JOURNAL OF BACTERIOLOGY, Jan. 1969, p. 97-106 Vol. 97, No. ICopyright © 1969 American Society for Microbiology Printed in U.S.A.

Aldohexuronic Acid Catabolismby a Soil AeromonasJ. J. FARMER III' AND R. G. EAGON

Department of Microbiology, University of Georgia, Athens, Georgia 30601

Received for publication 24 July 1968

Bacteria which utilize mannuronic acid as an energy source were isolated fromnature. One of the organisms, identified as a member of the genus Aeromonas,used glucuronate, galacturonate, and mannuronate as the sole source of carbonand energy. Glucuronate- and galacturonate-grown resting cells oxidized bothglucuronate and galacturonate rapidly, but mannuronate slowly. Mannuronate-grown cells oxidized all three rapidly, with the rate of mannuronate utilizationsomewhat lower. Cell-free extracts from glucuronate-, galacturonate-, and man-nuronate-grown Aeromonas C1 1-2B contained glucuronate and galacturonateisomerases, fructuronate, tagaturonate, and mannuronate reductases, and man-nonate and altronate dehydratases, with the exception of glucuronate-grown cellswhich lacked altronate dehydratase. Thus, the pathway for glucuronate and galac-turonate catabolism for Aeromonas was identical to Escherichia coli. Glucuronateand galacturonate were isomerized to D-fructuronate and D-tagaturonate whichwere then reduced by reduced nicotinamide adenine dinucleotide to D-mannonateand D-altronate, respectively. The hexonic acids were dehydrated to 2-keto-3-deoxy gluconate which was phosphorylated by adenosine triphosphate to 2-keto-3-deoxy-6-phospho gluconate. The latter was then cleaved to pyruvate and glycer-aldehyde-3-phosphate. Mannuronate was reduced directly to D-mannonate by areduced nicotinamide adenine dinucleotide phosphate-linked oxidoreductase.D-Mannonate was then further broken down as in the glucuronate pathway. Themannuronate reducing enzyme, for which the name D-mannonate:nicotinamideadenine dinucleotide (phosphate) oxidoreductase (D-mannuronate-forming) wasproposed, was shown to be distinct from altronate and mannoate oxidoreductases.This is the first report of a bacterial oxidoreductase which reduces an aldohexuronicacid to a hexonic acid. The enzyme should prove to be a useful analytical tool fordetermining mannuronate in the presence of other uronic acids.

Pathways for the degradation of glucuronateand galacturonate have been established in anumber of bacteria (2, 15, 18). The inability ofany of these organisms to utilize mannuronicacid and the inertness of mannuronate to theirenzymes suggested that the breakdown of thiscompound involves a different mechanism. Thereis little information in the literature concerningmannuronate catabolism, probably because thiscompound is not available commercially andbecause it is thought not to occur widely innature. The experiments described herein wereundertaken, therefore, to provide some insightinto the microbial pathway of mannuronatecatabolism.

X Present address: Environmental Services Branch, NationalInstitutes of Health, Bethesda, Md. 20014.

97

MATERIALS AND METHODS

Preparation of reagents. Alginic acid (Kelco Co.,Clark, N.J.) was washed exhaustively in cold water,once in 95% ethyl alcohol, and once in acetone. Afterdrying, it was found to be free from reducing sugarsas evidenced by thin-layer chromatographic tech-niques.

D-Mannuronic-y-lactone, D-mannonolactone, andD-mannaric acid dialactone were provided by H. S.Isbel of the National Bureau of Standards. Substrateswere converted to sodium salts by the addition ofstoichiometric amounts of 0.1 N NaOH. Mannuronic--y-lactone was also prepared by formic acid hydrolysisof alginic acid according to the method of Spoehr(30) and was recrystallized 2 to 4 times from ethylalcohol. The lactone chromatographed as a singlespot and co-chromatographed with authentic man-nuronic--y-lactone in nine different solvent systems.In addition, the lactone had the same melting point as

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mannuronic--y-lactone; when the two were mixedtogether, no change in the melting point was noted.

Calcium fructuronate was synthesized by themethod of Ehrlick and Guttman (7) as modified byAshwell, Wahba, and Hickman (3). A sample ofcalcium D-tagaturonate was generously provided byJoy Williams (Department of Microbiology, Uni-versity of Georgia). These were converted to theirsodium salts by treatment with Dowex 50 (H+)followed by neutralization with 0.1 N NaOH.

D-Altronate was synthesized enzymatically fromD-tagaturonate as described by Hickman and Ashwell(12), and 2-keto-3-deoxy gluconate was synthesizedenzymatically from D-mannonate as described bySmiley and Ashwell (28).

All other compounds were obtained from commer-cial sources.

