Crystalline D-Mannitol:NAD Oxidoreductase from Leuconostoc Mesenteroides

[Agr. Biol. Chem., Vol. 32, No. 7, p. 894•`899, 19681

Crystalline D-Mannitol:NAD Oxidoreductase from Leuconostoc mesenteroides

Part II. Substrate and Coenzyme Specificity

By Shuzo SAKAI* and Kei YAMANAKA

Department of Agricultural Chemistry, Faculty of Agriculture, Kagawa University, Miki-tyo, Kagawa-ken Received January 16, 1968

The crystalline D-mannitol dehyrogenase (D-mannitol:NAD oxidoreductase, EC 1.1.1.67) catalyzed the reversible reduction of D-fructose to D-mannitol. D-Sorbitol was oxidized only at the rate of 40 of the activity for D-mannitol. The enzyme was inactive for all of four pentitols and their corresponding 2-ketopentoses. The apparent optimal pH for the reduction of D-fructose or the oxidation of D-mannitol was 5.35 or 8.6, respectively. The Michaelis constants were 0.035M for D-fructose and 0.020M for D-mannitol. The enzyme was also found to be specific for NAD. The Michaelis constans were 1 x 10-5M for NADH2 and 2.7 x 10-4M for NAD.

The n-mannitol dehydrogenase was first enzyme. The sufficient dilution of enzyme was made crystallized from the n-fructose-grown cells of in acetate buffer (pH 5.35) to give an absorbance Lactobacillus brevis, ATCC 367 by Martinez, change of about 0.10 in a minute. For oxidation of Barker and Horecker.1) The authors also D-mannitol, activity was also determined in the reported the crystallization of similar enzyme reaction mixture (0.3ml) which contained 6 moles of Tris-HCI buffer (pH 8.60), 0.2ƒÊmole of NAD, 30ƒÊ from the glucose-grown cells of Leuconostoc moles of D-mannitol and enzyme. The formation or mesenteroides, ATCC 9135 (IFO* 3426).2) disappearance of NADH2 was followed up by measur The present paper reports the substrate ing absorption at 340mƒÊ with a model 139 Hitachi specificity and coenzyme specificity of this Perkin-Elmer spectrophotometer equipped with micro

crystalline D-mannitol dehydrogenase. cuvette chamber and with thermospacer in the cell compartment. The temperature was maintained 30•Ž MATERIALS AND METHODS through a circulating water bath of constant tem Enzyme assay. The activity of D-mannitol de perature. A microcuvette has 1.0cm-light path and hydrogenase was assayed by measuring the rate of 0.45ml capacity. They were purchased from Pyrocell NADH2 disappearance with fructose in the reaction Manufacturing Co., Westwood, New Jersey, U. S. A.

system described below. The reaction mixture (0.3 Protein was determined from absorbances at 280mƒÊ ml) contained 6ƒÊoles of acetate buffer (pH 5.35), and 260mƒÊ3) A unit of enzyme is defined as the 0.03ƒÊmole of NADH2, 30ƒÊmoles of D-fructose and amount of enzyme required to produce a change in absorbance at 340mƒÊ of 1.0 per minute at 30•Ž

* Present address: Research Laboratory in the reaction mixture of the standard assay with , Hayashib ara Co., Okayama, Japan. D-fructose. The specific activity is defined as units * Abbreviations: IFO , Institute for Fermentation, O of enzyme per milligram of protein. saka, Japan; EDTA, ethylenediamine tetraacetate; pCMB, p-chloromercuribenzoate. Enzyme preparation. D-Mannitol dehydrogenase 1) G. Martinez, A. Barker and B. L. Horecker, J. Biol. Chem., 238, 1598 (1963). 2) S. Sakai and K. Yamanaka, Biochim. Biophys. 3) O. Warburg and W. Christian , Biochem. Z., 310 Acta, 151, 684 (1968). , 384 (1941). Crystalline D-Mannitol:NAD Oxidoreductase from Leuconostoc mesenteroides 895

