THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 240, No. 3, March 1965

Printed in U.S.A.

Polyol Dehydrogenases of Gluconobacter oxydans*

K. KERSTERS, W. A. WOOD, AND J. DE LEP

From The Laboratory for Microbiology, State University, Gent, Belgium, and the Department of Biochemistry, Michigan State University, East Lansing, Michigan

(Received for publication, illarch 13, 1964)

Acyclic polyols and related substances are oxidized by acetic acid bacteria according to the well known rule of Bertrand and Hudson (l-3) (for a review, see Reference 3). This rule states that only polyols possessing Configuration I are oxidized to the corresponding ketoses (Configuration II) by strains of the subozydans group of acetic acid bacteria.

CHzOH CHzOH

HO-C-H ~ LO I

HO-C-H HO-C-H I I

(1) (11)

The secondary hydroxyl group involved in the oxidation, and the cis-vicinal secondary hydroxyl group, must have a D con- figuration jvith respect to t,he primary alcohol group adjacent to the site of oxidation.

This specific mode of otida,tion facilitated the synthesis of several new ketoses. In a series of papers, Hudson and Richt- myer and their a.ssociates described the microbiological synthesis of several new heptuloses (448), and the oxidation of n-cry&o- n-gala&o-octitol to an octulose (9).

Several w-deoxysugar alcohols, which do not possess the Bertrand-Hudson configuration, are nevertheless oxidized by growing cultures of Gluconobacter ox@ans (suboxydans) ATCC 621 (10, 11). By considering t,he CH&HOH- group as a substituted -CH,OH group, the Bertrand-Hudson rule was extended to predict the action of the organism upon w-deoxy- sugar alcohols (10).

Virtually no attention has been paid to the enzymology of polyol oxidations by acetic acid bacteria. According to Arcus and Edson (la), glycerol-grown G. oxydans (suboxydans) produces two types of mannitol dehydrogenase of low specificity. One is a particulate, cytochrome-linked system that oxidizes polyols with the Bertrand-Hudson configuration. The second dehy- drogenase is a soluble, nicotinamide adenine dinucleotide-linked enzyme with broad specificity. Cummins (13) reported on an NAD-linked sorbitol dehydrogenase in G. oxydans ATTC 621 which oxidized sorbitol to o-fructose. However, sorbitol was also oxidized by an NADP-linked enzyme to L-sorbose. By- grave and Shaw (14) partially purified an NADP-linked D-

mannitol dehydrogenase, which oxidized n-mannitol, D-glUCitOl

(to L-sorbo,se), and n-arabitol (to n-xylulose). The enzyme seems to be specific for polyols of the D-&X0 configuration. The

* Supported by research grants from the National Science Foundation. Contribution No. 3330 from the Michigan Agricul- tural Experiment Station.

occurrence and nature of the polyol dehydrogenases have recently been reviewed by Touster and Shaw (15).

The present paper provides an enzymological interpretation of the Bertrand-Hudson rule, based on exact knowledge of the specificity, cofactor requirement, and localization of the dehy- drogenases involved. The data indicate that not one, but a series of enzymes is responsible for the oxidation of polyols by acetic acid bacteria.

EXPERIMENTAL PROCEDURE

Bacteriological-G. oxydans (suboxydans), strain SU (originally isolated by Dr. A. H. Stouthamer, Utrecht, The Netherlands), was grown in Roux flasks at 30” on a solid medium which con- tained 5% n-glucitol (sorbitol), 0.5% yeast extract, and 2.5% agar. For some experiments liquid cultures were grown in Fernbach flasks at 30” on a rotary shaker. In this case the medium consisted of 5% D-&Kit01 and 0.5y0 yeast extract in 0.04 M potassium phosphate buffer, pH 6.0. No apparent difference in enzymatic activity was observed between cells grown on liquid or solid medium. The cells were harvested after 2 to 3 days of growth and washed with 0.01 M phosphate buffer, pH 6.2 Cell-free extracts were prepared by suspending 20 g of cells in 50 ml of 0.01 M phosphate buffer, pH 6.2, and disrupting them in a IO-kc, 250-watt oscillator for 20 minutes at 4” under an atmosphere of hydrogen. Intact cells were removed by centrifugation at 4” at 10,000 X g for 20 minutes. The super- natant solution was then centrifuged for 2 hours at 4” and 105,000 x g in a Spinco model L centrifuge. The yellowish supernatant solution after centrifugation contained the soluble enzymes, which were derived from the cytoplasm. This fraction will be called ‘LsoIuble enzymes” or “supernatant.” The gelat- inous, brown-red precipitate consisted mainly of fragments of the cytoplasmic membrane and cell wall (16). This fraction (referred to as “particles”) was washed twice by resuspension in 0.01 M phosphate buffer, pH 6.2, and recentrifugation as above.

Chemicals-Most sugar alcohols were commercially available preparations of high purity. L-Threitol was synthesized by lithium-aluminim hydride reduction of L-tartaric acid (17). n-Gulono-y-lactone was reduced to L-glucitol, as well as L-

rhamnose to L-rhamnitol and n-allose to meso-allitol, by sodium borohydride. n-Allose was a gift from Dr. M. Bernaerts, Central Laboratory, Ministry of Economic Affairs, Brussels (18). n- Iditol was prepared by catalytic reduction of L-sorbose (19). L-Lyxomethylitol (5.deoxy-n-lyxitol) and L-fucitol (6-deoxy-n- galactitol) were prepared by refluxing, respectively, L-arabinose diethylmercaptal tetraacetate and o-galactose diethylmercaptal pentaacetate with large amounts of Raney nickel in dilute ethanol

by guest on August 28, 2017

http://ww

w.jbc.org/

Dow

nloaded from

966 PolydE B&ydrogenases in G. oxydaw

(11). %@9&~-$$#&$o~ was prepared by se&p ;:boro- hydridem:zdtct ‘-& @f P-g&cr~-n-gulo-heptono-y-la&one @O). n-glycero-,,-g~aeto -Zl&&to~ @w&tol) was isolated from :a,vocado (21). The other he,*9 b,I&&fW L-mannitol, and D&i@d

F@‘&CTION NUMBER

FIG. 1, Chrmatographie separation of polyol dehydrogenazes from G. sz&oz&uns. An monium sulfate fraction (25 to 50% saturation) of soluble enzyw was chromatographed as describd in “Expc~rnent:~ Proceduna..” Or&n&a at the lefl expree# protein and a&iv’* of xylitol&hydrogenase (NADP-Xditol-Deb) Axylitol as substr&e) and ~~-&rythro dehydrogenase (NA n-r Erythro-Deh) (ribitolabstrate). ,The ordinates at the right express elution concentratim of NaCl & activity of ~-zyh dehydro- genase (NAD-D-Xyt~&&) (D-gh&@l as substrate).

