Vol. Z-17, No.

7, Issue of nyril10,
Printed in U.S.A.

pp. 2222-2227, 1872

D-hcose
I. OXInATION

Metabolism
OF D-JUCOSE

in a Pseudomonad
TO D-FUCOXO-6-LACTONE BY A D-ALDOHEXOST~: DEHYDKOGESASE November 19, 1971)

(Received for publica.tion, A. STEPHEX Fwnz

L)AHIZIS$ ANI) RICHARD

L. ANDERSON Michigan State university,

East Lansing,

the Department

of Biochemistry,

Michigan

48823

SUMMARY A soluble, NAD-dependent dehydrogenase which is specific for D-aldohexoses, including D-fucose, D-glucose, D-galactose, D-mannose, D-altrose, D-allose, Z-deoxy-D-glucose, and 2-deoxy-D-galactose, has been purified 335-fold from a pseudomonad capable of using D-fucose as a sole carbon source. Forty-five other sugars and related compounds tested did not serve as substrates and did not affect the rate of n-glucose oxidation. The pH optimum was 8 to 8.5 in Tris-HCl buffer and 9 to 10 in glycine-NaOH buffer. The enzyme was insensitive to thiols and thiol group reagents and was not activated by divalent metal ions. Representative apparent K, values were 5.8 XnM for D-fucose, 0.86 mM for D-glucose and 0.08 mM for NAD+. The ,3 anomer of D-glucose was preferred over the a anomer. D-Fuconate was isolated as the apparent product of D-fucose oxidation, indicating that the unstable D-fucono-fi-lactone rather than D-fucono-y-lactone was the immediate product. This was confirmed by ,the demonstration that D-glucono-6-lactone, or D-galactono-y-lactone, could but not D-fuCOnO-y-kXtOIU3 serve as a substrate in the reverse reaction. Thus, it is concluded that 8-D-glucopyranose and /3-D-fucopyranose are the actual substrates for the enzyme.

find that Eschericlzia coli metabolizrs D-fucose very slowly (8) or not at all (9). We have isolated a pseudomonad which ca.n utilize n-fucose as a sole source of carbon and energy. This pa.per provides evidence that one of the initial reactions in the metabolism of n-fucose in this bacteriuln is the oxidation of u-fucopyranose to n-fucono-&lactone, which spontaneously hydrolyzes to n-fuconate (Fig. I), and reports some of the propertics of the o-aldohexose dchydrogenase that catalyzes the oxidation. Subsequent papers report the osidat,ion of n-fucofurnnose to n-fuconate yia n-fucollo-y-lactone (lo), the dehydration of o-fuconatc t,o 2-keto3.deoxy-n-fuconate (II), and the cleavage of 2-keto-3-deosy-ufuconate to pyruvate and n-lactaldehyde (12). -1 preliminary presentation of some of these data has appeared (13).
3UTERISLS BND MEl'HODS

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n-Fucosc (6.dcos)--n-galactose) is widely distributed in nature, occurring as a component ol numerous heterosides and of the antibiotic chartreusin (1). Hydrolases apparently specific for the @-n-fucosidic linkage have been found in the tissues of various nmmmals (2-4), in the yiaceral hump of the limpet. (n), and iI1 the digestive juices of snails (6). However, despite the common occurrence of n-fucosyl compounds and of enzymes which catalyze the hydrolysis of P-u-fucosides, the pathway of n-fucose metabolism has not been previously dcscribcd for any organism. Although an early report. (7) indicated that the coli-aerogenes group of bacteria could metabolize n-fucosc, recent investigators * This investigat,ion was supported by Research Grant AI 080GG from the National Institute of Allergy and Infectious 11iscasrs, United St.nt,es Public Ilealth Service: Journal Art,icle 5688 from the ?vlichigen Agricultural I;xperiment Station. $ Predoctoral Fellow of the Xational Institutes of Health; present address, Department of Chemistry, University of California, Los Angeles, California 90024.

