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Applied and Environmental Microbiology, August 2003, p. 4438-4447, Vol. 69, No. 8
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.8.4438-4447.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Purification and Characterization of a Novel Mannitol Dehydrogenase from a Newly Isolated Strain of Candida magnoliae

Jung-Kul Lee,1* Bong-Seong Koo,1 Sang-Yong Kim,2 and Hyung-Hwan Hyun3

BioNgene Co. Ltd., Jongro-Ku, Seoul,1 Bolak Co. Ltd., Hwasung-Si,2 Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies, Yongin-Si, Kyunggi-Do, Korea3

Received 18 November 2002/ Accepted 5 May 2003


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ABSTRACT
 
Mannitol biosynthesis in Candida magnoliae HH-01 (KCCM-10252), a yeast strain that is currently used for the industrial production of mannitol, is catalyzed by mannitol dehydrogenase (MDH) (EC 1.1.1.138). In this study, NAD(P)H-dependent MDH was purified to homogeneity from C. magnoliae HH-01 by ion-exchange chromatography, hydrophobic interaction chromatography, and affinity chromatography. The relative molecular masses of C. magnoliae MDH, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size-exclusion chromatography, were 35 and 142 kDa, respectively, indicating that the enzyme is a tetramer. This enzyme catalyzed both fructose reduction and mannitol oxidation. The pH and temperature optima for fructose reduction and mannitol oxidation were 7.5 and 37°C and 10.0 and 40°C, respectively. C. magnoliae MDH showed high substrate specificity and high catalytic efficiency (kcat = 823 s-1, Km = 28.0 mM, and kcat/Km = 29.4 mM-1 s-1) for fructose, which may explain the high mannitol production observed in this strain. Initial velocity and product inhibition studies suggest that the reaction proceeds via a sequential ordered Bi Bi mechanism, and C. magnoliae MDH is specific for transferring the 4-pro-S hydrogen of NADPH, which is typical of a short-chain dehydrogenase reductase (SDR). The internal amino acid sequences of C. magnoliae MDH showed a significant homology with SDRs from various sources, indicating that the C. magnoliae MDH is an NAD(P)H-dependent tetrameric SDR. Although MDHs have been purified and characterized from several other sources, C. magnoliae MDH is distinguished from other MDHs by its high substrate specificity and catalytic efficiency for fructose only, which makes C. magnoliae MDH the ideal choice for industrial applications, including enzymatic synthesis of mannitol and salt-tolerant plants.


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INTRODUCTION
 
Mannitol, a naturally occurring acyclic hexitol, is widely distributed in nature and is found in bacteria, algae, fungi, and higher plants (34, 51). Acyclic polyols are important for growth, carbon storage, recycling of reductants, and efficient carbon fixation (48). Mannitol also plays an important role in the stress tolerance of microorganisms, lichens, and higher plants because of its function as a compatible solute, a compound that accumulates in the cytosol and prevents the inactivation of metabolic processes (31, 62, 66). In fungi, the osmoregulatory function of mannitol might also be critical in providing an influx of water from the environment to support turgor (25, 27). Other physiological roles have been postulated for mannitol in fungi, including serving as the main and most efficient respiratory source (24).

Mannitol is about half as sweet as sucrose and is not metabolized by humans; it is considered a low-calorie sweetener (11). Due to its favorable properties, mannitol is extensively used in the pharmaceutical and food industries and is produced primarily by the catalytic reduction of fructose with hydrogen gas and nickel catalyst. Due to the low selectivity of the catalyst, the isomer sorbitol is a major by-product of the process and is produced in almost equal amounts (63). Mannitol can also be produced by microbial methods by using osmophilic yeasts and some bacteria (64, 73, 76). Recently, a high-mannitol-producing yeast strain was isolated from fermentation sludge and identified as Candida magnoliae HH-01, KCCM-10252 (65). In a previous paper (64), Song et al. reported the production of mannitol from the newly isolated C. magnoliae HH-01 strain when grown in appropriate environmental conditions and fructose concentrations. There are several reports of erythritol production from glucose with C. magnoliae cultures (54, 75); however, mannitol synthesis in C. magnoliae has never been reported.

In C. magnoliae, mannitol is thought to be synthesized from fructose by a reaction catalyzed by NAD(P)H-dependent mannitol dehydrogenase (MDH). MDH is present in a number of organisms, and it catalyzes the oxidation and reduction of D-mannitol and D-fructose.

