Journal of Biochemistry and Molecular Biology, Vol. 35, No. 5, September 2002, pp. 443-451

© BSRK & Springer-Verlag 2002

Review

Malonate Metabolism: Biochemistry, Molecular Biology, Physiology,and Industrial Application

Yu Sam Kim*Department of Biochemistry, College of Science, Protein Network Research Center, Yonsei University, Seoul 120-749, Korea

Received July 3, 2002

Malonate is a three-carbon dicarboxylic acid. It is wellknown as a competitive inhibitor of succinatedehydrogenase. It occurs naturally in biological systems,such as legumes and developing rat brains, which indicatesthat it may play an important role in symbiotic nitrogenmetabolism and brain development. Recently, enzymes thatare related to malonate metabolism were discovered andcharacterized. The genes that encode the enzymes wereisolated, and the regulation of their expression was alsostudied. The mutant bacteria, in which the malonate-metabolizing gene was deleted, lost its primary function,symbiosis, between Rhizobium leguminosarium bv trifoliiand clover. This suggests that malonate metabolism isessential in symbiotic nitrogen metabolism, at least in clovernodules. In addition to these, the genes matB and matC havebeen successfully used for generation of the industrial strainof Streptomyces for the production of antibiotics.

Keywords: Bradyrhizobium japonicum, Malonamidase,Malonate, Malonate decarboxylase, Malonyl-CoA synthetase,Malonamate shuttle, mat operon, mdc operon, Rhizobiumleguminosarium bv trifolii, Streptomyces coelicolor

Introduction

Biochemical interest in malonate has been largely directedtoward its role as a competitive inhibitor of succinatedehydrogenase; little attention has been given to itsmetabolism. However, several investigators have suggestedthat malonate may also be important in the metabolism ofmulti-cellular organisms. Stumpf and Burris suggested thatmany plants accumulate malonate, but not as a dead-end

product of metabolism, as in soybean tissues. Also, thepathway of malonate biosynthesis in young soybean roottissue is via acetyl-CoA carboxylase (Stumpf and Burris,1981b). On the other hand, Koeppen and his co-workersstudied malonate-related enzymes in the developing rat brain.They indicated that malonate may be involved in the braindevelopment (Koeppen et al., 1974; Mitzen et al., 1976;Koeppen, et al., 1978). This review describes malonatemetabolism in view of the biochemical discovery of enzymesthat are related to metabolism, characterization of operonsencoding malonate-metabolizing enzymes, physiologicalsignificance of mutants knocked-out key enzyme, andindustrial application of the malonate metabolism.

Occurrence of Malonate

Malonate has been known to exist in plant tissues since 1925when it was characterized in alfalfa leaves. A survey of 27species of Leguminoseae revealed malonate in 18 species thatwere taken from the following genera: Medicags, Phaseolus,Vicia, Astragalus, Trifolum, Luhinus, Anthyllis, Lotus,Melilotus, Ononis, Colutea, Sophor, Thermopsis, andTrigonella (Bentley, 1952). In the soybean plant, malonate ispresent in high concentrations in all of its parts. Itsconcentration in the stem and root are related to age, but thatof the leaf is quite different. The level of this organic acid isalso affected by the nitrogen status of the plant. The source ofnitrogen has little effect on the content of malonate in the leaftissue. However, its root tissue level decreases by 34% in thenitrate-grown tissue and by 56% in the ammonia-growntissue, as compared to the controls without the added nitrogen.In the nodule, however, the malonate concentration decreasesby 68% in nitrate-grown plants and by 50% in ammonia-grown plants. These results suggest that the fixed nitrogencompounds may influence the nodule induction and growth,as well as a variety of other processes in the plants, throughthe regulation of the malonate concentration (Stumpf andBurris, 1981ab).