FIG. 1. Sedimentation Patterns of the D-Mannitol Dehydrogenase. The recrystallized enzyme was dissolved in 0.06M phosphate buffer (pH 7.0). The pho tographs from left of right were taken at 0, 8, 16, 24, and 32min after reaching 59,780rpm.

was isolated from the glucose-grown cells of Leuconostoc RESULTS mesenteroides(IFO 3426) and recrystallized three times Ultracentrifugal analysis as described in the previous paper.2) The crystalline The ultracentrifuge pattern obtained with enzyme with specific activity of 8140 was used throu the recrystallized enzyme showed a single ghout this investigation. and symmetric peak (Fig. 1). The so,, value Ultracentrifuge run. Ultracentrifugation was was calculated as 7.37s. carried out by a Spinco model E analytical ultracentrifuge at the Institute for Virus Research, Kyoto Effect of pH on dehydrogenase activity University. The temperature was maintained at The oxidation of NADH2 with D-fructose 21.0•Ž. by the enzyme was observed within the pH Paperchromatography. Paperchromatography of range of 5.0 to 6.5 and was maximal at about sugars was performed by the ascending technique 5.35. The enzymatic reduction of NAD with on No. 50 filter paper (Tdyo) with the use of n- D-mannitol was maximal at pH 8.6 (Fig. 2). butanol-acetic acid-water (4:1:2) as the developing solvent. The silber nitrate spray was used for detection.4)

Determination of ketose. Ketose was assayed by the cysteine-carbazole methods) and expressed as fructose. Color was developed for 30 min at 50•Ž. 6)

Chemicals. D-Mannitol was obtained from E. Merck, Darmstadt, Germany. D-Sorbitol was the product of Mann Research Laboratories, Inc., New York, U. S. A. Ribitol, D- and L-arabitols, xylitol, and i-erythritol were obtained from General Bio- chemicals Inc., Chagrin Falls, Ohio, U. S. A. These hexitols and pentitols were used without further crystallization. D-Tagatose, D-xylulose, D- and L- ribuloses were prepared by epimerization of cor responding aldoses in dry pyridine7) and purified as in the previous paper.$) NAD, NADH2, NADP, FIG. 2. Effect of pH on Enzyme Activity.

NADPH2 and the crystalline lactic dehydrogenase The enzyme activity is expressed as a change of type 1 (from rabbit muscle) were purchased from absorbance at 340mƒÊ per minute per 0.25 unit Sigma Chemical Co., St. Louis, Missouri, U. S. A. of the enzyme in 0.05M acetate buffer ((D) and 0.05M Tris-maleate buffer (9).

4) S. M. Partridge, Biochem. J., 42, 238 (1948). 5) Z. Dische and E. Borenfreund, J. Biol. Chem., Effect of pH on enzyme stability 192, 583 (1951). 6) K. Yamanaka, Agr. Biol. Chem., 27, 265 (1963). The enzyme was stable within the pH range 7) G. Glatthaar and T. Reichstein, Helv. Chim. of 5.0 to 9.0 at 35•Ž for one hour or at pH Acta, 18, 80 (1955). 6.0 to 8.0 at 35•Ž for five hours without any 8) K. Yamanaka and S. Sakai, Can. J. Microbiol., 14, 391 (1968). loss of activity (Fig. 3). 896 Shuzo SAKAI and Kei YAMANAKA

TABLE I. SUBSTRATE SPECIFICITY

The reaction mixture contained 30ƒÊmoles substrate . FIG. 3. Effect of pH on Stability of Enzyme. Enzyme protein, 0.06ƒÊg was used for D-mannitol or D-fructose; 3.0ƒÊg for other substrates. For other The crystalline enzyme (protein 5.93ƒÊg) was conditions see the text. dissolved with 20ƒÊmoles of buffer (acetate for pH 4•`5; Tris-maleate for pH 6•`8; glycine for pH Table I, of the sugar alcohols tested, the 9•`10) and 0.5ƒÊmole mercaptoethanol in total enzyme was most active on D-mannitol, but volume of 0.5ml. Incubated at 35•Ž. At inter vals, 4ƒÊl of aliquots were taken for the enzyme D-sorbitol was also oxidized at less than 4% of assay. The remaining activity was determined the rate for D-mannitol. Dulcitol, all of four by the standard method and expressed as the pentitols and i-erythritol were scarecely percentage of the original activity. The activity oxidized. D-Fructose was reduced by NADH2. after 1hr's incubation (•›) and 5hr's incubation

