JOURNAL OF BACTERIOLOGY, Sept. 1969, p. 667-673Copyright © 1969 American Society for Microbiology

Vol. 99, No. 3Printed in U.S.A.

Hexuronic Acid Dehydrogenase ofAgrobacterium tumefaciens

YUNG FENG CHANG' AND DAVID SIDNEY FEINGOLD

Department of Microbiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania

Received for publication 24 April 1969

Growth of Agrobacterium tumefaciens on D-glucuronic acid (GlcUA) or D-

galacturonic acid (GalUA) induces formation of hexuronic acid dehydrogenase [D-aldohexuronic acid: nicotinamide adenine dinucleotide (NAD) oxidoreductase].The dehydrogenase, which irreversibly converts GlcUA or GalUA to the corre-

sponding hexaric acid with the concomitant reduction of NAD, but not of nicotin-amide adenine dinucleotide phosphate was purified 60-fold by MnCl2 treatment,(NH4)2SO4 fractionation, chromatography on diethylaminoethyl Sephadex andnegative adsorption with Ca3(PO4)2 gel. The pH optimum is 8.0. Other uronicacids, aldohexoses, aldopentoses, and polyols, are not substrates. Reducednicotinamide adenine dinucleotide is an inhibitor strictly competitive with NAD.Kinetic data indicate that the dehydrogenase induced by growth on GlcUA may

not be identical with that induced by growth on GalUA.

In 1959, Zajic (11) reported the nicotinamideadenine dinucleotide (NAD)-linked dehydro-genation of D-glucuronic acid (G1cUA), D-galacturonic acid (GalUA), D-glucuronolactone,D-mannuronic acid, and D-mannuronolactoneby cell-free extracts of GlcUA-grown Agro-bacterium tumefaciens and suggested that thereaction product was either a dicarboxylic acidor uronic acid-ulose. Subsequently, we showedthat A. tumefaciens converts GlcUA to a-keto-glutaric acid by a multistep pathway in which thefirst step is the formation of D-glucaric acid(Y. F. Chang, 1966, Ph.D. Thesis, University ofPittsburgh; Y. F. Chang and D. S. Feingold,1967, Abstracts 7th Int. Cong. Biochem. 4:711).In this paper we describe the enzyme whichcatalyzes the first reaction in this pathway, hexu-ronic acid dehydrogenase (D-aldohexuronic acid: -NAD oxidoreductase).

MATERIALS AND METHODSMaterials. Diethylaminoethyl (DEAE) Sephadex

A-50 (coarse) was purchased from Pharmacia Inc.,New Market, N.J. Aged calcium phosphate gel was aproduct of Nutritional Biochemicals Corp., Cleveland,Ohio. Ammonium sulfate, enzyme grade, was aproduct of Mann Research Laboratories, New York,N.Y. Nicotinamide adenine dinucleotide (NAD), re-duced nicotinamide adenine dinucleotide (NADH2),nicotinamide adenine dinucleotide phosphate(NADP), and lactate dehydrogenase (EC 1 .1 .1.27;

' Present address: Department of Biological Chemistry, Uni-versity of Maryland, School of Medicine, Baltimore, Maryland.

crystalline suspensions in ammonium sulfate from rab-bit muscle type 1) were purchased from Sigma Chemi-cal Co., St. Louis, Mo. Analogues of NAD weregenerously donated by N. 0. Kaplan of BrandeisUniversity. All other chemicals were reagent gradecommercial products unless otherwise stated. Allstudies were carried out with strain It BN V6 ofAgrobacterium twnefaciens, which was maintained onyeast-extract potato dextrose agar (Difco).

Methods. Paper electrophoresis was performed asdescribed by Feingold et al. (1). Detection of sugarswas with periodate benzidine (2) or silver nitrate (9)reagents, or both. Protein was estimated by the ultra-violet spectrophotometric method of Waddel (10).Manometric studies were carried out by using theconventional Warburg apparatus.