Oryalzisnz-The bacteriunk used in this investigation was isolated in our laboratory by incubating nonsterile commercial D-fucosc in a mineral medium. Subsequent standard bacteriological techniques yielded a pure culture of an organism which could grow with n-fucose as the only carbon source. The organism is a small, nonmotile, aerobic, n eraln-negative rod, and thus is considered to be a pseudomonad. I-ntil further tasonomic studies have been carried out, we \vill refer to it by the trivial designation, pseudomonad AISU-1. The pnt,hwap of r,-arabinose metabolism by this organism has been described (14). Culture Conditions-Tile organism xTas grown aerobically in Fernbach flasks containing 1 liter of medium. The flasks were agitated on a rotary sha.ker at 32. The mctliurn consisted of: 1.35% nazHPOa.7HLO; O.lScr/, KH#O*; 0.3% (SH&SO,; 0.02% Y!gS04.7Ha0; 0.0005(x YeSOd; and 3.27; sugar (u-fucosc unless indicated otherwise). The sugnr was autoclaved separately and added aseptically to the mineral Inedium. Stepwise xldition of the sugar was necessary because 1 i;! sugar markedly reduced the growth rate. The first portion of sugar (20 ml of a 2.501~ (w/v) solut.ion) wa.s added immediately aft,cr inoculat.ion. Six additional 20.ml portions of 25 y0 (w/v) sugar were added at B-hour intervals. Thick cell suspensions (Am = 18 to 22) were obtailled; turbidity measuremcnt,s were nlade wit,h a Coleman Junior Spectrophotometer on appropriate dilut,iolls of the cultures in 18.mm diameter test tubes. The cells were harvested 4 hours after final addition of the sugar, which wa:: approximately 40 hours after inoculation. To determine growth rates on various sugars, cultures were grown in B-mm diameter culture tubes containing 7 ml of min-

2222

Issue of April

10, 1(3X

A, X. Ilahms
coo-

and R. L. ilnrlerson
TA~LI:

2223 I clehydrogenase
Total .ctivity Specific activity Ansa: 4260

(SpOntaneoUS) r 2
J

H:OH H&H HO:H HiOH bH, D - FUCONATE

Purification
Fraction

of o-ddohexo.se
VOlUIlX Total protein i %i

1111

,B -D- FUCOPY

RANOSE

D- FUCONO-JLACTONE

FIG. 1. Rcaotion for the oxidation the D-aldohesosc dchydrogenasc~.