*To whom correspondence should be addressed.Tel: 82-2-2123-3448; Fax: 82-2-392-3488Email. yskim@yonsei.ac.kr

444 Yu Sam Kim

Biosynthesis of Malonate

Malonate is formed from oxaloacetate in pig heartpreparations and in bush bean root homogenates (Veneslandan Evans, 1944). Recently, Stumpf and Burris investigated 14Cincorporation into malonate in young soybean root tissue thatwas supplied by [1-14C]acetate, [2-14C]acetate, and NaH14CO3.

A TLC analysis of the organic acid fraction revealed that14C malonate was present in a concentration of 50% or more.When [2-14C]acetate was supplied, the labeling pattern of thecomponent fractions was essentially the same as those withthe [1-14C]acetate. From these results, it was suggested that forthe malonate biosynthesis in young soybean root tissue,acetate is more of an immediate precursor than oxaloacetate,and that it may be through malonyl-CoA that is formed by thecatalysis of acetyl-CoA carboxylase (Stumpf and Burris,1981b). In addition, it was reported that in microorganisms,malonate is formed as a degradation product of pyrimidines(Hayaishi and Kornberg, 1952). However, none of theseproposals have been fully elucidated.

Malonate Metabolizing Enzymes

Three enzymes are known to have malonate as their substrate.The genes that encode these enzymes were isolated andcharacterized.

Malonyl-CoA Synthetase

Malonyl-CoA synthetase catalyzed the formation of malonyl-CoA directly from malonate and CoA with the hydrolysis ofATP into AMP and PPi in the presence of Mg2+.

Malonate + CoA + ATP -> Malonyl-CoA + AMP + PPi

This enzyme was first discovered in the bacteroids,Bradyrhizobium japonicum, of soybean nodules (Kim andChae, 1990a). The malonate-specific enzyme has long beenexpected to exist in nodules since free malonate is known tooccur in legumes, and its level increases under symbioticconditions. It was also reported that in the symbiotic host-plant cell, malonate is passively transported into bacteroids(Reibach and Streeter, 1984). However, nothing is knownabout the fate of malonate in bacteroids.

This enzyme was first purified from the symbiotic bacteria B.japonicum that is grown on a GYP medium (Kim and Chae,1990b), and later from Rhizobium leguminosarium bv trifolii,which has symbiosis with clover (Kim et al., 1993). The highsubstrate specificity of malonate, CoA and ATP, has been

revealed, but Mn2+ could be substituted for Mg2+ with nodifference in activity. A kinetic study showed that this catalysisinvolved three substrates and three products, which proceededwith the Bi Uni Uni Bi Ping Pong Ter Ter mechanism. Also,during the catalysis, malonyl-AMP is formed as a reactionintermediate. The formation of malonyl-AMP was confirmedby the an analysis of 31P NMR spectra of the AMP product thatwas isolated from an 18O transfer experiment using[18O]malonate (Kang et al., 1994; Kim and Kang, 1994).

Mat Operon

Mat operon in R. leguminosarium bv trifolii consists of 4genes that encodes malonyl-CoA decarboxylase (matA),malonyl-CoA synthetase (matB), a putative malonate carrierprotein (matC), and a regulatory protein (matR). A genecluster that consists of three consecutive genes, matABC, wasfirst isolated using a probe that was prepared from the aminoacid sequence information of malonyl-CoA synthetase, andwas subsequently sequenced. The matA and matB sequenceswere overlapped by four base pairs; whereas, the intergenicregion between matB and matC had 95 base pairs. Theribosome binding sites were found 7 to 12 base pairs upstreamof each gene. The MatA gene encoded a polypeptide of 462amino acid residues with a deduced molecular mass of 51,414

Fig. 1. Bi Uni Uni Bi Ping Pong Ter Ter mechanism of malonyl-CoA synthetase catalysis (Kim and Kang, 1994; Kang et al., 1994).