Assays for hexuronic acid dehydrogenase activitywere performed at 30 C in reaction mixtures contain-ing the following concentrations (mM): GalUA, 4;MgCl2, 4; NAD, 0.4; tris(hydroxymethyl)amino-methane (Tris)-hydrochloride (pH 8.0) 50; and 25,uliters of enzyme (about 0.03 to 0.16 units) in a totalvolume of 1 ml. The absorbancy increase at 340 nmwas followed with the Gilford Multiple SampleRecorder in absorption cells with a 1-cm light pathfor 1 min after adding the enzyme and mixing thecuvette contents; the latter operation took no longerthan 10 sec. A control without substrate was used tocompensate for nonspecific reduction of NAD. Aunit of activity is defined as the amount of enzymerequired to reduce 1 ,umole of NAD per min at 30 C.The relation between protein concentration and reac-tion rate was linear over the range used in this assay.

Growth of cells. A. tumefaciens was grown in themedium described by Kraght and Starr (4), with somemodification. The medium was prepared in three

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parts. A 1-liter amount of medium contained thefollowing: (a) (NH4)2SO4, 4 g; KH2PO4, 13 g;Na2HPO4, 4 g; (b) MgSO4.7H20, 1.42 g; H3BO3,1 mg; (c) FeC3* 6H20, 20 mg; carbohydrate (D-glucose, GlcUA, or GalUA as indicated), 10 g.Solutions of (a) and (b) were autoclaved separately,and solution (c) was sterilized by filtration. The com-ponents were combined when cool and brought topH 6.9 with sterile NaOH. Cells transferred from ayeast-extract potato dextrose-agar slant grew slowlyin the synthetic medium; however, rapid growth couldbe achieved by addition of yeast extract to a concentra-tion of 0.01%. The adapted cells from this populationgrew readily upon transfer into completely syntheticmedium. They were grown in the synthetic mediumfor 24 hr on a rotary shaker at 28 C in 500-ml Erlen-meyer flasks containing 200 ml of medium, harvestedby centrifugation, and washed three times with 0.02 MTris buffer (pH 8.0). These washed cells were usedthroughout the study, unless otherwise indicated.

Preparation of cell-free extracts. All subsequentoperations were performed at 0 to 4 C. In (NH4)2SO4precipitations, 30 min elapsed between addition of(NH4)2SO4 and centrifugation. The packed, washedcells were suspended in about three volumes of 0.02M Tris buffer (pH 8.0) and disrupted for 20 min in theRaytheon 6 kc sonic oscillator at maximal output.The broken cell suspension was centrifuged at 6000 Xg for 10 min and the pellet was discarded. The super-natant fluid was dialyzed overnight in 0.02 M Trisbuffer (pH 8.0) preparatory to enzyme purification(crude extract).Enzyme purification. Crude extract (46 ml) was

mixed with 9.2 ml of ice-cold 1 M MnCI2; after stir-ring for 1 hr, the precipitate was removed by centri-fugation, and the supernatant liquid was brought to35% saturation with 10 g of solid (NH4)2SO4. Aftercentrifugation, the precipitate was discarded and thesupernatant fluid was brought to 55% saturation with6.23 g of solid (NH4)2SO4. The precipitate was col-lected by centrifugation and dissolved in 10 ml of0.02 M Tris buffer (pH 7.5) [0.01 M in ethylenediamine-tetraacetate (EDTA); (NH4)2SO4 fraction]. Thissolution was dialyzed overnight against 400 ml of0.005 M Tris buffer at pH 7.5 (0.001 M in EDTA and0.005 M in 2-mercaptoethanol) loaded onto a 500-miDEAE Sephadex A-50 (coarse) column, equili-brated with the same buffer, and washed into theDEAEwith an additional 100 ml of buffer. The columnthen was eluted at a flow rate of about 3 ml/min with1 liter each of 0.1, 0.2, and 0.3 N NaCl in the samebuffer; 10- to 11-ml fractions were collected, and thespecific activity of hexuronate dehydrogenase in eachfraction was determined. Enzyme activity was elutedwith 0.3 N NaCl. Protein was precipitated from theactive fractions (approximately 490 ml) by addition of262 g of solid (NH4)2SO4 to 80% saturation. Theprecipitate was dissolved in 20 ml of 0.005 M Trisbuffer at pH 7.5 (0.001 M in EDTA and 0.005 M in2-mercaptoethanol) and the solution was dialyzedovernight against 1 liter of the same buffer (DEAESephadex). Aged calcium phosphate gel suspensionwas added to the dialyzed enzyme preparation [2mg of gel (dry wt) per mg of protein]. After occasional

stirring for 15 min, the gel was spun down and dis-carded. The supernatant fluid contained the enzymeactivity (calcium phosphate gel supernatant fluid).