Enzymes. Lactic dehydrogenase (EC 1.1.1.27;L-lactate: nicotinamide adenine dinucleotide (NAD)oxidoreductase) type I from rabbit muscle was ob-tained from Sigma Chemical Co., St. Louis, Mo.Extracts containing fructuronate reductase (EC1.1.1.57; D-mannonate: NAD oxidoreductase), tag-aturonate reductase (EC 1.1.1.58; D-altronate: NADoxidoreductase), D-mannonate dehydratase (EC4.2.1.8; D-mannonate hydro-lyase), D-altronate de-hydiatase (EC 4.2.1.7; D-altronate hydro-lyase),2-keto-3-deoxy-D-gluconokinase [EC 2.7.1.45; adeno-sine triphosphate (ATP): 2-keto-3-deoxy-D-gluconate6-phosphotransferase], and 2-keto-3-deoxy-6-phos-phogluconate aldolase (EC 4.1.2.14; 6-phospho-2-keto-3-deoxy-D-gluconate D-glyceraldehyde-3-phos-phate lyase) were prepared from Escherichiia coliATCC 9637 as described by Ashwell et al. (3).

Media. An enriched basal salts medium of thefollowing composition was used for the cultivation ofmicroorganisms: Na2HPO4, 3.0 g; KH2PO4, 2.0 g;NH4Cl, 0.5 g; (NH4)2SO4, 0.5 g; MgSO4 7H2O,0.05 g; dehydrated nutrient broth, 0.5 g; dehydratedyeast extract, 0.5 g; and distilled water to I liter. Thefinal pH was 7.0. Carbohydrate solutions weresterilized by filtration and were aseptically added tothe autoclaved medium to give a final concentrationof 0.5%. Nutrient broth and yeast extract wereomitted in experiments carried out to determinegrowth on uronates as the sole source of carbon andenergy.

Isolation and characterization of organism. Bacteriacapable of utilizing mannuronate as a sole source ofcarbon and energy were isolated by the elective cul-ture technique with the above enriched medium con-taining mannuronate. One of the isolates, designatedC11-2B, was selected for study because it grew onglucuronate, galacturonate, and mannuronate as thesole source of carbon and energy.

Standard determinative tests for characterizing theisolate were performed as described in the Manualof Microbiological Methods (29), and the organismwas classified as a member of the genus Aeromonasaccording to Bergey's Manual and Skerman's Guide(27). In Hugh and Leifson's scheme for the differentia-tion of various gram-negative bacteria (13), it fellinto group IIlb which includes the genus Aeromonas.The tentative designation Aeromonas Cl 1-2B was

assigned the isolate because its characteristics werenot identical to any of the Aeromonias species describedin Bergey's Manual.

Growth of organism. The microorganism wasgrown at 30 C on a rotary shaker in 500-ml Erlen-meyer flasks, each containing 100 ml of medium.The cells were harvested by centrifugation and werewashed twice in 0.067 M phosphate buffer (pH 7.0)before use.

Manometric studies. Oxygen uptake was measuredin a Warburg constant volume respirometer. Washedcells were suspended in 0.067 M phosphate buffer(pH 7.0) to give 5% transmittancy at 650 nm. Eachreaction flask contained 2.0 ml of resting cells, 10.0,umoles of substrate, and 0.2 ml of 40%0 KOH in thecenter well. The final volume was 3.2 ml. Endogenousoxygen uptakes were subtracted.

Preparation of cell-free extracts. Cell-free extractswere prepared by rupturing cells, which were sus-pended in 0.067 M phosphate buffer (pH 7.0) in a1/1 (v/v) ratio, with a French pressure cell at 10,000lb/in2, or by grinding a cell paste with alumina andextracting with 0.067 M phosphate buffer (pH 7.0).Extracts were then centrifuged at 17,000 X g for20 min to remove whole cells and cellular debris.Enzyme purification. Particulate enzymes were

removed by centrifugation at 105,000 X g for 2 hr.The supernatant solution was dialyzed against 100volumes of 0.067 M phosphate buffer (pH 7.0).Ammonium sulfate fractionation was performed withneutralized saturated ammonium sulfate solutionsas described by Seaman (26). Glucuronate and gal-acturonate isomerase (EC 5.3.1.12; D-glucuronateketo-isomerase) appeared in the 60 to 80% fraction.Fructuronate and tagaturonate reductases and man-nuronate reductase [a hitherto undescribed enzymefor which the name D-mannonate: nicotinamideadenine dinucleotide phosphate (NADP) oxidore-ductase (D-mannuronate-forming) is proposed] werefound in the 30 to 40% ammonium sulfate fractionas were D-mannonate and D-altronate dehydratases.The dehydratases were inactivated by dialysis in0.067 M phosphate buffer (pH 7.0) followed by storageat 0 C for 96 hr. Reductases free from dehydrataseactivities were prepared in this manner.

Colorimetric assays. Protein was determined bythe biuret method (10), uronic acids were determinedby the carbazole procedure (5), keturonic acids weredetermined by the cysteine-carbazole procedure (6),2-keto-3-deoxy gluconate was determined by theperiodate-thiobarbituric acid method (12), and re-ducing sugars were determined by the Folin-Wureagent (9).Enzyme assays. A unit of enzyme was defined as

that amount of enzyme which catalyzed the trans-formation of 1 ,umole of substrate per min under theconditions of the particular assay at 30 C.

