PATHWAY FOR THE DISSIMILATION OF ITACONIC AND MESACONICACIDS

VERNON BRIGHTMAN' AND WILLIAM R. MARTINDepartment of Microbiology and the Walter G. Zoller Clinic, The University of Chicago, Chicago 37, Illinois

Received for publication February 27, 1961

ABSTRACT

BRIGHTmAN, VERNON (The University of Chi-cago, Chicago), AND WILLIAM R. MARTIN. Path-way for the dissimilation of itaconic and mesa-conic acids. J. Bacteriol. 82:376-382. 1961.-Studies on the oxidation of itaconic and mesa-conic acids by a Pseudomonas sp., adapted toutilize either of these acids as a sole carbon source,have provided evidence for a pathway convertingboth itaconate and mesaconate to succinate. Ametabolic interconversion of itaconate, mesaco-nate, and citramalate has also been demon-strated by whole cell and cell-free enzyme studies.

Succinate derived from methylene-labeled ita-conate was found to be labeled in the inside car-bon atoms, a fact which indicates that thebranched chain compound was converted into astraight chain molecule by a shift of the methyl-ene carbon (C-5) from the side chain of itaconateto a position between C-2 and C-3 in an, as yet,unknown straight chain intermediate prior to itsconversion to succinate.

Reports in recent years (Adler, Wang, andLardy, 1957; Munch-Petersen and Barker, 1958;Barker, Weissbach, and Smyth, 1958; Arnon etal., 1960; Isenberg, Seifter, and Berkman, 1960a,b) provide evidence for the participation of ita-conic (methylene succinic) acid and mesaconic(methyl fumaric) acid in a number of importantbiological reactions. Prior to these reports, thetwo acids were considered as obscure productsassociated with mold metabolism, in the case ofitaconic acid (Calam, Oxford, and Raistrick,1939; Haskins, Thorn, and Boothroyd, 1955), orwith plant metabolism, in the case of mesaconicacid (Buston, 1928; Roberts, Fort, and Martin,1953). The distinctive accumulation of itaconicacid by Asper4llus terreus and its further oxida-

' Present address: University of PennsylvaniaDental School, 401 Spruce Street, Philadelphia 6,Pa.

tion by this organism, combined with the evidencefor its origin from cis-aconitate (Bentley andThiessen, 1957), suggested that an alternatepathway for carbohydrate oxidation might exist,one that deviates from the Krebs cycle at thelevel of cis-aconitate. This paper presents a studyof itaconic and mesaconic acid metabolism bymicroorganisms induced to utilize these acids asa sole source of carbon and energy.

MATERIALS AND METHODS

The microorganisms used in this study wereobtained by routine soil enrichment techniques.They were grown on a basal salts medium com-posed of KH2PO4, 3 g; Na2HPO4, 6 g; NH4Cl, 1 g;MgSO4 7H20, 0.12 g; and distilled water to 1liter. Sodium mesaconate or itaconate was addedas a sole carbon source to a final concentrationof 0.2%. During the course of this study, all mi-croorganisms were maintained on agar slants ofthe medium from which they were isolated,stored at 5 C, and transferred at least every 2weeks. For experimental purposes, the bacterialcells were grown on a reciprocating shaking ma-chine for 18 hr at 30 C in liquid basal mediumcontaining 0.02% Basamin, with either mesac-onate or itaconate as a sole carbon source. Se-quential induction studies were conducted withresting itaconate- or mesaconate-induced wholecells in a conventional Warburg apparatus byobserving 02 uptake in the presence of varioussubstrates. All respiratory studies were conductedat 30 C.

Itaconic acid was purchased from NutritionalBiochemicals Corporation, and mesaconic acidfrom H. M. Chemical Company. DL-Methylsuccinic acid was prepared by catalytic hydroge-nation of itaconic acid. Melting point values ob-tained both with the free acid and with thebromphenacyl derivatives of each of the aboveacids were in good agreement with literaturevalues reported for each compound. C"4-uniformlylabeled and methylene-labeled itaconic acids were

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prepared biosynthetically in cultures of A.terreus NRRL 1960 (Bentley and Thiessen, 1957).DL-Citramalic acid was synthesized by a combi-nation of the methods of Morawski (1875) andCarius (1864). It was purified from traces ofmesaconic acid by passing through a celite*column with ether as an eluant. The product wasa chromatographically pure, yellowish, viscoussubstance which defied all attempts at crystal-lization.