Fig. 2. Mat operon (An and Kim, 1998). matR, matA, matB,and matC indicate genes for a regulatory protein, malonyl-CoAdecarboxylase, malonyl-CoA synthetase, and a putative malonatetransporter, respectively.

Malonate metabolism 445

Da. It was confirmed to be a malonyl-CoA decarboxylase.MatB encoded a polypeptide of 504 amino acid residues witha deduced molecular mass of 54,612 Da. This gene wasexpressed in E. coli and characterized to be essentiallyidentical to the native malonyl-CoA synthetase (An et al.,1999). MatC encoded a 46,453 Da protein with a high contentof hydrophobic residues. It showed similarities to thedicarboxylate carrier protein, indicating that it might be amalonate carrier protein (An and Kim, 1998). These resultsstrongly suggest that the gene cluster encodes proteins that areinvolved in the malonate-metabolizing system, malonateout->malonatein->malonyl-CoA->acetyl-CoA, in R. leguminosariumbv trifolii. Also, the metabolic pathway in the malonate-richclover nodule might play an important role in symbiosis.

In addition to matABC, a novel gene (coined matR) wasdiscovered on the upstream region of R. leguminosarium bvtrifolii mat operon. The matR gene product (MatR) interactsspecifically with the DNA fragment that contains theupstream region of the promoter. MatR has a N-terminalDNA-binding domain that employs a helix-trun-helix motifand the C-terminal domain that is involved in malonatebinding. The addition of malonate increased the association ofMatR and the DNA fragment. DNaseI footprinting assaysidentified the MatR binding site that encompasses 66nucleotides near the mat promoter. The mat operator region

included an inverted repeat (TCTTGTA/TACACGA) that wascentered 46.5 relative to the transcription start site (Lee et al.,2000; Lee and Kim, 2001).

The malonyl-CoA synthetase gene was also isolated fromB. japonicum, but this gene was not clustered like the gene inR. leguminosarium bv trifolii mat operon. However, the genesequence was highly homologous with matB (Koo and Kim,2000). This suggests that B. japonicum may not have the genefor the malonate carrier, which indirectly supports thesupposition that malonate may be transported passively intobacteroids that were isolated from soybean plant nodules(Reibach and Streeter, 1984). The enzyme from B. japonicumwas expressed in E. coli, and characterized to be essentiallyidentical to the native enzyme (Koo and Kim, 2000). Theessential residues on the active sites were also identified bychemical modification, site-directed mutagenesis, and kineticanalysis (Lee and Kim, 1993a; Lee and Kim, 1993b; Lee andKim, 1993c; An et al., 1999; Koo and Kim, 2000).

Structure and Function of Malonyl-CoA Synthetase

The malonyl-CoA synthetase catalyzes the formation ofmalonyl-CoA in a two-step reaction that consists of theadenylation of malonate with ATP, followed by the malonyltransfer from malonyl-AMP to CoA. In order to identify

Fig. 3. Modeling structure of malonyl-CoA synthetase (A) and active site (B) (An et al., 1999).

(A)

(B)

446 Yu Sam Kim

amino acid residues that are essential for each step of theenzyme, catalysis was prepared. This was based on a chemicalmodification and database analysis, as well as mutantenzymes-R168G, K170M, R168G/K170M, and H206L. Akinetic analysis of the mutants revealed that Lys-170 and His206 play critical roles in the ATP binding and malonyl-AMPformation; whereas, Arg-168 is critical for the formation ofmalonyl-CoA and specificity for malonyl-AMP. Molecularmodeling provided the enzyme structure that could fullyexplain all of these biochemical data, even though thestructure of malonyl-CoA synthetase was generated bycomparative modeling. The active site and substrate-bindingmode of the enzyme was determined by transferred nuclearoverhauser effect spectroscopy. It was found that malonate isplaced between ATP and His 206, supporting His 206 in itscatalytic role as it generates the reaction intermediate,malonyl-AMP (An et al., 1999).