Glucuronate isomerase was detected by measuringthe fructuronic acid formed. The reaction system,in a total volume of 1.0 ml, contained: borate buffer(pH 8.0), 40.0 umoles; sodium glucuronate, 20.0,umoles; and diluted extract, 0.1 ml. A reaction sys-tem for the isolation and identification of the isom-erization product contained, in a total volume of

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5 ml: borate buffer (pH 8.0), 240.0 Jmoles; sodiumglucuronate, 200.0,smoles; and glucuronate isomerase(60 to 80% ammonium sulfate fraction), 20 units.The reaction was terminated after 1 hr.

Galacturonate isomerase was determined bymeasuring the tagaturonic acid formed. The assaysystem was identical to the one for glucuronateisomerase except that galacturonate was the substrate.The isomerization product was isolated and identifiedfrom a reaction mixture containing, in a total volumeof 5 ml: borate buffer (pH 8.0), 240.0,umoles; sodiumgalacturonate, 200.0 pmoles; and galacturonate isom-erase (60 to 80% ammonium sulfate fraction), 22units. The reaction was terminated after 1 hr.

Experiments to detect the presence of a man-nuronate isomerase (an undescribed enzyme forwhich the name would be D-mannuronate ketolisom-erase) were carried out by assaying for the forma-tion of the suspected keturonic acid in an assay sys-tem identical to glucuronate isomerase, but withmannuronate as substrate.

Fructuronate reductase and all other nicotinamideadenine dinucleotide (phosphate) [NAD(P)I-linkedoxidoreductases were determined by the change inoptical density at 340 nm. The reaction system, in atotal volume of 3 ml, was: phosphate buffer (pH 7.0),100.0 ,umoles; sodium fructuronate, 30.0 timoles;reduced nicotinamide adenine dinucleotide (phos-phate) [NAD(P)H], 0.3 ,imole; and diluted extract,0.1 ml. The reduction product was isolated andidentified from the following system: phosphatebuffer (pH 7.0), 100.0 Jmoles; sodium fructuronate,50.0 ;smoles; reduced nicotinamide adenine dinu-cleotide (NADH), 60.0 ,umoles; and fructuronatereductase (30 to 40% ammonium sulfate fractionfree from mannonate dehydratase activity), 5 units;in a total volume of 5 ml. The reaction was terminatedafter 1 hr.

Tagaturonate reductase was measured in a systemidentical to the one for measuring fructuronatereductase except that tagaturonate was the substrate.The reduction product was isolated and identifiedfrom a system containing, in a total volume of 5 ml:phosphate buffer (pH 7.0), 100.0 pmoles; sodiumtagaturonate, 50.0 ;moles; NADH, 60.0 jAmoles;and tagaturonate reductase (30 to 40% ammoniumsulfate fraction free from altronate dehydratase ac-tivity), 3.2 units. The reaction was terminated afterI hr.Mannuronate reductase was measured in a system

containing, in a total volume of 3.0 ml: phosphatebuffer (pH 7.0), 100.0,umoles; sodium mannuronate,30.0 Mmoles; reduced nicotinamide adenine dinu-cleotide phosphate (NADPH), 0.3 jmole; and dilutedextract, 0.1 ml. The reduction product was isolatedand identified from a system containing, in a totalvolume of 5 ml: phosphate buffer (pH 7.0), 100.0pmoles; sodium mannuronate, 50.0 smoles; NADPH,60.0 ,pmoles; and mannuronate reductase (30 to 40%ammonium sulfate fraction free from mannonatedehydratase activity), 9.0 units. This reaction wasterminated after 1 hr.

Mannonate dehydratase was determined by themethod described by Smiley and Ashwell (28) with

the following modification. The reaction system, in atotal volume of 0.5 ml, contained: phosphate buffer(pH 6.0), 17.0 ,Amoles; sodium mannonate, 5.0;,moles; and diluted extract, 0.1 ml. Altronate de-hydratase, D-glucarate hydro-lyase (no EC numberlisted), D-galactarate hydro-lyase (no EC numberlisted), and D-mannarate hydro-lyase (no EC numberlisted) were determined in the same manner with theappropriate substrate. The mannonate dehydrationproduct (i.e., 2-keto-3-deoxy gluconate) was identifiedfrom a system containing, in a total volume of 3 ml:phosphate buffer (pH 6.0), 100.0 jrmoles; sodiummannonate, 50.0 moles; and mannonate dehydratase(from a freshly prepared extract), 2.1 units. Whensodium altronate and altronate dehydratase were thesubstrate and enzyme, respectively, in the above reac-tion, the same intermediate was produced.

2-Keto-3-deoxy-D-gluconokinase (KDG kinase)and 2-keto-3-deoxy-6-phosphogluconate aldolase(KDPG aldolase) were determined qualitativelyby a modification of the method of Cynkin andAshwell (4). The reaction system contained, in avolume of 0.5 ml: phosphate buffer (pH 7.0), 50.0umoles; 2-keto-3-deoxy gluconate, 3.0 JAmoles; MgSO4,6.0 jsmoles; adenosine triphosphate (ATP), 12.0Mmoles; and extract, 0.1 ml. The reaction was stoppedby boiling, and the pyruvate formed was measuredby the oxidation of NADH in the presence of addedlactic dehydrogenase.