OL 60

3

FRACTION NUMBER

FIG. 2. Chromatographic separation of polyol dehydrogenases from G. suboxyhzs. An ammonium sulfate fraction (50 to 75% saturation) of soluble enzymes was chromatographed as in Fig. 1. Ordinates at the left express protein and activity of D-1~x0 de- hydrogenase (mannitol as substrate). Ordinates on the right express molarity of eluting agent, NaCl, and activity of 2-k&o- gluconic and Lketogluconic dehydrogenases (gluconate ss sub- strate).

were gifts of Dr. N.IK. Richtmyer, National fuistitutiesof Health. The pentuloses were(obtained as reported previ&& (22).

Sephadex G-25 (Pharmacia) was washed repetlbdig before use with 0.01 M phosphate:buffer, pH 6.5, in order to remove the fine particles. DEAE-c&lose (Serva-Entwicklung&bor, Heidel- berg) was washed widh (distilled water to remove the fines and

converted to the free ibase with 1 N NaOH. The exchange, agent was then thorougb?y washed with distilled water, treated @ith 0.1 M phosphate bu@er, pH 6.5, and equilibrated with 0.01 M phosphate buffer, pH 65..

AmaWcal Methods-The (oxidation of the sugar alcohols by &e ~grarticles was followed t&n the Warburg apparatus at 30”. %be @%rticles were suspended i&(0.05 M phosphate buffer, pH 6.2, ti ,&@:a *turbidity reading of &out 400 on the Klett calorimeter v&& No.. 66 filter. This correspcmds to about 2 mg of protein perr ti “?&e main compartment of&he Warburg vessel contained 1.7 & & micle suspension and 36 pmoles of MgClz in 0.1 ml. The @&@‘ti contained 0.1 ml of ag% KOH, and the side arm cont&@ad 10 moles of substrate in 0.1 ml. For large scale, prepar%ti@ms dtidation products, 1oaP vies of substrates were used. T& o~yge~~ nptake was corrected $m Rndogenous respira- tion, whi& WZB iwr&ly negligible.

Dehydrogenase essays were conducted ~IQ q-2 microcuvettes (reaction volume, cO,U ml) by observing &anges in NADH or NADPH coneentr&ion at 340 rnl.c in a &&nnan DU spectro- photometer. The assays were facilitated b pbotomultipher aad cuvette-positioning awhments which permitted automatic rew,rding of absorbance changes in four cuvet* simultaneously (23). Each cuvette contamed 0.01 to 0.04 ml of enzyme prepara- tion, @I rmole of NAB or NADP, 4 pmoles of substrate, and 0.05 M rpis-HCl buffer, pH 6.6, to a volume of 0.15 ml. The peactiop rn~ initiated by adding the substrate. One unrt of g&lvity was established as the amo@ntl of enzyme causing an absorbance w of 1.0 per &nute at room temperature. Speoififtg g&vity ia expressed as unita of enzyme per mg of protein

(24). The orcinol test for pentoses was performed as described by-

Horecker, Smyrniotis, and Klenow (25). Ketopentoses were- determined by the cysteine-carbazole method (26) with a 20-- minute incubation time for D- or n-ribulose, and a lO@minute- incubation time for D- or n-xylulose. L-Ribulose o-nitrophen& hydrazone and monoacetone n-xylulose were used as standard& Hexuloses were determined by the resorcinol method of Roe,, Epstein, and Goldstein (27), and heptuloses by the orcind? method of Dische (28) with n-fructose and n-gluco-heptulose, respectively, as standards. The cysteine-sulfuric acid reaction for hexuloses was performed according to Dische and Devi (29). The spectra were recorded with a Beckman DK-2 automatic spectrophotometer. Kinases were assayed spectrophotometri- tally as described previously (30). Chromatographic methods involving paper and Dowex l-borate were those described pre- viously (31), except for size and volume modifications. Optical rotation of several oxidation products was determined with a Zeiss polarimeter ( 3t0.010°), usually in a 20-cm tube at 589 mp. The blank, consisting of the endogenous reaction mixture, did not show optical activity.

Enzyme PreparationsXyliiol (+ n-xylulose) dehydrogenase free of xylitol (-+ D-xylulose) dehydrogenase was purified from guinea pig liver (32). n-Arabitol (- n-xylulose) dehydrogenase and ribitol (+ n-ribulose) dehydrogenase were preparations described by Wood, McDonougb, and Jacobs (22). L-Xylulo-

by guest on August 28, 2017

http://ww

w.jbc.org/

Dow

nloaded from

March 1965 K. Kersters, W. A. Wood, and J. De Ley 967

kinase was purified from Aerobacter aerogenes grown on n-xylose saturation contained NAD-specific dehydrogenases for polyols (30). L-Ribulokinase was purified from A. aerogenes grown on with the Bertrand-Hudson configuration, i.e. for ribitol, all&l, n-arabinose (33). Lactic dehydrogenase (containing pyruvate n-glucitol, and n-mannitol. n-Arabitol and meso-xylitol, which kinase) and yeast hexokinase were commercial crystalline en- lack the Bertrand-Hudson configuration, were also quickly zymes. oxidized by this fraction. Two explanations are possible for this

phenomenon: either the soluble enzyme fraction contains one RESULTS NAD-linked polyol dehydrogenase with a very broad specificity,

Soluble Polyol Dehydrogenases as suggested by Arcus and Edson (12), or several polyol dehydro- genases with differing specificities are present. Therefore, this

Separation and Purification of Soluble Polyol Dehydrogenases- ammonium sulfate fraction after dialysis was added to a DEAE- Several polyols are oxidized by soluble enzymes with accom- cellulose column which had been washed with 0.01 M phosphate panying reduction of NAD or NADP. In order to determine buffer, pH 6.5. Three different polyol dehydrogenase peaks the number and the specificity of the dehydrogenases, these were eluted by an increasing NaCl gradient (Fig. 1). From enzymes were fractionated as follows. MnC& was added to the specificity studies to be described, these were identified as an crude extract to a final concentration of 0.05 M in order to remove NADP-linked xylitol dehydrogenase (1Bfold purified); an the nucleic acids. After centrifugation, the supernatant solu- NAD-linked werythro dehydrogenase (12-fold purified) ; and an tion was fractionated with ammonium sulfate to obtain two NAD-linked D-zylo dehydrogenase (go-fold purified). active fractions. The precipitate obtained between 20 and 50% Another ammonium sulfate fraction obtained between 50 and

TABLE I

Substrate speci$city of several polyol dehydrogenases

Polyol Oxygen consumption by membrane fraction= ‘ADP-linked xylito

dehydrogenase

Glycerol ....................................... meso-Erythritol................................ L-Threitol. ....................................

meso-Xylitol................................... meso-Ribitol................................... L-Arabitol. .................................... D-Arabitol..................................... L-Lyxomethylitol ..............................

