Purification, characterization and structure analysis of NADPH-dependent d -xylose reductases from Candida tropicalis

JOURNALOF FERMENTATION ANDBIOENGINEERING Vol. 79, No. 3, 211-223. 1995

Purification, Characterization and Structure Analysis of NADPHDependent D-Xylose Reductases from Candida tropicalis SHIN-ICHIRO YOKOYAMA,’ TOHRU SUZUKI,2 KEIICHI KAWAI,2* HIROYUKI HORITSU,2§ AND KAZUHIRO TAKAMIZAWA2 United Graduate School of Agricultural Science’ and Department of Biotechnology, Gifu University, 1-I Yanagido, Gifu 501-11, Japan

Faculty of Agriculture,2

Received 5 October 1994IAccepted 15 December 1994

NADPH-dependent D-xylose reductases (XRs) from Can&da tropicalis IF0 0618 were purified and cbaracterlzed. Mono Q HPLC revealed three XR isomers. The K,,, values of XRl, XR2 and XR3 for Pxylose were 37,30 and 34 mM, and for NADPH 14,18 and 9 @l, respectively. NADII did not act as a cofactor. The specificities of the three XRs for several aldoses were essentially the same. Gel filtration and cross-linking analysis showed that both XRl and XR2 were dimers composed of identical subunits. The p1 values of XRl and XR2 were estimated to be 4.15 and 4.10, respectively. Comparlson of the peptlde maps of XRl and XR2 showed that the molecular weights of 8 fragments of lysylendopeptidase-digested XRl and XR2 were essentlally the same as each other. The amino acid composition of each XR was also very similar. The molecular weights of XRl and XR2 by mass spectra analysis were 36,497.91 and 36,539.68, respectively. The amino acid sequences of two XRl peptlde fragments (Nos. 4 and 5) were highly homologous with those of fkhia stipitis XR and mammalian aldose reductases. [Key words:

Candida tropicalis, xylose reductase,

isomer, NADPH, mass spectra]

Xylitol, one of the sugar alcohols, is an insulin-independent carbon source for diabetics (1, 2). It also has been reported to reduce the incidence of dental caries by inhibiting the growth and metabolism of bacteria causing dental plaque (3). Xylitol is now widely used in chewing gums as a sweetner, and its use is likely to increase in the future. Xylitol is an intermediary metabolite of D-xylose utilization by microorganisms, and many studies on D-xylose metabolism have been conducted, mainly with the aim at producing ethanol. Most of D-xylose-assimilating bacteria and a few yeasts such as Rhodotorula gracilis (4) and Candidu utilis (5) have D-xylose isomerase (XI), which directly converts D-xylose to D-xylulose. Most yeasts, however, does not have XI. In these yeasts, D-xylose is metabolized as follows. D-Xylose is reduced to xylitol by D-xylose reductase (XR) and then xylitol is dehydrogenated to D-xylulose by xylitol dehydrogenase (XDH). After phosphorylation by D-xylulokinase, D-xylulose-5phosphate enters the pentose phosphate pathway. In this reaction, XR and XDH require NAD(P)H and NAD(P)+ as cofactors, respectively. In an attempt to obtain bioconversion from xylan to xylitol, we have studied xylanases that hydrolyze xylan to xylose and xylooligosaccharides (6). Furthermore, we have reported that a maximum xylitol production rate of 2.67 g/l.h was obtained when the culture conditions were optimized by the Box-Willson method (7), using Candidu tropicalis IF0 0618 (8). For xylitol production from D-xylose, XR is a key enzyme. Here, we report purification and characterization of NADPH-dependent XRs from C. tropicalis IF0 0618, showing that it contains three XR isomers, and investigate their structural