Based on sequence analysis data, protein size, and coenzyme-binding motifs, the dehydrogenase reductases fall into three main groups that are referred to as short-chain dehydrogenase reductases (SDRs), medium-chain dehydrogenase reductases (MDRs), and long-chain dehydrogenase reductases (LDRs). The SDR (with subunits typically of 250 residues) enzymes have a typical coenzyme-binding site, GXXXGXG, and an active site, YXXXK (42). The MDR (with subunits typically of 350 residues) enzymes are zinc dependent and have the N-terminal coenzyme-binding motif GXGXXG (45). In spite of an overall sequence identity that can be as low as 10%, the LDR (350 to 560 residues) enzymes have a KXXXXNXXG motif (47). Several groups have reported the purification and characterization of MDH from plants and microbial sources (13, 19, 37, 42, 53, 55, 60). MDHs from plants and fungi have been characterized as members of the MDR family (68, 71). Other MDHs from fungi are members of the SDR family (26, 43). Often, bacterial MDHs do not share significant similarity with either of these families (58) but instead belong to a family of LDRs that includes 66 recognized members. To our knowledge, however, MDH from Candida species has never been purified and characterized.

In this study, we purified a novel NAD(P)H-dependent MDH from recently isolated C. magnoliae HH-01 (KCCM-10252) to homogeneity and characterized its physiological and kinetic parameters. Since MDH is presumed to be a key enzyme in the biosynthesis of mannitol from fructose, we undertook this study to determine whether the kinetic parameters of MDH reflect this physiological role. The properties of the enzyme, including its molecular form, reaction mechanism, stereospecificity of hydride transfer, and partial amino acid sequence, revealed that this enzyme is an SDR.


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MATERIALS AND METHODS
 
Materials.
Glucose, ribose, glucose-6-phosphate, fructose-6-phosphate, D-fructose, D-galactose, D-mannose, D-arabinose, xylose, and column chromatographic support media, including DEAE-cellulose and Cibacron-3GA dye resin, were obtained from Sigma Chemical Co. (St. Louis, Mo.). Enzyme cofactors (NAD, NADH, NADP, and NADPH), phenyl Sepharose, and various proteins used for calibration and assay runs were obtained from Pharmacia (Piscataway, N.J.). Diaflo YM 10 ultrafiltration membranes (cutoff, 10 kDa) were purchased from Amicon, Inc. (Danvers, Mass.). All other chemicals were of analytical grade or higher and purchased from Sigma Chemical Co., Fischer Scientific Co., or Difco Co., Ltd.

Microorganism and culture conditions.
C. magnoliae HH-01 (KCCM-10252) is an isolate that was produced in this laboratory (56). The growth medium contained 20 g of glucose/liter, 10 g of yeast extract/liter, and 20 g of peptone/liter. The fermentation medium consisted of 30 to 120 g of fructose/liter, 50 g of glucose/liter, 5 to 10 g of yeast extract/liter, 3 g of (NH4)2SO4/liter, and 3 g of KH2PO4/liter. The culture of C. magnoliae was performed as described in the previous report (64).

Preparation of cell extracts.
Cells from the culture broth were harvested by centrifugation at 10,000 x g for 30 min. After washing with 50 mM potassium phosphate buffer (pH 7.5), harvested cells were resuspended in homogenization buffer containing 50 mM potassium phosphate (pH 7.5), 10 mM MgCl2, and 1 mM phenylmethylsulfonyl fluoride. The cell suspension was incubated for 1 h at room temperature and then homogenized by grinding with 0.5-mm-diameter glass beads (Sigma) in a bead-beater (Biospec Products Co., Bartlesville, Okla.). Cell extracts were obtained by removing the ruptured cells by centrifugation at 10,000 x g for 30 min. The supernatants were combined and then concentrated and desalted by ultrafiltration through a YM10 membrane in a stirred cell (Amicon, Inc.).

