JOURNAL OF BACTERIOLOGY, May, 1965Copyright @ 1965 American Society for Microbiology

Vol. 89, No. 5Printed in U.S.A.

Enzymatic Basis for D-Arabitol Productionby Saccharomyces rouxii'JORDAN M. INGRAM AND W. A. WOOD

Department of Biochemistry, Michigan State University, East Lansing, Michigan

Received for publication 27 November 1964

ABSTRACTINGRAM, JORDAN M. (Michigan State University, East Lansing), AND W. A. WOOD.

Enzymatic basis for D-arabitol production by Saccharomyces rouxii. J. Bacteriol. 89:1186-1194. 1965.-The enzymatic steps in D-arabitol synthesis by Saccharomyces rouxiiwere studied. The fermentation of D-glucose-6-C14 gave rise to D-arabitol labeled at C-5;D-ribose of ribonucleic acid had the same isotope pattern. Crude extracts were able toreduce D-ribulose with reduced nicotinamide adenine dinucleotide phosphate(NADPH2) and D-xylulose with reduced nicotinamide adenine dinucleotide (NADH2).These extracts also oxidized D-arabitol with nicotinamide adenine dinucleotide phos-phate and xylitol with nicotinamide adenine dinucleotide. No reduction of D-ribulose-5-phosphate or D-xylulose-5-phosphate was observed. An enzyme which reducedD-xylulose with NADH2 was purified 33-fold and characterized as a xylitol (- > D-xylu-lose) dehydrogenase. Similarly, an enzyme reducing D-ribulose with NADPH2 was

purified 12-fold and characterized as a D-arabitol (-- D-ribulose) dehydrogenase. Alka-line and acid phosphatases were purified 50- and 40-fold, respectively, and their speci-ficities were determined. Only the acid phosphatase had detectable activity on D-ri-bulose-5-phosphate. The data support the postulate that D-arabitol arises bydephosphorylation of D-ribulose-5-phosphate and reduction of D-ribulose by aNADPH2-linked D-arabitol (-* D-ribulose) dehydrogenase.

Certain osmophilic yeasts grown in the pres-ence of high glucose concentrations produce, inaddition to ethanol and C02, a variety of poly-hydric alcohols; glycerol, erythritol, D-arabitol,and mannitol have been identified (Spencer andSallans, 1956). The effects of environmentalconditions, salt concentrations, nitrogen sources,and aeration upon production of polyols havebeen studied in detail (Spencer and Shu, 1957;Onishi, Saito, and Koshiyama, 1961). Sac-charomyces rouxii P8a, grown on specificallylabeled glucose, produced D-arabitol with labelingpatterns consistent with the postulate that thepentitol arose from D-ribulose-5-phosphate (Spen-cer et al., 1956). With the demonstration of apyridine nucleotide-linked D-arabitol (-* D-xylulose) dehydrogenase in bacteria (Wood,McDonough, and Jacobs, 1962), attention hasbeen directed to the mechanism of D-arabitolformation by these yeasts. Recently, two con-flicting reports have appeared stating that D-xylulose (Blakely and Spencer, 1962) and D-ribulose (Weimberg, 1962) were immediateprecursors of D-arabitol. It is currently unclear

1 Contribution No. 3520 of the Michigan Agri-cultural Experiment Station.

whether D-arabitol arises by reduction of aketopentose phosphate followed by a subsequentdephosphorylation, or by reduction of a keto-pentose produced by dephosphorylation or otherprocesses. The present report deals with theenzymatic steps leading to the biosynthesis ofD-arabitol in S. rouxii P3a.

MATERIALS AND METHODSBacteriological. The P8a strain of S. rouxii

was obtained from the National Research Councilof Canada, Prairie Regional Laboratory, Saska-toon, Saskatchewan, Canada. The yeast wasmaintained on a solid medium composed of 60%honey, 1% yeast extract, 0.23% urea, and 2%agar (Spencer and Sallans, 1956). The fermenta-tion medium was identical, except that 20% glu-cose was substituted for the honey. The yeastwas grown aerobically for 96 hr prior to harvesting.

Leuconostoc mesenteroides MAC 246, used forthe degradation of D-xylulose, was supplied byA. C. Blackwood, MacDonald College of McGillUniversity, Montreal, Quebec, Canada. Thecultures were maintained and grown on APTmedium (Difco).