Malonamidase

Three novel malonamidases (E1a, E1b, and E2) were purifiedand characterized from B. japonicum (Kim and Kang, 1994).These enzymes catalyze all, or some, of the followingreactions: malonyl-transfer to hydroxylamine, −OOCCH2COO−

+ NH2OH -> −OOCCH2CONHOH + H2O; hydroxylaminolysisof malonamate, −OOCCH2CONH2+ NH2OH -> −OOCCH2

CONHOH + NH3 and hydrolysis of malonamate, −OOCCH2

CONH2 + H2O -> −OOCCH2COO− + NH2OH. Among them,malonamidase E2 is highly specific for thehydroxylaminolysis and hydrolysis of malonamate. Theseenzymes were first found in B. japonicum bacteroids and the

plant cytosol of soybean nodules (Kang and Kim, 1996). Itwas proposed that they may be involved in the transport offixed nitrogen from bacteroids to the plant cell in symbioticnitrogen metabolism (Kim and Chae, 1990). The gene thatencodes malonamidase E2 has been cloned, sequenced, andexpressed in E. coli. The biochemical properties of therecombinant enzyme were essentially identical to those fromthe wild-type B. japonicum (Koo et al., 2000). A databasesearch showed that the enzyme has a high sequence similaritywith the common signature sequence of the amidase family. Inorder to identify amino acid residues that are essential forenzyme activity, a series of site-directed mutagenesis studiesand steady-state kinetic experiments were performed. Fromthese experiments, it was found that Ser199, Lys213, andGln195 are involved (Koo et al., 2000). Also, malonamidaseE2 was crystallized, and its three-dimensional structure waselucidated (Shin et al., 2002). This is the first report on thethree-dimensional structure of the amidase signature familyenzymes. The amidase signature sequence comprises thecatalytic core scaffold, and the whole structure defines a newprotein folding class. Surprisingly, the structure revealed thatthe amidase signature family enzymes contain a novel Ser-cisSer-Lys catalytic machinery. This novel catalytic triad bearsa gross mechanistic similarity to the classical Ser-His-Aspcatalytic triad, despite the very different combination of aminoacids constituents (Shin et al., 2002).

Malonamidase was also discovered in Rhizobium meliloti,but not in R. leguminosarium bv trifolii. It is interesting to notethat B. japonicum have malonyl-CoA synthetase andmalonamidases, but not malonyl-CoA decarboxylase. R.leguminosarium bv trifolii has malonyl-CoA synthetase and

(A) (B)

Fig. 4. Structure of malonamidase E2 (A) and a novel catalytic triad, Ser155-cisSer131-Lys62(B) (Shin et al., 2002). The AS (amidasesignature) sequence is in blue and the catalytic triad is in magenta. Ser132 and Thr150 are involved in hydrogen bonds with Lys62.Wat1 is one of the multiple water molecules on the hydrogen-bonded network.

Malonate metabolism 447

malonyl-CoA decarboxylase, but not malonamidases (Kim etal., 1990). Based on the distribution of these enzymes indifferent species of symbiotic bacteria, it has been proposedthat malonate may be a major carbon source for bacteroids.

In B. japonicum, it has been proposed that malonamidasesparticipated in the synthesis of the essential intermediatemalonamate and in the transport of ammonia. Malonyl-CoAsynthetase may be involved in regulating the concentration ofmalonate and malonamate in the bacteroids of nodules.Malonate is passively transported from the host plant tobacteroids. Malonate in the bacteroids will then be converted tomalonamate by malonamidase E1 with ammonia, and some ofthe malonate will be converted to malonyl-CoA by malonyl-CoA synthetase. The ammonia is from dinitrogen bynitrogenase. Malonamate that is formed may be transported tothe host plant by a carrier system, which is unknown at thepresent. The hypothetical transport system may be active ratherthan passive. In host plants, the malonamate that is transportedfrom bacteroids is hydrolyzed to malonate and ammonia bymalonamidase E2. In fact, the malonamate may possibly be anamine donor like glutamine. This proposed system was coinedas the ³malonamate shuttle´ (Kim and Chae, 1990).