Aconic acid was prepared from itaconic acidby the method of Campbell and Hunt (1947),and formyl propionic acid (succinic semialde-hyde) was prepared by the method of Ellinger(1904). Melting point values obtained withaconic acid and with the p-nitrophenyl- and2 ,4-dinitrophenylhydrazone derivatives of formylpropionic acid were in agreement with literaturevalues reported for each of the above compounds.Epoxycitramalic acid was prepared from mesa-conic acid by the method of Morawski (1875).Itatartaric (hydroxymethyl malic) acid wasobtained by hydrolysis of a purified sample ofepoxycitramalic acid at 120 C for 12 hr. a-Hy-droxyglutaric acid was prepared from glutamicacid by the method of Wolff (1890).

Detection of intermediates. Nonvolatile acidintermediates produced in the course of enzymereactions were detected by ascending paperchromatography. The various solvent systemsused for these and other compounds studied willbe noted in "Experimental Results." Isolablelevels of acid intermediates were separated andpurified on celite and silica gel columns by themethods of Phares et al. (1952) and Marvel andRands (1950), respectively.

Succinic acid was detected and assayed withbeef heart succinic dehydrogenase (Umbreit,Burris, and Stauffer, 1957). Itaconic acid wasdegraded by the method of Bentley and Thiessen(1957), and succinic acid by the method ofPhares and Long (1955). The Van Slyke-Folchwet oxidation (Calvin et al., 1949) was used fortotal combustion of the various intermediates.C'4 was assayed as barium carbonate at a constantgeometry with a windowless, gas flow Geiger-Miiller counter. All samples were corrected forbackground and selfabsorption.

EXPERIMENTAL RESULTS

Selection of microorganinms. The bacterialstrains isolated were surveyed in terms of their

* Johns Manville Co. trademark.

oxygen uptake on the two substrates, itaconateand mesaconate. All itaconate-induced bacteriawere observed to attack mesaconate noninduc-tively and vice versa. One of these micro-organisms (strain 111), a gram-negative, motile,short, rod-shaped bacterium, was selected forfurther study. The organism utilizes glucose andorganic acids oxidatively, produces a greenishblue diffusible pigment, and is a unipolar flagel-late. It was classified as a member of the genusPseudomonas.

Sequential induction studies. When strain 111was grown on any of the four acids, itaconic,mesaconic, DL-citramalic, or DL-methyl succinic,as a sole source of carbon and energy, the cellswere observed to oxidize each of these acids andsuccinic semialdehyde without lag. Citraconate,the cis-isomer of mesaconate, was not utilizedby cells grown on any of these four acids. Glucose-grown cells began to utilize the same four acids,itaconic, mesaconic, methyl succinic, and citra-malic, at a slow rate after a lag of 2 to 3 hr. Al-though the disappearance of the various non-volatile acid substrates was apparent bychromatography of the flask contents after thecompletion of the reaction, no newly formedcompounds were detected.

Cell-free enzyme studies. Cell-free extractsshowing activity on itaconate and mesaconatewere obtained by sonic rupture of itaconate-grown cells. Washed itaconate- or mesaconate-grown cells from 12 liters of growth medium weresuspended in 50 ml of deionized distilled watercontaining 0.002% reduced glutathione andexposed to maximal sonic vibration in a 10 kcoscillator for 40 min at 15 C. Cell debris andunbroken cells were removed by centrifugation at1700 X g. The faintly turbid supernatant wasdiluted to 100 ml with deionized distilled waterand centrifuged at 100,000 X g for 45 min in anultracentrifuge. Enzyme activity on mesaconateand itaconate was confined to the clear superna-tant fraction obtained by this last procedure. Thiscell-free enzyme preparation could be stored at-60 C for 2 to 3 months without detectable lossof activity.Enzyme reactions were carried out in Warburg

vessels with shaking at 30 C after gassing withnitrogen. The components of the reaction mixturewere present in the following amounts: substrate,10 ,umoles; enzyme fraction, 1.5 ml; 0.06 Mphosphate buffer, pH 6.8, 0.2 ml; and deionized

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TABLE 1. Characterization of nonvolatile acid intermediates produced by cell-free enzyme preparation

Mesaconic Itaconic Citramalic Succinic

Paper Chromatography*Authentic product Authentic product Authentic product Authentic product

