Báo cáo khoa học: A novel isoform of pantothenate synthetase in the Archaea potx

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A novel isoform of pantothenate synthetase inthe ArchaeaSilvia Ronconi, Rafal Jonczyk and Ulrich GenschelLehrstuhl fu¨r Genetik, Technische Universita¨tMu¨nchen, Freising, GermanyPantothenate is the essential precursor to CoA, whichis of central importance for all parts of metabolism.This is shown by the fact that more than 400 enzyme-catalyzed reactions are known to involve CoA (KEGGdatabase [1]). Many more enzymes utilize acylatedforms of CoA or require the CoA-derived phospho-pantetheine as a prosthetic group. Typically, plants,fungi and microorganisms are able to synthesize panto-thenate de novo, whereas animals rely on pantothenatein their diet.Pantothenate synthetase (PS) catalyzes the last step inthe biosynthesis of pantothenic acid, also known as vita-min B5. The enzyme (EC 6.3.2.1) has been extensivelystudied in Escherichia coli [2,3], Mycobacterium tubercu-losis [4,5], and Arabidopsis thaliana [6], and is highlyconserved in the Bacteria and Eukaryota. Bacterial PS(Eqn 1) generates pantothenate from pantoate andb-alanine. It is an AMP-forming synthetase thatproceeds via an acyl-adenylate intermediate and belongsto the HIGH superfamily of nucleotidyltransferases [3].Keywordsarchaeal metabolism; CoA biosynthesis;evolution of metabolism;Methanosarcina mazei; pantothenatesynthetaseCorrespondenceU. Genschel, Lehrstuhl fu¨r Genetik,Technische Universita¨tMu¨nchen, AmHochanger 8, 85350 Freising, GermanyFax: +49 8161 715636Tel: +49 8161 715644E-mail: genschel@wzw.tum.de(Received 7 February 2008, revised 17March 2008, accepted 19 March 2008)doi:10.1111/j.1742-4658.2008.06416.xThe linear biosynthetic pathway leading from a-ketoisovalerate to panto-thenate (vitamin B5) and on to CoA comprises eight steps in the Bacteriaand Eukaryota. Genes for up to six steps of this pathway can be identifiedby sequence homology in individual archaeal genomes. However, there areno archaeal homologs to known isoforms of pantothenate synthetase (PS)or pantothenate kinase. Using comparative genomics, we previously identi-fied two conserved archaeal protein families as the best candidates for themissing steps. Here we report the characterization of the predicted PS genefrom Methanosarcina mazei, which encodes a hypothetical protein(MM2281) with no obvious homologs outside its own family. Whenexpressed in Escherichia coli, MM2281 partially complemented an auxo-trophic mutant without PS activity. Purified recombinant MM2281 showedno PS activity on its own, but the enzyme enabled substantial synthesis of[14C]4¢-phosphopantothenate from [14C]b-alanine, pantoate and ATP whencoupled with E. coli pantothenate kinase. ADP, but not AMP, wasdetected as a coproduct of the coupled reaction. MM2281 also transferredthe14C-label from [14C]b-alanine to pantothenate in the presence of panto-ate and ADP, presumably through isotope exchange. No exchange tookplace when pantoate was removed or ADP replaced with AMP. Our resultsindicate that MM2281 represents a novel type of PS that forms ADP andis strongly inhibited by its product pantothenate. These properties differsubstantially from those of bacterial PS, and may explain why PS genes, incontrast to other pantothenate biosynthetic genes, were not exchangedhorizontally between the Bacteria and Archaea.AbbreviationsCOG, clusters of orthologous groups; KPHMT, ketopantoate hydroxymethyltransferase; KPR, ketopantoate reductase; PANK, pantothenatekinase; PS, pantothenate synthetase.2754 FEBS Journal 275 (2008) 2754–2764 ª 2008 The Authors Journal compilation ª 2008 FEBSD-pantoate þ b-alanine þ ATP ! D-pantothenate þ AMP þ PPið1ÞCoA biosynthesis is best understood in the Bacteria,where eight steps lead from a-ketoisovalerate to CoA(Fig. 1) [7,8]. The eukaryotic CoA pathway has beenstudied to various degrees in fungi, plants, and animals[9], and consists of both highly conserved bacterial-typeenzymes and divergent isoforms. Much less is knownabout CoA biosynthesis in the Archaea. We previouslyused comparative genomics to reconstruct the universalCoA biosynthetic pathway in the Bacteria, Eukaryota,and Archaea [10]. Archaeal genes for the ultimate foursteps can be identified by homology in all archaealgenomes, and experimental confirmation of this assign-ment is available for three of these steps [11,12]. Inaddition, bacterial-type genes for the first two steps areobvious in a number of the nonmethanogenic Archaea.However, homologs to bacterial PS or any of the threeestablished isoforms of pantothenate kinase (PANK)[13] are generally missing from archaeal genomes. Weapproached this problem by using a nonhomologysearch strategy based on conserved chromosomal prox-imity, a method that exploits the tendency of function-ally related genes to cluster along the chromosome [14].Using the archaeal CoA biosynthetic genes with homol-ogy to bacterial or eukaryotic genes on the CoA path-way as a starting point, this identified the clusters oforthologous groups (COG)1701 and COG1829 proteinfamilies as the best candidates for the PS and PANKsteps in archaeal CoA biosynthesis [10]. (COG proteinfamily identifiers cited in this report are defined in theCOG database [15]).Here we report the characterization of the predictedPS gene from Methanosarcina mazei. We conclude thatthe COG1701 family represents the archaeal isoformof PS, which utilizes the same substrates as bacterialPS, but forms ADP instead of AMP and has distinctkinetic properties. This supports the view that theintermediates