(•œ). L-Sorbose was also reduced by NADH2 with the excess amount of the enzyme. D-Tagatose, Substrate specificity D-ribulose, L-ribulose, and n-xylulose were Since many hexitol dehydrogenases are hardly reduced. The Michaelis constants from regarded as the enzymes which act on several the Lineweaver-Burk's plots were 0.035M and hexitols or pentitols, a variety of sugar alcohols, 0.02M for n-fructose and D-mannitol, respec

FIG. 4. Effect of Substrate Concentration on Enzyme Activity.

A: The reaction mixture contained 6ƒÊmoles acetate buffer (pH 5.35), 0.03ƒÊmole NADH2 0 , .06ƒÊg crystalline enzyme and D-fructose. B: The reaction mixture contained 6ƒÊmoles Tris-HC1 buffer (pH 8.6), 0.2ƒÊmole NAD , 0.12ƒÊ g crystalline enzyme and D-mannitol. ketohexoses and ketopentoses are tested as the tively (Fig. 4). substrate for the D-mannitol dehydrogenase. Coenzyme specificity In preliminary observation, the partially The enzyme was found to require NADH2 purified enzyme preparation exhibited a high for the reduction of D-fructose . As seen in degree of substrate specificity. As shown in Table II, NADPH2 could replace NADH2, Crystalline D-Mannitol:NAD Oxidoreductase from Leuconostoc mesenteroides 897

TABLE II. COENZYME SPECIFICITY

The reaction mixture for the reduction of ketohexose contained 6ƒÊmoles acetate buffer (pH 5.35), 0.03ƒÊmole reduced coenzyme, and 30ƒÊmoles ketohexose. Reaction mixture for the oxidation of hexitol contained 6ƒÊmoles Tris-HC1 buffer (pH 8.60), 0.2ƒÊmole oxidized co enzyme, and 30ƒÊmoles hexitol. The three times crystallized enzyme was used, * 0.03ƒÊg, ** 0.06ƒÊg, and *** 1.20ƒÊg.

TABLE III. SUBSTRATE SPECIFICITY ribulose and D-xylulose the enzyme was almost WITH NADPH2 inactive with either NADH2 or NADPH2

(Tables I and III). In the reaction with oxidized coenzyme and D-mannitol or D-sorbitol, only NAD was effective (Table II). The increase of absorbance at 340mƒÊ was not observed with D-sorbitol and NADP at pH 7.0, 7.5, 8.0 and 8.5 in 0.05M Tris-HC1 buffer and at pH 9.5 and

The reaction mixture contained 6ƒÊmoles acetate 10.6 in 0.05M glycine-NaOH buffer (30ƒÊmoles buffer (pH 5.35), 30ƒÊmoles ketose, 0.03ƒÊmole NADPH2 D-sorbitol, 0.2ƒÊmole NADP and 14 units (2ƒÊg) and enzyme (* 0.1ƒÊg, ** 2.01ƒÊg, and *** 4.02ƒÊg). of enzyme). It can rule out the possibility The reduction of D-fructose with NADPH2 was ex of the NADP-linked D-sorbitol dehydrogenase pressed as 100. being present in this crystalline enzyme but the enzyme activity with NADPH2 was preparation. The Michaelis constants were about 20% of that with NADH2. L-Sorbos( obtained as 1 X 10-5 for NADH2 and 2.7 X was reduced with NADH2 at 2% the rate for 10-4M for NAD (Fig. 5). D-fructose, or at 1.35% of the rate for D- NADP was found, moreover, not to inhibit fructose with NADPH2. However, for D- the oxidation of D-mannitol and of D-sorbitol

FIG. 5. Effect of Coenzyme Concentration on Enzyme Activity.