of u-fucose to D-fuconate by

era1 medium supplemented with 0.5$; of the test sugar. The tubes were agitated at an angle on a reciprocal shaker at 32. Turbidity measur(mcnts were made at suitable time intervals on appropriate dilutions. Prepnrafion 01 Cell Rx&acts--Cells \vcrc harvested by centrifugation, washed once bv resuspension in distilled water, and were finally r~uspcndcd inO.10 M sodium phosphate buffer (pH 7.0) The cells were broken by treating unless othertvise illdi(*at.ed. the suspension lot. 13 min in a Rsytheoil IO-KHz sonic oscillator cooled with circulating ice water. The superllatan t fluid rcsulting from lo-mill ccntrifugation of the broken ccl1 suspension at 40,000 x g was used as the cell extract. n-dldohezose IMydrogenase Standard Assay-The reaction mixture (0.15 ml) consisted of 2.5 Fmoles of u-glucose, 0.3 pmole of X*441)+, 15 pmoles of Tris-HCl buffer (pH 8.1), and D-aldo,I control was used in the early steps herosr dchydrogcnase. of purification to c>orrect for S1DH osidase; this control was minus n-glucose and cont,ainrd 0.05 pmole of NADH in place of XX)+. (Later in the investigation, WC discovered that NA&DH osidsse acti\-ity could bc eliminated by preparing the crude extracts in a solution conta,ining 0.15 rntil Z-t.hiocthanoI alld 0.10 M Bicine buffer, pH 7.4.) The reaction was monitored at 340 nm with a Gilford model 2400 absorbance-recording spectrophot~ometer thrrmostated at 25. The rate was constant with time and proportional to the nlzyme concentration in the ranges used. A mlit of n-aldohesose dehydroacnasc was defined as the amount of enzyme that catalyzed the reduction of 1 kmole of ;\J=lD+ per min in the standard assay. 11nalyticnl ~I,lethods~~educ~ing sugar was dctcrmined by the method of Sumner and Ho\vell (15). Mdonic acids mere detcrminrd after conversion to the corresponding la&ones by boiling in 1 N HCl for 5 min. La&ncs were determined as the hydrosamic acids by the method of He&n (16). Pyruvate was determilled with lactic acid dehydrogcnasc and NADH, alld by the calorimetric method of Friedeman a11d Haugen (17), as modified by &I;\-re and Greenberg (18). Protein was usually determined by the method of Varburg and Christian (19) ; in crude rxbracts and other preparations high in nucleic acid content, the biurct method (20) was used, with bovine serum albumin as the stalldard. Descending paper c~hl,orllato~r:Lphy was performed on Whatman No. 1 f&r paper, using the followjug solvents: Solvent 1, water-saturated 2.butanone; Solvent, 2, l~butanol-~~~vater-nj 5; ethanol (5:4: 1). Lactoncs were visualized by their formation of hydrosamic acids (21) ; aldonic acids lvere detcctcd by t,he same method after itl sifu lactonization by spraying with 0.2 nHCI and heating in an oven for 15 min at 100. Reagenls--u-Fucosc (22, 23), u-fucollate (24), L-mannosc (25), L-glucose (26, 27), L-galactose (28), L-fructose (29), and (i-iodo6-deorv-u-galact,oxc (30) were prcparrd as described in the in-

Ccl1 extract. Prot.arnine sulfate sripernat)ant (NHJZSOI precipitate;. Sephadex G-200 DEAE-cellulosea Calcium phosphate gel.

6c50 780 53 133 330 66

I 4220 4060 1010 i 109 ~ 14.1 1.7 755 459 243 0.186 i 0.434 I 2.24

0.63 0.86 1.17 1.25 1.48 1.58

a The values given for these fractions have been corrected for the proportions of the previous steps not subjected to further purification dicated references. 6.Dcoxy-D-allosc and 6-deoxy-D-glucose (D-quinivose) were the gifts of Dr. T. Reichstein, TJl1iversit.y of Basel, Base& Switzerla.nd. 3,6-T>ideosy~u~galactose (abequose) \\*as the gift of Dr. Otto Luderitz, Max Plan& Institute, Freiburg, Germany. 2.hcetamido-2.deosy-D-allose and 2-acetamido-2-dcoxy-u-altrose were the gift,s of Dr. Xl. n. Perry, National Research Council of Canada, Ott,awa. D-Allosc, D-altrose, D-sylulose, and lo-glucose-free 2-deoxy-n-glucose were the gifts of Dr. W. A. Wood, Michigan Ht,:lt,e I-niversity. Other chemicals were from commercial sources. D-&Iannose (31) and Dgalactose (32) were twice recrystallized before use as substrates.
RESULTS

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General Observations
Growth. Rates-The organism grelv equally well on u~fucoar, n-glucose, L-arabinose, and u-galactosc, with a generation time of about 3 hours at 32. Initial Investigations with Crude Cell fixtracts-In prrliminary experiments with crude extracts, nc were unable to detect any modification of -D-fucose by means of isomerization or cpimerization reactions wheu assayed by various chemical and chromatographic procedures. Furthermore, w could not detect, the phosphorylation of n-fucose or u-fuconate with hTP, nor the reduction of D-fucose with N,%DH or N;\DPH. However, extracts readily catalyzed a n-fucose-tlependeiit reduction of NhD+ and NlYDP+. Subsequent, invest.igations revealed the presence of two enzymes which catalyzed pyridine nucleotidc-depcndellt oxidations of D-fUc0z-X. The purification and characterization of one of these dchydrogcnnses is described below.