Analytical. Ketopentoses and pentitols wereseparated on Dowex-1-borate, as described byKhym and Zill (1952). Ketopentoses were assayed

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by the cysteine-carbazole method (Anderson andWood, 1962a; Dische and Borenfreund, 1951).Pentitols were determined by the periodate-formaldehyde method of West and Rappoport(1949). Inorganic phosphate was determined bythe method of Fiske and SubbaRow (1925). Pro-tein was determined by the 280:260 ratio methodof Warburg and Christian (1942).Enzymatic. Crude extracts of S. rouxii were

prepared from cells washed twice by treatmentin a 10-kc, 250-w oscillator for 25 min, followed bycentrifugation to remove cell debris. Dehydrogen-ase assays were performed spectrophotometricallywith the aid of a Gilford Log Converter (Wood andGilford, 1961) by observing the rate of reducednicotinamide adenine dinucleotide (NADH2) orreduced nicotinamide adenine dinucleotide phos-phate (NADPH2) oxidation in the presence ofketopentose. One unit is described as the amountof dehydrogenase producing an absorbancy changeof 1.0 per minute at 340 m,u in a reaction volume of0.15 ml (1-cm light path). Kinase assays wereperformed in a manner described by Andersonand Wood (1962b).The activity of alkaline phosphatase eluted

from a diethylaminoethyl (DEAE)-cellulosecolumn was determined from the hydrolysis ofp-nitrophenyl phosphate, as described by Torriani(1960). Acid phosphatase was assayed by the re-lease of inorganic phosphate from the varioussubstrates (Fiske and SubbaRow, 1925).

Ribitol (-* D-ribulose) and D-arabitol (-* D-xylu-lose) dehydrogenases were prepared from Aerobac-ter aerogenes, as described previously (Wood et al.,1961). Xylitol (-* D-xylulose) dehydrogenase wasprepared by gradient elution with KCl of a crudeextract of Acetobacter suboxydans from DEAEcellulose (Kersters, Wood, and DeLey, J. Biol.Chem., in press). Xylitol (-* L-xylulose) dehy-drogenase from guinea pig liver was prepared bythe method of Hickman and Ashwell (1959).

Chemical. D-Xylulose was isolated after growthof A. suboxydans on D-arabitol (Reichstein, 1934).D-Ribulose was isolated as the o-nitrophenylhy-drazone after isomerization of D-arabinose (Glat-thar and Reichstein, 1935). D-Xylulose-5-phos-phate and D-ribulose-5-phosphate were preparedby phosphorylation of the ketoses by use of D-xylu-lokinase and D-ribulokinase from A. aerogenes(Mortlock et al., 1965), according to the methodof Simpson and Bhuyan (1962). Glucose-6-C14was obtained from Volk Radiochemical Co.,Skokie, Ill. All other preparations were from com-mercial sources.

Radiochemical. D-Ribose from ribonucleic acid(RNA) was isolated after growth on glucose-6-C'4,as described by Bernstein (1953). The D-ribose wasnot converted to D-ribonate, but was chroma-tographed on Whatman no. 3 filter paper in ethylacetate-acetic acid-water (3:1:3, v/v). TheD-ribose was detected with silver nitrate, elutedand rechromatographed in butanol- acetic acid-water (4:1:5, v/v). After a(ldition of carrier, theD-ribose-C'4 was fermented to acetate and lactate

by Lactobacillus arabinosus (Bernstein and Wood,1957).

D-Arabitol-C'4 produced from the fermentationof glucose-6-C14 was converted to D-xylulose-C'4with D-arabitol (-* D-xylulose) dehydrogenasefrom A. aerogenes. The D-arabitol was incubatedin the presence of nicotinamide adenine dinucleo-tide (NAD), pyruvic acid, lactic acid dehydro-genase, and tris (hydroxymethyl)aminomethane(Tris) buffer, pH 10.0 (Wood et al., 1961). Thereaction was followed to completion by determin-ing D-xylulose formation with the cysteine-car-bazole test. D-Xylulose-C'4 was chromatographedin butanol-acetic acid-water, eluted, and dilutedwith carrier D-xylulose. D-Xylulose-CI4 was fer-mented to acetate and lactate with L. mesen-teroides (Spencer et al., 1956).The acetate was separated from lactate by