of pantothenate biosynthesis are univer-sally conserved, whereas the corresponding enzymeswere recruited independently in the Bacteria andArchaea.ResultsPrediction of conserved archaeal protein familiesfor PS and PANKGenomic context and phylogenetic pattern analysispreviously identified the COG1701 and COG1829 pro-tein families as the best candidates for the missingsteps leading from pantoate to 4¢-phosphopantothenatein archaeal CoA biosynthesis [10]. Meanwhile, manymore archaeal genomes have been completed, and thecomparative genomics search for the missing steps wasrepeated by using the STRING tool [16]. This analysisrevealed additional, previously undetected, linksbetween established archaeal CoA genes and theCOG1701 and COG1829 families, confirming that thelatter are strong candidates for archaeal PS andPANK (Fig. 1).Fig. 1. The CoA biosynthetic pathway and its reconstruction in theArchaea. The linear pathway leading from a-ketoisovalerate to CoAcomprises eight steps in the Bacteria and Eukaryota. It proceedsvia pantoate, pantothenate (vitamin B5), and 4¢-phosphopantothe-nate. The remaining intermediates, as well as the branch for pro-duction of b-alanine, are left out for clarity. For six of these steps,homologs can be established in the Archaea, and the correspondingCOG families are shown in color to indicate the average level ofsequence identity to the respective E. coli or human CoA biosyn-thetic enzymes [10]. Nonhomologous functional links to the archa-eal homologs were obtained from the STRING database [16], asdescribed in Experimental procedures. Taken together, the links toCOG1701 and COG1829 clearly support these protein families asthe best candidates for the missing steps in archaeal CoA biosyn-thesis. The functional assignments for COG1701 (archaeal PS) andCOG1829 (archaeal PANK) are explained in the main text. PPCS,phosphopantothenoylcysteine synthetase; PPCDC, phospho-pantothenoylcysteine decarboxylase; PPAT, phosphopantetheineadenylyltransferase; DPCK, dephospho-Co A kinase; P-pantothe-nate, 4¢-phosphopantothenate.S. Ronconi et al. Pantothenate synthetase from Methanosarcina mazeiFEBS Journal 275 (2008) 2754–2764 ª 2008 The Authors Journal compilation ª 2008 FEBS 2755Consistent with the assumption that both proteinfamilies represent archaeal isoforms of CoA biosyn-thetic enzymes, their members are found in nearly allarchaeal genomes, but not outside the archaealdomain. Furthermore, COG1701 and COG1829 sharea strictly conserved phylogenetic profile and frequentlyoccur in tandem in potential operons (e.g. in Me. maz-ei; Fig. 2). A straightforward general function predic-tion is possible for the COG1829 family, whichbelongs to a superfamily of small molecule kinases(GHMP kinases [17]) and is therefore proposed to rep-resent archaeal PANK. This leaves COG1701, anorphan family with no obvious links to other proteinfamilies, as the best candidate for archaeal PS. Usingthe hhpred prediction server [18], COG1701 wasfound to be a distant homolog of acetohydroxyacidsynthase, which ligates two molecules of pyruvate toyield acetolactate. Specifically, there is approximately20% sequence identity between COG1701 proteins andthe b-domain of acetohydroxyacid synthases. Thisdomain has no specific catalytic function but isthought to be important for the structural integrity ofacetohydroxyacid synthase [19].Functional complementation of an E. colipanC mutantIn the genome of Me. mazei, the predicted ORF forPS (MM2281) is situated in a potential operontogether with the predicted PANK gene and the dfpgene (Fig. 2), and this cluster is therefore expected tocover the CoA biosynthetic steps leading from panto-ate to 4¢-phosphopantetheine. The MM2281 ORF wascloned by PCR and tested for its ability to comple-ment the E. coli panC mutant strain AT1371 in liquidminimal medium (Fig. 3). The panC gene, whichencodes PS in E. coli, and the empty pBluescript KSvector served as positive and negative controls in thisexperiment, respectively. All transformants grew wellin cultures supplemented with pantothenate (notshown). The cultures generally showed long lag peri-ods, during which the cells recovered from the starvingprocedure in pantothenate-free medium (see Experi-mental procedures). In the absence of supplements,AT1371 cells harboring MM2281 showed a shorter lagperiod and faster growth than the negative control,but did not grow as well as the positive control(Fig. 3A). The same pattern was observed in minimalmedium containing 1 mm pantoate, a substrate of PS,except that the growth of cells containing MM2281 orthe negative control was stimulated (Fig. 3B). In ourhands, the E. coli panC mutant carrying empty vector(negative control) showed minor growth in minimalFig. 2. Potential operon for CoA biosynthesis in Me. mazei. Thepredicted genes for PS and PANK, as well as the dfp gene encod-ing the bifunctional enzyme PPCS ⁄ PPCDC (MM2281 throughMM2283), occur in a cluster, which corresponds to the steps lead-ing from pantoate to 4¢-phosphopantetheine. PPCS, phospho-pantothenoylcysteine synthetase; PPCDC, phosphopantothenoylcysteine decarboxylase.Fig. 3. Functional complementation of an E. coli pantothenateauxotrophic mutant. The pantothenate-requiring E. coli mutant car-rying the Me. mazei MM2281 gene (d), the E. coli panC gene (h)or empty vector (s) was grown in liquid culture in the absence ofpantothenate, as described in Experimental procedures. The mini-mal medium contained no supplements (A) or an additional 1 mMpantoate (B). All transformants grew equally well when the mediumwas supplemented with 1 mM pantothenate (not shown).Pantothenate synthetase from Methanosarcina mazei S. Ronconi et al.2756 FEBS Journal 275 (2008) 2754–2764 ª 2008 The Authors Journal compilation ª 2008 FEBSmedium. This might be caused by an endogenous non-specific activity able to produce pantothenate or by theemergence of revertants. Nevertheless, regardless of theactual reason for this behavior, the observation thatMM2281-carrying cells recovered more quickly frompantothenate starvation and grew faster than the nega-tive control in two independent experiments indicatesthat expression of MM2281 partially complements theauxotrophic phenotype of E. coli AT1371.PS activity of recombinant MM2281The MM2281 protein was overproduced as an N-ter-minal His-tag fusion protein in E. coli and had a sub-unit molecular mass in good agreement with itspredicted size (30 kDa as judged by SDS ⁄ PAGE). Thenative molecular mass of MM2281 estimated by gelfiltration was 57 000 Da, indicating that the enzymeis apparently a dimer in solution.Purified recombinant MM2281 was checked for itsability to synthesize pantothenate from pantoate,b-alanine and ATP by using a sensitive isotopic assayprocedure. However, we were not able to demonstratePS activity of MM2281 alone. Even after incubationfor 3 h, the amount of [14C]b-alanine converted into[14C]pantothenate was below the lower limit of detec-tion (1% conversion). This means that PS activity ofMM2281 was absent or below 0.6 nmolÆmin)1Æmg)1inour assay.In an attempt to confirm pantothenate as a productof MM2281, we coupled the reaction with excessE. coli PANK, which converts pantothenate to 4¢-phosphopantothenate. MM2281, individual helperenzymes or combinations of these were assayed fortheir ability to convert [14C]b-alanine into [14C]panto-thenate or [14C]4¢-phosphopantothenate under stan-dard conditions. The14C-labeled reaction productswere separated by TLC, revealed by phosphoimaging(Fig. 4), and quantified to calculate specific PS activi-ties (Table 1). Whereas MM2281 alone had no signifi-cant pantothenate-synthesizing activity in this assay, itwas obvious that E. coli PS efficiently converted[14C]b-alanine into [14C]pantothenate (Fig. 4, lanes 2and 3). E. coli PANK alone did not act on [14C]b-ala-nine but was conducive to quantitative formation of[14C]4¢-phosphopantothenate when coupled with E. coliPS (Fig. 4, lanes 4 and 5). As the products of E. coliPS and E. coli PANK are firmly established, the reac-tions with these enzymes provide chromatographystandards for pantothenate and 4¢-phosphopantothe-nate and also confirm that the E. coli PANK prepara-tion used here was not contaminated with detectablePS activity. Therefore, the [14C]4¢-phosphopanto-Fig. 4. Synthesis of pantothenate or 4¢-phosphopantothenatethrough MM2281 (Me. mazei PS) or helper enzymes. Standardenzyme assays were carried out as described in Experimentalprocedures, containing no enzyme (control), individual enzymes,or enzyme combinations, as indicated. The figure shows the14C-labeled products after a reaction time of 3 h. Separation wasachieved by TLC. The enzyme abbreviations are as follows: EcPS,E. coli PS; EcPANK, E. coli PANK; PyrK, rabbit pyruvate kinase;bAla, b-alanine; PA, pantothenate; PPA, 4¢-phosphopantothenate.Table 1. Formation of pantothenate or 4¢-phosphopantothenatethrough MM2281 and helper enzymes. MM2281 and helperenzymes were tested individually or in combinations for their abilityto form [14C]pantothenate or [14C]4¢-phosphopantothenate from[14C]b-alanine, pantoate and ATP under the conditions of the stan-dard assay (Fig. 4). ND, not detectable; PyrK, rabbit pyruvatekinase.Enzyme(s)Specific pantothenate synthetaseactivity (nmolÆmin)1Æmg)1)[14C]Pantothenate[14C]4¢-PhosphopantothenateMM2281 ND NDE. coli PS > 900aNDE. coli PANK ND NDE. coli PS + E. coli PANK ND > 900aMM2281 + E. coli PANK ND 94 ± 11bMM2281 + PyrK ND NDMM2281 + PyrK +E. coli PANKND 140 ± 22baWith respect to E. coli PS. This value is a lower estimate becausethe reaction was complete within the first interval.bWith respectto MM2281.S. Ronconi et al. Pantothenate synthetase from Methanosarcina mazeiFEBS Journal 275 (2008) 2754–2764 ª 2008 The Authors Journal compilation ª 2008 FEBS 2757thenate produced by the combined action of MM2281and E. coli PANK clearly demonstrates the capacity ofMM2281 to synthesize pantothenate from pantoateand b-alanine (Fig. 4, lane 6). The rate of 4¢-phospho-pantothenate synthesis in this assay corresponds to aPS activity of 94 nmolÆmin)1Æmg)1with respect toMM2281, indicating that the E. coli PANK-mediatedremoval of pantothenate accelerated the synthesis ofpantothenate through MM2281 at least 100-fold.Principally, the above behavior can be explained byassuming either that MM2281 is potently inhibited bypantothenate or that equilibrium is reached after onlya small fraction of substrates has reacted. In bothcases, the reaction is expected to accelerate when pan-tothenate is removed, because this should abolish inhi-bition or displace the equilibrium. It should bementioned that active removal of pantothenate has nosignificant effect on the reaction rate of bacterial PS,essentially excluding the possibility that the MM2281preparation was contaminated with bacterial PS. Thisis because pantothenate was shown to be a very weakproduct inhibitor of My. tuberculosis PS [4] and theequilibrium of the reaction catalyzed by bacterial PSlies far on the product side. The latter statement canbe derived by considering the equilibrium constant ofthe reaction catalyzed by bacterial PS (Eqn 1). Theequilibrium constant for Eqn (1) has not been deter-mined