Purification

of D-dldohexose Dehydrogenase

Extracts were prepared from r)-glucose-grown cells. The following operations were performed at O-4. X summary of the purification procedure is given in Table I. Protarnine XulJate Treatment-To a cell extract containing 0.2 M ammonium sulfate was added an amount of 2?> (w/v) protamine sulfate solution (pH 7.0) t,o give a final concentration of 0.33%. L&fter 30 min, the prccipitatc was removed by ccntrifugation and discarded. Ammonium Sulfate Prcactior~ation~The protein in the supernatant, from the protamine sulfate step was fractionated by the addition of crystalline ammonium sulfate. The protein prccip,itat,irrg between 30 and 40y0 sat,uration was collect,ed by ccn-

2224

D-Fucose

Metabolism.

I. o-Aklohexose

Dehydrogenase

Vol. 247, No. 7

-FUCOSE

IMOLARITY]-' of D-fucose assay was concentra-

PH FIG. 2. Effect of pH and buffer composition on D-aldohexose dehydrogenase. The standard assay was used except that the pH and buffer (0.10 hf) were varied with the dehydrogenase concentration constant. pH measurements were made on duplicate reaction mixtures.
trifugat.ion and was dissolved in 0.01 M sodium phosphate, pH

FIG. 3. Lineweaver-Burk plot showing the effect concentration on the reaction velocity. The standard used except that the substrate was D-fucose at varying tions. TABLE II

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Substrate

specifkity

sf D-aldohexose

clehyclrogenase

7.0.
Gel Filtration-The above fraction was chromatographed on a column (6 X 60 cm) of Sephadex G-ZOO equilibrated with 0.01 M sodium phosphate buffer (pH 7.0). Fractious (15 ml) were collected during elution with the same buffer, and those with the highest specific activity were combined. Prior to the next purification step, the combined fractions were reduced in volume from 135 ml t,o 15 ml with a, Diaflo ultrafiltration cell (Amicon Corp., Cambridge, Massachusetts) containing a type UM-10

The standard assay was used except that the substrate was varied as indicated. K, and V,,, values were determined from Lineweaver-Burk plots, with at, least five concentrations of each substrate; deviation of the points from a straight line was essentially nil. <elative V,,, %
D-Glucose D-Galactose 188 142 124

Kll% ?nM
0.86 1.6 4.5 1.6 5.8 6.3 2.4 NDC 13.0 NDc

membrane. DRAE-celluEose Chronzatography-DEAE-cellulose (Sigma, exchange capacity = 0.9 meq per g) was pretreated as recommended by Sober et a.1. (33), and was equilibrated with 0.02 M sodium phosphate buffer, pH 7.0. A portion (2 ml) of the Sephades G-200 concentrate fraction was applied to a column which was then washed with (3 x 5 cm) of the DEAE-cellulose, 60 ml of the same buffer. The protein was cluted with a stepwise gradient of 60 ml each of 0.10, 0.20, 0.30, 0.40, and 0.80 M NaCl in the same buffer. D-Aldohexose dehydrogenase eluted at the
0.20 to 0.30 M NaCl range. Fractions containing most of the ac-

D-Mannose 2-Deoxy-D-glucose . D-Fucose __.._.. X-Deoxy-D-galactose D-Altrose 6-Deoxy-n-glucose D-Allose................ 3,6-Dideoxy-n-galactose

_.....

