Judgingfrom gel filtration chromatography, the protein was a hexamer.The protein (Tm-PanK) displayed PanK activity in the pres-ence of pantothenate and ATP, and the reaction product 4=-phos-phopantothenate was clearly detected by HPLC (see Fig. S2 in thesupplemental material). The specific activity of the protein was0.18 �mol min�1 mg�1 in the presence of 0.4 mM pantothenateand 6 mM ATP.

We next examined the effects of CoA on Tm-PanK activity.CoA or acetyl-CoA was added to the reaction mixture at concen-trations from 0 to 1 mM. Surprisingly, although Tm-PanK struc-turally resembled type III PanKs, we observed a dramatic inhibi-tion of activity in the presence of CoA (Fig. 7). Activity was almostcompletely abolished in the presence of 0.4 mM CoA. In contrast,no effects were observed with acetyl-CoA at concentrations up to1 mM.

Examination of the T. maritima PS. Our results suggestedthat Tm-PanK is the target of feedback inhibition in T. maritima.

FIG 3 Basic enzymatic properties of Tm-KPR/KARI. (A) The effects of temperature on KPR activity. Data are means standard deviations (SD); n 3. (B) AnArrhenius plot of the data shown in panel A. (C) The effects of pH on KPR activity. Data are means SD; n 3. �, MES (pH 5.5 to 6.5);o, PIPES (pH 6.5 to7.5); �, Tris (pH 7.5 to 8.0); Œ, Bicine (pH 8.0 to 9.0); Œ, CHES (pH 9.0 to 10.0). Data are means SD; n 3. (D) Thermostability of Tm-KPR/KARI. Symbols:�, 70°C; Œ, 80°C; �, 90°C. The 100% relative activity corresponds to 3.22 �mol min�1 mg�1.

FIG 4 Specific activity of Tm-KPR/KARI toward various ketoacids. Substratesand NADPH were added at a concentration of 10 mM and 0.2 mM, respec-tively, and the reactions were carried out at 80°C. Data are mean SD, n 3.

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In order to further support this, we examined whether the PS inthis organism (Tm-PS) was inhibited by CoA. The TM1077 genewas expressed in E. coli, and the recombinant protein was purifiedto apparent homogeneity by heat treatment, followed by anion-exchange chromatography and gel filtration chromatography.Only one band corresponding to the molecular mass of theTM1077 protein (32,756 Da) was observed after SDS-PAGE anal-ysis (Fig. 2C). Judging from gel filtration chromatography, theprotein was a tetramer. Tm-PS displayed PS activity in the pres-ence of pantoate, �-alanine, and ATP, with the generation of pan-tothenate clearly detected during the reaction (see Fig. S3 in the

supplemental material). The specific activity of the protein was11.1 �mol min�1 mg�1 in the presence of 4 mM pantoate, 4 mM�-alanine, and 4 mM ATP. When we added CoA or acetyl-CoA tothe reaction mixture at concentrations from 0 to 1 mM, no inhi-bition was observed (Fig. 7).

DISCUSSION

In this study, we have clarified that the three genes, TM0550,TM1077, and TM0883, from T. maritima encode proteins thatdisplay KPR, PS, and PanK activities, respectively, and have alsoexamined the effects of CoA on the activities of these three en-zymes. Only Tm-PanK was affected, and activity was almost com-pletely inhibited in the presence of 400 �M CoA. This was unex-pected, as Tm-PanK is structurally related to type III PanKproteins, which have been regarded to be enzymes that are unaf-fected by CoA (14). Our results strongly suggest that, at least at theenzyme activity level, CoA biosynthesis in T. maritima is regulatedby a feedback regulation mechanism targeting PanK. This raisesthe possibility that in addition to the organisms with type I andtype II PanK proteins, there are a number of bacteria with type IIIenzymes that regulate CoA biosynthesis via PanK inhibition.Whether there are additional mechanisms in T. maritima at thetranscription/translation level that regulate CoA biosynthesis willrequire further studies.

Concerning Tm-KPR/KARI, the protein exhibited both KPRactivity and KARI activity. As there is only one KPR that has beencharacterized from a hyperthermophile (17), it is difficult tojudge, from a biochemical viewpoint, whether the catalytic effi-ciency of Tm-KPR/KARI is sufficient to conclude that it can act as

FIG 5 Kinetic examination of Tm-KPR/KARI. (A) Initial velocities with various concentrations of ketopantoate and 0.2 mM NADPH. (B) Initial velocities withvarious concentrations of ketobutyrate, ketoisovalerate, or ketovalerate with 0.2 mM NADPH. Activity measurements were not possible with ketovalerate atconcentrations higher than 40 mM. (C) Initial velocities with various concentrations of acetolactate and 0.2 mM NADPH. (D) Initial velocities with variousconcentrations of NADH or NADPH in the presence of 10 mM ketopantoate.