Malonate Decarboxylase

Malonate decarboxylase catalyzes the decarboxylation ofmalonate to acetate (Byun and Kim, 1994). This enzyme wasdiscovered in microorganisms that were grown on malonate asa sole carbon source. It can be classified into two groups. Oneis a membrane-integrated, biotin-dependent, energy-conserving Na+ translocating enzyme. The other is cytosolicand biotin-independent. The enzyme from Malonomonasrubra (Hilbi et al., 1992) falls into the first category, whereas

the enzymes from Acinetobacter calcoaceticus (Kim andByun, 1994), Klebsiella pneumoniae (Schmid et al., 1996),Pseudomonas putida (Chohnan et al., 1998), andPseudomonas fluorescence (Byun and Kim, 1995) fall into thesecond. However, the feature that is common to both types ofmalonate decarboxylase is the formation of malonyl-S-ACPby an exchange reaction of malonate with an acetyl group thatis bound to an acyl-carrier protein, which yields acetate andsubsequent decarboxylation with the regeneration of theacetyl-S-ACP. The specific acyl-carrier protein subunit with acovalently attached 2’-(5́-phosphoribosyl)-3’-dephospho-CoA prosthetic group is also known to be common to bothtypes of the malonate decarboxylases (Berg et al., 1996;Hoenke et al., 2000).

In order to reveal the stereochemical course of the reactionsthat were catalyzed by the biotin-independent enzymes fromA. calcoaceticus and Pseudomonas fluorescens, a chiralsubstrate (malonate carring 13C in one carboxyl group and 3Hat one of the methylene positions) was prepared and used inthe reactions that were catalyzed by these two enzymes. Thedecarboxylation of (R)-[1-13C1,2-3H]malonate in 2H2O gave apseudo-racemate of chiral acetate, which was converted via

Fig. 5. Hypothetical “Malonamate shuttle” (Kim and Chae,1990). E1, E2, and MS indicate malonamidase E1,malonamidase E2, and malonyl-CoA synthetase, respectively.

Fig. 6. Stereochemical catalytic mechanism of malonatedecarboxylase (Handa et al., 1999). α, β, γ, and δ indicatesubunits for malonate decarboxylase.

Fig. 7. Catalytic cycle of malonate decarboxylation (Koo et al.,1997).

448 Yu Sam Kim

acetyl-CoA into malate with malate synthase. From thereactive proportions of the isotopomers of malate that werepresent (determined by 3H NMR analysis), it was concludedthat in the decarboxylation of malonate by these two biotin-independent enzymes COOH is replaced by H with retentionof configuration (Handa et al., 1999). The samestereochemical outcome was observed for the reaction thatwas catalyzed by the biotin-dependent malonatedecarboxylase from M. rubra (Micklefield et al., 1995).

Gene clusters from A. calcoaceticus (Koo et al., 1997), K.pneumoniae (Hoenke et al., 1997), M. rubra (Berg et al.,1997), and P. putida (Chohnan et al., 1999) that encode thecomponents of enzymes and proteins that are involved in thedecarboxylation of malonate were cloned and sequenced.

All of the genes that are involved in malonatedecarboxylation have been functionally evaluated. Recently,mdc operon from A. calcoaceticus was found to be regulatedby malonate and a transcriptional repressor, mdcY. This genewas knocked out by insertion of the IS3 element in A.calcoaceticus that was isolated from soil by using a malonateplate, but was reconstructed for the elucidation of its function(Koo et al., 2000).