107 100 94 79 61 47 23b

(D-quinivose) (abequose) not serve

a Forty-five compounds t,hat did listed in the text. b Assayed only at a concentration c Not determined.

as substrates 23 mM.

are

of about

tivity were combined. Cal&m Phosphate Gel Step-The pooled DEAE-cellulose fractions were adjusted to pH 6.5 with 0.05 N HCl and dialyzed for 24 hours against 0.01 M sodium cacodylate buffer (pH 6.5). To a portion (5.0 ml) of the dialyzed protein was added 1.0 ml of calcium phosphate gel (containing 6% solids) prepared as deThe gel suspension was centrifuged for scribed by Wood (34). 1 min in a clinical centrifuge and the centrifugate was successively eluted with 1.0 ml each of 0.01,0.02,0.03,0.04, and 0.05 M sodium phosphate buffer (pH 7.0). About half of the activity was reThis fraction was 335-fold covered in the 0.01 to 0.02 M range. purified with a 137 over-all recovery of activity (Table I). It was free from detectable ( <0.3 %) L-arabino-aldose dehydrogenase (lo), n-fucono-y-lactonase (lo), and n-fuconate dehydratase (11). Properties of D-Aldohexose Dehydrogenase

Except where noted otherwise, the most highly purified fraction was used in the following experiments.

Effect of pH and Bu$er Composition-D-Aldohexose dehydrogenase activity as a function of pH was maximal at pH 8.0 to 8.5 in Tris-HCI buffer and at pH 9 to 10 in glycine-XaOH buffer (Fig. 2). a Linewcaver-Burk E$ect oj D-FZLcose Concentrution-From plot, the apparent K, for D-fucose was determined to be 5.8 JIIM (Fig. 3). Substrate Specificity and Kinetic Co?Lstants-In addition to Dfucosc, several other D-aldohexoses were oxidized by this enzyme. Table II lists the apparent I<, and relative V,,, values for 10 Rates were not additive when sugars that served as substrates. four of the more active sub&&es were mixed in various combinations (Table III); t.his is consistent wit.h one enzyme acting on all four substrates. Forty-five compounds whirh did not serve as substrates ( < 1 y. of the rate with D-fucose) at 33.3 mM concentrations were: Laldohexoses, L-fucose, L-rhamnose, L-glucose, L-galactose, ancl L-mannose; ketohexoses, D-fructose, L-fructose, L-sorbose, and

Issue of April

10, 1972
TABLE

A. X. Dahms

uncl

R. L. Anderson

222s

Egect

III o-aldohexose dehydrogenuse activity The standard assay was used except that the substrate was varied. Each sugar was at a concentration of 33.3 mM.
of mixing substrates on
Substrate Rate of NAD reduction protein

-i i
7 4 I

pmoles/min/mg

D-Glucose.. D-Galactose
o-Mamma..

6.8
5.1

D-Fucose.. D-Mannose
o-Mannosc

+ D-fucose
+

4.5 3.6 4.0

5.7

D-glucose.

D-Mannose + D-galactose D-Glucose + D-galactose D-Glucose + D-fucose. D-Fucose + D-galactose.

4.8 5.8 5.0 4.3

MINUTES

MINUTES

AT

5!?