experimentally, but can be deduced from theequilibrium constants for the hydrolysis of pantothe-nate into pantoate and b-alanine (K¢ = 42 at pH 8.1and 25 °C [20]) and the phosphorolysis of ATP intoAMP and PPi(K¢ =3· 109at pH 8 and 25 °C [21]).Combining the above constants gives the overall equi-librium constant for Eqn (1) at approximately pH 8and 25 °C(K¢Eqn (1)= 7.2 · 107). The large valuemeans that the reaction in Eqn (1) will go to comple-tion under physiological conditions, including theenzyme assay used in this study (pH 8.0, 37 °C).Given the large effect of removing pantothenate onMM2281, we also tested the effect of removing thepossible coproduct ADP. ADP was removed by pyru-vate kinase, which generates ATP from ADP in thepresence of excess phosphoenolpyruvate. This systemdid not detectably accelerate pantothenate synthesis byMM2281 alone but, interestingly, increased the rate of4¢-phosphopantothenate formation through MM2281and E. coli PANK approximately 1.5-fold (Fig. 4,lanes 7 and 8).With a view to directly observing possible adenosinenucleotide coproducts of MM2281-catalyzed pantothe-nate synthesis, standard assays were analyzed by usinga TLC system that provides separation of ATP, ADP,and AMP (Fig. 5). Whereas MM2281 alone had nodiscernible hydrolytic activity towards ATP, the cou-pled reaction of MM2281 and E. coli PANK generateda substantial amount of ADP. AMP was not detectedas a coproduct, suggesting that MM2281 is not anAMP-forming PS according to Eqn (1). By compari-son, stoichiometric coupling of E. coli PANK, whichproduces ADP, with a bacterial, AMP-forming PSwould lead to the accumulation of equimolar amountsof ADP and AMP. The observations that synthesis ofphosphopantothenate through MM2281 and E. coliPANK was accelerated by removing ADP and notaccompanied by production of AMP gave rise to thehypothesis that MM2281 is an ADP-forming synthe-tase according to Eqn (2):D-pantoate þ b-alanine þ ATP ! D-pantothenate þ ADP þ Pið2ÞThe equilibrium constant for Eqn (2) can be calcu-lated in the same way as that for Eqn (1) (see above).Using the equilibrium constant for phosphorolysis ofATP into ADP and Pi(K¢ = 1.6 · 107at pH 8 and25 °C [21]), the overall equilibrium constant forEqn (2) at approximately pH 8 and 25 °C becomesFig. 5. Detection of adenosine nucleotides produced by MM2281(Me. mazei PS) and E. coli PANK. Standard assays containingMM2281 alone (lane 4) or together with E. coli PANK (lane 5) werecarried out as described in Experimental procedures. Reaction mix-tures were separated by TLC, and nucleotides were visualized underUV light. Authentic standards of ATP, ADP and AMP were cochro-matographed on the same plate (lanes 1–3). EcPANK, E. coli PANK.Pantothenate synthetase from Methanosarcina mazei S. Ronconi et al.2758 FEBS Journal 275 (2008) 2754–2764 ª 2008 The Authors Journal compilation ª 2008 FEBSK¢Eqn (2)= 3.9 · 105. Although K¢Eqn (2)is smallerthan K¢Eqn (1), the reaction shown in Eqn (2) will stillessentially go to completion in our enzyme assay orunder physiological conditions.MM2281-catalyzed pantothenate–b-alanineisotope exchangeThe role of ATP and ADP in the MM2281-catalyzedde novo synthesis of pantothenate could not be investi-gated independently, because the assay for this forwardactivity required the presence of E. coli PANK, whichutilizes ATP and generates ADP. In order to circum-vent this problem, we assayed MM2281 alone for itsability to catalyze an isotope exchange between [14C]b-alanine and pantothenate (Table 2). The cosubstratedependence of this exchange activity then allowed con-clusions about the role of adenosine nucleotides andthe mechanism of MM2281. Generally, isotopeexchange between a given substrate–product pairoccurs in the presence of all cosubstrates and coprod-ucts (complete system) or, if the enzyme catalyzes apartial reaction, in the presence of a subset of reac-tants. Each cosubstrate may either be indispensable forthe exchange reaction to occur or merely affect theexchange rate [22].The MM2281-catalyzed incorporation of14C-labelfrom [14C]b-alanine into pantothenate was investigatedin the presence of full sets or subsets of the reactantsin Eqn (1) or Eqn (2), respectively (Table 2, Experi-ment I). In the presence of the full set of reactants(complete system), the assay based on Eqn (2) revealeda five-fold higher rate than that based on Eqn (1).Removing pantoate reduced the incorporation of14C-label into pantothenate to negligible levels in boththe Eqns (1,2) systems. In contrast, removing ATPabolished the accumulation of [14C]pantothenate onlyin the Eqn (1) system, whereas the Eqn (2) systemretained approximately 50% exchange activity. Thismeans that the pantothenate–b-alanine exchange in theEqn (2) system has no absolute requirement for ATP,and this result was confirmed by a second set of iso-tope exchange assays (Table 2, Experiment II). Wheninorganic phosphate (Pi) was removed from theEqn (2) system in addition to ATP, there was no sig-nificant further reduction in exchange activity, showingthat both ATP and Piare dispensable. However, whenboth ATP and ADP were removed, the resultingexchange activity was negligible. In summary,MM2281 catalyzed significant transfer of14C-labelfrom b-alanine to pantothenate in presence of pantoateand ADP. When pantoate was removed, the resultingpantothenate–b-alanine exchange was negligible. Also,the exchange reaction occurred only in the presence ofadenosine nucleotide, and ADP but not AMP couldsatisfy this requirement.Again, this behavior shows that the MM2281 prepa-rations were not contaminated with E. coli