steam distillation, and the lactate was recoveredby continuous ether extraction. Each was thenpurified by Celite chromatography (Bernsteinand Wood, 1957). Lactate was oxidized with per-manganate to give acetate from C-2 and C-3 oflactate and CO2 from C-1 (Abraham and Hassid,1957). The acetate was purified by Celite chroma-tography and degraded by the Schmidt procedure,as described by Phares (1951) in the apparatusdescribed by Krichevsky and Wood (1961). C02from C-1 of acetate was collected and counted in0.25 M NaOH. The methylamine from C-2 ofacetate or C-3 of lactate was distilled and isolatedas the hydrochloride salt. Radioactivity was de-termined in the Packard Tri-Carb liquid scintil-lation spectrometer in the counting system de-scribed by Baldwin, Wood, and Emery (1963).

RESULTS

Labeling patterns of D-ribose from RNA andD-arabitol produced from glucose-6-C'4. Theoreti-cally, D-arabitol could arise either by reductionof D-ribulose, D-xylulose, or their respective phos-phate esters. Production of either D-ribulose orD-xylulose from glucose-6-C'4 via the hexosemonophosphate pathway or from fructose-6-phosphate via transketolase and transaldolasewould yield these structures labeled at C-5.However, reduction of D-ribulose-o-C'4 wouldyield D-arabitol-5-C'4, whereas reduction of D-xylulose-5-C'4 would yield D-arabitol-1-C"4. D-Ribose-C"4 from RNA was examined as an indica-tor of the labeling pattern of the metabolic poolof pentose phosphates. The D-ribose and D-arabitol were isolated, fermented to lactate andacetate, and the acids degraded carbon by carbonas described in Materials and Methods. Table 1shows the distribution of radioactivity in thepentose and pentitol. It is evident that the C-6of glucose becomes C-5 of both D-ribose andD-arabitol, and that virtually no activity is foundin the other carbon atoms. Also, the recovery ofthe activity at the completion of the procedure

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TABLE 1. Isotopic patterns in D-ribose and D-arabitolfrom Saccharomyces rouxii grown on glucose-6-C14

Specific activity X 104*Carbon no.

D-Ribose D-Arabitol

1 0.07 0.02 0.0 0.03 0.0 0.04 0.0 0.05 1.11 1.40

* Expressed as disintegrations per minute permillimole. Initial specific activity: D-ribose, 1.29 X104; D-arabitol, 1.60 X 104.

E

o 2lw

zc]

ca.0

MINUTES

FIG. 1. Reduction of ketopentoses in the presenceof NADPH2 (left) and NADH2 (right). Each reac-

tion contained: 0.1 ,umole of NADPH2 or NADH2;6.0 ,umoles of substrate; 25 ,umoles of cacodylatebuffer (pH 6.0); and enzyme and water to a totalvolume of 0.15 ml (1-cm lightpath).

was 85%, indicating the acceptability of theprocedure. The absence of cross-contaminationin the degradation procedures for lactate andacetate has been established (Baldwin et al.,1963).

Pentitol dehydrogenases of crude extracts. Crudeextracts of S. rouxii catalyzed the reduction ofD-ribulose with NADPH2 and D-xylulose withNADH2 (Fig. 1). These represent the principalpolyol-producing reactions of extracts and,hence, probably of whole cells (Table 2). It isparticularly significant that reductions of D-

ribulose-5-phosphate and D-xylulose-5-phosphatewere not observed. Table 3 illustrates the abilityof crude extracts to oxidize the four pentitols.These reactions performed against an unfavorableequilibrium were aided by trapping the keto-pentose formed in semicarbazide buffer, therebyfavoring the accumulation and measurement ofreduced nicotinic acid adenine dinucleotide. In

TABLE 2. Nicotinic acid adenine dinucleotide-dependent ketose reductions by crude extracts

of Saccharomyces rouxii

Specific activitytSubstrate*

NADH2 NADPHS

D-Ribulose ......... ........... 0.0 21.0D-Ribulose-5-PO4......... .... 0.0 0.0D-Xylulose ....................39.5 1.9D-Xylulose-5-PO4.O . 0.0 0.0Dihydroxyacetone ..............0. 1.6Dihydroxyacetone-PO4.2.5 0.0D-Fructose ....................2.0 0.0D-Fructose-6-PO4.............. .0 0.0

* Reaction mixture contained: 7.5 ,ug of crudeextract; 5,moles of substrate; 0.1 ,umole of NADH2or NADPH2 , and 25,moles of cacodylate buffer,pH 6.0, in a volume of 0.15 ml (1-cm light path).

t Specific activity as described in text.