TABLE 1 Kinetic parameters of Tm-KPR/KARI toward varioussubstratesa

SubstrateVmax (�molmin�1 mg�1) Km (mM) kcat (s�1)

kcat/Km

(s�1 mM�1)

Ketobutyrate 15.8 0.8 91.1 8.4 9.96 0.53 0.11Ketovalerate 7.62 0.78 40.2 6.5 4.83 0.49 0.12Ketoisovalerate 5.73 0.13 38.8 2.1 3.63 0.08 0.09Ketopantoate 2.74 0.04 0.696 0.038 1.74 0.02 2.50NADH 2.29 0.05 0.0292 0.0018 1.45 0.03 49.7NADPH 4.26 0.06 0.0138 0.0007 2.70 0.04 195Acetolactate 11.6 0.4 0.77 0.06b 7.38 0.28 9.58c

16.7 1.6d

a All acid substrates were examined in the presence of 0.2 mM NADPH. Values aremeans standard deviations or ratios. Activity with acetolactate displayed substrateinhibition.b Ks1.c kcat/Ks1.d Ks2.

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a KPR, in addition to its role as a KARI. When we compare theKPR activities of Tm-KPR/KARI and the KPR protein from T.kodakarensis, the kcat/Km values are 2.50 s�1 mM�1 and 3,830 s�1

mM�1, respectively (17). The catalytic efficiency of the KPR activ-ity of Tm-KPR/KARI is much lower than that of the KPR proteinfrom T. kodakarensis, mostly due to the extremely low Km value (6�M) of the T. kodakarensis KPR toward ketopantoate (17). Whenwe compared the catalytic efficiencies of KPR activity and KARIactivity of Tm-KPR/KARI, the kcat/Km value toward ketopantoate(2.50 s�1 mM�1) was �26% of the kcat/Ks1 value toward acetolac-tate (9.58 s�1 mM�1). In the case of KARI from E. coli, whichharbors a classical KPR gene in addition to KARI, the kcat/Km forketopantoate (1.104 s�1 mM�1) was �12% of that for acetolactate(9.020 s�1 mM�1) (27). As T. maritima lacks the classical KPRgene, the relatively higher ratio of KPR activity against KARI ac-tivity in Tm-KPR/KARI than that in the E. coli KARI may reflectthe fact that the protein does function as a KPR. In any case, themajority of KPR activity that was detected in T. maritima cells wasshown to derive from the Tm-KPR/KARI protein, indicating thatit is the major source of KPR activity in T. maritima cells.

The crystal structure of Tm-PanK has been determined, andstructural characteristics have been proposed to explain the rea-son why type III PanKs, including Tm-PanK, are not affected byCoA (25, 26). However, to our knowledge, a direct examination ofthe effects of CoA on Tm-PanK activity has not been previouslyreported. As our analysis indicated that CoA inhibits Tm-PanK,this suggests the presence of an unidentified CoA binding site onthis protein. Structural studies of Tm-PanK crystallized in thepresence of CoA will be important to clarify the mechanisms re-sponsible for this inhibition and reveal the specific structural ele-ments that distinguish the two kinds of type III PanKs: those thatare affected by CoA and those that are not.

One notable feature of Tm-PanK is the stark difference in ef-fects observed between CoA and acetyl-CoA. We did not observe

FIG 6 KPR activity in T. maritima cell extracts. (A) T. maritima cell extractswere applied to a Resource Q column, and the rate of decrease in absorption ofNADPH at 340 nm was monitored for each fraction. The rate of decrease in areaction mixture without ketopantoate was subtracted from each result.mAbs, milli-absorbance units; ��mAbs (decrease in absorbance at 340nm) � 103. (B) SDS-PAGE analysis of fractions B3 to C2. Aliquots (10 �l)from each fraction were applied, and the gel was stained with Coomassie bril-liant blue. (C) Western blot analysis of the samples described in panel B usinganti-TM0550 protein antisera.

FIG 7 The effects of CoA and acetyl-CoA on KPR, PS, and PanK activities.CoA or acetyl-CoA was added to the KPR, PS, or PanK reaction mixture. TheKPR reaction mixture contained 5 mM ketopantoate and 0.2 mM NADPH.The PanK reaction mixture contained 0.4 mM pantothenate and 6 mM ATP.The PS reaction mixture contained 4 mM pantoate, 4 mM �-alanine, and 4mM ATP. The 100% relative activities of KPR, PS, and PanK correspond to2.43 �mol min�1 mg�1, 11.1 �mol min�1 mg�1, and 0.13 �mol min�1 mg�1,respectively.

Regulation of CoA Biosynthesis in Thermotoga maritima

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any inhibitory effects on Tm-PanK in the case of acetyl-CoA (Fig.7). In the case of the type I PanK from E. coli, the thiol group ofCoA interacts with the side chains of aromatic residues (28). How-ever, when acetyl-CoA binds to PanK, the interaction becomesweaker due to the bulkiness of the acetyl group, resulting in alower inhibitory effect (28). Similarly, the free thiol group of CoAmay be required for interaction with Tm-PanK, and the bulkyacetyl moiety of acetyl-CoA might be completely disrupting thisrecognition with Tm-PanK.

ACKNOWLEDGMENT

This study was funded by the CREST program of the Japan Science andTechnology Agency within the research area Creation of Basic Technologyfor Improved Bioenergy Production through Functional Analysis andRegulation of Algae and Other Aquatic Microorganisms (to H.A.).

FUNDING INFORMATIONThis work, including the efforts of Haruyuki Atomi, was funded by JapanScience and Technology Agency (JST).

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