PS, becausebacterial PS requires only AMP to catalyze the panto-thenate–b-alanine isotope exchange [4,6]. The data inTable 2 also show that, apart from pantoate, bothATP and ADP have a strong effect on the rate of theMM2281-catalyzed pantothenate–b-alanine exchangereaction. The simplest explanation for this behavior isthat pantoate, ATP and ADP are all substrates orproducts of MM2281, which is consistent with thenotion that the enzyme is a synthetase that drivespantothenate formation by hydrolysis of ATP.MM2281 alone showed no detectable net synthesisof pantothenate in the forward assay (see above;Fig. 4), and the forward rate would be expected to beeven lower in the presence of products. We assume,therefore, that the transfer of14C-label from b-alanineto pantothenate was due to isotope exchange. Maximalexchange activity occurred in the complete system ofEqn (2), pointing to Eqn (2) as the basic reaction forMM2281. Moreover, our data suggest that MM2281is able to catalyze an ADP-dependent, but not anAMP-dependent, pantothenate–b-alanine exchangeTable 2. MM2281-catalyzed isotope exchange between [14C]b-ala-nine and pantothenate. MM2281 was assayed for its ability totransfer14C-label from [14C]b-alanine to pantothenate in the pres-ence or absence of cosubstrates. The cosubstrates in the completesystem are ATP, AMP, PPi, and pantoate (Eqn 1), or ATP, ADP, Pi,and pantoate (Eqn 2). Different preparations of MM2281 were usedin two independent experiments (Experiments I and II). Missing val-ues indicate that the cosubstrate combination indicated was nottested. ND, not detectable.ReactantsInitial exchange ratea(%)Experiment I Experiment IIEqn (2)Complete system 100 100Minus pantoate 2Minus ATP 46 50Minus ATP, minus pantoate 3Minus ATP, minus Pi45Minus ATP, minus ADP 3Eqn (1)Complete system 20Minus pantoate 1Minus ATP NDMinus ATP, minus pantoate NDaNormalized to the value in the complete system of Eqn (2), whichwas equal to 2.5 and 1.5 · 10)3Æmin)1in Experiments I and II,respectively.S. Ronconi et al. Pantothenate synthetase from Methanosarcina mazeiFEBS Journal 275 (2008) 2754–2764 ª 2008 The Authors Journal compilation ª 2008 FEBS 2759reaction in the presence of pantoate. This is difficult toreconcile with Eqn (1), and further supports Eqn (2) asthe overall reaction catalyzed by MM2281. The resid-ual exchange activity in the complete system ofEqn (1) may be due to contamination of the commer-cial ATP preparation with ADP.We also considered the theoretical possibility thatMM2281 is a hydrolase, which facilitates the equilib-rium between pantoate, b-alanine and pantothenateaccording to Eqn (3):D-pantoate þ b-alanine $ D-pantothenate ð3ÞAlthough there is no simple argument to rule outEqn (3) as the basic reaction of MM2281, explainingthe overall behavior of MM2281 in this way is verydifficult and also generates a conflict with the reportedvalue for K¢Eqn (3). Most importantly, Eqn (3) impliesthat ATP and ADP are not substrates of MM2281.The observed effects of ADP and ATP on the isotopeexchange rate can then be explained by assuming thatADP and ATP effectively promote a switch from inac-tive to active enzyme. However, this is at odds withthe observation that enzymatic removal of ADP accel-erated MM2281 in the forward direction (see above).Second, considering the equilibrium constant ofEqn (3) (K¢Eqn (3)=1⁄ 42 at pH 8.1 and 25 °C [21]),this reaction clearly favors hydrolysis of pantothenate.Thus, based on Eqn (3) and K¢Eqn (3), the majority ofthe pantothenate in the isotope exchange assay usedhere would be converted to pantoate and b-alanine.Using K¢Eqn (3)and the initial concentrations of panto-ate, b-alanine and pantothenate in the assay, themaximum fraction of14C-label associated with panto-thenate at equilibrium would be 35%. However, weobserved that [14C]pantothenate accumulated up to60% of the total14C-label during the assay. This cor-responds to an equilibrium constant of ‡ 1 ⁄ 15, whichis much larger than the reported value for K¢Eqn (3).DiscussionExperimental confirmation of computationallypredicted archaeal PSMetabolic reconstruction of the CoA biosyntheticpathway in representative organisms previouslyrevealed that the Archaea lack known genes for theconversion of pantoate into 4¢-phosphopantothenate.The protein families COG1701 and COG1829 werethen identified as the best candidates for the missingsteps by comparative analysis of the 16 completelysequenced archaeal genomes available at the time [10].The STRING database, which currently integrates 26archaeal genomes, revealed additional functional asso-ciations that support a role for COG1701 andCOG1829 in archaeal CoA biosynthesis (Fig. 1). Onthe basis of distant homology relationships, we tenta-tively assigned the PS and PANK functions to theCOG1701 and COG1829 protein families, respectively.Three lines of experimental evidence support thecomputational prediction of archaeal PS. First, thecloned COG1701 member from Me. mazei (MM2281)partially complemented the auxotrophic phenotype ofan E. coli mutant lacking PS activity (Fig. 3). Second,the recombinant proteins MM2281 and E. coli PANKtogether facilitated the synthesis of 4¢-phosphopantoth-enate from pantoate, b-alanine, and ATP (Fig. 4).Arguably, the enzyme preparations were not contami-nated with bacterial PS, allowing the conclusion thatpantothenate synthesis in the coupled assays was dueto MM2281. Third, MM2281 catalyzed the transfer of14C-label from b-alanine to pantothenate, presumablyby isotope exchange, in a cosubstrate-dependent man-ner (Table 2). Given that function is typically con-served