TABLE 3. Pentitol dehydrogenations by crude extractsof Saccharomyces rouxii

ActivitytPentitol*

NAD NADP

D-Arabitol 0.03 0.42

Ribitol ................... 0.15 0.05Xylitol .................... 1.05 0.22L-Arabitol ..0.05

* Reaction mixture contained: 0.1 ,umole ofNAD or NADP; 1 ,Mmole of pentitol; 48,ug of crudeextract; 0.1 ml of semicarbazide buffer, pH 9.2(Kaplan and Ciotti, 1957), in a total volume of0.15 ml.

t Change in absorbancy at 340 ma.s per 60 min.

harmony with the results shown in Fig. 1, xylitolwas oxidized in the presence of NAD; D-arabitol,and to a lesser extent xylitol, were oxidized in thepresence of nicotinamide adenine dinucleotidephosphate (NADP).

Purification and properties of xylitol (- D-

xylulose) dehydrogenase. Since D-arabitol is theproduct formed by the reduction of D-xyluloseand NADH2 in extracts of A. aerogenes (Woodet al., 1962), the D-xylulose-reducing activityfrom S. rouxii was purified to facilitate identifica-tion of the reduction product. A 33-fold purifica-tion was obtained by: (i) precipitation of thenucleic acids with 0.4% protamine sulfate in thepresence of 0.1 M ammonium sulfate; (ii) pre-

cipitation of the activity between 50 and 70%saturation of ammonium sulfate; (iii) adsorptionon calcium phosphate gel (3.4 mg of gel per mg ofprotein) and elution with 0.2 M phosphate buffer,

KETOPENTOSE REDUCTIONS BY CRUDE EXTRACTS

OF S. rOUXiiWITH NADPR2 WITH NADH1

-NO SUBSTRATE LD-RIBULOSE, L-RIBULOSE,-J L-RRIBULOSE OR L-XYLIULOSE

1.5 \ 4 0

D XYLULE

I.C j OR L-XYLULOSE

D.St I C

0 2 4 6 0 2 4 6

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TABLE 4. Identification of reduction and oxidation products of purified xylitol (-* D-xylulose)dehydrogenase from Saccharomyces rouxii

Test Pentitol from D-Xylulose Authentic Ketose from Authenticreduction xylitol xylitol oxidation D-xylulose

Periodate-formaldehyde test ............ + + *Cysteine-carbazole test (time for maxi-mal color) ........................ Negative Negative <60 min <60 min

Paper chromatography Rp, butanol-acetic acid-water (4:1:5) ............. 0.39 . 0.39 -

p-Dimethylphenaline sprayt.-_ Rose-gray Rose-grayDowex-l-borate chromatography........ Cochromatographed - _

with xylitolEnzyme tests

D-Arabitol (-* D-xylulose) dehydro-genase ........................... Negative Negative + +

Xylitol (-v D-xylulose) dehydrogenase. + + + +Xylitol ( L-xylulose) dehydrogenase. + +Ribitol ( D-ribulose) dehydrogenase.. Negative NegativeD-Xylulokinase.--.+ +

* A dash indicates that the test was not run.t Koch, Geddes, and Smith (1951).

pH 7.0; (iv) precipitation with ammonium sul-fate between 60 and 75% saturation.

Reaction characteristics of the dehydrogenasewere examined with the partially purified prepara-tion. The enzyme exhibited a sharp pH optimumof pH 6.0 for D-xylulose reduction. The Michaelisconstant (Km) for D-xylulose in cacodylatebuffer (pH 6.0) was 1.2 X 10-2 M.For identification of the pentitol and keto-