Purification of MDH.
All procedures were performed at 4°C. Cell extracts were fractionated by ammonium sulfate precipitation. The supernatant was brought to 40% saturation with ammonium sulfate, and the pellet obtained after centrifugation at 20,000 x g for 20 min was discarded. The supernatant was then brought to 80% saturation with ammonium sulfate, and the pellet was collected by centrifugation (20,000 x g for 20 min) and suspended in 50 mM potassium phosphate buffer (pH 7.5). The enzyme solution was dialyzed against the same buffer at 4°C for 24 h. The dialyzed enzyme solution was concentrated and loaded onto a DEAE-cellulose column (1.4 by 15.0 cm) equilibrated with 50 mM potassium phosphate buffer at pH 7.5, and protein was eluted with a linear gradient of 0 to 0.5 M NaCl in the same buffer at a flow rate of 20 ml/h. Active fractions were pooled, dialyzed against the same buffer, and concentrated by ultrafiltration. The enzyme was further purified with a hydrophobic interaction chromatography column (1.4 by 10 cm) of phenyl Sepharose (Amersham Pharmacia Biotech, Uppsala, Sweden) previously equilibrated with 1.5 M ammonium sulfate in 50 mM potassium phosphate. The enzyme was eluted with a linear gradient of 1.5 to 0 M ammonium sulfate in 50 mM potassium phosphate (pH 7.5) at a flow rate of 30 ml/h. Active fractions were pooled, concentrated, and dialyzed against the 10 mM potassium phosphate (pH 7.5). The enzyme was further purified with an affinity column (1.4 by 5.0 cm) of Cibacron Blue 3GA previously equilibrated with 10 mM potassium phosphate (pH 7.5). The enzyme was eluted with a linear gradient of 0 to 1.0 M NaCl in potassium phosphate buffer (pH 7.5) at a flow rate of 30 ml/h. The combined active fractions were pooled, concentrated, and dialyzed against the same buffer and concentrated with a Centricon (Millipore Corp., Bedford, Mass.) ultrafiltration device with a molecular mass cutoff of 10 kDa and then used as a purified enzyme in the following experiments. Protein was measured by the method of Lowry et al. (35), with bovine serum albumin as a standard. All chromatographic separations and monitoring (A280) of protein in the column effluents were performed by using a BioLogic LP system (Bio-Rad, Hercules, Calif.).

MDH activity assay.
The activity of MDH was determined spectrophotometrically by monitoring the change in A340 upon oxidation or reduction of NADP(H) at 37°C (8). Unless indicated otherwise, the MDH assay mixture (1 ml) for reduction consisted of 0.25 mM NADPH, 0.1 M fructose, and enzyme solution in 50 mM potassium phosphate (pH 7.5). This reaction mixture was allowed to stand for 1 min to eliminate the endogenous oxidation of NADPH. The MDH assay mixture for oxidation consisted of 0.5 mM NADP, 0.1 M mannitol, and enzyme solution in 50 mM Tris-HCl (pH 10.0). The reaction was started by the addition of substrate. The effects of various salts and reducing agents on the activity of MDH were studied in the standard assay conditions. For assays at different pH values, the reactions were performed with the following buffers (50 mM) and pH values (in parentheses): sodium citrate (4.5 to 6.0), potassium phosphate (6.0 to 8.0), Tris-HCl (8.0 to 9.0), and glycine-NaOH (9.0 to 10.0). One unit of enzyme activity represents 1 µmol of NADPH consumed or produced per min. Activities were expressed as units per milligram of protein, and the results presented show the means of triplicate assays.

Molecular mass determination by size-exclusion chromatography.
The molecular mass of the native enzyme was determined by size-exclusion chromatography with a Superose 12 (Amersham Pharmacia Biotech) column attached to a BioLogic LP system (Bio-Rad). The column was equilibrated and eluted with 50 mM Tris-HCl buffer (pH 7.5) and calibrated with ß-amylase (Mr = 200,000), alcohol dehydrogenase (Mr = 150,000), bovine serum albumin (Mr = 66,000), carbonic anhydrase (Mr = 29,000), and cytochrome c (Mr = 12,400).

PAGE and activity staining.
For the determination of subunit molecular mass, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed as described by Laemmli (32) with 10% gels. Protein bands were visualized with Coomassie brilliant blue R-250 (Sigma Chemical Co.). Before electrophoresis, the enzyme samples were boiled for 5 min in sample buffer containing 2% (wt/vol) SDS and 5% (vol/vol) 2-mercaptoethanol. Standard proteins (Bio-Rad) used for estimation of molecular mass were phosphorylase B (Mr = 97,400), bovine serum albumin (Mr = 92,000), ovalbumin (Mr = 45,000), glyceraldehyde 3-phosphate dehydrogenase (Mr = 35,000), carbonic anhydrase (Mr = 21,500), and lactalbumin (Mr =14,200).

Native PAGE was performed with 10% polyacrylamide gels without SDS. MDH activity staining on the polyacrylamide gel was performed by using a modification of the method described by Birken and Pisano (12). The staining mixture used for the detection of NADP-mannitol activity consisted of 40 ml of 0.1 M Tris buffer (pH 10.0), 25 mg of nitroblue tetrazolium, 3 mg of phenazine methosulfate, 30 mg of NADP, and 500 mg of mannitol. Gels were incubated in staining solution for 15 min, washed in water, and stored in 7% acetic acid.