within orthologous groups [15], demonstrationof PS activity for one member of the group providesstrong support for the prediction that COG1701 repre-sents the archaeal PS protein family.Properties of MM2281 (Me. mazei PS)Our data suggest that MM2281 is an ADP-formingpantothenate synthetase (Eqn 2) that is subject tostrong product inhibition by pantothenate. The behav-ior of MM2281 in the isotope exchange experimentsclearly suggests that MM2281 is an adenosine nucleo-tide-dependent pantothenate synthetase and not areversible pantothenate hydrolase. On the basis of thelarge values of the equilibrium constants for Eqns (1,2)(see above), the equilibria of both reactions can beassumed to lie on the side of pantothenate formation.In other words, regardless of the type of synthetasereaction, coupling of pantothenate synthesis from pan-toate and b-alanine to the hydrolysis of ATP will drivethe equilibrium to the product side. Therefore, thestrong acceleration of MM2281-catalyzed pantothenatesynthesis by the removal of pantothenate is very prob-ably not due to a shift in the equilibrium of the reac-tion, leaving potent inhibition of MM2281 bypantothenate as the best explanation. We propose thatMM2281 is an ADP-forming synthetase according toEqn (2), because this is consistent with the observationthat the synthesis of 4¢-phosphopantothenate throughMM2281 and E. coli PANK was accompanied by theaccumulation of ADP but not AMP (Fig. 5) and accel-erated by removing ADP. Furthermore, the isotopePantothenate synthetase from Methanosarcina mazei S. Ronconi et al.2760 FEBS Journal 275 (2008) 2754–2764 ª 2008 The Authors Journal compilation ª 2008 FEBSexchange data can readily be accounted for by Eqn (2)but not by Eqn (1).The highest PS activity of MM2281 observed in thisstudy was 140 nmolÆmin)1Æmg)1, which is equivalent toa turnover of 0.07 s)1. This is significantly below typi-cal values for kcat, which range from about 0.8 s)1to2 · 105s)1[22]. More specifically, the MM2281 activ-ity reported here was more than 15-fold lower thanthat of E. coli PS under optimal conditions (calculatedfrom data in [2]) and nearly 50-fold lower than the kcatreported for My. tuberculosis PS [4]. Given the com-paratively low activity of MM2281, it is possible thatthe enzyme requires an activator or cofactor that wasabsent from the standard assay. One attractive candi-date for this role is the predicted PANK in Me. mazei(MM2282; Fig. 2), which may be more effective thanE. coli PANK in accelerating MM2281. Moreover,conserved phylogenetic profiles and chromosomalproximity indicate a strong functional link betweenarchaeal PS (COG1701) and archaeal PANK(COG1829) (Fig. 1). Also, lack of an interacting pro-tein required for optimal activity could explain whyexpression of MM2281 achieved only partial comple-mentation of the E. coli panC mutant (Fig. 3). How-ever, our attempts to express and purify MM2282 didnot meet with success (data not shown), so thishypothesis could not be tested.The observation that MM2281 facilitated the panto-thenate–b-alanine exchange in the absence of ATP andPimay be taken to indicate that MM2281 is a PingPong enzyme able to catalyze a partial reaction. How-ever, the isotope exchange data in Table 2 show clearlythat the kinetic mechanism of MM2281 is differentfrom the Ping Pong system of bacterial PS. The latterconsists of two half-reactions, which proceed via anenzyme-bound pantoyl adenylate intermediate [2–5].As a result, the pantothenate–b-alanine exchange reac-tion of bacterial PS is independent of pantoate andhas an absolute requirement for only AMP. By com-parison, the cosubstrate dependence of the MM2281-catalyzed exchange reaction differs on several counts(see above) and is inconsistent with the Ping Pongsystem of bacterial PS. This shows that archaealPS evolved a distinct mechanism to synthesizepantothenate.Evolution of phosphopantothenate biosynthesisWe hypothesize that the entire upstream portion ofCoA biosynthesis, leading from common precursors tophosphopantothenate, evolved independently in theBacteria and Archaea. This view was initially based onthe finding that many archaeal genomes contain nohomologs to any of the corresponding bacterialenzymes and on the prediction of distinct archaealforms of PS and PANK [10]. The experimentalevidence in this study provides strong support for thepredicted identity of archaeal PS (COG1701). This, inturn, also supports the prediction of archaeal PANK(COG1829), because there are strong nonhomologouslinks between the two families (Fig. 1).The linear pathway from a-ketoisovalerate to phos-phopantothenate comprises four steps in the Bacteria(Fig. 1). So far, unrelated archaeal genes on this path-way have been computationally predicted for the thirdand fourth steps (i.e. PS and PANK), but not for thefirst and second steps [i.e. ketopantoate hydroxymeth-yltransferase (KPHMT) and ketopantoate reductase(KPR)]. Interestingly, the phyletic distribution of PSgenes indicates that they were not subject to horizontaltransfer, in either direction, between the archaeal andbacterial domains. Archaeal PS and PANK isoformsshow strict co-occurrence and are present in most ofthe Archaea, except in the Thermoplasmata class ofthe Euryarchaeota and in Nanoarchaeum equitans.Also, they are absent from the Bacteria andEukaryota. All of the Archaea that have archaeal-typePS and PANK universally lack homologs to bacterialor eukaryotic PS and PANK isoforms. In fact, bacte-rial PS is entirely absent from the archaeal domain,and archaeal homologs to bacterial PANK are limitedto the Thermoplasmata class.A