0.50 0 0.52 0 0.22c 0

0 100 0.50 0 0 0 0.52 0 0 0

D-Mannitol .................................... 0.51 0 L-Mannitol .................................... 0 D-Glucitol..................................... 0.49 0 L-Glucitol..................................... 0 0 meso-Allitol .................................... 0.50 0 D-Altritol...................................... 0.47 meso-Galactitol. ............................... 0 0 L-Iditol........................................ 0 0 L-Rhamnitol................................... 0 0 D-Fucitol...................................... 0 0

meso-glycero-allo-Heptitol ...................... 0.23c D-glycero-D-altro-Heptitol. ...................... 0.46 D-glycero-D-manno-Heptitol .................... 0.47 D-glycero-D-gko-Heptitol ...................... 0.27~ D-glycero-D-galacto-Heptitol .................... 0.48 0 meso-glycero-g&o-Heptitol ...................... 0.50 0 meso-glycero-ido-Heptitol ....................... 0 L-glycero-D-galacto-Heptitol. .................... 0 L-glycero-L-ido-Heptitol ........................ 0

D-Ribonate, D-xylonate ......................... n&luconate ................................... n-Glucose ......................................

0.48

NAD-linked n-zylc dehydrogenase

0 0 0

21 0 0 0 0

2 0

100 0 0

0 15

0 0

0 0 0

20 0 0 9

37 3

0 0

a Amount of oxygen (micromoles) consumed in 5 hours per pmole of substrate added. b Relative rates. c Oxidation continued after measurement period (5 hours).

Pyridine nucleotide reduction”

NAD-linked werythro

dehydrogenase

0 0 0

23 loo

50 0

11

0 15 0 2 5

0 0

15 0

0 0

0 0 0

0

NADD;;oked

dehydrogenase

0 0 0

0 0 0 3 0

100

16 0 cl

0 0 0 0

by guest on August 28, 2017

http://ww

w.jbc.org/

Dow

nloaded from

968 Polyol Dehydrogenases in G. oxydans Vol. 240, No. 3

70y0 saturation was absorbed on a similar DEAE-cellulose column. Three different dehydrogenases were eluted with an increasing NaCl gradient (Fig. 2) as follows: an NADPH-linked 2-ketogluconic reductase forming n-gluconate; an NADP-linked

D-Zyxo dehydrogenase (20-fold purified); and an NADPH-linked 5-ketogluconic reductase forming n-gluconate.

Specificity of Polyol Dehyclrogenases-The specificity of oxida- tion by the most purified fractions of the soluble polyol dehy- drogenases, and by the insoluble or membrane fraction, is sum- marized in Tables I and II. The oxidation products were subsequently identified in order to establish which secondary alcohol group of the polyol was oxidized.

Identijcation of Pentitol Oxidation Products-As an example of the procedure used, the oxidation of xylitol by the D-xylo dehy- drogenase will be described in detail. Although the equilibrium of xylitol oxidation by this dehydrogenase lies far in the direction of xylulose reduction, xylitol oxidation proceeds almost to comple- tion by coupling with the reduction of pyruvate by lactic dehy- drogenase. This is the case with either NAD or NADP as the carrier. The fractions from the DEAE-cellulose column con- taining the highest specific activity for the D-xylo dehydrogenase (Fig. 1) were pooled and dialyzed or passed through a Sephadex G-25 column to remove salts. The dehydrogenase was then absorbed on a small amount of DEAE-cellulose and concentrated IO-fold by elution with 1 M NaCl-0.01 M phosphate buffer, pH 7.0. For large scale isolation of the oxidation product, 100 pmoles of xylitol, 300 pmoles of sodium pyruvate, and 5 pmoles of NAD were incubated for 2 hours at 30” at pH 8.5 (Tris buffer) with about 400 units of crystalline lactic dehydrogenase and 160 units

TABLE II Substrate specijkity of NADPH-linked ketogluconic reductases

Pyridine nucleotide reductionb

oxygen consumption ,y membrane

fraction5 NADPH-

linked J-keto-

gluconic reductase

Substrate

4 0

46 100 -

37 200 -

0 0 0 0 0

Sodium n-ribonate. Sodium n-arabonate.. Sodium n-xylonate. Sodium n-gluconate Sodium n-galactonate. Sodium 5-ketogluconate .

0 0 0

100

Sodium 2-ketogluconate . Glucose ................. Glycerol ................ meso-Erythritol .......... nzeso-Ribitol ............ n-Mannitol .............. n-Glucitol.. ..............

0 0 0.1” 0.48 0.20” 0

-a

- -

- -

84

of purified (30.fold) NAD-specific D-xylo dehydrogenase. Ma- I

a Amount of oxygen (micromoles) consumed per rmole of sub- terial reacting in the cysteine-carbazole test (97 pmoles) was

strate added per 5 hours. produced. The proteins were precipitated with perchloric acid,

b Relative rates. and the perchloric acid was removed as potassium perchlorate. c Oxidation continued after the measurement period (5 hours). The reaction mixture was then deionized by passage through d Dash represents reactions not tested. Amberlites IR-120 (H+) and IR-45 (OH-), and the effluent was

TABLE III

Identification of pentuloses formed in oxidation of xylitol and o-arabitol

Dehydrogenases Authentic pentuloses

1. Test - 2. NAD-linked

D-zylo

Xylitol 0.01

100

0.59

Gray

Rose 0.47

+

0

0

0

3

-_

-

4. NADP-linked D-lyXO 5. D-Rib&se 6. L-Ribulose D-xylulosl NADP-linkee

xylitol

Xylitol 0.01

100

0.58

Gray

Rose 0.49

0

+

0

+

0.01

100

0.59

Gray

Rose 0.49

L-Xylulose

0.01

100

0.59

Gray

Rose 0.49

0

n-Arabitol 0.01

100

0.59

Gray

Rose 0.47

+

0

0

0 0

Substrate oxidized Dowex l-borate, elution concentra-

tion, molarity Cysteine-carbazole test (time in

minutes for full color develop- ment)