Amino acid composition.
Approximately 500 µg of enzyme was precipitated with 30% (wt/vol) trichloroacetic acid, and the precipitate was washed with ice-cold acid acetone (0.1% [vol/vol] concentrated HCl in acetone). The amino acid composition of the dried acetone powder was analyzed by ion-exchange chromatography (10) on an amino acid high-performance liquid chromatography (HPLC) system (Waters, Milford, Mass.). The compositional relatedness between MDH from C. magnoliae and MDH from other sources was assessed by using the method of Metzger et al. (39).

Peptide sequencing and enzymatic digestion.
The purified protein was resolved by SDS-PAGE and then electroblotted onto a polyvinylidene difluoride (PVDF) membrane (Bio-Rad) by the methods of Lauriere (33). For the internal amino acid sequence analysis, the digestion of MDH (20 µg) by trypsin (type XIII; Sigma) was performed in 50 mM (NH4)2CO3 (pH 8.5) at an enzyme-to-substrate ratio of 1:55 (wt/wt) for 3 h at 25°C. The digestion was stopped by lowering the pH to approximately 3 by the addition of acetic acid. For digestion with endoproteinase Asp-N (sequencing grade; Boehringer-Mannheim, Mannheim, Germany), the MDH (2 mg) was dissolved in 8 M urea and then incubated with 6 µg of proteinase in 2 M urea-50 mM sodium phosphate buffer (pH 8.0) for 22 h at 30°C. For digestion with endoproteinase Lys-C (Roche, Indianapolis, Ind.), the MDH (50 µg) was dissolved in 25 mM Tris-HCl (pH 8.5) containing 1 mM EDTA, 3 µl of endoproteinase Lys-C (1 mg/ml) was added, and the reaction mixture was incubated at 37°C for 4 h. A second 3-µl portion of enzyme was added and incubated for a further 2 h at 37°C. The resulting peptide fragments were separated by SDS-PAGE (12.5% polyacrylamide), and the separated peptides were transferred to a PVDF membrane by electroblotting. Peptide bands were visualized by 0.1% Coomassie brilliant blue R-250 staining in 50% methanol. The amino acid sequences were determined with an automatic protein sequencer model 491A (Applied Biosystems, a Division of Perkin-Elmer) at the National Instrumentation Center for Environmental Management (Suwon, Korea). The partial amino acid sequence was used to identify analogous proteins through a BLAST search of the nonredundant protein database (2).

Stereospecificity of hydride transfer from NADPH.
The stereospecificity of hydride transfer from NADPH was studied by 1H nuclear magnetic resonance (NMR) performed with an ARX Fourier transform spectrometer (Bruker Instruments, Inc., Billerica, Mass.) operating at 400 MHz in the pulsed Fourier transform mode. Spectra were recorded at 27°C by using D2O (99.9%) and 3-(trimethylsilyl)-1-propanesulfonic acid (sodium salt) as the references. The (R) and (S) isomers of [4-D]NADPH were prepared as previously described (40). The deuterium contents of (4R)-[4-D]NADPD and (4S)-[4-D]NADPD were determined to be greater than 98%. A reaction mixture contained 2 mg of (4R)-[4-D]NADPD or (4S)-[4-D]NADPD, 1 U of MDH, and 100 mM D-fructose dissolved in 50 mM Tris-HCl buffer (pH 7.5). When the oxidation of NADPD was complete (monitored spectrophotometrically at 340 nm), the sample was used for NMR analysis without further treatment. The stereospecificity for the hydride transfer for MDH was determined by comparing the integrated peak area of C4-H ({delta}, 8.75 ppm) for the reaction mixtures containing B- or A-side-labeled NADPH as the coenzyme.


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RESULTS
 
Purification of MDH.
MDH was purified as described in the Materials and Methods, and the results are summarized in Table 1. Fractionation with ultrafiltration and ammonium sulfate increased the specific activity about fourfold, with 47% recovery of MDH activity. The active fractions were applied to a DEAE-cellulose column, and MDH was eluted with approximately 0.1 M NaCl (Fig. 1A). Three peaks containing protein were observed with hydrophobic interaction chromatography. The second peak, eluted with approximately 0.8 M ammonium sulfate, showed MDH activity. The third protein peak, eluted with approximately 0.6 M NaCl, in Cibacron Blue 3GA affinity chromatography, showed MDH activity (Fig. 1B). This method resulted in a 604-fold purification of MDH with a recovery of 14.5%. The yield of the purified enzyme (specific activity, 314 U/mg of protein) was 2.32 mg starting from 120 g (wet weight) of C. magnoliae cells. As shown in Fig. 2, the purified enzyme produced a single protein band on PAGE both in the absence (Fig. 2A) and presence (Fig. 2B) of SDS. Activity staining of the nondenaturing gel showed a single band with the same mobility as that of the protein band (Fig. 2A-a). These results clearly indicate that the purified enzyme is homogeneous. Analysis of the enzyme by gel electrophoresis in the presence of SDS (Fig. 2A) revealed one band with an Mr of 35,000 ± 1,000 (n = 3). Size-exclusion chromatography on Superose 12 resulted in the elution of the enzyme activity as a symmetrical peak corresponding to an Mr of approximately 142,000 (Fig. 2C). These results indicate that the enzyme migrates as a tetramer in gel filtration under the mild conditions used and thus may also be present and active as a tetramer in solution. The yeast MDHs that have been purified and characterized in some detail occur chiefly as homotetramers with a subunit molecular mass of 26 to 39 kDa (28).