D-Glucuronate: NAD(P) oxidoreductase (no ECnumber listed) was assayed in both the forward andthe reverse direction. The "forward" reaction systemcontained, in a total volume of 3.0 ml: phosphatebuffer (pH 7.0), 100.0 ,moles; sodium glucuronate,30.0,moles; NAD or NADP, 0.3 jmole; and extract,0.1 ml. The "reverse" reaction system contained, in atotal volume of 3.0 ml: phosphate buffer (pH 7.0),100 gmoles; sodium glucarate, 30.0 ,umoles; NADHor NADPH, 0.3 jumole; and extract, 0.1 ml. D-Galac-turonate: NAD(P) oxidoreductase (no EC numberlisted) and D-mannuronate: NAD(P) oxidoreductase(no EC number listed) were similarly measured withthe appropriate substrates.

Alginase (EC 3.2.1.16; poly-,B-1 ,4-mannuronideglycanohydrase) and polygalacturonase (EC 3.2.1.15;poly-a-1,4 - galacturonide glycanohydrase) were as-sayed by measuring the liberation of reducing sugars.Alginate lyase (EC 4.2.99.4; poly-jg-1 ,4-D-man-nuronide lyase) and pectate lyase (EC 4.2.99.3;poly-a-1 ,4-D-galacturonide lyase) were assayed bythe method of Preiss and Ashwell (24). Each reactionmixture contained, in a volume of 1 ml: phosphatebuffer (pH 7.0), 100.0 ;&moles; sodium alginate orpoly-galacturonate, 10.0 jAmoles; and extract, 0.1ml.

Assays for NAD(P) transhydrogenase (EC 1.6.1.1;reduced NADP: NAD oxidoreductase) and NADPHphosphatase (no EC number listed) were done bytaking advantage of the NAD-linked lactic dehydro-genase of Aeromonas Cl 1-2B. In these assays, cell-freeextracts from uronic acid- or glucose-grown Aero-monas were incubated with pyruvate, NADPH,and NAD. The reaction system for the NADPHphosphatase contained, in a total volume of 3.0 ml:

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phosphate buffer (pH 7.0), 10.0 umoles; sodiumpyruvate, 10.0 ,moles; NADPH, 0.3 jAmole; andextract, 0.1 ml. The transhydrogenase assay systemwas identical, except that 1.0 ,Amole of NAD wasadded to the system. The failure to detect oxidationof NADPH under these conditions indicated theabsence of both the NAD(P) transhydrogenase andNADPH phosphatase.

Fructuronic acid in the presence of a large excess ofmannuronic acid was determined enzymatically withE. coli extracts containing fructuronate reductaseaccording to the assay devised by Ashwell et al. (3).

Thin-layer chromatography. Ascending thin-layerchromatography was performed on 250-,Om layers ofMN-cellulosepulver 300 (Brinkman Industries, Inc.,Westbury, N.Y.). The following eluents were used:solvent 1, ethyl acetate-acetic acid-water (3:1:3); sol-vent 2, n-butyl alcohol-acetic acid-water (4:1:5);solvent 3, pyridine-ethyl acetate-acetic acid-water(5:5:1:3); solvent 4, n-butyl alcohol-acetic acid-water (4: 1: 3); solvent 5, ethyl acetate-acetic acid-water(3:3:1); solvent 6, n-butyl alcohol-pyridine-water(6:1:2); solvent 7, ethyl acetate-acetic acid-pyridine-water (5:1:5:5); solvent 8, propanol-acetic acid-pyridine-water (8:1:8:4); and solvent 9, propanol-water (3:1). All nine solvents were used for eachmetabolic product, which was characterized and iden-tified chromatographically.The following spray reagents were used: anisidine-

phthalate for uronic acids (25), alkaline periodate-permanganate for hexonic acids (16), ferric-hydroxyl-amine for lactones (1), periodate-thiobarbituric acidfor 2-keto-3-deoxy gluconate (32), and ammoniacalsilver nitrate for all types of carbohydrates (25).

Isolation and detection of intermediary catabolicproducts. The reactions previously described wereterminated by the addition of 0.1 volume of 50%trichloroacetic acid. The precipitated protein wasremoved by centrifugation and washed twice with

5.0 ml of water. The supernatant solution and thewashings were then extracted three times with diethylether to remove trichloroacetic acid. When nicotin-amide adenine nucleotides had been included, theywere removed by treatment with 0.5 g of acid-washedcharcoal at 0 C for 20 min. The charcoal was removedby filtration, and the filtrate desalted by passagethrough a column (1 by 35 cm) containing Bio Rad ionretardation resin (Ag IA8, 50 to 100 mesh; Bio RadLaboratories, Richmond, Calif.). The fractions wereassayed for intermediates according to the techniquespreviously described, and tubes containing the desiredproducts were pooled and concentrated by lyophiliza-tion.