pentose involved, the reactions were performedat pH 6 in the presence of D-xylulose andNADH2, or at pH 9.2 with xylitol, NAD, sodiumpyruvate, and lactic dehydrogenase. The reac-tions were terminated by the addition of per-chloric acid, and the perchlorate was removed byneutralization with KOH. The supernatantliquid was deionized by passage through IR-120(H+) and IR-45 (OH-). The ketopentose orpentitol was purified by chromatography onDowex-1-borate, and the eluates containing thepeak were treated with methanol to remove theborate, as described by Khym and Zill (1952).The products of ketopentose reduction andpentitol oxidation were examined after purifica-tion. Paper chromatography, Dowex-1-boratechromatography, and specific enzymes were usedfor positive identification of the pentitol. Thecysteine-carbazole test, as well as paper chro-matography and specific enzyme tests, was usedto identify the ketopentose formed on pentitoloxidation. The product formed by reduction ofD-xylulose gave a positive periodate-formaldehydetest and a negative cysteine-carbazole test(Table 4). The Rp value on paper chromatog-raphy (detected with silver nitrate) was identical

to that of authentic xylitol and the product co-chromatographed with xylitol on Dowex-1-borate. When tested as a substrate for specificenzymes, it again behaved as did xylitol. Thus,it was evident that D-xylulose is reduced toxylitol.

In a similar manner, the compound isolatedafter oxidation of xylitol behaved identically toD-xylulose in the same series of tests. Based uponthese identifications, the enzyme is a xylitol(-*) D-xylulose) dehydrogenase.

Purification and properties of D-arabttol (D-rzbulose) dehydrogenase. The dehydrogenasereducing D-ribulose with NADPH2 was purified12-fold by the following steps: (i) precipitationof nucleic acids with 0.4% protamine sulfate inthe presence of 0.1 M ammonium sulfate; (ii)precipitation of the activity between 50 and 75%saturation of ammonium sulfate; and (iii) adsorp-tion to calcium phosphate gel and elution with0.2 M phosphate buffer, pH 7.0.

Characteristics of the partially purified enzymepreparations were examined as previously de-scribed. The enzyme for D-ribulose reduction hada broad pH optimum at pH 6.0. The Km forD-ribulose in cacodylate buffer (pH 6.0) was2.0 X 10-2 M. Products of D-ribulose reductionand D-arabitol oxidation were prepared andpurified as described above for D-xylulose andxylitol, except that NADP was regenerated in asystem containing glutamic dehydrogenase,which was present in the crude extract, ac-keto-glutarate, and ammonium hydroxide-ammoniumchloride buffer, pH 9.4 (Weimberg, 1962). In acombination of chemical, chromatographic, and

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TABLE 5. Identification of reduction and oxidation products of D-arabitol (-s D-ribulose)dehydrogenase from Saccharomyces rouxii

Pentitol from D-ribulose Authentic Ketose from D- AuthenticTest reduction D-arabitol arabitol oxida- D-ribulosetion

Periodate-formaldehyde test ............ + + *Cysteine-carbazole test (time for maxi-mal color) ............................ Negative Negative >60 min >60 min

Paper chromatography RF, butanol-acetic acid-water (4:1:5) . ............ 0.41 0.41 0.53 0.53

Enzyme testsD-Arabitol (-* D-xylulose) dehydro-

genase ........................... + + Negative NegativeRibitol ( D-ribulose) dehydrogenase. Negative Negative + +Xylitol ( D-xylulose) dehydrogenase Negative Negative -

* A dash indicates that the test was not run.

enzymatic tests (Table 5), the reduction productcorresponded closely to authentic D-arabitol, andthe oxidation product to D-ribulose. These identi-fications strongly suggested that the enzyme is aD-arabitol (-k D-ribulose) dehydrogenase.

Formation of D-ribulose by phosphatase action.Since D-ribulose, rather than D-xylulose or aketopentose phosphate, appeared to be theimmediate precursor of D-arabitol, the path offree D-ribulose formation in S. rouxii was inves-tigated. Both alkaline and acid phosphataseswere found in crude extracts, and both werepurified and examined for activity on D-ribulose-5-phosphate. All attempts to demonstrate theexistence of a specific phosphatase for D-ribulose-5-phosphate have failed.