analyses. MATERIALS

AND METHODS

Organism and cultivation The organism used in this study was C. tropicalis IF0 0618. It was maintained on YPD agar plates (2.0% glucose, 0.5% yeast extract, 0.5% Polypepton, 0.1% KH2P0,, 0.05% MgS04, and 2.0% agar, pH 5.5). C. tropicalis was cultivated by inoculating cells into 500 ml Erlenmeyer flasks containing 250ml of growth medium (3% D-xylose, 0.3% yeast extract, 0.3% K2HP04, and 0.1% MgS04.7H20, pH 5.0). The cells were grown on a reciprocal shaker (6 cm, 133 strokes/min, 3O”C, 24 h). They were harvested by centrifugation at 8,000 xg for 20 min at 4°C and washed twice. The washed cells were suspended in 20ml of 0.5 M potassium phosphate buffer (pH 7.5) and then disrupted with sea sand in a mortar. The cell debris was removed by centrifugation at 10,000X g for 30 min at 4°C. The supernatant fluid obtained was used as cell-free extract (each liter of cells yielded about 30 g of cell paste). Purification of xylose reductase XR was purified as follows. All purification procedures were carried out at 8°C. After cell-free extract (1OOml) was dialyzed for 10 h against TEM buffer (20mM Tris-HCl (p&I7.5), 0.5 mM EDTA, and 0.5 mM 2-mercaptoethanol) with several replacements of fresh buffer, the enzyme solution was applied on to a DEAE-cellulose column ($23 x 250 mm) that had been previously equilibrated with TEM buffer. The column was washed with two bed volumes of the buffer. XR was eluted from the column by a linear gradient of 0 to 0.5 M NaCl in the same buffer at a flow rate of lOOml/h. The active fractions were pooled. The enzyme solution was put on a Blue Sepharose CL6B affinity column (9) (418 X 16Omm), which had been equilibrated with TEM buffer. After washing with two bed volumes of TEM buffer, the column was eluted by a linear gradient of 0 to 50 PM

* Corresponding author. s Present address: Faculty of Home Economics, Department of Food and Nutrition, Chukyo Women’s University, 55 Natakayama, Yokone-cho, Ohbu, Japan. 217

218

YOKOYAMA ET AL.

NADP+ in the same buffer. The active fractions were pooled, concentrated, and dialyzed against the same buffer by ultrafiltration. The concentrated enzyme solution was purified by high performance liquid chromatography (HPLC) using a Mono Q column (Pharmacia Co., Sweden). Enzyme assay The activity of XR was determined by monitoring the oxidation of NADPH in a spectrophotometric cuvette at 340 nm at room temperature. The cuvette contained 0.2 ml of 1 M phosphate buffer (pH 7.0), 0.2 ml of 0.1 M 2-mercaptoethanol, 0.1 ml of enzyme solution, 0.1 ml of 3.4 mM NADPH and 1.2 ml of deionized water. This reaction mixture was allowed to stand for 1 min to eliminate the endogenous oxidation of NADPH. The reaction was started by addition of 0.2 ml of 0.5 M substrate (DL-glyceraldehyde, D-xylose, Larabinose or D-glucose). One unit (U) of enzyme was defined as the amount of enzyme that caused an initial rate of decrease of 1 pmol NADPH per minute. Specific activity was expressed as units of the enzyme per mg of protein. Protein was measured by the Lowry method (10) with bovine serum albumin as a standard. Gel electrophoresis of protein Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed as described by Laemmli (11). Native PAGE was done without SDS. Protein bands were stained with Coomasie brilliant blue R2.50 or silver staining. Isoelectric focusing was carried out with Pharmalyte (Pharmacia Co., pH 2.5-5.0) at a constant voltage of 500V at 10°C for 3 h. Cross-linking reaction The constituent polypeptide chains of XR were cross-linked by employing a bifunctional reagent, dimethylsuberimidate, according to the procedure of Davies and Sterk (12). The extent of crosslinking was monitored by SDS-PAGE. The molecular weight of XR was Mass spectrum measured by the ion-spray mass spectrum (13-15) of API III mass spectrometer (Perkin Elmer Sciex, USA). To prepare peptide maps of XRl and XR2, both XRs were digested with lysylendopeptidase for 12 h at 37°C and analyzed by liquid chromatography linked with mass spectrometry (LWMS), which was combined with the reversed phase HPLC using a solvent containing 0.1% trichloroacetic acid-H,O-acetonitrile (1% gradient). Analysis of amino acid composition and amino acid The amino acid composition was analyzed sequences with a PICO-TAGTM automatic amino acid analyzer (Applied Biosystems Co., USA). The samples were hydrolyzed in the gas phase for 24 h by using 6M HCl before analysis. The amino acid sequences of lysylendopeptidase-treated XR fragments were determined by a protein sequencer system (Model 473-A; Applied Biosystems Co.). A homology search was carried out by DNASIS, version 7.00.