Physiological Significance of Malonate Metabolism in Symbiosis

The role of malonate in symbiotic nitrogen metabolism haslong been controversial, even though large amounts of it occurin legume roots, especially in the nodules. In some cases,malonate accounts for as much as 4% of the dry weight andup to 50% of the total acid of legume tissues. Therefore, thehigh concentration of malonate in root nodules of legumes hasled to the suggestion that it has an essential role in nitrogen-fixing symbiosis (Stumpf anf Burris, 1981b). However, therole of malonate as a significant carbon source for bacteroid

metabolism was ruled out by studies on the utilization ofmalonate, and the metabolite uptake with intact symbiosomes(Streeter, 1991). Moreover, in recent studies on thedistribution of organic acids and the organic acid contentunder different kinds of abiotic stress, malonate was suggestedas a plant defensive chemical (Li and Copeland, 2000).Nevertheless, it has been suggested that malonate may play arole in symbiosis since there are elevated levels of malonate

Fig. 8. Gene clusters encoding proteins for malonate decarboxylation (Koo et al., 1997; Hoenke et al., 1997; Berg et al., 1997;Chohnan et al., 1999). Mrmad, KPmdc, PPmdc, and ACmdc indicate gene clusters from Malonomonas rubra, Klebsiella pneumoniae,Pseudomonas putida, and Acinetobacter calcoaceticus KCCM40902, respectively. TR, transcription factor; MT, malonate transporter;MAT, malonate:ACPtransferase; ACP, acyl carrier protein; DCD and DCE, β and γ subunit of decarboxylase; HAS, holo-ACP synthase;PF, a protein for the formation of the prosthetic group precursor; MCT, malonyl-CoA:ACP transacylase.

Fig. 9. Growth of white clover (Trifolium). (A). Trifoliuminfected by R. leguminosarium bv trifolii, (B). Trifolium infectedby ∆matB R. leguminosarium bv trifolii mutant, (C). Trifoliumuninfected (An et al., 2002, Mol & Cell, in press).

Malonate metabolism 449

that remain in the nodules during the most active nitrogen-fixing period in legumes. Also, bacteroids cannot survivewithout the malonate metabolizing system in the presence of ahigh concentration of malonate. As described previously,malonate metabolizing enzymes, malonyl-CoA synthetaseand malonamidases, were discovered in symbiotic bacteria. InR. leguminosarium bv trifolii that symbiosis with clover, matoperon (consisting of three consecutive genes, matABC, thatare related to malonate metabolism) was discovered (An andKim, 1998). In order to investigate whether malonate plays arole in Rhizobium, clover symbiosis (a matB-deleted mutantR. leguminosarium bv trifolii ) was constructed by the insertionof the kanamycin-resistant gene cassette into the codingregion of matB. ∆matB R. leguminosarium bv trifolii in thefree-living state displayed no obvious difference from thewild-type strain. However, clover that was infected with themutant bacteria showed a significant reduction of growth,even though they formed nodules.

The nodules formed by wild and mutant strains wereindistinguishable by their shape or site on root. Therefore, thinsections of longitudinal cuts of nodules were prepared andanalyzed by light microscopy. In the nodules that were formedby the infection of mutant bacteria, most of the nodule partspace were primarily composed of senescent zones, and packedwith vacuoles and starch granules instead of bacteroids.

The starch granules, which are energy reservoirs of plantcells, are used for the nitrogen fixation process in bacteroids. Inthe case of the partial or complete deficient mutants in nitrogen

fixation, the starch level in the mutant modules was reported tobe higher than in wild-type nodules (Vance et al., 1980).Presumably, malonate is compartmentalized in the abundantvacuoles of these nodules (Mellor, 1989). These facts suggestthat malonate metabolism is more important for endocytosisand bacteroids differentiation than for root hair deformation orinfection thread development. This is the first report ofmalonate metabolism being important in biological systems.