Alkaline phosphatase was purified by: (i)treatment of the crude extract in 0.1 M ammoniumsulfate with 0.2% protamine sulfate, (ii) chro-matography on DEAE cellulose previouslyequilibrated with 0.02 M Tris buffer (pH 8.0).One major and two minor peaks were batch-eluted with 0.05 to 0.2 M NaCl-0.02 M Trisbuffer (pH 8.0). The major peak was diluted to0.02 M and rechromatographed on DEAE cellu-lose. One peak was obtained when the Trisconcentrations varied (0.1 to 0.3 M, pH 8),and the NaCl concentration was constant at 0.1M. This procedure yielded a 50-fold purifiedalkaline phosphatase. The phosphatase wasactive on a large number of phosphate esters,especially nucleotides, but no activity wasobserved with D-ribulose-5-phosphate (Table 6).The acid phosphatase was purified 40-fold by:

(i) adjusting the pH of the crude extract to 3.5with HCI and removing foreign protein by cen-trifugation; (ii) precipitation of the majority ofremaining protein by ammonium sulfate, 90%saturation; (iii) exhaustive dialysis against 0.02M Tris buffer (pH 7.0); (iv) lyophilization to

dryness and solution of the residue in a smallvolume of water. The specificity towards variousphosphate esters is shown in Table 6. A series ofcarbohydrate phosphate esters, including D-ribulose-5-phosphate and D-xylulose-5-phosphate,were hydrolyzed; however, the activity wasconsiderably higher on hexose phosphates.

Coupled phosphatase-pentitol dehydrogenase reac-tions. In exploratory experiments, it was foundthat the phosphatase could be assayed spec-trophotometrically in the presence of D-ribulose-5-phosphate, NADH2, and excess ribitol (--D-ribulose) dehydrogenase purified from A.aerogenes. The reaction was dependent uponD-ribulose-5-phosphate, ribitol (-- D-ribulose)dehydrogenase, and phosphatase (Fig. 2).

In a large scale reaction, ribitol was identifiedenzymatically as the final end product with ribitol(-k D-ribulose) dehydrogenase. The product didnot react with D-arabitol (- D-xylulose) dehy-drogenase from A. aerogenes. The Km for D-ribulose-5-phosphate, as determined in this assay,was 4.0 X 10-8 M. The phosphatase was in-activated by heating at 70 C for 10 min, andinhibited 96% by fluoride ion (6.7 X 10-2 M)and 90% by phosphate ion (6.7 X 10-2 M).

In a similar manner, the phosphatase wascoupled to the D-arabitol (-* D-ribulose) dehy-drogenase purified from S. rouxii. Figure 2 showsthe dependence of the reaction upon substrate,dehydrogenase, and phosphatase. The product ofthe coupled reaction was identified as D-arabitolwith D-arabitol (-k D-xylulose) dehydrogenase.The product gave no reaction in the presence ofribitol (-> D-ribulose) dehydrogenase.

DISCUSSIONThe specificity of the pentitol dehydrogenases

and identification of the products formed in boththe oxidation of pentitols and the reduction of

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TABLE 6. Substrate specificity for purified alkalineand acid phosphatases from Saccharomyces routxii

Activity

Substrate*Alkaline Acid

phosphataset phosphatasel

Adenosine monophosphate. 1.22 4.60Adenosine diphosphate 0.87Adenosine triphosphate.... 0.03Cytosine monophosphate... 0.85Guanosine monophosphate. 1.35Uridine monophosphate.... 1.00Inosine monophosphate 1.85D-Ribulose-5-phosphate .... 0.0 2.00D-Xylulose-5-phosphate 1.15Glucose-6-phosphate ....... 0.01 3.006-Phosphogluconate . .... 0.01Fructose-6-phosphate .6.60Fructose-1, 6-diphosphate . 6.25D-Ribose-5-phosphate ...... 0.03 0.0a-Glycerophosphate ..... . 0.01 0.0

* Reaction mixture contained for alkaline phos-phatase: 5 ,umoles of substrate; 5 Amoles of MgCl2;80,umoles of Tris buffer, pH 9.0; enzyme and waterto 1.0 ml. For acid phosphatase, the reaction mix-ture contained: 2.5,umoles of substrate; 2.5,umolesof MgCl2 ; 80,umoles of acetate buffer, pH 5.5; en-zyme and water to 1.0 ml. The same amount ofalkaline phosphatase, eluted from DEAE cellulose,was used for each substrate. Similarly, a constantamount of lyophilized acid phosphatase was usedfor the assay of each substrate.

t Activity of alkaline phosphatase expressed asmicromoles of phosphate released per 30 min.

t Acid phosphatase activity expressed as thatamount of enzyme which caused an absorbancychange of 1.0 at 660 mu in a reaction volume of 1.0ml in 5 min.