J. FERMENT.BIOENG.,

1 0.4 g

0.3

C

3E

9 g 8 f 5 B

1 3

> 2 .= 0.06 .2 - 0.13 00 2 G z”

0.2 0.1 0.0 10

15

20

0.02 25

Retention time (mid B

FIG. 1. Elution pattern of XRs on Mono Q HPLC and polyacrylamide gel electrophoresis of XRl, XR2 and XR3. Panel A: Blue Sepharose eluent (300,ul) was injected into a Mono Q HR 5/S column. TEM buffer was used at a flow rate of 1.0 ml/min at room temperature. 0, XR activity; -, protein concentration; ----, NaCl concentration. Panel B: The separation gel contained 7.5% acrylamide.

lOO-140mM NaCl gradient, three active peaks were observed in agreement with the protein peaks. Thus, three types of XR isomers were present in C. tropicalis IF0 0618, which were named XRl, XR2 and XR3 according to the order of elution. The results of the purification are summarized in Table 1. XRl was purified 25.9-fold with a recovery of 15%. At this purification step, XRl was found to be homogenous by SDS-PAGE. Since fractions containing XR2 and XR3 had some impurities, the two XRs were further chromatographed on a Mono Q column in the

RESULTS Purification of XR from C. tropicalis The crude extract was applied to a DEAE-cellulose column, and XR was eluted by a O-O.5 M NaCl gradient. XR activities were recovered in approximately 0.3 M NaCl-containing fractions. In the O-50 (zM NADP+ gradient of Blue Sepharose CLdB column chromatography, XR was eluted as a single peak at approximately 13 ,nM NADP+. Active fractions were then pooled, concentrated, and applied on to a Mono Q HPLC column (Fig. 1). In the

0.10 1 - 0.14

TABLE 1.

Purification of XRl, XR2 and XR3

Total T?fa’ ‘pecific Purification Yield protein acttvtty activity (fold) (%) (mg) W) Wmg) Cell-free extract 60.7 133.0 2.2 1.0 100.0 DE-32 cellulose 11.3 84.5 7.5 3.4 63.6 Blue sepharose CL-6B 0.8 41 .o 51.3 23.3 30.8 Mono Q HPLC XRl 0.35 19.9 56.9 25.9 15.0 XR2 0.21 17.0 81.0 36.8 12.7 XR3 0.02 0.92 47.8 21.8 0.7 Step

NADPH-DEPENDENT

VOL. 79, 1995 TABLE 2.

C. tropicalis

(native) IF0 0618

XRl XR2 XR3 C. shehatae ATCC 22984 C. Milk ATCC 9950 Pachysolen tannophilus IF0 1007 CBS 4044 A B NRRL Y-2460 NRRL Y-2460 Pichia stipitis CBS 5773 P. quercuum IF0 0949 I IIa IIb Rhodotorula sp. a mM. b ,uM. c NADH as a cofactor.

(subunit)

36,497.91 58,000 36,539.68 58,000 58,000 33,000_+2,000 33,000+2,000 70,000 40,ooo 41,000

40,000 -

37,000

-

35,000 38,000 36,000 36,QOO 65,OOOf4,000 34,C@Of2,000 160,000 61,000 61,000 62,000

same manner as described above. In this operation, XR2 and XR3 were purified to homogeneity. Kinetic properties The kinetic parameters of the three types of XR were estimated on S/V-S plots (Table 2). The K,,, values of XRl, XR2 and XR3 for n-xylose were 37, 30 and 34 mM, and those for NADPH were 14, 18 and 9 ,uM, respectively. However, activities of the three XRs for NADH were not detectable. The XRs are thus completely NADPH dependent. On the other hand, xylitol, as a consequent product, was a competitive inhibitor for each enzyme. The Ki values of XRI and XR2 for xylitol were 0.42 and 0.34M, respectively. The activities of the XRs on several substrates were tested. In each case, the activity for Dr.-glyceraldehyde was highest among the five substrates examined, followed by L-arabinose and n-xylose. The XRs were less active for n-galactose and n-glucose (Table 3). Thus, the three types of XR were almost the same with respect to their substrate specificity. The optimal pH for both XRl and XR2 activities was around pH 6.0, though XR2 showed higher activity over a wider pH range than XRl did. Treatment of purified XRl and XR2 for 1 h at various temperatures resulted in the progressive loss of enzyme activities, the activities of both XRl and XR2 being completely lost after 1 h at 60°C. Structure of XRl and XR2 XRl and XR2 could both be purified sufficiently to be subjected to structural analyses. The molecular masses of the native XRs were estimated by gel filtration and calculated to be approximately 58,000 (data not shown). From the results of TABLE 3.

Substrate specificity of XRl, XR2 and XR3

Substrate D-Xylose rx-Glyceraldehyde L-Arabinose D-Galactose D-Glucose

Relative activity (%) XRl

XR2

XR3

100 200 II7 49 10

100 268 I20 40 11

100 143 101 33 11

The rate for D-XylOSewas taken as 100%.