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  		TABLE 1. Purification of MDH from the cell extract of C. magnoliae HH-01



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  		FIG. 1. (A) Separation of MDH from C. magnoliae on DEAE-cellulose. The ammonium sulfate precipitate of C. magnoliae cell extracts was loaded onto a DEAE-cellulose column. (B) Separation of MDH from C. magnoliae on Cibacron Blue 3GA affinity resin. Active fractions from hydrophobic interaction chromatography were loaded onto a Cibacron Blue 3GA column. The proteins were eluted with a linear NaCl gradient, and each fraction (2 ml) was collected. Fractions were assayed for MDH activity by using fructose as a substrate. Bars I and II indicate the fractions used for native PAGE (inset). {circ}, protein; {blacktriangleup}, MDH activity; -, NaCl gradient.



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  		FIG. 2. PAGE and determination of molecular mass of MDH purified from the C. magnoliae. (A) Native PAGE; (A-a) activity staining after native PAGE; (B) SDS-PAGE. The enzyme solution was run on a 10% (wt/vol) polyacrylamide slab gel as described in Materials and Methods. The arrow indicates the protein band containing MDH. (C) Determination of the Mr of native C. magnoliae MDH, purified according to the present method, by gel filtration chromatography. The chromatography runs were performed as described in Materials and Methods. The arrow indicates the position for the MDH Mr from C. magnoliae. Kav = (Ve - Vo)/(Vt - Vo); Ve, elution volume of protein; Vo, elution volume of Blue Dextran 2000; Vt, total bed volume.

HPLC analysis of the reaction products formed after the enzyme was incubated in a mixture of buffer and fructose confirmed the nature of the reaction product under examination, with and without NADPH. The HPLC chromatogram showed a significant decrease in fructose content in the sample containing NADPH, accompanied by the formation of a new peak with the retention characteristics of mannitol; this substance was absent when the enzyme and NADPH were incubated without fructose.

Amino acid sequence and amino acid composition.
The pure enzyme (1.5 µg) was separated by SDS-10% PAGE and blotted onto a PVDF membrane. Automated Edman degradation of the enzyme protein was unsuccessful, implying that the N terminus of the enzyme is blocked. C. magnoliae MDH was partially digested with trypsin, endoproteinase Asp-N, and endoproteinase Lys-C, separated by SDS-12.5% PAGE, and blotted onto a PVDF membrane. Three fragments were sequenced on an automatic protein sequencer (Fig. 3). One Lys-C fragment, one Asp-N fragment, and one trypsin fragment were sequenced. The Lys-C fragment contained a GXXXGXG segment which generally characterizes coenzyme-binding folds (30, 70). The trypsin and Asp-N fragments were similar to ßF and ßD of the SDR family, respectively (Fig. 3), which form one side of the active site cavity (26, 28).



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  		FIG. 3. Comparison of internal amino acid sequences of C. magnoliae MDH with those of other SDRs, including MDHs (A. bisporus [26], Drosophila ADH [38], Bactrocera oleae ADH [7], E. coli gluconate DH [5], and Klebsiella aerogenes ribitol DH [18]). The domain names {alpha}B, ßD, and ßF were assigned by Jörnvall et al. (28). Residues given against a black background are those conserved in more than 90% of the sequences aligned, and boxed residues are conserved in more than half of the aligned sequences. The percentages are given according to the results of complete alignment with the 57 enzymes in reference 28.

The amino acid compositions of C. magnoliae MDH and MDHs from various sources were compared by using the difference index (DI), where 0 represents two identical proteins and 100 represents two proteins with no identical amino acids (39). The overall amino acid composition of C. magnoliae MDH is quite similar to that of SDRs from Agaricus bisporus (DI = 9.0), Uromyces fabae (DI = 10.0), and Candidatropicalis (DI = 9.1) (Table 2).