ketopentoses by crude extracts or purified prep-arations demonstrate the presence of only oneroute of D-arabitol production; this route is thereduction of D-ribulose in the presence ofNADPH2. No reduction of D-ribulose-5-phos-phate or D-xylulose-5-phosphate with NADH2or NADPH2 has ever been observed. Contrary tothe specificity of the D-arabitol (-k D-xylulose)dehydrogenase of A. aerogenes, the reduction ofD-xylulose by the dehydrogenase of S. rouxiiyields xylitol, as is the case in mammalian liver(Hickman and Ashwell, 1959).That D-arabitol (- D-ribulose) dehydrogenase

functions in vivo in D-arabitol formation isstrongly supported by isotope data from thefermentation of D-glucose-6-C'4. C-6 of glucosegives rise to C-5 of both D-ribose of RNA andD-arabitol, each with similar specific activities.Thus, it is likely that the precursors of each arein equilibrium with, or identical to, D-ribose-,

E NADH2 NAD NADPHj NADP

t) 2.0 NO DEHYDROGENASE OR - --NO DEHYDROGENASE OR->ID-RIBULOSE-5-P04 HID-RIBULOSE-5-P04

-50 PHOSPHATASE-NO PHOSPHATASE

1.0

.-COMPLETE COMPLETE

0 2 4 6 0 2 4 6MINUTES

FIG. 2. Coupled reactions of acid phosphatasewith ribitol (-+ D-ribulose) dehydrogenase (left)and D-arabitol (-* D-ribulose) dehydrogenase(right). Each reaction mixture contained: 0.1 jAmoleof NADH2 or NADPH2; 1.5 MAmoles of D-ribulose-5-phosphate; 9.4 units of ribitol dehydrogenase, or 5

units of D-arabitol dehydrogenase; acid phosphatase;25 MAmoles of cacodylate buffer (pH 5.5); and waterto a total volume of 0.15 ml (1-cm light path).

D-ribulose-, and D-xylulose-5-phosphates. Inaddition, the labeling patterns have unique valuein determining which of the possible ketopentosesis directly responsible for D-arabitol synthesis.In the reduction of D-ribulose, C-5 would becomeC-5 of D-arabitol, whereas, in the reduction ofD-xylulose to D-arabitol, C-5 would become C-1 ofD-arabitol. Clearly, the latter possibility is notsupported by the isotope data.

D-Ribulose can arise by the action of a phos-phatase on D-ribulose-5-phosphate, but a specificD-ribulose-5-phosphate phosphatase has not beenfound. Furthermore, the probability of theexistence of a specific D-ribulose-5-phosphatephosphatase seems to be diminished by the factthat mannitol, erythritol, and glycerol are alsooften produced, presumably from the ketosesderived from phosphate esters. This suggests thata number of phosphate esters are attacked by a

nonspecific phosphatase and then reduced to apolyol.The acid phosphatase of baker's yeast is

reported to be associated primarily with theexternal cell surface (Schmidt et al., 1963). If a

similar situation existed in S. rouxii, D-ribulose-5-phosphate would be unavailable for dephos-phorylation. However, Suomalainen, Linko, andOura (1960) reported that approximately 30%of baker's yeast acid phosphatase was locatedintracellularly. In a similar study, Tonino andSteyn-Parv6 (1963) concluded that the presenceand location of nonspecific phosphatases de-pended upon the type of yeast, the age of the

D-RIBULOSE-5-P04 +H2 -> D-RIBULOSE + Pi

-RIRULILOSF , RIRITOI in-lRI 11OCRF- n-ARA ITOI-

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INGRAM AND WOOD

culture, and the composition of the growthmedium. Preliminary experiments with wholecells of S. rouxii indicated an inability to dephos-phorylate D-ribulose-5-phosphate, as measuredby the liberation of inorganic phosphate. Sec-ondly, the optimal pH of whole baker's yeast acidphosphatase is between 3 and 4 (Schmidt et al.,1963). The activity at pH 6.0 is negligible,whereas the optimum of the acid phosphatase ofS. rouxii is between 5.5 and 6.5. Apparently, theacid phosphatase of S. rouxii does not resemblethat of baker's yeast and, hence, may not belocated entirely at the cell surface. Although nottested directly, it is considered likely that D-ribulose-5-phosphate is available for attack byacid phosphatase.The demonstrated activity of an acid phos-