Paper chromatography (water-sat- urated phenol), RF value

Orcinol-trichloroacetic acid spray

(34) Dimethylphenaline spray (35) Orcinol reaction, A 54~1: A670 Enzyme tests

n-Arabitol (+ n-xylulose) dehy- drogenase

Xylitol (- n-xylulose) dehydro- genase

Ribitol (+ n-ribulose) dehydro- genase

L-Ribulokinase L-Xylulokinase

0.02

10

0.66

Gray-brown

Pinka 0.96

0

0

+

+ 0

0.02

10

0.66

Gray-brown

Pinka 0.96

0

0

0

++ 0

a Orange fluorescence.

by guest on August 28, 2017

http://ww

w.jbc.org/

Dow

nloaded from

March 1965 K. Kersters, W. A. Wood, and J. De Ley 969

concentrated by lyophilization. The residual syrup, dissolved in 3 ml of water, contained 54 pmoles of ketopentose (as xylulose) by the cysteine-carbazole test. Data used for the identification of the ketose are summarized in Column 1 of Table III. The ob- served elution from a Dowex l-borate column with 0.01 M borate, and the complete cysteine-carbazole color development in 100 minutes rather than in 10 minutes, are typical of xylulose. The Rp value (0.59) on paper chromatograms run in water-saturated phenol, and the ratio (0.47) of the absorbances at 540 rnp and 670 rnp in the orcinol reaction, are in agreement with the values

FRACTION NUMBER

FIG. 3. Separation on Dowex l-borate of products of n-arabitol oxidation by D-erythro dehydrogenase. A ketopentose mixture (10 pmoles) in 10 ml of 0.005 M borate was absorbed on a Dowex l-borate column (1.4 cm2 X 10 cm) and eluted with potassium borate (- - -, right ordinate). Fractions (5 ml) were collected and assayed for ketopentose.

for authentic xylulose (Table III). The configuration of the isolated xylulose was established as the D isomer by its ability to serve as substrate for a specific n-arabitol (- D-XylUlOSe) dehy- drogenase and by its failure to serve as substrate for specific xylitol (+ n-xylulose) dehydrogenase and for n-xylulokinase.

By similar identification procedures, it was shown that the NADP-linked xylitol dehydrogenase oxidized xylitol t,o n-xylulose and the NAD-specific-n-lyzo dehydrogenase oxidized n-arabitol to n-xylulose (Columns 2 and 3 of Table III).

Preliminary experiments indicated that n-arabitol was oxidized by the NAD-linked n-erythro dehydrogenase to a mixture of xylulose and ribulose. For large scale isolation of the oxidation products, 50 pmoles of n-arabitol, sodium pyruvate, NAD, lactic dehydrogenase, and 10 units of D-erythro dehydrogenase were incubated for 6 hours as described previously; 20 pmoles of cysteine-carbazole-positive material were formed. After depro- teinization and deionization, solid sodium tetraborate was added to 0.005 M concentration, and the solution was applied to a Dowex l-borate column. Analysis of the eluted fractions re- vealed two cysteine-carbazole-reactive peaks (Fig. 3). After removal of the borate as methyl borate from the eluates contain- ing each peak, the residue of each was dissolved in a small amount of water for analysis (Columns 2 and 3 of Table IV). Based upon the cysteine-carbazole reaction rate, elution position in Dowex l-borate chromatography, orcinol reaction, paper chromatog- raphy, and enzyme tests, Peak 1 corresponded to L-xylulose, and Peak 2 to n-ribulose.

Formation of the second ketose by a 3-epimerase in the D-

erythro dehydrogenase fractions was ruled out since incubation of

TABLE IV

Identijkation of pentdoses formed by NAD-linked o-erythro-dehydrogenase and particles

NAD-linked D-erythro-dehydrogenase Particles

1. Test

2

For comparison with authentic ketopentoses, refer to Table III.

n-Arabitol Peak 2

0.02

Gray-brown

Substrate oxidized L-Arabitol Peak 1

Dowex l-borate, elution concentra- 0.01 tion, molarity

Cysteine-carbazole test (time in 100 10 minutes for full color develop- ment)

Paper chromatography (water-sat- 0.58 0.66 urated phenol), RF value

Orcinol-trichloroacetic acid spray Gray (34)

Dimethylphenaline spray (35) Rose Pinka Orcinol reaction, ASH,: A670 -b Enzyme tests

n-Arabitol (+ n-xylulose) dehy- 0 0 drogenase

Ribitol (+ n-ribulose) dehydro- 0 0 genase

n-Ribulokinase 0 + n-Xylulokinase + 0 Xylitol (- n-xylulose) dehydro- + 0

genase

a Orange fluorescence.

4

Xylitol Ribitol o-Arabitol

0.01 0.02 0.01

100 10 100

0.58 0.66 0.59

Gray Gray-brown Gray

Rose Pink5 Rose 0.50 0.94 0.49

+

0

- 0 0

0

+

+ 0 0

+

0

- 0 0

6 7

Ribitol

0.02

10

0.66

Gray-brown

Pinka 0.97

0

0

+ 0 0

6 Dash represents reactions not tested.

by guest on August 28, 2017

http://ww

w.jbc.org/

Dow

nloaded from

970 Polyol Dehydrogenases in G. oxydans Vol. 240, No. 3

n-xylulose or n-ribulose with the werythro deh produce detectable amounts of the epimeric pentitol oxidation by the different dehydrog follows.

ase did not gar. Thus ,roceeds as

meso-Xylitol dehydrogenase (NADP-specific) Xylitol + L-xylulose

D-crythro dehydrogenase (NADspecific) Itibitol 4 o-ribulose Xylitol --f D-xylulose L-Arabitol 4 L-xylulose + I,-ribulose

D-xylo dehydrogenase (NAD-specific) Xylitol 4 D-xylulose

D-Zyxo dehydrogenase (NADP-specific) n-Arabitol + D-xylulose

Ident$cation of Oxidation Products of Hexitols-These oxida- tion products were obtained by a procedure similar to that described for the pentitols. The hexuloses were identified by paper chromatography in water-saturated phenol, and by an enzyme test with crystalline yeast hexokinase which for the ketohexoses is specific for n-fructose. In addition, sorbose, and not the other hexuloses, produces a blue color (maximum, 605 rnp) in the cysteine-sulfuric acid reaction according to Dische and Devi (29).