RESULTS

Warburg respirometry. Cells grown on glu-curonate oxidized both glucuronate and galac-turonate rapidly and without a lag. Approximately50%N of the theoretical oxygen uptake for com-plete oxidation to CO2 was observed. Man-nuronate was oxidized at less than 10% of therate of the other uronates. Galacturonate-growncells showed an almost identical response. Glu-curonate and galacturonate were again rapidlyoxidized, but mannuronate was oxidized onlyslowly. However, mannuronate-grown cells oxi-dized all three uronic acids rapidly and with nolag. Cells grown on nutrient broth or glucose didnot oxidize uronic acids.Enzymes of known uronic acid pathways de-

tected in cell-free extracts from uronic acid- andglucose-grown cells. Dialyzed cell-free extractswere assayed for the key enzymes in uronic acidcatabolic pathways. The specific activities of theenzymes are listed in Table 1. The correspondinghexonic and hexuronic acid lactones were tested

TABLE 1. Enzymes detected in cell-free extracts from cells grown on uronic acids and glucosea

Specific activity of extracts from cells grown onEnzyme

Glucuronate Galacturonate Mannuronate Glucose

Glucuronate isomerase 2.20 0.90 4.0 0.00(ND) (ND) (ND) (ND)

Galacturonate isomerase 1.60 1.20 2.00 0.00Fructuronate reductase 0.88 1 .10 0.56 0.00Tagaturonate reductase 0.74 0.75 0.57 0.07Mannuronate reductase 0.18 0.58 0.63 0.00

(ND) (ND) (ND) (ND)Mannonate dehydratase 0.05 0.04 0.06 0.00

(0.01) (0.01) (0.015) (ND)Altronate dehydratase 0.00 0.002 0.00 0.00

a The values shown parenthetically are the specific activities (units per milligram of protein) with theuronic or hexonic acid lactone as substrate. ND indicates not detected. The following enzymes were notdetected: D-mannuronate isomerase, D-glucarate hydro-lyase, pectate lyase, D-glucuronate: NADoxidoreductase, D-galactarate hydro-lyase, alginate lyase, D-galacturonate: NAD oxidoreductase,NADPH phosphatase, D-mannarate hydro-lyase, polygalacturonase, alginase, D-mannuronate: NADoxidoreductase, NAD(P) transhydrogenase.

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as substrates; however, the specific activity waslower or zero in each case (Table 1). Altronatedehydratase was detected in freshly preparedcrude extracts from mannuronate-grown cells,but, as the data in Table 1 indicate, the dehy-dratase was not detected after dialysis. Fructu-ronate reductase and tagaturonate reductase alsoused NADPH as a cofactor, but the rate was20% of that when NADH was used. The con-verse was true with mannuronate reductase;the rate with NADH was 25% of that withNADPH. The enzymes which were not detectedare also listed in the footnote to Table 1.

Production of intermediates of uronic acidpathways. The intermediates that were detectedin the dissimilation of glucuronate, galacturonate,and mannuronate are listed in Table 2. Once anintermediate was identified, it was isolated andused as a substrate in the next sequential reaction.

Characterization of the glucuronate and galac-turonate isomerization products. Cell-free extractsfrom mannuronate-grown cells converted glu-curonate to fructuronate and galacturonate totagaturonate to an extent of 48 and 30%, re-spectively (Table 2). Thin-layer chromatographyconfirmed the identification of the isomerizationproducts. In addition, when fructuronate and theglucuronate isomerization product or taga-turonate and the galacturonate isomerizationproduct were chromatographed together, re-spectively, a single spot was observed in eachcase. The products had maximal absorptions inthe cysteine-carbazole assay which correspondedto fructuronate and tagaturonate; i.e., 510 and525 nm, respectively. Finally, both productscaused the oxidation of NADH, as did authenticfructuronate and tagaturonate, when incubatedwith extracts from glucuronate-grown E. coli(Table 3).Reduction of mannuronate. Mannuronate iso-

merase activity was not detected in glucuronate-,

galacturonate-, or mannuronate-grown cells(Table 1). Prolonged incubation of mannuronatewith both crude and dialyzed extracts did notresult in the formation of fructuronate, but addedfructuronate still remained after incubation (Table4). In addition, the kinetics of nicotinamideadenine nucleotide oxidation were typical of aone-step reduction when mannuronate was thesubstrate (Fig. 1). Preincubation of mannuronatewith extract did not increase the reaction rate.When glucuronate and galacturonate were usedas substrates, a noticeable lag was present; thislag was eliminated by preincubating the sub-strate with extract, indicating isomerization be-fore reduction.

Characterization of the fructuronate, taga-turonate, and mannuronate reduction products.Fructuronate and tagaturonate were quantita-tively reduced to hexonic acids in the presence ofan excess of NADH and cell-free extracts frommannuronate-grown cells, as was mannuronatewith an excess of NADPH (Table 2). The RFvalues of the fructuronate and mannuronatereduction products corresponded to those ofauthentic D-mannonate and those of the taga-turonate reduction product corresponded toauthentic D-altronate. Indentification was con-firmed when the unknowns co-chromatographedwith authentic products in each of the solvents.In addition, when the fructuronate and man-nuronate reduction products were incubatedwith extracts from glucuronate-grown E. coli,2-keto-3-deoxy gluconate could be detectedchromatographically. When the altronate reduc-tion product was used, no keto-deoxy compoundwas found since altronate dehydratase activitywas not present at the time of testing.