phatase on D-ribulose-5-phosphate provides ameans for D-ribulose formation. However, thisphosphatase has high activity in vitro on anumber of other sugar phosphates, and an in-discriminate dephosphorylation of all phosphateesters might be expected. Indeed, the productionof mannitol, erythritol, and glycerol may resultfrom this lack of specificity, as well as from thepresence of the necessary dehydrogenases. Theextent of indiscriminate phosphatase action maybe regulated by the steady-state levels, the Kmvalues of the phosphatase, and the turnovervelocity of the various esters. For example, it isdoubtful that D-xylulose-5-phosphate is hy-drolyzed, perhaps due to one of the above param-eters, because neither D-xylulose nor xylitol hasbeen found in fermentation liquors or cell-freeextracts. On the other hand, D-fructose, ratherthan D-fructose-6-phosphate, is reduced withNADH2, presumably to mannitol. Furtherstudies of the relative quantities of the phosphateesters present during the fermentation will benecessary to answer this question.The stimulatory effect of a low concentration

of phosphate ion and a high oxygen tension onthe conversion of glucose to D-arabitol, reportedby Spencer and Shu (1957), may now be partiallyunderstood. Weimberg and Orton (1963) reportedthat the synthesis of acid phosphatase by S.mellis is inhibited by a high extracellular in-organic phosphate concentration. This in vivoeffect was not investigated in S. rouxii, but theactivity of the purified acid phosphatase wasfound to be markedly inhibited by inorganicphosphate. Thus, it appears that both the syn-thesis and activity of the acid phosphatase maybe under regulation of the intracellular inorganicphosphate concentration. The stimulation ofD-arabitol synthesis by a high oxygen tension isconsidered to result from an increased glucoseoxidation through the hexose monophosphate

D-GLUCOSE

D- GLUCOSE-6-PO4

NON-OXIDATIVE OXIDATIVE

6- PHOSPOD- FRUCTOSE-6-PO4 GLUCONATE

D- RIBULOSE-5-PO4

4HPi 2(NADPH)

D-RIBULOSE

D-ARABITOL

FIG. 3. Metabolic reactions leading to synthesisof D-arabitol from D-glucose by Saccharomycesrouxii.

pathway, accompanied by an accumulation ofNADPH2. This would be accompanied by anaccumulation of phosphate esters and a decreasein the level of inorganic phosphate. The acidphosphatase, now more active at low phosphateconcentration, is able to dephosphorylate thefirst ketopentose formed, D-ribulose-5-phos-phate, to D-ribulose, with regeneration of in-organic phosphate. D-Arabitol (I D-ribulose)dehydrogenase then regenerates NADP by the re-duction of D-ribulose to D-arabitol. Since the oxida-tion of 1 mole of glucose to 1 mole of D-ribulose-5-phosphate generates 2 moles of NADPH2,sufficient NADPH2 is available for both biosyn-thetic and maintenance reactions and for produc-tion of D-arabitol, as described. The stoichiometryof D-ribulose-5-phosphate formation vs. NADPH2formation may be balanced through an inde-pendent route to D-ribulose-5-phosphate fromD-fructose-6-phosphate via transaldolase andtransketolase reactions.An increased yield of D-arabitol has been

reported to be coupled to a decreased yield ofethanol under aerobic conditions (Spencer andShu, 1957). This would be expected for increasedparticipation of the hexose monophosphatepathway and the regeneration of NADP viaD-arabitol formation. These considerations in-dicate that NADPH2 is not readily oxidized bymolecular oxygen, but that NADP is regeneratedby a system analogous to the anaerobic produc-

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D-ARABITOL PRODUCTION BY S. ROUXII

tion of ethanol and regeneration of NAD byalcohol dehydrogenase.The overall sequence of reactions for D-arabitol

synthesis is illustrated in Fig. 3. The plausibilityof this scheme is further supported by the ob-served synthesis of D-arabitol from a mixture ofpurified acid phosphatase, D-arabitol (-* D-ribulose) dehydrogenase, NADPH2, and D-ribulose-5-phosphate.

ACKNOWLEDGMENTThis investigation was supported by a grant-

in-aid from the National Institutes of Health.

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