RESULTS

Manometric studies. Oxygen uptake by restingcells grown in different carbon sources wasmeasured with a number of substrates. Cellsgrown in GalUA immediately oxidized bothGalUA and galactaric acid; galactaric acid-growncells immediately oxidized galactaric acid, butnot GalUA. GalUA was oxidized only after alag period of about 1 hr (Fig. 1). Glucose-grown cells, on the other hand, failed to oxidizeeither GalUA or galactaric acid during the periodof experiment (about 3 hr). Crude extracts of

120

100

C,)

. 80-J-i

, 60y

a. 40

20

00 40 80 120 160 180

TIME (MIN)FIG. 1. Oxygen uptake of GalUA- and galactaric

acid-grown cells on different carbon sources. A 20-hrculture of GalUA- or galactaric acid-grown cells waswashed twice with 0.03 M (pH 7.0) phosphate bufferand was suspended in the same buffer to give a Klettreading of 350 (Filter 42). A 0.2-ml amount of 0.1 Msubstrate was placed in the side arm, 2 ml of the cellsuspension (containing 1.4 mg of cells, dry weight) wasplaced in the main compartment of a Warburgflask,and 0.2 ml of10% KOH was placed in the center well.The experiment was started by mixing the substratewith the cell suspension. All experiments were per-formed at 30 C with air as the gas phase. Resultsshown are net oxygen uptake after substracting theappropriate endogenous oxygen uptake (0.4 to 0.5,liter/min). Symbols for GalUA-grown cells: 0,GalUA as carbon source; A, galactaric acid as carbonsource. Symbols for galactaric acid-grown cells: 0,GalUA as carbon source; *, galactaric acid as carbonsource.

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both Ga1UA- and galactaric acid-grown cellsreduced NAD in the presence of GalUA, but nosuch activity was found in extracts from glucose-grown cells. These results show that the enzymesfor hexuronic acid utilization are inducible, andsuggest that galactaric acid is the product ofGalUA oxidation.Enzyme purification. The results of the purifica-

tion procedure for enzyme from GalUA-growncells are presented in Table 1. Sixty-fold purifica-tion was achieved with a total recovery of 35%.

D-Mannuronolactone and L-iduronic acid alsoreact, but only at 1 to 2% the rate of GalUA. It isnot known whether the observed activity withD-mannuronolactone and L-iduronic acid is dueto contamination of the substrates with GIcUAor to a contaminating enzyme. The followingcompounds are not substrates when tested atconcentrations of 4 mM: L-guluronic acid, L-arabinose, D-xylose, D-glucose, glycerol, D-arabitol, L-arabitol, ribitol, xylitol, D-glucitOl,D-galactitol, and D-mannitol.

TABLE 1. Purification of hexuronate dehydrogenase from sonicates of cells grown on GalUA

Specific activity of substrate(units/mg of proteina)

Fraction Volume Total units Recovery

GalUA GlcUA GlcUA/

ml

Crude extract ................. 46 85 0.067 0.122 1.8 100MnCl2 supernatant fluid .. .... 50 104 0.164 0.294 1.8 120(NH4)2SO4 fraction ............ 15 78 0.485 0.825 1.7 96DEAE Sephadex............... 25 51 1.800 3.420 1.9 60Calcium phosphate gel......... 25 30.4 4.300 8.160 1.9 35

a Assay conditions are described in the text. Activities are given with both GalUA and GlcUA assubstrate.