Characterization of the hexonic acid dehydra-tion product. When either mannonate or al-tronate was incubated with cell-free extractsfrom mannuronate-grown cells, 2-keto-3-deoxy

TABLE 2. Accumulation of intermediates when various substrates were incubated with cell-free extracts frommannuronate-grown cells

MannuronateMannuronateGlucuronateGalacturonateFructuronateTagaturonateMannonateAltronate2-Keto-3-deoxygluconate

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TABLE 3. Reactions of intermediauronic acid catabolism of Aeron

with cell-free extracts jglucuronate-grown E. t

Aeromonasintermediary

product

Glucuronateisomeriza-tion product

Galacturonateisomeriza-tion product

Fructuronatereductionproduct

Tagaturonatereductionproduct

Mannuronatereductionproduct

Mannonatedehydrationproduct

Altronate de-hydrationproduct

Addition

NADH

NADH

ATP + Mg++

ATP + Mg++

ites produced innonas CJ1-2Bfromcoli

Reaction withE. coli extracts

Oxidation ofNADH

Oxidation ofNADH

Production ofKDG

KDG not pro-duced

Production ofKDG

Production ofpyruvate

Production ofpyruvate

TABLE 4. Amount offructuronate detected after thereaction of mannuronate and fructuronate with

cell-free extracts from mannuronate-vrowncells

Fructuronate (umoles) mea;ured:Substrate added

Colorimetrically Enzymatically

Sodium mannu- 0.0 0.00ronate, 10.0 Mmoles

Sodium fructu- 1.0 0.99ronate, 1.0 Amole

gluconate (KDG) accumulated. The yield was88 and 9%, respectively (Table 2). The RF valuesfor both dehydration products closely resembledthose for authentic KDG. The identification wasconfirmed when both unknowns co-chromato-graphed with KDG in each of the solvents. Inaddition, when each of the unknowns was in-cubated with ATP, Mg+, and extracts fromglucuronate-grown E. coli, pyruvate was formed(Table 3). Under these conditions, authenticKDG was also converted to pyruvate.

Evidence for KDG kinase and KDPG aldolaseactivities. When KDG was incubated with ex-tracts from mannuronate-grown cells, ATP,and Mg++, pyruvate was produced in a 37%

E0v)

L0c0a

'-C.)0.Q

MINUTESFIG. 1. Reaction of glucuronate, galacturonate,

and mannuronate with reduced nicotinamide adeninenucleotides and cell-free extracts from mannuronate-grown cells.

yield (Table 2). The reaction had an absoluterequirement for ATP, as no pyruvate was formedunless this cofactor was included (Table 5).These results were interpreted as evidence thatKDG was first phosphorylated to 2-keto-3-deoxy-6-phospho gluconate (KDPG) by KDGkinase, and that KDPG was then cleaved byKDPG aldolase to pyruvate and, presumably,to glyceraldehyde-3-phosphate. Extracts fromglucuronate-grown E. coli, known to containthese two enzymes, behaved identically in thatpyruvate production was absolutely ATP-de-pendent.

Separation of reductase activities. The effectof thermal denaturation on fructuronate, taga-turonic, and mannuronate reductases is shown inTable 6. Heating for 20 min at 45 C resulted inthe complete loss of fructuronate and tagaturonatereductase activities. However, mannuronate re-ductase activity was still present.

DISCUSSIONThe catabolism of glucuronate and galac-

turonate has been studied by culturing the bac-teria directly on these substrates. However, thedegradation of mannuronate has been studiedonly in bacteria which produce this compoundby the hydrolysis of alginic acid. Alginic acid isreadily available, but mannuronate must bechemically synthesized in a lengthy process.Unexpectedly, none of the 11 species of alginolyticbacteria that we have previously isolated grew

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TABLE 5. Formation of pyruvatefrom 2-keto-3-deoxy gluconate, A TP, and cell-free ex-tracts from mannuronate-grown cells

Reaction system Product Conversionto product

KDG None detected 0KDG + Mg++ None detected 0KDG + Mg-+ Pyruvate 37+ ATP

TABLE 6. Per cent of activity remaining afterthermal inactivation offructuronate,

tagaturonate, and mannuronatereductasesa

Time Fructuronate Tegaturonate Mannuronateat 45 C reductase reductase reductase

min

0 100 100 1005 44 46 10010 26 29 9420 0 0 64

a The extracts were placed in a water bath at45 C for the indicated period, removed, cooled to0 C, and then assayed for each activity.

on mannuronate or produced mannuronate fromalginate. Presumably alginate was degradedeliminatively as first described by Preiss andAshwell (24), and later by Nakada and Sweeny(19) and by Lynn and co-workers (17), ratherthan hydrolytically as in Alginomonas alginicum(8, 33). For this reason, a bacterium which grewon mannuronate was isolated from nature. Theisolate, which was classified in the genus Aero-monas, resembled most uronic acid-utilizing bac-teria in that it grew on both glucuronate andgalacturonate. However, it was unique amongthe species described since it could also usemannuronate.