As shown in Table V, n-glucitol was oxidized by the D-1~x0

dehydrogenase to a hexulose which produced a sharp peak at 605 rnp with the cysteine-sulfuric acid reaction, whereas the oxidation product of n-glucitol by the D-xylo dehydrogenase does not (Fig. 4). This indicates that the product of the former oxidation is n-sorbose and that of the latter oxidation is probably n-fruc- tose. Similarly, n-glucitol was oxidized by the D-erythro dehy- drogenase to a compound which produced a sharp peak at 605 rnl.c in the cysteine-sulfuric acid reaction, indicating that L-

TABLE V

Identijication of hexuloses after oxidation of several hexitols by particulate and soluble enzymes

Dehydrogenase and substrate

NADP-linked D-lyX0 n-Mannitol n-Glucitol

NAD-linked D-erythro L-Glucitol.. L-Mannitol. Allitol

NAD-linked D-xylo

n-Glucitol n-Iditol n-Mannitol

Particles n-Glucitol n -Mannitol . Allitol

None n-Fructose............... L-Sorbose................

--

Paper chroma- tography’

Cysteine- Yeast sulfuric text hexokinase

RF

0.48 0.37

0.38 0.49 0.56

0.49 0.37 0.49

0.37 0.49 0.55

0.49 0.38

A606

-

+

+ - -

-

+ -

+ -

-

-

+

+ -

- - -

+ - +

- + -

+ -

a Green color and green fluorescence with orcinol-trichloro- acetic acid spray (34) and brown color with dimethylphenaline spray (35).

WAVELENGTH ImA)

FIG. 4. Ultraviolet absorption spectra of hexuloses in cysteine-sulfuric acid test (23). For details, see the text.

the

glucitol is not oxidized to n-fructose, but to n-sorbose. From these and similar tests with other hexitols (Table V) it is con- cluded that the hexitols are oxidized by the several NAD- and NADP-linked dehydrogenases as follows.

n-erythro dehydrogenase L-Mannitol L-Glucitol Allitol

D-xylo dehydrogenase n-Glucitol L-Iditol n-Mannitol

~4~x0 dehydrogenase n-Mannitol n-Glucitol

4 L-fructose + n-sorbose --f allulosei

---t n-fructose 4 L-sorbose + n-fructose

+ n-fructose + L-sorbose

Identijication of Oxidation Products of Heptitols-These oxida- tion products were synthesized enzymatically as described for the pentitols. The configuration of the heptuloses could be determined in most cases by paper chromatography in water- saturated phenol (Table VI). On the assumption that only the C-2 or C-6 hydroxyl groups of the heptitols are oxidizable, L-

glycero-n-galacto-heptitol can theoretically be o‘xidized to L-

gulo-heptulose or n-galacto-heptulose. On the chromatograms, both the oxidation product of n-glycero-n-galacto-heptitol by the D-xylo dehydrogenase and authentic n-galacto-heptulose yielded one spot with Rxyiuiose = 0.79 (Table VI), whereas n-g&o- heptulose has an Rxylulose of 0.89. Thus the NAD-linked D-

xylo dehydrogenase oxidized n-glyeero-n-gala&o-heptitol to L-

gal&o-heptulose. From the Rp values for the product of several heptitol oxidations (Table VI) it is concluded that the oxidation by the soluble D-xylo dehydrogenase proceeds as follows.

1 No configuration could be determined because the amount of sample was small.

by guest on August 28, 2017

http://ww

w.jbc.org/

Dow

nloaded from

March 1965 K. Kersters, W. A. Wood, and J. De Ley 971

D-xylo dehydrogenase L-gZycero-D-gaZacto-Heptitol ---t L-galacto-heptulose D-gZycero-D-gZuco-Heptitol + D-aZtro-heptulose L-glycero-r-ido-Heptitol 4 L-gluco-heptulose meso-glycero-ido-Heptitol 4 D-ido-heptulose

The oxidation product of meso-glycero-ido-heptitol was levoro- tatory (a, -0.07” =t 0.01”). Specific rotation could not be determined since the concentration of the heptulose could not be estimated, owing to lack of standards. The reported [c$,” for D-ido-heptulose is -20” (7), indicating that the obtained ido- heptulose has a D configuration.

Particulate Polyol Dehydrogenuse

The ultramicroscopic particles oxidize numerous polyols (Tables I and II). Accumulation of large amounts of oxidation products was facilitated by the ability of the particles to couple oxidation of polyols to oxygen via cytochrome carriers. The resulting ketoses were identified as described for the soluble enzymes.

The heptulose formed after oxidation of meso-glycero-gulo- heptitol by the particulate dehydrogenase had the L configuration ([ali -69” (c, 0.37 in water)); the [LY]$’ of n-gluco-heptulose, the expected product, was reported as -67.8” (4).

meso-glycero-allo-Heptitol was oxidized to a levorotatory heptulose (crD -0.08” f 0.01”). No specific rotation could be determined since allo-heptulose was not available as a standard. The reported [a!]:’ for n-allo-heptulose is -52.1” (8); thus the obtained allo-heptulose has the L configuration.

It can be concluded from Tables IV, V, and VI that pentitols, hexitols, and heptitols are oxidized by the particulate-linked dehydrogenase or dehydrogenases as follows.

D-habit01 + D-xylulose Ribitol + D-ribulose D-Mannitol + D-fructose D-Glucitol + L-sorbose meso-Allitol ---t ?-allulose2 D-glycero-D-glUCO-HeptitOl + L-gulo-heptulose D-glycero-D-gUlUCtO-HeptitOl --f D-galucto-heptulose meso-glycero-gulo-Heptitol + D-gluco-heptulose meso-glycero-allo-Heptitol + D-allo-heptulose D-gZycero-D-munno-Heptitol ---f D-munno-heptulose

+ D-uZtro-heptulose

DISCUSSION

As shown in this communication, chromatography of extracts of G. oxydans (suboxydans), strain SU, on DEAE-cellulose yielded at least six different soluble polyol dehydrogenases, each with distinct substrate and cofactor specificities.3

Soluble Dehydrogenases

NADP-linked xylitol dehydrogenase resembles an NADP- specific xylitol dehydrogenase isolated from guinea pig liver mitochondria (36) in that both oxidize xylitol to n-xylulose. The G. oxydans dehydrogenase does not attack other polyols, but does oxidize D-xylonate to an unidentified product.

NAD-specific o-erythro dehydrogenase does not oxidize any

2 The configuration could not be determined owing to the low specific rotation of allulose ([oL]~ of allulose = -3.3”).

3 In the following discussion, the secondary hydroxyl group which is oxidized will always be considered as C-2.

TABLE VI Chromatographic identijkation oj heptuloses following oxidation OJ

heptitols by particulates and NAD-linked D-xylo dehydrogenuse of G. oxyduns (suboxydans)

The solvent used was water-saturated phenol. The spots were detected by the orcinol-trichloroacetic acid test (34).