When GlcUA-grown cells were used as an enzymesource, the procedure described yielded the samedegree of purification; however, the purified frac-tion obtained from GlcUA-grown cells had only85% the specific activity with GalUA as enzymefrom GalUA-grown cells. The GalUA/GlcUAvalue (Table 1) for enzyme from GlcUA-growncells is approximately 1.6, as opposed to 1.9 forGalUA-grown cells. The purified enzyme gaveonly one reaction product from GalUA orGlcUA, detectable by paper electrophoreticanalysis at pH 5.8; less-purified preparations gaveseveral reaction products.Enzyme stability. The enzyme purified through

the step with calcium phosphate gel is unstableand loses up to 25% of its initial activity in 24 hrat 0 C, or frozen at -20 C. Unless otherwiseindicated, this preparation was used in thestudies described below. Less-purified enzymepreparations are considerably more stable;(NH4)2SO4 fraction retains full activity for atleast 2 years when kept frozen at -20 C.

Optimal pH. The optimal pH for the dehy-drogenation of GlcUA or GalUA is 8.0, withenzyme prepared from cells grown on eitherGalUA or GlcUA (Fig. 2).

Specificity. Enzyme obtained from cells grownon GlcUA or GalUA catalyzes the dehydrogena-tion of GlcUA, GalUA, and D-glucuronolactonewhen tested under standard assay conditions.

24

cm0CoI-x

z

%-

20

16

12

8

4

07 8 9

pHFIG. 2. Dependence of activity on pH. The condi-

tions of assay are those described in the text exceptthat pH of the buffer (0.05 M Tris) was varied. DEAESephadex preparation from GalUA-grown cells.Symbols for substrates: 0, GkcUA; *, GalUA.DEAE Sephadex preparation from GlcUA-grown cells.Symbols for substrates: A, GIcUA; A, GalUA.

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CHANG AND FEINGOLD

When tested in the standard assay with GalUAas substrate and with enzyme from GalUA-growncells, the following NAD analogues (0.3 mM)were reduced at 11, 15, 21, and 55% the rate,respectively, of NAD: ethylnicotinate-NAD,ethylpyridine - ketone -NAD, 3 - acetylpyridine-NAD, and thionicotinamide-NAD. The follow-ing were inhibitors when incorporated intostandard reaction mixtures together with, andat the same concentration as, NAD (0.3 mM),inhibiting 6, 7, and 35%, respectively: ,B-NADP,a-NAD, and 3-pyridine-aldehyde-NAD.

Activators and inhibitors. When the standardassay was conducted by using enzyme (dialyzedDEAE Sephadex step) from GalUA-grown cellsin the presence of added divalent cations (con-centration 0.5 mM) and GalUA as substrate,hexuronic acid dehydrogenase activity was in-creased to the extent of 30, 15, and 10% byMgCl2, CaC12, and MnCl2, respectively; itwas inhibited 30% by CoCl2, or CuS04. How-ever, EDTA (0.5 mM) inhibited less than 10%.Involvement of an -SH group in enzyme ac-tivity was shown by the effect of p-mercuribenzoicacid, which inhibited 90% at a concentration of0.2 mm. The same concentration of iodoaceticacid, on the other hand, was not inhibitory.

Effect of substrate concentration. The effect ofGlcUA and GalUA concentration on reaction

V

I-

z

U4

0 4 8 12 16

ES) (mM URONIC ACID)-'FIG. 3. Effect of GalUA and GkcUA concentration

on hexuronic acid dehydrogenase activity. Reactionconditions are those described in the text for the assayexcept that the concentration ofGalUA or GlcUA wasvaried; the enzyme was prepared from GalUA-growncell.s. Symbols: 0 GalUA; 0, GlcUA.

rate determined with enzyme from GalUA-growncells is shown in Fig. 3; that determined withGlcUA-grown cells is shown in Fig. 4, whereasthe effect of NAD concentration with enzymefrom GalUA-grown cells is shown in the lowercurve of Fig. 5. The determination of Km valuesby the method of Lineweaver and Burk (6) forGalUA or NAD did not differ significantly withenzyme source, being 0.15 mm and 0.07 mm forGalUA, and 0.06 mm and 0.13 mm for NAD,when determined with enzyme from GalUA-grown and GlcUA-grown cells, respectively.Interestingly, Km for GlcUA differed significantlywith enzyme source, being 0.25 mm for enzymefrom GlcUA-grown cells and 2.4 mm for enzymefrom GalUA-grown cells.