Cells of Aeromonas C1 1-2B grown on glu-curonate or galacturonate oxidized both at thesame rate. This was unlike Serratia marcescens,which oxidized glucuronate much more rapidly(21). The rate of mannuronate oxidation wasvery low in comparison. All of the enzymes forthe complete catabolism of mannuronate werepresent in extracts from glucuronate- or galac-turonate-grown cells, suggesting that it was themannuronate transport system which was notfully induced. In contrast, cells grown on man-nuronate oxidized this compound rapidly, in-dicating that the necessary transport system wasformed.The enzymes for uronic acid catabolism were

clearly shown to be inducible rather than con-stitutive, since glucose-grown cells neither oxi-dized uronic acids nor contained the enzymes fortheir degradation. This was not the case withXanthomonas malvacearum, which contains con-stitutive enzymes (22).Glucuronate and galacturonate isomerase ac-

tivities were detected in extracts from bothglucuronate- and galacturonate-grown cells ofAeromonus C11-2B. Both activities were also in-duced in cells grown on mannuronate, althoughmannuronate itself was not isomerized. It is pos-sible that a single enzyme is responsible for bothactivities as it is in E. coli (3, 31) and that Aer-monas Cl 1-2B is similar to most uronic acid-metabolizing bacteria in that cells grown on eitheruronate isomerized both. Pseudomonas natriegensis the only species studied that isomerizes onlyglucuronate but not galacturonate (18).The isomerization of mannuronate for fruc-

turonate was shown not to occur in AeromonasC11-2B. The possibility that fructuronate isformed but then utilized in a second reaction waseliminated when it was shown that the amount ofadded fructuronate remained unchanged afterprolonged incubation with partially purified ex-tracts. This same experiment ruled out the possi-bility that mannuronate isomerase was presentbut had an equilibrium greatly favoring man-nuronate formation. Since fructuronate did notdisappear, the equilibrium for glucuronateisomerase, which was shown to be present, musthave been far to the right. This was the case withthe enzyme from E. coli; with this enzyme, thisreverse reaction could not be demonstrated (3).The enzymatic assay used to detect fructuronatehad a sensitivity of about 0.01 ,umole, but fruc-turonate conversion from mannuronate was notobserved. Preincubation of substrate with enzymewas used by McRorie and co-workers (18) todemonstrate the isomerization of glucuronate andgalacturonate before reduction. This techniqueindicated that in Aeromonas Cl 1-2B glucuronateand galacturonate were isomerized, but that man-nuronate was reduced without isomerization.Thin-layer chromatography, a colorimetric test,and enzyme assays indicated that the glucuronateand galacturonate isomerization products wereidentical to fructuronate and tagaturonate, re-spectively.

Cells grown on all three uronates containedfructuronate, tagaturonate, and mannuronate re-ductase activities. Assays with partially purifiedpreparations showed that both NADH andNADPH could be used as cofactors. Fruc-turonate and tagaturonate were reduced morerapidly with NADH, but mannuronate was re-duced more rapidly with NADPH. Aeromonas

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Cl 1-2B was like Erwinia carotovora (14) and S.marcescens (23) in its cofactor requirement. How-ever, the two reductases from E. coli were abso-lutely NADH specific (12). In Aeromonas C1 1-2B,no NAD(P) transhydrogenase or NADPH phos-phatase activity was detected, implying that thereductases had nonspecific cofactor requirements.After 10-fold purification, mannuronate reductasestill used both cofactors. All three reductase ac-tivities appeared in the 30 to 40% ammoniumsulfate fraction, but mannuronate reductase couldbe distinguished from the other two by heat in-activation. Mannuronate reductase was still 64%active after being incubated at 45 C for 20 min,whereas fructuronate and tagaturonate reductaseactivities were completely destroyed. Fructuronateand tagaturonate reductases were inactivated atthe same rate, suggesting the possibility that asingle enzyme was catalyzing both reactions. In E.coli fructuronate and tagaturonate reductaseswere distinct enzymes; however, fructuronate re-ductase was active with both fructuronate andtagaturonate, making the above hypothesis lessattractive. It could only be concluded that man-nuronate reductase was a distinct enzyme in Aer-omonas Cl 1-2B. The exact nature of fructuronateand tagaturonate reductase activities must awaitfurther investigation. Glucose-grown cells con-tained a slight amount of tagaturonate reductaseactivity but no fructuronate or mannuronate re-ductase activity. The low level of tagaturonatereductase was not considered significant, sincenone of the other enzymes on the pathway werepresent.The product of both fructuronate and man-

nuronate reduction was in every way identical toD-mannonate. The configuration at the secondcarbon atom of fructuronate could be restored intwo ways by reduction, producing either D-man-nonate or L-gulonate. L-Gulonate did not causereduction ofNAD in the presence of extract whenthe reverse reaction was tested, but D-mannonatedid. L-Gluconate was not dehydrated to 2-keto-3-deoxy gluconate when it was incubated with ex-tracts from glucuronate-grown cells of E. coli, butthis latter compound was produced from the fruc-turonate and mannuronate reduction productsand from authentic D-mannonate. These observa-tions constituted further evidence against thepathway that mannuronate would presumablyfollow if it were metabolized in a manner anal-ogous to glucuronate and galacturonate by E.coli:

mannuronate ; fructuronate NADH, D-mannOnate

Chromatographically, the tagaturonate reductionproduct in Aeromonas C11-2B was identical to