Rx~1ulose

Reference heptuloses D-manno-Heptulose. _.... _..__...__.

L-gal&o-Heptulose D-uZtro-Heptulose D-gluco-Heptulose..................... L-gulo-Heptulose

NABlinked D-xyZo-dehydrogenase oxi- dation products

0.64” 0.79 0.74 0.69 0.89

L-gZycero-D-g&-&o-Heptitol. D-gZycero-D-gZuco-Heptitol meso-glycero-ido-Heptitol. L-glycero-r>-ido-Heptitol

Particulate dehydrogenase oxidation products

0.79 0.73 0.84 0.69

D-glycero-D-gZuco-Heptitol . 0.89 D-gZycero-D-manno-Heptitol 0.64= D-gZycero-D-munno-Heptitol . . . . . . 0.74 D-gZycero-D-aZtro-Heptitol.. 0.91 meso-glycero-allo-Heptitol 0.92 meso-glycero-gulo-Heptitol 0.79 D-gZycero-D-gaZacto-Heptitol.. 0.69

a Green-yellow color with the orcinol-trichloroacetic acid spray. The other heptuloses produced a blue-green color.

tetritol or heptitol. Pentitols are only oxidized when the second- ary hydroxyl group on C-2 possesses a D configuration (e.g. D-

arabitol is not oxidized, Table I). n-Arabitol has the required configuration both at C-2 and C-4; thus the dehydrogenase oxidizes n-arabitol to n-xylulose (77%; relative rate, 38.5) and to n-ribulose (23%; relative rate, 11.5). Crystalline n-iditol dehydrogenase from sheep liver also oxidizes n-arabitol to both D-xylulose and n-ribulose (37). However, this L-iditol dehydro- genase differs from D-erythro dehydrogenase since the former oxidizes n-iditol and D-glucitol, whereas the latter does not. The reaction rate is influenced by the steric configuration of the hydroxyl groups at C-3 and C-4; the more hydroxyl groups in the D configuration, the higher the oxidation rate of the C-2 hydroxyl group. Thus ribitol is oxidized faster than L-arabitol is oxidized to n-xylulose, and xylitol is oxidized faster than L-

arabitol is oxidized to n-ribulose (Fig. 5). Increasing chain length decreases the activity (compare ribitol and allitol; L-

arabitol and L-mannitol, Fig. 5). Hexitols are oxidized when the hydroxyl groups at C-2 and C-3 possess a D-erythro configura- tion. A slow oxidation of hexitols such as L-iditol and meso- galactitol, which possess only the C-2 hydroxyl group in the D

configuration, cannot be excluded since very weak activities might not be detected. Thus, the D-erythro dehydrogenase from G. oxydans is similar to the ribitol dehydrogenase of A. uerogenes

(22, 38, 39), which also oxidizes ribitol, xylitol, and n-arabitol to D-ribulose, D-xylulose, and n-xylulose, respectively.4

4 D. Fossitt, R. P. Mortlock, and W. A. Wood, unpublished results.

by guest on August 28, 2017

http://ww

w.jbc.org/

Dow

nloaded from

972 Polyol Dehydrogenases in G. oluydans Vol. 240, Ko. 3

different dehydrogenase. The presence of a primary alcohol group at C-l is necessary, since a carboxyl group or an aldehyde group completely prevents enzyme activity (e.g. o-gluconate and o-glucose; Table I). This NAD-specific n-xylo dehydrogenase from G. oxydans does not have the exactly same substrate specific- ity as the NAD-linked L-iditol dehydrogenase isolated from rat liver (37, 40), or from Azotobacter ugilis (41). Polyols of both the D-ribo and the n-xylo configuration are oxidized by these L-iditol dehydrogenases.

NADP-linked D-1~x0 dehydrogenase oxidizes pentitols and hexitols with the D-Zyxo configuration (Fig. 7). Heptitols are not oxidized. The presence at C-6 of a carboxyl function pre- vents the activity of this dehydrogenase (n-gluconate; Table I). This enzyme is probably the same as that described by Bygrave and Shaw (14).

n-Clucitol is thus oxidized by two different soluble dehydro- genases as already reported (13, 14). D-xylo Dehydrogenase oxidizes o-glucitol to n-fructose, and D-Zyxo dehydrogenase oxidizes it to L-sorbose.

Two different, soluble NADP-linked dehydrogenases oxidize n-gluconate (Figs. 2 and 8). The oxidation product of D-gh-

SUBSTRATE PRODUCT EL.RATE SUBSTRATE PRODUCT -L RATE L

1 e-

100

t t

2

RIBITOL D-RIBULOSE L-GLUCITOL WSORBOSE

-I t

23 t

15

3 XY LITOL D-XYLULOSE -RHAMNITOL

4 t 1

(38.5) (11.5)

k

5

L-ARABITOL .-XYLULOSE ALLITOL L-RIBULOSE

$ t

J II

15 Ch

L-LYXO- L-MANNITOL L-FRUCTOSE AETHYLITOL

FIG. 5. Substrate specificity of soluble NAD-linked n-erythro dehydrogenase. Tests were performed with a 12-fold purified fraction. The relative rates are expressed in comparison to the rate of ribitol as 100.

EL.RAl m

20

9

3

2

-L.RATE L

100

16

3

SUBSTRATE PRODUCT

1 D-MANNITOL

i D-GLUCITOL

1 D-ARABITOL

PRODUCT

i D-ALTRO-

EPTULOSE

c D-IDO-

IEPTULOSE

i L-GLUCO-

IEPTULOSE

i D-FRUCTOSE

SUBSTRATE

% D-GLYCERO- -GLUCC-HEPTlTC(

t ;LYCERO-IDO-

HEPTITOL

f -GLYCERO-L- SO-HEPTITOL

1 D-MANNITOL

PRODUCT

4

o-XYLULOSE

I-

D-FRUCTOSE

t

L-SORBOSE

i

e-GALACTO- IEPTULOSE

SUBSTRATE

+

XY LITOL

i

D-GLUCITOL

i

L-IDITOL

c

L-GLYCERO- I-GALACTO- HEPTITOL

D-FRUCTOSE

$ L-SORBOSE

-e D-XYLULOSE

FIG. 7. Substrate specificity of NADP-linked ~4~x0 dehydro- genase. A 20-fold purified fraction was used. The rates are compared to that of n-mannitol at 100.

i :ONO REDUCT SE PRODUCT EL RATI

COO”

1 D-2-KETO-

;LUCONATE

100

37

45

195

ASE ?.RATE -

100

c ” 5-KETO-GI :ONO REDUI SUBSTRATE PRODUCT

coon

i I-GLUCONATE

COO”

T

coon

f

D-5-KETO- GLUCONATE

-C&AClWATE

2-KETO-GL SUBSTRATE

coon

i D-GLUCONATE

COOH

f ,GALACTONATE

COO”

f D-XYLONATE

COCJH

$

rKETOGLlKCIVATl

.uc

,

FIG. 6. Substrate specificity of NAD-linked D-XYZO dehydro- genase. Tests were made with a 60-fold purified fraction and the rates are compared to n-glucitol as 100.