Characterization of reaction products. Understandard conditions of assay, the reduction ofNAD was linear in respect to time only for thefirst few minutes of reaction; thereafter, the in-creasing concentration of inhibitory NADH2quickly brought the reaction to a halt. Therefore,in order to obtain sufficient reaction product tocharacterize, it was necessary to maintain a lowlevel of NADH2 throughout the dehydrogenation.This was accomplished by coupling the oxidationof NADH2 to the reduction of pyruvic acid in thepresence of lactic dehydrogenase. The reactionmixture contained (mmoles) GalUA or GlcUA,0.28; potassium pyruvate, 1.2; MgC92, 0.4;NAD, 0.01; Tris buffer (pH 8.0), 3.0; hexuronicacid dehydrogenase from GalUA-grown cells

v

I-z

-

4

100

75

50

25

00 10 15 20

S(mM URONIC ACID)

Is)FIG. 4. Effect of GalUA and GlcUA concentration

on hexuronic acid dehydrogenase activity. Reactionconditions are those described in the text except thatthe concentration of GalUA or GlcUA was varied; theenzyme was prepared from GkcUA-grown cells.Symbols: *, GalUA; 0, GlcUA.

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(DEAE Sephadex step), 1.67 mg; and lactatedehydrogenase, 3 mg, in a total volume of 40 ml.A few drops of toluene were added, and the reac-tion mixture was incubated at 25 C for 18 hr. Thesolution was then held at 100 C for 5 min; pre-cipitated protein was removed by centrifugationand 1 g of acid-washed charcoal (Norit A) wasadded to the supernatant solution to adsorbnucleotides. After 30 min at 0 C, the suspensionwas filtered; the filtrate was condensed at 45 Cto 0.1 its volume, and loaded onto a 1.5 x 25cm Dowex 1 (x 1)-formate column. The columnwas eluted at the rate of 3 ml/min with an in-creasing gradient of formic acid obtained with areservoir containing 6 N formic acid and a mix-ing vessel containing 200 ml of water. Fractions(3 ml) were collected and examined by paperelectrophoresis at pH 5.8. The reaction productemerged after residual substrate. Whereas thereaction product from GalUA formed colorlesscrystals when its formic acid solution was cooledin ice, no crystals were obtained from the productderived from GlcUA. The crystals were purifiedby washing with 5% sulfuric acid, cold water, and50% alcohol, were recrystallized from alkalinesolution, and were dried in vacuum; the meltingpoint and mixed melting point (225 to 235 C)were identical to those found with authenticgalactaric acid. The identity of the product ofGalUA dehydrogenation was further confirmedaccording to the procedure of Kessler et al. (3)by conversion to dimethyl galactarate (meltingpoint, 188 to 190 C) and by NaBH4-reduction ofthe latter to galactitol. Similarly, the product ofGlcUA dehydrogenation was shown to be D-glucaric acid by conversion to D-glucitol.

Inhibition by reaction products. Neither galac-taric acid nor D-glucaric acid inhibits the dehy-drogenation of substrate when tested in standardassay mixtures (enzyme from GalUA-grown cells)at concentrations of 40 mm (10 times substrateconcentration). On the other hand, NADH2is a potent inhibitor of the reaction, strictly com-petitive with NAD (Fig. 5), Ki = 10-4 mM. Theinhibition is not dependent on uronic acid con-centration.

Reversibility of reaction. When 0.4 mM NADH2was incubated with 4 mM D-glucaric acid or galac-taric acid, and enzyme (from GalUA-grown cells)under standard conditions, there was no observa-ble change in absorbancy at 340 nm. When reac-tion mixtures which contained NADH2 and 14Clabeled-D-glucaric acid [specific activity 4.78,lc/,umole, prepared from "IC-labeled GlcUAaccording to Kessler et al. (3)] were subjected topaper-electrophoretic analysis at pH 5.8 andexamined by radioautography, no radioactivecompounds other than substrate were found.

I

600

T

U)- 400-

z

0 40 80 120 160 200

s (mM NAD)IS]FIG. 5. Inhibition of hexuronic acid dehydrogenase

activity by NADH2. Reactioni conditions are thosedescribed in the text for the assay except that theconcentration of NAD was varied and the concentra-tion of NADH2 in the reaction mixtures was as indi-cated; enzyme used was prepared from GalUA-growncells. NADHa concentration (mM): (0) 0; (0) 0.2;(A) 0.3. Symbols for NADH2 concentration: (0) 0;(0) 0.2; (A) 0.3.