n-ribose, D-fructose-6-l; pentoses, D-xylose, L-xylose, D-l~xosc, 2.deoxy-D-ribose, o-arabinose, L-arabinose, and D-xylulose; trios&s, DL-glyceraldehydc; polyols, D-mannitol, D-glucitol, nayoinositol, L-arabitol, n-arabitol, xylitol, and ribitol; disaccharides, maltose, cellobiose, sucrose, lactose, melezitose, turanose, meliraffinose; and derivafives of biose, and t,rehalose; trisaccharide, D-a.ldohexoses, D-glucuronic acid, D-galacturonic acid, D-galactoseB-P, n-glucose-6-P, u-glucosamine, Ai-acetyl-D-glucosamine, (Ymethyl-glucoside, 6-deoxy-D-allose, 6-iodo&deoxy-D-galactose, and 2-acetamido-6-deoxy-r-al2.acetamido-6-deoxy-D-allose, trose. Also, none of these compounds reduced the rate of oxidation of 33.3 rnM D-glucose when added at equimolar concentrat,iolls, further substantiating that they have little or no affinity for the enzyme. Anomer Preference-The rate at which the enzyme catalyzed the oxidation of an equilibrium solution of cr,/?-D-glucose was compared to the rate at which it catalyzed the oxidation of a freshly prepared solution of a-D-glucose at 15.6 and pH 7.5. Under these conditions, in which the mutarotation of D-glUCOSe would be the rate-limiting step (35, 36), ar,P-D-glucose was oxidized at a rate 5 times that of a-~-glucose (Fig. 4), indicating that the @ anomer is preferred over the cy anomer. Since the products of D-glucose and D-fucose oxidation by this enzyme are the b-lactones (see below), the actual substrates have to be P-Dglucopyranose and P-D-fucopyranose. Nucleotide SpeciJicity-From a Lineweaver-Burk plot, the apNADP+ parent K, for NAD+ was determined to be 0.08 DIM. was completely ineffective as a cofactor for the D-aldohexose dehydrogenase at concentrations up to 20 mM. Also, 4 mM NADP+ did not inhibit the reaction when added to the standard assay. Activators rind Inhibitors-The effects of various thiols, thiol group reagents, and salts are shown in Table IV. The dehydrogenase activity was not affected by thiols or thiol group reagents. Also, there was no inhibition by 6.7 mM EDTA or activation by divalent metal ions at 6.7 mu; on the contrary, NH4+ and several divalent metal ions caused inhibition. St&My-Sephadex G-200 fractions (in 0.01 M sodium phosphate buffer, pH 7.0) were stable to freezing at -20 for at least 6 months. The half-life of the enzyme at 55 was about 40 set (Fig. 5). The heat inactivation profiles of the enzyme assayed with either n-fucose, D-galactose, D-glucose, or n-mannose as the substrate were superimposable, which is consistent with all four activities being the result of a single enzyme.

FIG. 4 (Iejt). Anomer specificity of D-aldohexose dehydrogenase. The standard assay (0.15 ml) was used except that the substrate was 0.026 Mumole of D-glucose, the Tris-HCl buffer was pH 7.5, and the temperature was 15.6. The cuvettcs containing the reaction mixture minus D-glucose were equilibrated to 15.6, and then a freshly prepared solution of or-o-glucose or an equilibra.ted solution of a&D-glucose was added. FIG. 5 (right). Thermal inactivation of D-aldohexose dehydrogenase. A DEAE-cellulose fraction (specific activity, 13.8) was used. The enzyme (1.8 mg of protein per ml) was heated in 0.01 M sodium phosphate buffer (pH 7.0) at 55. Samples mere withdrawn at time intervals and assayed for D-aldohexose dehydrogenase activity witho-glucose, D-fucose, D-ma.nnose, and D-galaCtose as substrates. The rate of inactivation was identical with all four substrates.
T.&ISLE Effect oj

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IV
dehyokogenase
Relative

various reagents
Reagent

on o-aldohexose
Concentrstion

activity
activity

None................... p-Chloromercuribenzoate Sodium iodoacetate. 2-Thioethanol . GSH EDTA. MgClz : MnC12. NH&l.. ..: : . (NHd)zSOd CoClz. NiC12 cuso4. :. .I II. FeSO* CaC12 ZnClz IclentiJication

0.5 0.5 1.0

1.0

6.7 6.7 6.7 6.7 6.7 6.7 6.7 6.7 6.7 6.7 6.7

100 100 100 100 100 100 100

97 86 86 51 35 16 16 12

10

of the Reaction Product

oxidation Attempts to isolate a lactone resulting from D-fUCOSe by this D-aldohexose dehydrogenase, using the same techniques that were employed for the isolat,ion of D-fucono-y-lactone resulting from D-fucose oxidation by the L-arabino-aldose dehydrogenThis suggested that t,he ase (10) were consistently unsuccessful. D-fucose oxidation product of the D-aldohexose dehydrogenasecatalyzed reaction was the &lactone, which is known to be unstable (37-39). Thus, the apparent product of D-fucose oxidation by this enzyme would be expect,ed to be D-fUCOUate.

o-Fucose TABLE Reversibility of the u-aldohexose V

Metabolism.