219

REDUCTASES FROM C. TROPICALIS

Properties of XRs from various organisms

M.W.

Strain

D-XYLOSE

K, NADPHb

NADHb

31 30 34 28

14 18 9 16

12 $5’)

PI

Opt. pH

Reference

N.D. N.D. N.D. -

4.15 4.10 5.2 -

6.0 6.0 5.5-1.6

This study This study This study (29) (30)

21

40

-

5-6 -

(31) (26)

180

-

-

-

-

162 42 330

59 9 10 8 8 -

21 N.D. N.D. N.D. -

4.87 4.6 -

7.0 6.0 6.0 6.0 6.3 6.3 -

D-xylose”

78 78 -

5.05

(32) (33) (34) (28) (35)

SDS-PAGE, the molecular weights of both XRs were estimated to be approximately 36,000. These data suggested that the XRs were composed of two identical subunits. These findings were confirmed by cross-linking experiments (Fig. 2). The pI values of XRl and XR2 were 4.15 and 4.10, respectively (Fig. 3), showing that some difference in charge exists between them. Amino acid composition To investigate the divergence between XRl and XR2, their amino acid compositions were determined (Fig. 4). The disruption rates in

kDa

49.6 37.2 24.8 FIG. 2. Analysis of the cross-linking of XRl and XR2 by dimethylsuberimidate. The enzymes were cross-linked as described in Materials and Methods. SDS-PAGE represents the cross-linking pattern obtained with protein concentrations of 1 mg/ml and dimethylsuberimidate concentrations of 2 mg/ml. 1, Dimer; 2, monomer; 3, noncross-linked sample.

220

J. FERMENT. BIOENG.,

YOKOYAMA ET AL.

PI 4.55 4.15 a.50

DESGHRTAPYVMCILFK

FIG. 4. Amino acid composition of XRI and XR2. Both XRs were hydrolyzed with 6 N HCl at 105°C for 24 h and analyzed by the PICO-TAGTM amino acid analysis system. Tryptophan residue was not determined. n , XRI; 0, XR2.

2.80

DISCUSSION FIG. 3. Estimation of isoelectric point @I) of XRl and XR2. Proteins for standard pI markers were as follows; soybean trypsin inhibitor (PI =4.55), glucose oxidase (4.15), amyloglucosidase (3.50) and pepsinogen (2.80).

the hydrolysis were not revised. The amino acid compositions of the two enzymes were found to be essentially the same. Mass spectra The molecular masses of XRs were also analyzed by the LC/MS. The molecular weights of XRl and XR2 were calculated to be 36,497.91 and 36,539.68 Da, respectively (Fig. 5). These results correspond with the molecular weights estimated by SDSPAGE. The difference in molecular mass between XRl and XR2 was 41.77. Peptide map The LC/MS elution patterns of both XRs are shown in Fig. 6. No distinct difference between XRl and XR2 was observed. Moreover, both XRs agreed in molecular weight at 8 fragment peaks (Fig. 6, nos. 2, 4, 5, 6, 8, 11, 12 and la), indicating that they are very similar to each other in protein structure. Partial amino acid sequence The N-terminal amino acid sequences of XRl and XR2 were not determined because Edman degradations did not proceed. To determine the amino acid sequences of XRl peptide fragments, XRl was partially digested with lysylendopeptidase for 2 h. Then, two small molecular fragments (Peptide 4, M.W. 15,000; Peptide 5, M.W. 13,500) were applied to the protein sequencer. Thirteen amino acid residues of Peptide 4 and 20 residues of Peptide 5 were determined (Fig. 7). The similarities of the XRl peptide fragments are shown in Fig. 7. Highly homologous regions were found in Pichia stipitis XR (16, 17) for Peptide 4 and mammalians aldose reductases (18-25), as well as P. stipitis XR (16, 17) for Peptide 5. From the primary structure of P. stipitis, Peptides 4 and 5 are considered to be linked sequentially by (NVETA LNKTLSDLNLDYVDLFLIHFPIA). This agrees with the fact that the C-terminus of lysine was digested with lysylendopeptidase.