The substitution of the primary alcohol group in L-mannitol by a methyl group to give L-rhamnitol does not influence enzyme activity (Fig. 5). The presence of a carboxyl group at C-l com- pletely prevents the activity of the enzyme (o-ribonate, D-

xylonate; Table I). NAD-linked o-xylo-dehydrogenase has a much narrower

specificity than werythro dehydrogenase. This enzyme oxidizes pentitols, hexitols, and heptitols with the D-xylo configuration (Fig. 6). The weak oxidation of n-mannitol is an exception. Rechromatography on DEAE-cellulose could not separate the activities for o-glucitol and n-mannitol. Acetobacter aceti (Zique- fuciens) does not possess a soluble NAD-linked D-xylo dehydro- genase but has a soluble NAD-linked n-mannitol dehydrogenase. Tentatively we may conclude that for G. oxyduns, the activity of D-xylo dehydrogenase fractions on n-mannitol is due to a

FIG. 8. Substrate specificities of NADPH-linked 2-ketogluconic reductase and NADPH-linked 5-ketogluconic reductase. Rela- tive rates are compared to gluconate oxidation set at 100.

by guest on August 28, 2017

http://ww

w.jbc.org/

Dow

nloaded from

March 1965 K. Kerstem, W. A. Wood, and J. De Ley 973

SUBSTRATE

L-THREITOL

4

EAYTHRITOL

:c

D-ARABITOL

3 RIBITOL

1

D-MANNITOL

+

D-GLUCITOL

4

ALLITOL

PRODUCT

t

:ERYTHRULOSE

4

.-ERYTHRULOSE

c

D-XYLULOSE

4 L-RIBULOSS

i

D-FRUCTOSE

fl

L-SORBOSE

1

L-ALLULOSE

SUBSTRATE

i D-ALTRITOL

t GLYCERO-GULO

HEPTITOL

rc D-GLYCERO-D-

;ALACTO-HEl=WOI

s

D-GLYCERO-D- HANNO-HEPTITOI

1 GLYCERO-ALL0

HEPTITOL

PRODUCT

t D-TAGATOSE

I- L-GLUCO-

HEPTULOSE

$ L- GALACTO-

HEPTULOSE

$1 MANNOHEPTULOSE

-ALTROHhJLOSE

4

L-ALLO- HEPTULOSE

FIG. 9. Substrate specificity of the particulate polyol dehy- drogenase fraction of G. ozydans.

conate with the 2-ketogluconic reductase is 2-ketogluconate, since 2-ketogluconate (and methyl-2-ketogluconate), but not 5-ketogluconate, is reduced by this enzyme. n-Galactonate, n-xylonate, and 5-ketogluconate are also oxidized by this enzyme (Table II). This would indicate that the hydroxyl groups in the o( and in /? position with regard to the carboxyl function require an L-threo configuration. The oxidation product of D-

gluconate with the 5-ketogluconic reductase is 5-ketogluconate, since 5-ketogluconate and not 2-ketogluconate is reduced in the presence of the enzyme and NADPH. Galactonate is also oxidized, whereas 2-ketogluconate is not (Table II). Neither gluconic dehydrogenase oxidizes n-mannitol, n-glucitol, ribitol, etc. (Table II). Both enzymes have previously been reported in crude extracts of the same strain of G. oxydans (42). A 2- ketogluconic reductase has been detected in Corynebacterium helvolium, Debaryomyces, and Aspergillus (43), and 5-ketogluconic reductase was discovered in Klebsiella and Escherichia (44).

In addition to the demonstrated six different polyol dehydro- genases an NAD-linked secondary alcohol dehydrogenase is present in the fraction obtained between 20 and 50% saturation of ammonium sulfate. This enzyme oxidizes secondary alcohols and glycols such as meso-2,3-butanediol and meso-3,4-hexanediol, but not polyols (45).

Particulate Dehydrogenases

The particles (fragments of the cytoplasmic membrane (16)) oxidize (with the exception of L-threitol) only polyols with the L-erytkro configuration, i.e. polyols with the Bertrand-Hudson configuration (Fig. 9). n-glycero-n-manno-Heptitol (volemitol) possesses the favorable configuration at both ends of the molecule and consequently is oxidized to both n-manno-heptulose and D- altro-heptulose (Fig. 9). It is not yet known whether polyol oxidation by the particles is due to one or to several enzymes.

The oxidation of polyols by the particles reflects oxidations

long observed for growing cultures of G. oxydans (suboxydans). The rule of Bertrand and Hudson was formulated with growing cultures of acetic acid bacteria. Since the particles are fragments probably derived from the cell envelope (16), it is likely that the commonly observed polyol oxidations by growing cells are due to the unique oxidative capacities of the cytoplasmic membrane. Xylitol is not oxidized by growing cultures, whereas the cells contain in the cytoplasm at least three different xylitol dehydro- genases. L-Arabitol, L-mannitol, L-rhamnitol, L-glycero-D- gatacto-heptitol, etc., are not oxidized by growing culture, but are similarly oxidized by soluble enzymes. Probably these soluble enzymes cannot display their activity in growing cells.

L-Fucitol is not oxidized by our strain SU, either by intact resting cells or by any of the enzymes studied. The reported oxidation of some w-deoxysugar alcohols (11) and of L-fucitol to L-fuco-4-ketose (10) is probably due to an enzyme different from the several dehydrogenases described in this paper.

SUMMARY

Gluconobacter oxydans (suboxydans) contains at least six different polyol dehydrogenases, each with unique structural specificity. These have been defined as a nicotinamide adenine dinucleotide phosphate-specific xylitol dehydrogenase, an NAD- linked D-erythro dehydrogenase, an NAD-linked D-xylo dehydro- genase, an NADP-linked D-&X0 dehydrogenase, and two different NADP-linked gluconate dehydrogenases.

One or more L-erythro dehydrogenases (with the Bertrand- Hudson specificity) are attached to the cytoplasmic membrane.