Industrial Application of Malonate Metabolism

R. legunminosarum bv trifolii malonyl-CoA synthetaseprovides an attractive route to expand the lexicon ofprecursors that are available for polyketide biosynthesis,because it can use various malonate analogs, includingmethylmalonate as its substrate (Pohl, 2001). Polyketides, alarge family of bioactive natural products, are synthesizedfrom building blocks that are derived from α-carboxylatedCoA thioesters, such as malonyl-CoA and (2S)-methylmalonyl-CoA. The productivity of polyketidefermentation processes in natural and heterologous hosts isfrequently limited by the availability of these precursors invivo. The genes matB and matC from R. legunminosarum bvtrifolii encode malonyl-CoA synthetase and the putativedicarboxylate transport protein, respectively. These proteinscan directly convert exogenous malonate and methylmalonateinto their corresponding CoA thioesters with an ATPrequirement of 2 mol per mol of produced acyl-CoA. Theheterologous expression of matBC in a recombinant strain ofStreptomyces coelicolor that produces the macrolactone 6-deoxyerythronolide B results in a 300% enhancement ofmacrolactone titers (Lombo et al., 2001).

Fig. 10. Electron microscope of nodule sections induced by wildtype R. leguminosarium bv trifolii (A, B) and by mutant ∆matBR. leguminosarium bv trifolii (C, D) (An et al., 2002).

Fig. 11. Production of macrolides by recombinant Streptomycescoelicolor. S. coelicolor CH999/pCK7 produces 6-deoxyerythronolide B, the macrocyclic core of the antibioticerythromycin (Lombo et al., 2001).

450 Yu Sam Kim

The direct conversion of inexpensive feedstocks, such asmalonate and methylmalonate, into polyketides represents themost carbon- and energy-deficient routes to these high-valuenatural products. This approach may have implications forother commercial fermentation processes (e.g., avermectin,monensin, tylosin, rifamycin, amphotericin, tetracycline,doxorubicin, and lovastatin). It may also be implied in theimprovement of processes that yield high-valuedevelopmental stage products, such as the recently discoveredanticancer agent epothilone.

Conclusion

Malonate is a simple three-carbon dicarboxylate. It is wellknown as a competitive inhibitor of succinate dehydrogenase invitro. Until recently, little was known about the biologicalimportance of this naturally occurring biomolecule.Biosynthesis of malonate is still unknown. Bacterialassimilation of malonate through its decarboxylation bymalonate decarboxylase has been studied extensively. In thestudy of malonate, a lot of progress has been made in symbioticbacteria, such as B. japonicum and R. legunminosarum bvtrifolii. Novel enzymes (malonyl-CoA synthetase andmalonamidases) and proteins, including the transporter andtranscriptional regulator, were discovered and characterized. Inaddition to these, the genes that encode the proteins were clonedand characterized to be an operon or single gene. From thesediscoveries, novel metabolic pathways (³malonamate shuttle´ inB. japonicum and malonate->malonyl-CoA->acetyl-CoA in R.legunminosarum bv trifolii) were proposed. It was especiallysurprising to discover that the ∆matB R. leguminosarium bvtrifolii mutant infected clover and induced empty nodules. Thisindicates that malonate metabolism is essential, at least for thesymbiosis between R. leguminosarium bv trifolii and clovers.This is the first proof that malonate metabolism is important forsome biological systems. Some of the novel metabolicpathways, malonate (or methylmalonate)out-> malonate(methylmalonate)in -> malonyl-CoA were applied verysuccessfully in developing an industrial strain for the polyketidedrugs. More basic studies about malonate metabolism arerequired in plant and animal systems for the elucidation ofmalonates role in biological systems.

Acknowledgments This work was supported by a grant(2000-2-0607) from the Korea Science and EngineeringFoundation through the Protein Network Research Center atYonsei University. I thank my former students who wereinvolved in this malonate project for their dedicatedcontributions - Drs. Ho Zoon Chae, Sang Won Kang, Hye SinByun, Jae Hyung An, Hyun Min Koo, Jae Hyung Koo, andHwan Young Lee.

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