RF solvent system A ........... 0.84 0.84 0.75 0.77 0.54 0.53 0.70 0.69RF solvent system B ........... 0.87 0.87 0.74 0.74 0.51 0.48 0.61 0.62RF solvent system C ........... 0.96 0.94 0.87 0.87 0.63 0.63 0.78 0.78

mp and mixed mp............. 199 194 162 162

* Solvent system A, chloroform-ethanol-formic acid (65:10:1); solvent system B, n-pentanol-formicacid (equal volumes); solvent system C, ether-formic acid-water (16:1:1).

distilled water to 2.0 ml. After incubation withthe substrate for 2 to 3 hr, the contents of thereaction vessel were analyzed for the appearanceof new nonvolatile acids by ascending paperchromatography.When itaconate, mesaconate, or citramalate

was added to the enzyme mixture, three addi-tional acids were formed, whereas no change wasobserved when any of these same substrates wasadded to boiled enzyme controls. For identifica-tion, the products were extracted from the reac-tion mixture with ether, separated, and purifiedby passage through celite and silica gel columns.The compounds were identified by paper chro-matography (three solvent systems) and meltingpoint determination (Table 1). The three acidsproduced from itaconate by the cell-free enzymepreparation were identified as mesaconate,citramalate, and succinate. When mesaconate wasused as a substrate, itaconate was formed alongwith citramalate and succinate; with citramalateas substrate, succinate and small amounts ofitaconate and mesaconate were produced.Whereas whole cells grown on itaconate, mesaco-nate, or citramalate were adapted to methylsuccinate as described under "Sequential Induc-tion Studies," the cell-free fraction showed noproduction of methyl succinate or utilization ofthe synthetic DL-compound (Table 2).Under the experimental conditions, 85% of the

itaconate or mesaconate supplied as substratewas found to be converted to the three otherproducts listed in Table 2. These were present inthe following percentages: citramalate, 78%;itaconate or mesaconate, each 11%; and succi-nate, 11%. Repeated attempts failed to show theproduction of any stream-volatile acids. Smallamounts of a compound with an RF identical to

TABLE 2. Intermediates accumulated bycell-free enzyme preparation on various

substrates

Substrate Intermediates produced

Itaconate MesaconateCitramalateSuccinate

Mesaconate ItaconateCitramalateSuccinate

Citramalate SuccinateMesaconateItaconate

Succinic semialdehyde Succinate

Methyl succinate None

that of succinic semialdehyde 2,4 dinitrophenyl-hydrazone (Bessman, Rossen, and Layne, 1953)were detected chromatographically when itacon-ate and the enzyme were used in large amountsand 2,4 dinitrophenylhydrazine added. We wereunable to demonstrate the presence of any ac-tivated intermediates, following the methods ofLipmann and Tuttle (1945) and Stadtman andBarker (1950) with our active cell-free extracts.Neither glucose- nor itaconate-grown cells (orenzyme preparations derived from them) oxi-dized epoxycitramalate, itatartrate, aconate,a-hydroxyglutarate, or glutarate.

C14-uniformly labeled itaconic acid was pre-pared by adding uniformly labeled glucose to A.terreus cultures actively producing itaconic acid.The labeled itaconic acid was extracted fromculture filtrates with ether and purified by column

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chromatography. Uniformly labeled itaconatewas incubated with the enzyme preparation andlabeled mesaconate, itaconate, and succinate wereisolated. The purified products were titrated withstandard alkali and a small portion of each driedon a copper planchet and counted as an infinitelythin sample. The specific activities of itaconate,mesaconate, and citramalate were found to bethe same. Succinic acid showed a lower specificactivity as would be expected if it were derivedby the loss of one carbon from a five-carbon com-pound (Table 3).Attempts to fractionate the enzyme mixture

with ammonium sulfate and ethanol resulted incomplete irreversible inactivation of the system.Norit-adsorbed preparations retained full ac-tivity, and short periods of dialysis against 0.06M phosphate buffer, pH 6.8, had no effect on theactivity of the enzyme preparation. Longerperiods (6 to 8 hr) resulted in some loss of ac-tivity but this activity could not be restored byadding boiled enzyme or a variety of coenzymesand divalent ions. It is assumed that the observedinactivation obtained over these periods ofdialysis was due to denaturation. Dialyses against0.05 M tris buffer, pH 7.0, for similar periodsproduced no inactivation.Sodium itaconate labeled predominantly in the

methylene carbon was produced biosyntheticallyby adding C14-methyl-labeled sodium acetate toa culture of A. terreus, actively producing itaconicacid (Corzo and Tatum, 1953). After a 3-dayperiod of incubation, the medium was freed of un-incorporated acetate by steam distillation and theitaconate purified. The radioactive itaconate wasdiluted with carrier to a specific activity of 82.5counts per min per,mole and degraded by themethod of Bentley and Thiessen (1957).