D-altronate; but incubation of this product withextracts from glucuronate-grown cells of E. colidid not result in the production of 2-keto-3-deoxygluconate, since altronate dehydratase is ex-tremely labile and was not active in these extracts.D-Mannonate dehydratase was present in cells

of Aeromonas C11-2B grown on all three uronicacids, but D-altronate dehydratase was detectedonly in extracts from mannuronate- and galac-turonate-grown cells. Mannonate and altronatedehydratases were two distinct enzymes in cell-free extracts from mannuronate-grown cells. Thiswas evident because extracts which had beenstored at -60 C for several months containedmannonate dehydratase but no altronate de-hydratase activity. Aeromonas Cl 1-2B was similarto E. coli in this respect (28). Altronate dehy-dratase was not detected in dialyzed cell-free ex-tracts from mannuronate-grown cells. However,it was detected in fresh crude extracts, indicatingthat the enzyme was inactivated by dialysis in thiscase. It must be pointed out that the specific ac-tivities of both dehydratases were very low and theoptimal assay conditions were not experimentallydetermined. Both enzymes were inducible toAeromonas C11-2B, as well as in E. coli (12),since glucose-grown cells contained no activity.The compound formed when either mannonate oraltronate was dehydrated was identical to authen-tic 2-keto-3-deoxy gluconate in its chromato-graphic migration and reactions with extracts ofcells of E. coli.The formation of pyruvate when 2-keto-3-deoxy

gluconate was incubated with ATP, Mg, andextracts from mannuronate-grown cells was inter-preted to mean that 2-keto-3-deoxy gluconate wasphosphorylated to 2-keto-3-deoxy-6-phospho glu-conate which was then cleaved to pyruvate andglyceraldehyde-3-phosphate. The direct cleavageofKDG to pyruvate and glyceraldehyde was con-sidered unlikely, since pyruvate production wasabsolutely dependent on the presence of ATP.However, some organisms cleave keto-deoxy com-pounds without prior phosphorylation. P. sac-charophila converts D-arabinose to 2-keto-3-deoxyarabonic acid which in turn gives rise to pyruvateand glycolaldehyde (20). Aeromonas C11-2B wassimilar to E. coli (4) in that phosphorylation pre-ceded cleavage. We concluded from these experi-ments that mannuronate-grown cells containedKDG kinase and KDPG aldolase activities.The enzymes for the oxidation of uronic acid

to hexaric acid were not detected in extracts fromcells grown on uronic acids, and uronate-grownresting cells did not oxidize hexaric acids. Theuronic acids did not cause the reduction ofNAD(P), nor did the hexaric acids cause theoxidation on NAD(P)H when they were in-

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ALDOHEXURONIC ACID CATABOLISM

Mannuronate

1NADPH

NADH

Glucuronate ;± Fructuronate I Mannonate

Galacturonate i:± Tagaturonate Altronate

2-Keto-3 deoxy ATP, 2-Keto-3 deoxy-t-4 gluconate 6-phospho gluconate

IPyruvate+

Glyceraldehyde-3-phosphate

FIG. 2. Reactions demonstrated in cell-free extracts from mannuronate-grown cells and the proposed pathwayfor uronic acid catabolism in Aeromanas C11-2B.

cubated with cell-free extracts from uronate-grown cells. This strongly suggested that thepathway described by Kilgore and Starr (15)for uronic acid utilization in phytopathogenicPseudomonads was not present in uronate-grown Aeromonas C11-2B. Further evidence wasobtained when it was shown that hexaric acidswere not converted to keto-deoxy hexaric acids.Growth on uronic acids did not induce en-

zymes for the breakdown of alginate or poly-galacturonate, and Aeromonas C11-2B did notuse these substrates for growth.

In Aeromonas Cl 1-2B, the catabolism ofglucuronate and galacturonate is identical to theknown sequence in E. coli (2). This is the secondorganism in the order Pseudomonadales in whichthis pathway has been shown. For the first time,a pathway for the bacterial degradation ofmannuronic acid has been established. Man-nuronate is first reduced by an NAD(P)H-linkedoxidoreductase to D-mannonate which then isbroken down further as an intermediate of theglucuronate pathway. This reduction closelyresembles the first step in the metabolism ofglucuronate and galacturonate in mammalian tis-sue preparations; in these preparations, NADPH-linked oxidoreductases reduce glucuronate toL-gulonate and galacturonate to L-galactonate(11). Although the name D-mannonate: NAD(P)oxidoreductase is appropriate for the new en-zyme, this name has been assigned to the enzymewhich catalyzes the reduction of fructuronate tomannonate and tagaturonate to altronate. There-fore, the name D-mannonate: NAD(P) oxidore-ductase (D-mannuronate-forming) is proposedfor this enzyme which reduces mannuronate butnot fructuronate or tagaturonate. The enzymemay prove to be a useful analytical tool fordetermining mannuronate in the presence ofother uronic acids or similar substances. Allof the reactions that were demonstrated in cell-free extracts from mannuronate-grown cells and

the proposed pathway for uronic acid catabolismin Aeromonas C11-2B are presented in Fig. 2.

ACKNOWLEDGMENT

This investigation was supported by Contract Nonr 3677(03),ONR Project NR 103-624, between the Office of Naval Research,Department of the Navy, and the University of Georgia.

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