The oxidations by growing acetic acid bacteria are mainly due to polyol dehydrogenases localized on the cytoplasmic membrane. The formulation of the rule of Bertrand and Hudson is the result of the unique oxidative properties of the dehydrogenases att,ached to the cytoplasmic membrane of the acetic acid bacteria.

Acknowledgment-We are grateful to Dr. N. K. Richtmyer for generous gifts of heptitols and heptuloses.

REFERENCES

1. BERTRAND, G., Ann. chim. et phys., 3, 181 (1904). 2. HANN, R. M., TILDEN, E. B., AND HUDSON, C. S., J. Am.

Chem. Sot., 60, 1201 (1938). 3. FULMER, E. I., AND UNDERKOFLER, L. A., Iowa State Coil.

J. Sci., 21, 251 (1947). 4. MACLAY, W. D., HANN, R. M., AND HUDSON, C. S., J. Am.

Chem. Sot., 64, 1606 (1942). 5. STEWART, L. C., RICHTMYER, ;2;. I<., AND HUDSON, C. S.,

J. Am. Chem. Sot., 71, 3532 (1949). 6. STEWART, L. C., RICHTMYER, N. K., AND HUDSON, C. S.,

J. Am. Chem. Sot., 74, 2206 (1952). 7. PRATT, J. W., RICHTMYER,N. Ii., AND HuDsoN,C.S.,J. Am.

Chem. Sot., 74, 2210 (1952). 8. PRATT, J. W., AND RICHTMYER, N. K., J. Am. Chem. Sot., 77,

6326 (1955). 9. CHARLSON, A.J., AND RICHTMYER,N. Ii., J.Am.Chem.Soc.,

82, 3428 (1960). 10. RICHTMYER, N. K., STEWART, L. C., AND HUDSON, C. S.,

J. Am. Chem. Sot., 72, 4934 (1950). 11. BOLLENBACK.G.N.. AND UNDERKOFLER. L.A.. J. Am. Chem.

Sot., 72, 741 (1950,). 12. ARCUS, A. C., AND EDSON, N. L., Biochem. J., 64, 385 (1956). 13. CUMMINS, J. T., thesis, Oregon State College, Corvallis, 1958. 14. BYGRAVE, F. L., AND SHAM*, D. R. D., Proc. Univ. Otago Med.

Sch., 39, 15 (1961). 15. TOUSTER, O., AND SHOW. D. R. D., Physiol. Revs., 42, 181

(1962).

by guest on August 28, 2017

http://ww

w.jbc.org/

Dow

nloaded from

974 Polyol Dehydrogenases in G. oxydans Vol. 240, No. 3

1%

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

28. 29.

DE LEY, J., AND DOCHY, R., Biochim. et Biophys. Acta, 49, 177 (1960).

KLOSTERMAN, H., AND SMITH, F., J. Am. Chem. Sot., 74,5336 (1952).

BERNAERTS. M. J., FURNELLE, J., AND DE LEY, J., Biochim. et Biophyb. Acta; 69, 322 (1963):

CRAMER. F. B.. AND PACSU. E.. J. Am. Chem. Sot., 69. 1469 (1937): ’

I I

WOLFROM, M. L., AND WOOD, H. B., J. Am. Chem. Sot., 73, 2933 (1951).

HANN, R. M., AND HUDSON, C. S., J. Am. Chem. Sot., 61, 336 \1939).

WOOD, W. A., MCDONOUGH, M. J., AND JACOBS, L. B., J. Biol. Chem., 236. 2190 (1961).

WOOD, W. A., AND GILFORD, S. R., Anal. Biochem., 2, 589 (1961).

WARBURG, O., AND CHRISTIAN, W., Biochem. Z., 310, 384 (1941).

HORECKER, B. L., SMYRNIOTIS, P. Z., AND KLENOW, H., J. Biol. Chem., 206, 661 (1953).

DISCHE, Z., AND BORENFREUND, E., J. Biol. Chem., 192, 583 (1951).

ROE, J. H., EPSTEIN, J. H., AND GOLDSTEIN, N. P., J. Biol. Chem., 176,839 (1949).

DISCHE, Z., J. Biol. Chem., 204, 983 (1953). DISCHE, Z., AND DEVI, A., Biochim. et. Biophys. Acta, 39, 140

(1960).

30.

31.

32.

33.

34.

35. 34, 225 ‘(19f;l).

_

KOCH, R. B., GEDDES, W. F., AND SMITH, F., J. Cereal Chem., 28. 424 (1951).

36. HOL~MAN~, S.; AND TOUSTER, O., J. BioZ. Chem., 226, 87 (1957).

37. 38. 39.

40.

SMITH, M. G., Biochem. J., 83,135 (1962). FROMM, H. J., J. BioZ. Chem., 233, 1049 (1958). HULLEY, S. B., JORGENSEN, S. B., AND LIN, E. C. C., Biochim.

et Biophys. Acta, 67, 219 (1963). MC~ORKINDALE, J., AND EDSON, N. L., Biochem. J., 67, 518

(1954). 41. 42.

MARCUS, L., AND MARR, A. G., J. Bacterial., 82, 224 (1961). DE LEY, J., AND STOUTHAMER, A. H., Biochim. et Biophys.

Acta, 34, 171 (1959). 43. DE LEY. J.. AND DEFLOOR, J., Biochim. et Biophys. Acta, 33,

44. 45.

ANDERSON, R. L., AND WOOD, W. A., J. BioZ. Chem., 237, 1029 (1962).

SIMPSON, F. J., WOLIN, M. J., AND WOOD, W. A., J. BioZ. Chem., 230,457 (1958).

HICKMAN, J., AND ASHWELL, G., J. BioZ. Chem., 234, 758 (1959).

SIMPSON, F. J., AND WOOD, W. A., J. BioZ. Chem., 230, 473 (1958).

BEVENUE. A.. AND WILLIAMS. K. T.. Arch. Biochem. Biophys.,

47 (1959) .’ ,

DE LEY, J., Biochim. et Biophys. Acta, 27, 652 (1958). KERSTERS, K., AND DE LEY, J., Biochim. et Biophys. Acta, 71,

311 (1963).

by guest on August 28, 2017

http://ww

w.jbc.org/

Dow

nloaded from

K. Kersters, W. A. Wood and J. De LeyGluconobacter oxydansPolyol Dehydrogenases of

1965, 240:965-974.J. Biol. Chem. 

  http://www.jbc.org/content/240/3/965.citation

Access the most updated version of this article at

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/240/3/965.citation.full.html#ref-list-1

This article cites 0 references, 0 of which can be accessed free at

by guest on August 28, 2017

http://ww

w.jbc.org/

Dow

nloaded from