different situation is encountered for the first twoCoA biosynthetic steps. In the Bacteria, these stepsare catalyzed by KPHMT and KPR, which converta-ketoisovalerate into pantoate. A subset of the non-methanogenic Archaea acquired these enzymes, pre-sumably by horizontal gene transfer, fromthermophilic bacteria [10]. Individual archaealgenomes encode either both bacterial-type KPHMTand bacterial-type KPR or either one or none of them.This pattern suggests that some archaeal speciesproduce pantoate by combining an archaeal KPHMTisoform with bacterial-type KPR or by combining anarchaeal KPR isoform with bacterial-type KPHMT.In other words, the distribution of bacterial-typeKPHMT and KPR genes supports the view that themajority of the Archaea contain so far unidentifiedgenes that encode unrelated isoforms of KPHMT andKPR. Moreover, the observed distribution may wellbe the result of nonorthologous gene displacement[23], where the archaeal isoforms of KPHMT andKPR were individually replaced by their bacterialcounterparts in certain archaeal species. Verification ofthis hypothesis awaits, of course, identification of thearchaeal genes for pantoate synthesis.S. Ronconi et al. Pantothenate synthetase from Methanosarcina mazeiFEBS Journal 275 (2008) 2754–2764 ª 2008 The Authors Journal compilation ª 2008 FEBS 2761Given that horizontal gene transfer occurred exten-sively between the thermophilic Bacteria and Archaea[24,25], the question arises of why KPHMT and KPRgenes were transferred, whereas PS genes were not.The archaeal PS (MM2281) characterized in this studyproduces pantothenate from the same precursors asbacterial PS but has clearly distinct kinetic properties.The most striking difference is the apparent inhibitionof MM2281 by pantothenate, raising the possibilitythat this step has a role in regulating archaeal CoAbiosynthesis. In contrast, the most important controlpoint in bacterial CoA biosynthesis is PANK [9], andno regulatory function is known for PS. It is attractiveto speculate, therefore, that horizontal gene transferof PS, and possibly PANK, was suppressed by incom-patible regulatory properties.Experimental proceduresMaterialsE. coli strain AT1371 [panC4, D(gpt-proA)62, lacY1,tsx-29, glnV44(AS), galK2(Oc), LAM-, Rac-0, hisG4(Oc),rfbD1, xylA5, mtl-1, argE3(Oc), thi-1] [26] was obtainedfrom the E. coli Genetic Stock Center, Yale University.[3-14C]b-alanine (55 mCiÆmmol)1) was from AmericanRadiolabeled Chemicals ⁄ Biotrend Chemikalien (Cologne,Germany). Rabbit pyruvate kinase and all other reagentswere from Sigma-Aldrich (Munich, Germany) unless indi-cated otherwise. d-Pantoate was prepared from d-pantoyllactone as described elsewhere [27]. Genomic DNA fromthe Me. mazei strain Goe1 (DSM 3647) was a gift fromK. Pflu¨ger, Universita¨tMu¨nchen.Cloning of the Me. mazei and E. coli genes for PSThe Me. mazei ORF MM2281 (GenBank accession numberAE008384) was PCR-amplified from genomic DNA byusing Pfu polymerase (Stratagene, Amsterdam, the Nether-lands) and the primers dGCGCGCATATGACcGATATtCCGCACGAtCACCCGcGcTACGAATCC and dGCGCGCTCGAGTtAGTAgCCgGTTTCCGCGGCCATGGT. Thestart and stop codons are in bold. Lower-case letters desig-nate silent nucleotide changes that were introduced toreduce the number of rare codons for expression in E. coli.The amplified ORF was subcloned via NdeI and XhoIrestriction sites in the primers into the pET28-a vector(Novagen ⁄ Merck Chemicals, Darmstadt, Germany). Theresulting plasmid, pET–MM2281, contains the MM2281ORF in translational fusion with the vector-encodedN-terminal His-tag, leading to the expression of NH2-MGSSHHHHHHSSGLVPRGSH-MM2281.For functional complementation of the E. coli panCmutant (AT1371), the MM2281 ORF was reamplified frompET-MM2281 using the primers dGCGCGAGAAGGAGATATACCATGACCGATATTCCGCACGATCACCCGCGC and dGCGCGCTCGAGTTAGTAGCCGGTTTCCGCGGCCATGGT. The ribosome-binding site in the for-ward primer is underlined, and the start and stop codonsare in bold. The PCR product was inserted into thepGEM-T vector (Promega, Mannheim, Germany), and aclone carrying the MM2281 ORF in the correct orientationfor expression under the lac promoter was selected andnamed pGEM–MM2281. The E. coli panC ORF wasamplified from genomic DNA of E. coli strain XL1Blue(Stratagene) using Pfu polymerase and the primersdCGCGCCTCGAGGAGGAGTCACGTTATGTTAATTATCGAAACC and dGCGCGTCTAGATTACGCCAGCTCGACCATTTT. The PCR product was inserted into pBlue-script KS (Stratagene) via the restriction sites XhoI andXbaI. The resulting plasmid (pBKS–panC) harbors thepanC gene under the control of the lac promoter and servedas a positive control in the functional complementationexperiment. Automated DNA sequencing of the insertsin pET–MM2281, pGEM–MM2281 and pBSK–panC con-firmed the desired sequences.Functional complementation of E. coliAT1371 (panC–)The plasmids pGEM–MM2281 and pBKS–panC (positivecontrol) and the empty pBluescript KS– vector (negativecontrol) were introduced into the pantothenate-auxotrophicE. coli strain AT1371. Single colonies of the transformantswere grown overnight at 37 °C in 5 mL of liquid dYTmedium (1.6% tryptone, 1% yeast extract, 0.5% NaCl)containing 100 lgÆlL)1ampicillin. The E. coli cells werepelleted and washed twice in 5 mL of GB1 buffer [100 mmpotassium phosphate, pH 7.0, 2 gÆL)1(NH4)2SO4]. Thepelleted cells were resuspended in GB1 buffer, adjusted toan D600 nmof 0.3, and incubated at 25 °C for 1 h. Thestarved cells were then used to inoculate [0.5% (v ⁄ v)] theexperimental cultures (4 gÆL)1glucose, 0.25 gÆL)1MgSO4.10H2O, 0.25 mgÆL)1FeSO4.7H2O, 5 mgÆL)1thia-mine, 68 mgÆL)1adenine, 127 