I. o-Aldohexose

Dehydmgenase

Vol.

247, so.

dehydrogenase-catalyzed

reaction

The reaction mixtures (0.15 ml) contained 30 rmoles of sodium phosphate buffer (pH 6.5), 0.26 rmole of NADH, 10 pmoles of the indicated ketone, and 0.37 unit of D-aldohexose dehydrogenase (specific activit.y, 24.5). The lactone solutions were prepared in 0.10 M sodium phosphate buffer (pH 6.5) just prior to each assay.
Substrate Rate of NADH nmoles/min oxidation

V). Only the &lact,one served as the substrate for the reverse reaction, confirming that the enzyme must be operative on the D-Fuconoand n-gnlactono-ypyranose form of aldohexoses. lactones caused no inhibition of the rate of reduction of D-gluconod-lactonc by the enzyme, further substantiating that the enzyme has little or no affinity for the y-la&one or, presumably, for sugars containing the furanose ring.
Induction 0) ~-~4kiohexose Dehydrogenase

~-Gl~~cono-6-lacto~~e. D-Galnctouo-y-lactone.. . D-Fueono-y-lactonc . . n-Glucono-&lactone + D-galactono-y-lactone........ . .._._...._...._..._.,.... D-Glucono-G-la&tone + n-fucono-y-lactone

4.65 10.005 <0.005 4.61 4.61

The inducibility of D-aldohexose dchydrogenase Q-as test,ed by comparing specific activities in extracts of cells grown on various suga.rs and on nutrient broth (Table VI). D-Fucose and D-glucose were effective inducers, whereas D-galactose induced only slightly and L-arabinose not at all.
DISCUSSIOK

TABLE Induction

VI dehydrogenase

of o-aldohexose

Cells were grown in nutrient broth (Difco) or in mineral mesugar. Cell exdium supplemented with 0.5% of the indicated of cells suspended in tracts were prepared by sonic oscillation 0.01 M L\~,L~-bis(2-hgdroxgethyl)glycine buffer (pH 7.4) plus 0.14

rnM 2-thioethanol.
Growth

Protein
substrate

was estimated

by the biuret
Rate of NAD reduction

assay.

pmoles/min/mg

grotein

D-Glucose............ D-Fucose D-Galactose...................... L-Arabinose

.._......

0.27 0.092 0.015 0.002

Nutrient

broth..

0.003

To prepare the product for identification, the following reaction mixture (2.5 ml) was prepared: 400 pmoles of Tris-maleate buffer 125 pmoles of pyruvic acid (pH 6.5); 100 pmolcs of D-fUCOSe; (pH 6.5) ; 50 pg of lactic acid dehydrogenase; 1.0 pmole of IGAD+; and 25 units of n-aldohexose dehydrogenase. The low pH was chosen to favor stability of the originally anticipat,ed lactone product,. After 1 hour at 25, t,he pyruvate level was constant at. 27 ~molcs and 110 u-fucose or lactone could be detected. The cations in the reaction misture were exchanged for H+ by passing 50WH+ and t,he eluate the sol&on through a column of Donex wa.s chromatographed in Solvents 1 and 2. Color devclopmcnt revealed only one spot, which co-chromatographed with authentic D-fuconate in both solvent systems. A portion of the Dowex 5OW cluate ~-as chromat.ographed after lactouization. The lactonization product co-chromatographed with authentic D-fuconoy-lactonc in both solvent systems. Thus, D-fuconate, and not as the apparent reaction D-fu?Ol~o-y~lactol~e, was identified product, of this n~xldohexose dehydrogenase. Since the purified enzyme used in t,his experiment was free from D-fucono-y-lact.onase activity, it may be concluded that, u-fucono-Mactone was the immediate D-fucose dehgdrogenation product, and that this unstable lnctone spontaneously hydrolyzed to n-fuconate. Reversal oj the o-Aldohexose Dehydrogenase-catalyzed Reaction