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  		TABLE 2. Amino acid composition of MDHs and SDRs from several sources, including C. magnoliae MDHa

Optimum pH and temperature.
The optimum pH for reduction by MDH was 7.5, with 87 and 84% of the maximum activity at pH 7.0 and 8.0, respectively. The optimum pH for oxidation was 10.0, with 86 and 92% of the maximum activity at pH 9.0 and 11.0, respectively. Maximal reductase activity at about pH 7.0 and an alkaline pH optimum for mannitol oxidation are common features of similar enzymes isolated from diverse microbial systems (57, 60, 74).

The optimum temperatures for the reductive and oxidative reactions were 37 and 40°C, respectively. The stability of MDH was tested in standard buffer. Preparations were stored at 4, 20, 30, 45, and 55°C and retained 50% of their initial activities after 45 days, 14 days, 5 days, 10 h, and 30 min, respectively.

Substrate and cosubstrate specificity.
The oxidative reaction catalyzed by MDH was very slow; the rate for mannitol oxidation was less than 3.0% of that for D-fructose reduction. Most of the polyol oxidizing and reducing enzymes described to date are pyridine nucleotide linked, requiring either NADH or NADPH as a cosubstrate. With respect to the cosubstrate, the MDH from C. magnoliae HH-01 showed a much higher affinity for NADPH (Km = 57.8 µM) than for NADH (Km = 612 µM) in the presence of 100 mM fructose.

The MDH activities for various sugar substrates and polyols are shown in Table 3. Mannose, ribose, xylose, galactose, arabinose, glucose, glucose-6-phosphate, and fructose-6-phosphate (all at 50 mM), with NADPH as a cosubstrate, were examined as alternative substrates for C. magnoliae MDH. While arabinose, mannose, ribose, and xylose showed slight activity (2% or less, compared with fructose-NADPH), the other sugars and sugar phosphates did not serve as substrates for MDH in the presence of either NADPH or NADH. C. magnoliae MDH had a high preference only for fructose and mannitol. Other MDHs, such as Pseudomonas fluorescens DSM 50106 (14) and Rhodobacter sphaeroides MDH (58), reduce other sugar substrates with about 30 to 90% of their fructose reduction activity. The narrow substrate specificity and high catalytic efficiency of purified C. magnoliae MDH are apparent from Table 3. These results show that the enzyme has a high substrate specificity and will only catalyze the production of mannitol under physiological conditions.


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  		TABLE 3. Substrate and coenzyme specificity of MDH purified from C. magnoliae HH-01a

Stereospecificity of hydride transfer.
The stereospecificity of the hydride transfer step was examined by using stereospecifically labeled NADPH, and the oxidized NADP+ generated during the reaction was analyzed by 1H NMR. When (4S)-[4-D]NADPD was used as a coenzyme for the reduction of D-fructose, the H-4 signal at {delta} 8.75 was retained in the NADP+ species, indicating the transfer of the 4-pro-S deuterium at the C-4 position of the nicotinamide ring. By contrast, incubation with (4R)-[4-D]NADPD resulted in the absence of the {delta} 8.75 signal, which was due to the depletion of (4S)-hydrogen upon C. magnoliae MDH-catalyzed oxidation of NADPD. Therefore, C. magnoliae MDH specifically transfers the 4-pro-S hydrogen from the C-4 of the nicotinamide ring to the si face of the carbonyl carbon of the substrate, which is typical of members of the SDR family (28).

Kinetics.
Initial velocities were determined in the standard assay mixture at pH 7.5. All the substrates reported below had hyperbolic saturation curves, and the corresponding double-reciprocal plots were linear. The concentration of D-fructose varied from 1 to 300 mM. Figure 4 shows typical Michaelis-Menten-type kinetics for MDH activity, increasing with fructose concentrations. Maximum enzyme activity was obtained with a fructose concentration of about 200 mM under the experimental conditions. The Lineweaver-Burk plot (Fig. 4, inset) obtained for the conversion of D-fructose under standard assay conditions shows that the Km for D-fructose is 28.0 mM. The catalytic efficiency value (kcat/Km= 29.4 mM-1 s-1) of C. magnoliae MDH was greater than that the 13 mM-1 s-1 of A. bisporus MDH (26) or the 2.2 mM-1 s-1 of P. fluorescens (60). These properties may be important for supporting the large accumulation of mannitol observed in this yeast strain.



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  		FIG. 4. Effects of substrate concentration on the activities of MDH. MDH activity of the enzyme (1 U) was measured in the presence of the indicated concentrations of D-fructose and 0.25 mM NADPH, at pH 7.5. The inset shows a Lineweaver-Burk plot of initial velocity versus various fixed D-fructose concentrations. Each value represents the mean of triplicate measurements and varied from the mean by not more than 10%.