Were 1% or more of the D-glucaric acid convertedto GlcUA, it could have been detected under theconditions of the experiment.

Differential activity toward GlcUA and GalUA.Hexuronic acid dehydrogenase obtained fromGalUA-grown cells oxidized GlcUA 1.9 timesfaster than GalUA (GalUA/GlcUA; Table 1);the ratio was 1.6 for enzyme from GlcUA-grown cells. The ratio does not change apprecia-bly during the course of the purification, suggest-ing that the same enzyme acts on both substrates.Furthermore, in the presence of GalUA andGlcUA (4 mm each), the observed reaction ratewith enzyme from GalUA-grown cells was about40% higher than that with only 8 mm GalUA,and 50% lower than that with only 8 mm GIcUA.Addition of increments of GalUA (reaching afinal concentration of 4 mM) to a reaction mix-ture containing GlcUA caused a progressivedecrease in reaction rate. When this experimentwas repeated with 0.2 mm GlcUA, the reactionrates determined in the presence of incrementsof GalUA, although higher than those for GalUAalone, were lower than what would be expectedwere the rates for GlcUA and GalUA additive.

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CHANG AND FEINGOLD

All these results are consistent with a one en-zyme to two substrate situation.

DISCUSSION

The data presented in this paper confirm andextend the preliminary observations of Zajic(11). GIcUA or GalUA in the presence of NAD isoxidized to the corresponding hexaric acid byGlcUA- or GalUA-grown A. tumefaciens. Theenzyme which catalyzes the reaction is specificfor GlcUA, D-glucuronolactone, and GalUA;activity toward D-mannuronic acid noted withearlier preparations was probably due to con-taminating enzymes.

Failure to detect GlcUA upon incubation ofD-glucaric acid with NADH2 in the presence ofthe enzyme suggests that the reaction is irre-versible. A similar conclusion has been reachedin studies of the oxidation of GlcUA by an en-zyme from mammalian liver (7). Furthermore, thehigh pH optimum of the dehydrogenation wouldnot favor the reverse reaction because of theproton released during reduction of NAD.The results of the manometric experiments

show that the oxidation of GalUA is inducible,since GalUA-grown, but not glucose-grown, cellsutilize GalUA without lag. That galactaric acidis the product of the oxidation of GalUA is sug-gested by utilization of galactaric acid with nolag and at the same initial rate by GalUA-grownas by galactaric acid-grown cells. Galactaricacid-grown cells, on the other hand, only oxidizeGalUA after a considerable lag; the oxidationthen proceeds at a rate not greatly lower thanthat observed with GalUA-grown cells (Fig. 1).This lag is probably due to lack of a permeasefor GalUA in galactaric acid-grown cells, sincehexuronic acid dehydrogenase is present in crudeextracts of both GalUA-grown and galactaricacid-grown cells.On the basis of the available data, it is not

possible to decide whether dehydrogenase in-duced by growth on hexuronic acid differs fromthat induced by growth on the correspondinghexaric acid. However, more data are availablefor the dehydrogenases obtained from cells grownin GalUA or GlcUA. These enzymes differsignificantly in their Km for GlcUA; in addition,the ratio of activity with GalUA to that withGlcUA (GalUA/GlcUA; Table 1) is consistentlyhigher by approximately 20% for enzyme de-rived from GalUA-grown cells than for enzymefrom GlcUA-grown cells at pH values in therange of 7 to 8.6 (Fig. 2). Although these resultssuggest that the enzymes may not be identical,further work is needed to substantiate this hypoth-

esis. Nevertheless, it is evident that growth ofA. tumefaciens on GalUA induces a dehydrogen-ase which can catalyze the oxidation of eitherGalUA or GlcUA. This is shown by the constantlevel of (GalUA/GlcUA) during purification(Table 1), and by the nonadditive nature of thereaction rates in the presence of both compounds.The dehydrogenase induced by growth on GIcUA,although possibly not identical with the GalUA-induced enzyme, is also capable of catalyzing thedehydrogenation of both uronic acids.The conversion of hexuronic acids to hexaric