In this study, we examined the purification and characterization of XR from C. tropicalis IF0 0618. There were three types of XR were shown to exist in this yeast. It has been reported that P. tannophilus (26) and Aureobasidium pullulans (27) have two isomers and Pichia quercuum (28) has three. Although distinct differences were revealed with respect to cofactor dependency and molecular weight, the enzymatic properties of the three isomers from C. tropicalis showed no significant differences. It is thus suggested that these isomers are similar to each other in structure. These enzymes were also similar to other yeast XRs (Table 2) (26, 28-35). The question arises why the xylitol production of C. tropicalis is higher than that of the others, even though their XR properties are almost equal P. stipitis (34) and C. shehatae (29) have NADH-dependent XR activities and can maintain the redox balance in cells. Therefore, the requirement of XR for NADH is suitable for use in ethanol production. On the other hand, C. tropicalis has almost NADPH-dependent XR activity, and this dependency may be concerned in the accumulation of xylitol. From the results of gel filtration, SDS-PAGE and the cross-linking reaction, both XRl and XR2 were found to be dimers composed of identical subunits. The results of PAGE, isoelectric focusing and mass spectrum analysis showed a few differences, whereas the peptide map obtained by LC/MS and the amino acid components of the enzymes show the similarity between XRl and XR2. The difference in molecular weight was only slight at 41.77. A sugar chain was not detectable by PAS staining (data not shown). On the basis of these results, the most reasonable hypothesis is that XRl and XR2 are translated from the same gene, and XRl may be changed to XR2 by post translational modification such as by an acetyl group (M.W. =42). The N-termini of both XRs were considered to be blocked, because Edman degradation did not proceed. XR2 could therefore be modified to XRl at a site other than the N-terminus. From the partial homology of the XR peptide fragments, this enzyme could be classified into the aldose reductase super family. The histidine residue in the consensus sequence (the 16th histidine of Peptide 5) corre-

NADPH-DEPENDENT

VOL. 79. 1995

100

REDUCTASES FROM C. TROPZCALZS

D-XYLOSE

XRl

'2595,305 121e.c

1142.5 I

221

Avg. corn ound mass 364 gP 7.91

1522.0

16

5 1850.5

I

1730.0

L 1500 m/z

XR2 Avg. compound mass 36,539.08

1828.5 L

1000

1100

1200

1300

1400

1500

1800

1700

1800

1024.0

IQ00

ml2

FIG. 5. Mass spectra of XRl and XR2. Samples were dissolved in 0.1% trifluoroacetic spectrometer.

sponds to the 110th histidine of human aldose reductase, which is considered to play an important role in the orientation of substrates in the active site pocket (36). Probably, this sequence is the part of the substrate binding region. ACKNOWLEDGMENTS We thank Drs. Yoshihisa Umeda and Keiji Ogawa of Takara Shuzo Co. Ltd. for conducting the mass spectrometry. We also thank Miss Yukie Tanahashi for assistance in performing of the experiments. REFERENCES 1. Emodi, A.: Xylitol: its properties and food applications. Technol., 32, 20-32 (1978).

Food

acid solution and injected into API III mass

2. Pepper, T. and Olinger, P.M.: Xylitol in sugar-free confections. Food Technol., 10, 98-106 (1988). 3. Siiderling, E., Isokangas, P., Tenovuo, J., Mustaknllio, S., and Miikinen, K.: Long-term xylitol consumption and mutants streptococci in plaque and saliva. Caries Res., 25, 153-157 (1991). 4. Hofer, M., Betz, A., and Kotyk, A.: Metabolism of the obligatory aerobic yeast Rhodotoruka gracilis. IV. Induction of an enzyme necessary for D-xylose catabolism. Biochim. Biophys. Acta, 252, l-12 (1971). 5. Tomoeda, M. and Horitsu, H.: Pentose metabolism by Candida utilis. I. Xylose isomerase. Agr. Biol. Chem., 28, 139-143 (1964). 6. Kubata, B.K., Suzuki, T., Horitsu, H., Knwai, K., and Takamizawa, K.: Purification and characterization of Aeromonas caviae ME-l xylanase V, which produces exclusively xylobiose from xylan. Appl. Environ. Microbial., 60, 531-535

222

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XR2

XRl 100

Peak No. Compound mau, 887.88 9 1834.77

1

Peak No. 2’

Compound malo SS7.4S

4’ 6’

1634.77

1769&S 2259.74 3133.67 2584.24 3098.75 3681.79

6’ 8’ 11’

16’

25’

29.4

1

368 Sca&Ilme (mln)

;9”: 441

51

38 5008

z

51.5 700

5 Scan/Time (mln)

FIG. 6. LC/MS peptide maps of XRl and XR2 fragments by lysylendopeptidase digestion. Both XRs were digested with lysylendopeptidase (1 nmol/ml) for 12 h at 37OC. The proteolytic fragments were separated by an ODS column eluted by 0.1% trifluoroacetic acid-H20 and an acetonitrile gradient (l%/min) at a flow rate of 200 ,ul/min.