A: The reaction mixture was similar to that in Fig. 4A, but the concentration of NADH2 instead of D-fructose was varied as indicated. B: The reaction mixture was similar to that in Fig. 4B, but the concentration of NAD in stead of D-mannitol was varied as indicated. The substrate concentration was 30ƒÊmoles in both cases. 898 Shuzo SAKAI and Kei YAMANAKA

TABLE IV. DETECTION OF KETOSE FROM D-MANNITOL WITH NAD OR NADP

The coupled reaction system was employed to detect the product from D-mannitol. The incubation mix ture (0.3ml) contained 5ƒÊmoles Tris-HCl buffer (pH FIG. 6. Effect of NADP on the Oxidation of D- 8.0), 20ƒÊmoles D-mannitol, 10ƒÊmoles sodium pyru Mannitol and of D-Sorbitol with NAD. vate, 0.2ƒÊmole oxidized or reduced coenzyme, The oxidation of D-mannitol was carried out mannitol dehydrogenase and lactic dehydrogenase in in the reaction mixture (0.3ml) which contained the quantities indicated. The mixture was incubated 6 moles Tris-HCl buffer (pH 9.0), 30ƒÊmoles D- for 1 hr or 4 hr at 30•Ž. mannitol, and 0.2ƒÊg enzyme (1.4 units). 1, with * Experiment 1: with 0 .01mg lactic dehydrogenase 0.03ƒÊmole NAD (1 x 10-4M) and 0.30ƒÊmole NADP and 0.01mg D-mannitol dehydrogenase, incubated