mgÆL)1l-arginine, 16 mgÆL)1l-histidine, 230 mgÆL)1l-proline, and 100 mgÆL)1ampicil-lin in GB1 buffer), which were grown at 37 °C withshaking. For each transformant, three cultures were startedthat contained an additional 1 mm pantothenate, 1 mmpantoate, or no further supplements, respectively. D600 nmwas determined over an incubation time of 24 h.Overexpression and purification of MM2281 andhelper enzymesThe MM2281 protein was expressed in E. coli BL21(DE3)carrying the pET–MM2281 plasmid described above andpurified on Ni–nitrilotriacetic acid agarose (Qiagen, Hilden,Pantothenate synthetase from Methanosarcina mazei S. Ronconi et al.2762 FEBS Journal 275 (2008) 2754–2764 ª 2008 The Authors Journal compilation ª 2008 FEBSGermany) following the manufacturer’s standard protocol.After affinity chromatography, MM2281 was loaded onto aMonoQ anion exchange column equilibrated in 50 mmTris ⁄ HCl (pH 8.8). In a linear 0–1 m KCl gradient,MM2281 eluted at approximately 350 mm KCl. Theenzyme preparation was then dialyzed exhaustively against50 mm Tris ⁄ SO4(pH 8.0) and 5 mm dithiothreitol, frozenin liquid N2, and stored in aliquots at )70 °C. The nativemolecular mass of MM2281 was estimated by gel filtrationchromatography as previously described [6]. E. coli PS [6]and E. coli PANK [28] were overexpressed and purified aspreviously described. Protein concentrations were deter-mined using the Bradford protein assay kit (Bio-Rad,Munich, Germany) with BSA as standard.Enzyme assaysThe standard assay for PS activity contained 20 mm potas-sium d-pantoate, 1 mm b-alanine, 0.08 mm [3-14C]b-alanine(55 mCiÆmmol)1), 5 mm ATP, 10 mm MgSO4, 7.5 mmK2SO4,5mm dithiothreitol, 50 mm Tris ⁄ SO4(pH 8.0) and2.5 lg of MM2281 in a final volume of 25 lL. The reactionwas initiated by the addition of substrates, and incubatedat 37 °C, and 5 lL aliquots were removed at 30, 90 and180 min time points. Separation of reaction products(10 nCi aliquots) by TLC, quantitation of14C-label abovenonenzymatic activity and estimation of initial rates was aspreviously described [6]. The detection of14C-label was lin-ear between 0.1 and 20 nCi, covering a range of 1–100% ofthe amount analyzed per time point. The assay was carriedout in the absence or presence of E. coli PANK (2.5 lg) orpyruvate kinase from rabbit (2 units). Phosphoenolpyruvate(2 mm) was included in the assay when pyruvate kinase waspresent. Control reactions in the absence of MM2281 con-tained either or both of the helper enzymes E. coli PS(1 lg) and E. coli PANK (2.5 lg) or no enzymes.In order to detect possible adenosine nucleotide productsof MM2281, standard assays containing MM2281 alone ortogether with E. coli PANK were carried out as describedabove, except that [3-14C]b-alanine was omitted. The reac-tion was quenched after 180 min, and the products werecochromatographed with authentic ATP, ADP and AMPstandards (Sigma). Adenosine nucleotides were separatedon silica plates using dioxane ⁄ NH3(25%) ⁄ H2O(6:1:4)as a mobile phase and detected under UV light (254 nm).Isotope exchange assayThe pantothenate–b-alanine isotope exchange was assayedat 25 °C, and the standard reaction contained 1 mm b-ala-nine, 0.07 mm [3-14C] b-alanine (55 mCiÆmmol)1), 5 mmpantothenate, 5 mm ADP, 5 mm sodium phosphate, 5 mmATP, 20 mm potassium d-pantoate, 10 mm MgSO4,7.5 mm K2SO4,5mm dithiothreitol, 50 mm Tris ⁄ SO4(pH 8.0), and 1.1 lgÆlL)1MM2281. Individual reactionscontained all of the above components [Eqn (2), completesystem] or lacked one or more of the reactants ATP, ADP,pantoate, or sodium phosphate. A second set of exchangereactions was carried out with AMP and sodium pyrophos-phate replacing ADP and sodium phosphate in the abovescheme [Eqn (1), complete system]. Aliquots were removedfrom the reactions at 7, 24 and 48 h after initiation by theaddition of MM2281. Quantitation of14C-label associatedwith pantothenate and b-alanine and estimation of initialexchange velocities was as previously described [6].Nonhomologous functional linksIn order to verify the prediction of COG1701 andCOG1829 as the missing steps in archaeal CoA biosynthesis[10], the STRING database [16] was searched for nonho-mologous functional links using the criteria ‘Neighborhood’(conserved chromosomal proximity) and ‘Co-occurrence’(conserved phylogenetic profile). To this end, the archaealmembers of the protein families COG0413, COG1893,COG0452, COG1019, and COG0237, which are implied inarchaeal CoA biosynthesis through homology (Fig. 1), wereused as protein queries. Functional links to COG1701 orCOG1829 were classified as strong links (high or highestconfidence in STRING) or weak links (low or medium con-fidence in STRING).AcknowledgementsWe would like to thank Katharina Pflu¨ger, Universita¨tMu¨nchen, for genomic DNA from Me. mazei, andIshac Nazi, McMaster University, for the E. coliPANK expression plasmid pPANK. We also thankErich Glawischnig for critically reading this manu-script. S. Ronconi was funded by graduate scholar-ships from the German Academic Exchange Service(DAAD) and Technische Universita¨tMu¨nchen(Frauenbu¨ro). Work on pantothenate and CoAbiosynthesis in this laboratory was funded by theDeutsche Forschungsgemeinschaft.References1 Kanehisa M, Goto S, Hattori M, Aoki-Kinoshita KF,Itoh M, Kawashima S, Katayama T, Araki M & Hirak-awa M (2006) From genomics to chemical genomics:new developments in KEGG. 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However, there areno archaeal homologs to known isoforms of pantothenate synthetase (PS)or pantothenate kinase. Using
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