The product inhibition studies, under nonsaturating conditions, showed that the inhibition by mannitol was noncompetitive against NADPH and fructose (Fig. 5). The secondary plots for noncompetitive inhibition with NADPH and fructose are shown in Fig. 5C, indicating that the mannitol product binds to C. magnoliae MDH with a Ki of 188 mM. The inhibition by NADP+ was competitive with NADPH (Ki = 210 µM) and was noncompetitive with fructose (Ki = 180 µM). Plotting the y-axis intercepts against the NADPH concentrations gave a straight line. The results suggest that NADPH and NADP+ bind to the free form of the enzyme and rule out the possibility of a random mechanism (15). Under nonsaturating conditions, the pattern of product inhibition observed in a two-substrate reaction can be diagnostic for the mechanism of catalysis by that enzyme (52). Only an ordered Bi Bi reaction mechanism exhibits a pattern of inhibition in which one product is a noncompetitive inhibitor for both substrates and the other product is competitive and noncompetitive for the respective substrates. The results of the initial-velocity and product inhibition studies suggest that the enzyme reaction proceeds via a sequentially ordered Bi Bi mechanism in which NADPH binds first to the enzyme and is followed by D-fructose and mannitol leaves the enzyme before the release of NADP+ (15, 50). This type of reaction mechanism has already been suggested for the MDH of P. fluorescens (60).



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  		FIG. 5. Graphical analysis of the inhibition of C. magnoliae MDH by mannitol. The effects of increasing mannitol (product) concentration on the apparent Km and Vmax values for fructose and NADPH were examined. Analysis of these data by double-reciprocal plots indicated that mannitol inhibited MDH noncompetitively with respect to fructose (A) and NADPH (B). In panel C, the secondary plots for noncompetitive inhibition with fructose and NADPH are shown. The mannitol product binds to MDH with a Ki of 188 mM.

Effects of metal ions and various compounds.
The MDH activity was measured in the presence of metal ions (1 mM) or various other compounds (Table 4). MDH activity was not stimulated by MgCl2, MnCl2, ZnCl2, CaCl2, NiCl2, CoCl2, or FeCl2 (each at 1 mM concentration), and it was neither inhibited nor activated by EDTA at concentrations ranging from 1 to 10 mM. By contrast, a Cu2+ ion caused significant inhibition of C. magnoliae MDH (Ki = 1.1 µM) that appeared competitive with respect to the substrate fructose.


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  		TABLE 4. Effect of various chemicals on the activity of MDHa

Dependence of the enzyme activity on sulfhydryl compounds has been reported for the MDH from celery (67) and several reductases purified from Candida tenuis (41) and pig lens (13). We also examined the effects of sulfhydryl compounds on the MDH from C. magnoliae (Table 4). The addition of 0.5 mM 2-mercaptoethanol, glutathione, cysteine, or dithiothreitol to the reaction mixture fully inhibited the enzyme activity. This may merely indicate that MDH contains easily accessed disulfide bonds that are readily reduced and the activity of MDH might be directly regulated by the redox state in vivo (56).


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DISCUSSION
 
In a previous study, the isolation of the high-mannitol-producing C. magnoliae HH-01 strain was reported. Using this newly isolated strain, the highest yield of mannitol production ever reported by a mannitol-producing strain was obtained (65). Although the purification and properties of MDH from several strains have been reported (14, 19, 38, 42, 53, 55, 60), this is the first report on the purification and characterization of MDH from C. magnoliae, an organism currently used for industrial mannitol production. C. magnoliae MDH is the only yeast MDH next to Saccharomycescerevisiae MDH, and its strict specificity for fructose and high catalytic efficiency make C. magnoliae MDH the ideal choice for industrial applications.

Table 5 shows a comparison of the properties of NAD(P)H-linked MDH from various sources. These enzymes can be divided into three groups: SDR, MDR, and LDR. The first group, including C. magnoliae MDH, has the highest catalytic efficiency (kcat/Km). C. magnoliae MDH had a comparable Km value of 28.0 mM for fructose. In comparison, purified MDHs from other sources had fructose Km values of 200 mM (Cephalosporum chrysogenus) (12), 58.7 mM (A. bisporus) (26), 35 mM (Leuconostoc mesenteroides) (74), 16.3 mM (R. sphaeroides) (57), and 24.6 mM (P. fluorescens) (60). The S. cerevisiae MDH has been reported to have high substrate specificity, and the Km (29 mM) for fructose seems to be similar to that of C. magnoliae MDH (46). But in the case of S. cerevisiae MDH, the catalytic efficiency for mannitol oxidation (kcat/Km = 11.9 mM-1 s-1) is much higher than that for fructose reduction (kcat/Km = 2.5 mM-1 s-1). Since the oxidative reaction of C. magnoliae MDH is much slower than the reductive one and C. magnoliae MDH is oxidatively active at alkaline pH, the differences in the pH activity profile and substrate specificity in the oxidative reaction have no practical meaning in living cells, where the pH is weakly acidic. Therefore, this enzyme is thought to catalyze the reduction of D-fructose (formation of mannitol) in C. magnoliae exclusively. Unlike NADP(H) preferring the C. magnoliae enzyme, S. cerevisiae MDH activity was found to be NAD(H) dependent, required the presence of Mtl-1 as a regulatory gene, and was classified as an LDR. There was no sequence homology between C. magnoliae and S. cerevisiae MDH when the partial amino acid sequences of C. magnoliae MDH were aligned with those of the S. cerevisiae enzyme.