(1 X 10-3M); 2, with 0.03ƒÊmo1e NAD. The oxi for 1 hr. dation of D-sorbitol was carried out in the reaction ** Experiment 2: with 0 .02mg lactic dehydrogenase mixture (0.3ml) which contained 6ƒÊmoles Tris- and 0.04mg D-mannitol dehydrogenase, incubated HCl buffer (pH 9.0), 30ƒÊmoles D-sorbitol and 5ƒÊg for 4 hr. enzyme (35.4 units). 3, with 0.03ƒÊmole NAD and 0.30ƒÊmole NADP; 4, with 0.03ƒÊmole NAD. TABLE V. EFFECT OF INHIBITORS with NAD (Fig. 6). The most likely explanation of high ac tivity of the enzyme with NADH2 and D- fructose and of negligible activity of the enzyme with NADP and D-mannitol was ob tained from the following experiment. The oxidation of D-mannitol was coupled to the reduction of pyruvate with lactic dehydro genase (Table IV). A significant amount of * 10-4M a ketose was obtained from D-mannitol with NAD or NADH2 and accumulation of the Effect of inhibitors ketose from D-mannitol with NADP or Table V showed the effect of some inhibi NADPHZ was also found. The ketose formed tors on enzyme action. Among the tested from D-mannitol with NAD was identified as chemicals, p-chloromercuribenzoate complete fructose by paper chromatography. The ly inhibited enzyme action at the concentra reaction to form fructose from D-mannitol tion of 10-4M. with NAD was almost completed within one hour under the experimental conditions, DISCUSSION whereas the reaction velocity with NADP The crystalline D-mannitol dehydrogenase was very slow. The experiment 2 in Table reported in this paper seems to be analogous IVclearly showed that the oxidation of D- to that of L. brevis1) The apparent pH mannitol by NADP was also almost completed optima of L. brevis enzyme for the reduction after 4 hr incubation with more both de of fructose and for the oxidation of D-mannitol hydrogenases. are similar to those of Leuconostoc mesenteroides Crystalline D-Mannitol:NAD Oxidoreductase from Leuconostoc mesenteroides 899 enzyme in this paper. Both enzymes require enzyme and L. brevis enzymes' are quite differend NAD or NADHZ. The Michaelis constants from either D-arabitol dehydrogenase of both enzymes for substratesand coenzyme or D-mannitol dehydrogenase in other organ are similar: 0.035M (L. mesenteroides), 0.07M isms. However, there is a question on the (L. brevis) for D-fructose; 0.02M (L. mesenteroides), coenzyme specificity. NADPHZ was effective 0.06M (L. brevis) for D-mannitol; 2.7x10-4M on the reduction of D-fructose, but NADP (L. mesenteroides), 2.3x10-4M (L. brevis) for was almost ineffective on the reverse reaction NAD and 1.0 X 10-5M (L. mesenteroides), 1.3X (Table II). The other polyol dehydrogenase 10-4M(L. brevis) for NADH2. which acts on D-fructose may be contaminated Among the D-mannitol dehydrogenases from in this crystalline enzyme preparation, but bacteria, Azotobacter agilis enzyme oxidized the NADP-linked D-sorbitol (-4 D-fructose) D-mannitol, D-arabitol, D-rhamnitol and per dehydrogenase activity was not demonstrated seitol which has D-manno-configuration and it in this preparation. The ratio of activity in also reduced D-fructose and D-xylulose.9) The the reduction of fructose with NADH2 to that NAD-linked D-mannitol dehydrogenase in the with NADPH2, however, was found to be conidia of Aspergillus oryzae was reported to constant during the crystallization procedures oxidize D-mannitol, sorbitol and D-arabitol at of the first to the third (100:18 to 100:20.5). a rate of 100:35:41. The NADP-linked D- For L. brevis enzyme, NADPHZ could be mannitol dehydrogenase in the same fungus replaced with NADH2 in the reduction of was more specific to D-mannitol.10) The fructose at about 50 per cent activity, but NADP-linked D-mannitol dehydrogenase from the rate of the reaction with NADP was only Diplodia viticola catalyzed the oxidation of D- 2.5 per cent of that with NAD.1) Martinez mannitol, sorbitol and D-arabitol at a ratio of et al. suggested that NADP would be inhibi 100:24:10 and the reduction of D-fructose, D- tory.1) As seen in Fig. 6, however, NADP xylulose and L-sorbose at a ratio of 100:33:22.11) was not inhibitory on the oxidation of D- The D-arabitol dehydrogenase from Aerobacter mannitol by Leuconostoc mesenteroides enzyme. aerogenes oxidized D-mannitol at the rate of Fructose was obtained from D-mannitol with 45 per cent of the activity on D-arabitol.12) NAD or NADP in the coupled reaction of The D-arabitol dehydrogenase from Cellvibrio this enzyme and lactic dehydrogenase (Table polyoltrophicus ATCC 14774 also catalyzed the IV). These facts together with the data in dehydrogenation of D-mannitol to D-fructose.13) Tables I, II and III imply that the specific The dehydrogenation of D-mannitol and of D- D-mannitol dehydrogenase from Leuconostoc arabitol is also considered to be catalyzed by the mesenteroides requires NAD for its activity and same enzyme, D-arabitol dehydrogenase.11,12) that NADH2 can be replaced by NADPH2 The crystalline D-mannitol dehydrogenase of to some extent, but the enzyme has little Leuconostoc mesenteroides is inactive on dulcitol, affinity to NADP. all of four pentitols, i-erythritol, D-tagatose,

D- and L-ribuloses and D-xylulose. This Acknowledgements. The authors wish to express their gratitude to Dr. H. Katagiri, Emeritus professor of Kyoto University. 9) L. Marcus and A. G. Marr, J. Bacteriol., 82, 224 (1961). Thanks are due to Dr. H. Yamada, the 10) K. Horikoshi, S. Iida and Y. Ikeda, ibid., 89, Research Institute for Food Science, Kyoto 326 (1965). 11) A. G. Strobel and T. Kosuge, Arch. Biochem. University for his assistance on the ultra Biophys., 109, 622 (1965). centrifugation and to Dr. B. L. Horecker, 12) E. C. C. Lin, J. Biol. Chem., 236, 31 (1961). Albert Einstein College, New York, U. S. A, 13) E. M. Scolnick and E. C. C. Lin, J. Bacteriol., 84, 631 (1962) for his kind advice on the coenzyme specificity.