acids has been demonstrated in animals andhigher plants, as well as in bacteria. Mung bean(Phaseolus aureus) seedlings convert GlcUA andGalUA to the corresponding hexaric acids;furthermore, the seedlings contain D-glucaricacid and galactaric acid as natural products (3).An NAD-linked enzyme which converts D-glucuronolactone to D-glucaric acid (D-glucu-ronolactone:NAD oxidoreductase EC 1. 1. 1. 70)was partially purified from rat liver by Marsh(7). Subsequently, Sadahiro et al. (8) partiallypurified the enzyme from guinea pig liver, andshowed that it was active with D-mannuronolac-tone as well as with D-glucuronolactone. In addi-tion, these workers suggested that the aldehydeforms of the substrates may act as direct pre-cursors in the enzymatic dehydrogenations.

It is likely that a number of bacterial speciesutilize GlcUA and GalUA via an initial con-version to the corresponding hexaric acid. Thus,Kilgore and Starr demonstrated the NAD-linkeddehydrogenation of the uronic acids by Pseu-domonas syringae extracts (5); Kilgore and Beck-man showed subsequently that D-glucaric acid,as well as galactaric acid, is converted by the sameorganism to CO2 and a nonreducing, nonlactoniz-ing acid (4). Our preliminary results, which showthat GlcUA and GalUA are converted to a-ketoglutaric acid by A. tumefaciens (Chang,Y. F., 1966, Ph.D. Thesis, University of Pitts-burgh; Chang, Y. F. and Feingold, D. S., 1967,Abstracts, 7th Int. Cong. Biochem. 4:711) are inagreement with these findings.

ACKNOWLEDGMENTS

This investigation was supported by Public Health Servicegrants GM-08820 and 1K3-GM-28,296 from the National Insti-tute of General Medical Services.We are grateful to E. Adams for helpful criticism and sugges-

tions.

LITERATURE CITED

1. Feingold, D. S., E. F. Neufeld, and W. Z. Hassid. 1964. En-zymes of carbohydrate synthesis. P. 474-519. In Linskens,H. F., B. D. Sanwal and M. V. Tracey (eds.), ModemMethods of Plant Analysis, 7, Springer, Heidelberg.

2. Gordon, H. T., W. Thornberg, and L. N. Werum. 1956.

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Rapid paper chromatography of carbohydrates and relatedcompounds. Anal. Chem. 28:849-855.

3. Kessler, G., E. F. Neufeld, D. S. Feingold, and W. Z. Hassid.1961. Metabolism of D-glucuronic acid and D-galacturonicacid by Phaseolus aureus. J. Biol. Chem. 236:308-312.

4. Kilgore, W. W., and D. M. Beckman. 1962. Metabolism ofD-galacturonic acid by Pseudomonas syringae. Biochem.Biophys. Acta 58:631-637.

5. Kilgore, W. W., and M. P. Starr. 1959. Uronate oxidation byphytopathogenic Pseudomonads. Nature 183:1412-1413.

6. Lineweaver, H., and D. Burk. 1934. The determination ofenzyme dissociation constants. J. Am. Chem. Soc. 56:658-666.

7. Marsh, C. A. 1963. Metabolism of D-glucuronolactone in

mammalian systems. 2. Further studies of D-glucuronolac-tone dehydrogenase of rat liver. Biochem. J. 89:108-114.

8. Sadahiro, R., Y. Hinohara, A. Yamamoto, and M. Kaurada.1966. Some aspects of D-glucuronolactone dehydrogenationby guinea pig liver enzyme (EC 1.1.1.70). J. Biochem.59:216-222.

9. Trevelyan, W. E., D. P. Proctor, and J. S. Harrison. 1950.Detection of sugar on paper chromatograms. Nature166:444 445.

10. Waddel, J. J. 1956. A simple ultraviolet spectrophotometricmethod for the determination of protein. J. Lab. Clin. Med.48:311-314.

11. Zajic, J. E. 1959. Hexuronic acid dehydrogenase of Agrobac-terium tumefaciens. J. Bacteriol. 78:734-735.

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