This methylene-labeled itaconate was used

TABLE 3. Specific activity of intermediatesaccumulated by cell-free enzyme preparation

incubated with C14-uniformlylabeled itaconate*

Intermediate Specific activity

counts:min:4&mole dicar-boxylic acid

Mesaconate ............... 130Citramalate ............... 138Succinate ................. 105

* Specific activity, 137 counts per min per,umole of titratable dicarboxylic acid.

TABLE 4. Degradation of methylene-labeleditaconic acid and succinic acid derivedfrom methylene-labeled itaconic acid

Itaconic acid

Carbon atom no. Specific Total activity

counts :min: 5umole carbon

'COOH 1 5.2 5.9

5CH2=2C 2 8.2 9.2

3CH 3 20.3 22.8

4COOH 4 4.4 5.0

5 50.7 57.1

Specific Total activityCarbon atom no. activity C2 + C3 + C4aciiy + C* = 100%

counts:rnin:2 Amoles carbon

C2 + C4 12.6 15.1C3 + C5 71.0 84.9

* Total specific activity of itaconic acid, 16.5counts: min :/Amole carbon.

Succinic acid

Carbon atom no. Specific Total activityactivityt

counts:min:2 pmoles carbon

2 X mean carboxyl 5 10.2carbon (Cl + C4)

2 X mean methylene 44 89.0carbon (C2 + C3)

t Total specific activity of succinic acid, 13counts: min :Mmole carbon.

with the enzyme preparation in an attempt togain some insight into the origin of the enzymat-ically produced succinate. Carrier succinate wasadded to the reaction mixture and the resultingsuccinate was isolated and purified by elutionwith ether from a celite column. Since the peakeffluent volumes of itaconic and succinic acidsare close in this system, the succinic acid obtainedby elution from this column was redeveloped on acelite column with 5% butanol in chloroform, toinsure that the succinate was free of contami-nating radioactive itaconate.

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When the cell-free enzyme preparation wasincubated under identical conditions with anyone of the Krebs tricarboxylic acids (citrate,cis-aconitate, or isocitrate), small amounts ofsuccinate and fumarate accumulated but themain product of this reaction was an equilibriummixture between the substrate and the other twotricarboxylic acids. Because of these results, andthe lack of accumulation of any of the Krebstricarboxylic acids on incubation of enzyme withitaconate, citramalate, or mesaconate, it canreasonably be assumed that the succinate whichaccumulated under these conditions was deriveddirectly from these 5-carbon acids and not viacis-aconitate and the Krebs cycle.

In Table 4, the data on the distribution ofradioactivity in the succinate derived from theC14-methylene-labeled itaconate are listed. In thesame table, this distribution is compared withthat of the radioactivity in the labeled itaconate.

DISCUSSION

The experiments described in this study indi-cate that a close metabolic relationship existsbetween itaconate, mesaconate, and citramalate,and that one of these compounds is converted tosuccinic acid. Such a relationship could be ex-plained by a reaction sequence in which itacon-ate and mesaconate were converted via citramal-ate and succinic semialdehyde to succinate.This postulated pathway is illustrated below.

is incubated with itaconate or mesaconate, andby the production of succinate from citramalateor succinic semialdehyde in the same system.The formation of small amounts of mesaconatefrom itaconate, of itaconate from mesaconate,and of itaconate and mesaconate from citramal-ate is consistent with the sequential induction ofactivity on itaconate and mesaconate in citramal-ate-grown cells. The large amount of citramalateproduced from both itaconate and mesaconateindicates that the equilibrium for the first partof the reaction lies far on the side of citramalate.The data on the "back adaptation" of citra-malate-grown cells and on the intermediatesproduced from itaconate, citramalate, and mes-aconate indicate that the reaction may also pro-ceed in the reverse direction, as illustrated bybroken lines. Data on the specific activities ofcitramalate, mesaconate, and succinate l)roducedby the action of the cell-free enzyme preparationon uniformly labeled itaconate are taken asadditional evidence that these 5-carbon acids arerelated by simple reactions which do not involverupture of the carbon chain and that succinate isproduced from a 5-carbon acid by loss of a 1-car-bon unit.The relationship of methyl suceinate to mesaco-

nate, itaconate, and citramalate metabolism isnot clear. It was shown in our experiments thatitaconate- and mesaconate-grown cells are simul-taneously adapted to metabolize methy-l succinate