The validity of the conclusion that the product of the dehydrogenation reaction is the b-lactone was tested by an investigation of the reverse reaction, using n-fucono-y-lact,one, n-galacas the substrate (Table tono-y-lnctone, or D-~~UCOllO-6-~~C~Oll~

Pseudomonad MSU-1 can grolv on n-fucose as a sole carbon source, a compound for which the pathway of degradation has not been previously described in any organism. This paper describes one of two dehydrogenases active in the oxidat,ion of n-fucose in this organism. The other enzyme is an L-arabinoaldose dehydrogenase described in an accompanying paper (10). These two dehydrogenases differ in their sugar specificity, nucleotide specificity, and the nature of the product, formed from to n-fucorrate via D-fucosc oxidation. The oxidation of D-fucosc y- or &lactone or both represents the first step in the degradation of n-fucose in this organism. Of 55 carbohydrates and related compounds tested, only ualdohexoscs were oxidized by the dchydrogenase reported here. All five of the nonderivatized o-aldohexoses tested served as substrates. Slight modification of the sugars, as by deoxygcnation at C-2, C-6 (except n-allose), or C-3 and C-6, is tolerated by the Modificaenzyme, and thus n-fucose can serve as a substrate. tion of t,he n-aldohexoses by the addition of bulkier groups at C-6, however, as by oxidation to an acid, phosphorylatioll, or iodinntion, completely abolished detectable activity. Other classes of compounds that did not serve as substrates were L-aldohexoses, ketohexoses, pentoses, trioses, polyols, and di- and trisaccharides. Hence, this enzyme is apynqriately called a D-aklohexose dehydrogenase. Of the several soluble pyridinc-nucleotide-dependent aldose dehydrogenases of bacterial origin previously described (40-49, two have been reported t,o be active on n-fucose. These are an aldose dehydrogcnase and a D-galactose dehydrogenase, both of which were isolated from a single organism, a pseudomonad, by Cline and Hu (42). Both of these enzymes differed in substrate specificity from the n-aldohexose dehydrogenase report,ed here, the aldose dehydrogenase failed to oxidize D-mannose and oxidized the pentoses, L-arabinose and D-xylosc, and the D-galactose dehydrogenase used NhDP instead of KAD and oxidized only L-arabinose and 2-deoxy-D-galactose in addit.ion to n-galactose and n-fucose. Thus, the dehydrogenase described here is unique in that it is specific for u-aldohesoses. The identity of the product of D-fucosc oxidation by this enzyme as n-fuconate rather than n-fucono-y-lactone indicates that the immediate product must have been the unstable D-fueono-6lactone. This was supported by the demonstration that Dglucono-&lactone, but not n-fucono-y-lactone or n-galactono-ylactone, could serve as the substrate in the reverse react.ion. Thus, the preferred ring size of the n-aldohexose subst,rate must be the pyranose. This is consist,ent with other soluble, pyridine-

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Issue of April

10, 1972

A. X. Dahms and R. L. Anderson

2227

nucleotidc-linked dehgdrogenases that oxidize n-glucose (36, 41, 43) and L-fucoee (39). It seems likely that t.his n-ddohcxose dehydrogenase functions in the metabolism of bot,h D-fUCOse arid ~-glucose, since it is induced to a high level by both of these sugars and has a reasonably high affinity for both of them. However, it presumably does not function in the metabolism of u-galactose or n-mannose, even though it can catalyze their oxidation and has a fairly high affinity for them; n-galactose induces the enzyme only slightly, and t,he organism does not grow on n-mannose. REFERENCES 1. STAN&K, J., CERNP, M., KOCOUREK, J., END P;\c~~K, J. (1963) pp. 403-404, Academic Press, New York ?he monosaccharides 2. LEVVY, G. A. (1960) l\aLure 187, 1027 A. (1963) Biochem. J. 87,361-367 3. LEVVY, G. A., AXD McAI,L~N, 4. ESTERLY, J. R., STANDEN, A. C., AND PEARSON, B. (1967) J. Histochcm. Cytochem. 16, 470
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