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  		TABLE 5. Properties of MDHs from various sourcesa

The reaction mechanism of C. magnoliae MDH was determined to be an ordered Bi Bi mechanism, as proposed for many SDRs, such as Drosophila alcohol dehydrogenase (72), DHPR (49), and Leishmania PTR1 (36). The internal amino acid sequence analysis of C. magnoliae MDH showed the presence of a coenzyme-binding fold and a ß-sheet similar to the active site cavity of SDRs. Most SDRs are homodimers or homotetramers, and they possess a unique ß-{alpha}-ß unit that contains a coenzyme-binding fold (GXXXGXG), which is the coenzyme-binding region (29, 30, 44). The Asp-N and trypsin fragments of C. magnoliae MDH are similar to the ßD (DXXXXNAG) and ßF (N/SXXXPGXXXT) structure elements of SDR, respectively. The former stabilizes the central ß-sheet (20), and the latter is proposed to play roles in structure and reaction direction (21, 22). The stereospecificity of hydride transfer by C. magnoliae MDH is also very similar to that described for many SDRs. The preference of MDH for transfer of the pro-S hydrogen from NAD(P)H is typical of all members of the SDR family that have been studied (3, 4, 6, 9, 17). These results suggest that C. magnoliae MDH should be classified as an NAD(P)H-linked SDR.

The instability, low substrate affinity, low Vmax, and broad substrate specificity of most MDHs limit their practical applications. In general, polyol dehydrogenases, including MDHs, are characterized by relatively broad substrate specificities. The novel MDH from C. magnoliae HH-01, however, shows strict substrate specificity for fructose and mannitol and differs from other MDHs in that it possesses a significantly higher catalytic efficiency (kcat/Km = 29.4 mM-1 s-1) than do MDHs purified from other sources. These properties of C. magnoliae MDH may partially explain the high mannitol production without other by-products observed in this strain and make it useful for industrial applications. Such applications could include (i) quantitative analysis of mannitol concentration in serum and urine in a simple and sensitive enzymatic assay for clinical use (16), (ii) enzymatic production of mannitol from fructose to reduce downstream purification (59), and (iii) transgenic expression of MDH in plants to improve salt tolerance and resistance to oxidative stress in agricultural crops (69). A method for enzymatic pure synthesis of mannitol has been developed with the P. fluorescens MDH (42). The catalytic efficiency of C. magnoliae MDH is much higher than that of P. fluorescens MDH (2.2 mM-1 s-1), indicating that C. magnoliae MDH can be a good candidate for enzymatic pure synthesis of mannitol.

Our results improve the understanding of mannitol biosynthesis in C. magnoliae and should contribute to better industrial production of mannitol by biological processes and better industrial applications of MDH. However, definitive proof for the characteristics of C. magnoliae MDH requires further crystallographic analysis of the enzyme or enzyme-coenzyme complex.


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ACKNOWLEDGMENTS
 
We are indebted to Walter Niehaus for helpful advice and stimulating discussions. We thank Eun-Hee Kim (Korea Basic Science Institute, Taejon) for help in 1H NMR analysis and Ji-Hyun Ryu for thorough proofreading.

This work was supported by a grant (02-PJ1-PG11-VN01-SV02-0027) from the Ministry of Health and Welfare, Kwacheon-Si, Kyunggi-Do, Korea.


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FOOTNOTES
 
* Corresponding author. Mailing address: BioNgene Co. Ltd., 10-1, 1Ka Myungryun-dong, Jongro-Ku, Seoul, Korea 110-521. Phone: 82-2-747-0700. Fax: 82-2-747-0750. E-mail: jkrhee{at}biongene.com. Back


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Applied and Environmental Microbiology, August 2003, p. 4438-4447, Vol. 69, No. 8
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.8.4438-4447.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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