Itaconic

COOH

CH2~c"C\,-1

CH2

COOHtH +H20

CH3 COOH

C1/CH

COOHMesaconic

COOH

CH3-C-OHICH2COOH

Citramalic

CHO COOH

CH2 CH2---+?l --* -l

CH2 CH2

COOH COOHSuccinic SticcinicSemialdehyde

The sequential induction of activity on citra-malate and succinic semialdehyde in both itacon-ate- and mesaconate-grown cells provides thefirst evidence for the forward reaction. MIoreconclusive evidence is provided by the accumula-tion of citramalate and succinate when a cell-free enzyme preparation derived from these cells

and that, in addition, methyl succinate-growncells are simultaneously adapted to itaconate andmesaconate. Structurally, itaconate and mesaco-nate are similar, and an intermolecular shift ofone proton could convert one into the other.There is no evidence, however, that a directinterconversion of these two acids can occur

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without the participation of an intermediatecompound. Methyl succinate would seem to fillthe role of an intermediate between itaconate andmesaconate since saturation at the double bondof either acid will produce methyl succinate. Noevidence could be obtained, however, for theproduction of methyl succinate or for its utiliza-tion by the cell-free enzyme preparation, and, inthe absence of more definitive evidence, theproposed role is purely speculative.When methylene-labeled itaconate was incu-

bated with the cell-free enzyme preparation,succinate labeled predominantly in the insidecarbons was isolated from the reaction mix-ture. Referring to the data in Table 4, if weassume that the C-1 of itaconate is lost, theresulting radioactivity contained in the C-2 toC-5 moiety of itaconate is seen to be made up of15% in C-2 and C-4 and 85% in C-3 and C-5.Comparing this with the data on the degradationof the radioactive succinate, we find that thecarboxyl carbons of succinate make up 10% ofthe total radioactivity of the molecule and themethylene carbons, 89%. These data indicatethat, either prior to or after decarboxylation ofC-1 of itaconate (or citramalate), C-5 swings inand is located between C-2 and C-3; the linkbetween C-3 and C-4 remains intact. The pro-posed transformation is illustrated below.

'COOH 2COOH

5CH2_2C 5CH2_3H + 'CO2

3CH2 3CH2

4COOH 4100H

The reaction mechanism which suggests theconversion of a branched to a straight chaincompound is not entirely a novel one. It ispossible to describe this reaction in terms similarto those used for the propionate-transfer re-actions postulated in the a-methyl aspartateglutamate and methyl malonyl coenzyme Asuccinyl coenzyme A chain straightening reac-tions of Munch-Petersen and Barker (1958) andStern and Friedman (1960), respectively. Al-though both of the systems of transpropionationso far studied have involved transfer of un-substituted propionate groups, the transfer ofhydroxy propionate (which would be involvedhere) or other substituted products of propionatemight be included in the same general type ofreaction.

The nature of the intermediate(s) indicatedby the query in the postulated conversion ofcitramalate to succinic semialdehyde remainsunknown. The radioactive data, together withour inability to detect any "activated" inter-mediates, would seem to indicate a pathwayquite different from that studied by Adler et al.(1957) and Wang, Adler, and Lardy (1961) inmammalian tissues. As the enzyme showed noactivity on itatartaric acid and the closely re-lated compound, epoxycitramalic acid, a pathwayinvolving direct oxidation of the 5-carbon acids(Stodola et al., 1945; Arpai, 1958) seems unlikely.a-Hydroxyglutaric acid, a straight chain, 5-carbon compound, might well occupy the inter-mediary position between the 5-carbon, branchedchain citramalate and the 4-carbon, straightchain succinate, but neither whole cells nor cell-free preparations of this strain exhibited activityon this compound.

ACKNOWLEDGMENTS

This research was supported by grant no.G-4406 from the National Science Foundation.One of the authors (W. R. M.) is a USPHSSenior Research Fellow.

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