XRl-PEP.4 XR-Pi&a

Homology (96)

Amino acid sequence

Rotein Candida tropicalis st@itis

XRl-PEP.5

C. rropicalis

Reference

HVETALNKTLSDL DVEKALDRTLSDL

84.6

(16, 17)

TLSDLDLDYVDLFLIHFPIA

XR-P. stipitis

TLSDLQVDYVDLFLIEFPVT

80.0

AR-Human

TLSDLKLDYLDLXLIHWPT~

70.0

(16, 17) (lB-22)

AR-Rat

TLBDLQLDYLDLYLIEWPTQ

70.0

(22)

AR-Bovine

TLSDLKLDTLDLYLIENPTO

70.0

(23)

AR-Rabbit

TLDDLILDYLDLYLIDVPTO

65.0

(24)

AR-House mouse

TLSDLKLDYLDLYLVRUPQO

65.0

(25)

Consensus sequence

TLuDLklDIlDLFLiBwPtq

FIG. 7.

Amino acid sequences and homologous searches for Peptide fragments 4 and 5 of XRl.

(1994). 7. Box, G. and Hunter, W.: Multi-factor experimental design for exploring response surfaces. Ann. Math. Stat., 28, 195-241 (1957). 8. Horltsu, H., Yahashi, Y., Takamlsawa, K., Kawal, K., Suzuki, T., and Watanabe, N.: Production of xylitol from Dxylose by Candida tropicalis: optimization of production rate. Biotechnol. Bioeng., 40, 1085-1091 (1992). 9. Thompson, S. T., Cass, K. H., and Stellwagen, E.: Blue dextran-Sepharose: an affinity column for the dinucleotide fold in proteins. Proc. Nat. Acad. Sci. USA, 72, 669-672 (1975). 10. Lowry, 0. H., Rosebrougb, N. J., Farr, A. L., and Randall, R. J.: Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193, 265-275 (1951). 11. Laemmli, U.K.: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680685 (1970). 12. Davies, G. E. and Stark, G. R.: Use of dimethyl suberimidate, a cross-linking reagent, in studying the subunit structure of oligomeric proteins. Proc. Nat. Acad. Sci. USA, 66, 651-656 (1970). 13. DNinS, A. I’., Covey, T. R., and Henion, J. B.: Ion spray inter-

14.

15.

16.

17.

18.

19.

face for combined liquid chromatography/atmospheric pressure ionization mass spectrometry. Anal. Chem., 59, 26422646 (1987). Whitehouse, C. M., Dreyer, R. N., Yamashita, M., and Fenn, J. D.: Electrospray interface for liquid chromatographs and mass spectrometers. Anal. Chem., 57, 675-679 (1985). Fenn, J. B., Mann, M., Meng, C. K., Wong, S. F., and Wbitehouse, C. M.: Electrospray ionization for mass spectrometry of large biomolecules. Science, 246, 64-71 (1989). Amore, R., Kiitter, P., Kilster, C., Ciriacy, M., and P.Hollenberg, C.: Cloning and expression in Saccharomyces cerevisiae of the NAD(P)H-dependent xylose reductase-encoding gene (XYLI) from the xylose-assimilating yeast Pichiu stipitis. Gene, 169, 89-97 (1991). Hallborn, J., Walfrldsson, M., Airaksinen, U., Ojamo, H., Hahn-Hiigerdal, B., Penttilii, M., and Kerilnen, S.: Xylitol production by recombinant Saccharomyces cerevisiae. Bio/ Technol., 9, 1090-1095 (1991). Chung, S. and LaMendola, J.: Cloning and sequence determination of human placental aldose reductase gene. J. Biol. Chem., 264, 14775-14777 (1989). Bohren, K. M., Bullock, B., Wermuth, B., and Gabbay, K. H.:

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20.

21.

22.

23.

24.

25.

26.

27.

28.

NADPH-DEPENDENT

The aldo-keto reductase superfamily. J. Biol. Chem., 264, 9547-9551 (1989). Graham, A., Hedge, P. J., Powell, S. J., Riley, J., Brown, L., Gammack, A., Carey, F., and Markham, A. F.: Nucleotide sequence of cDNA for human aldose reductase. Nut. Acids Res., 17, 8368 (1989). Grundmann, U., Boho, H., Obermeier, R., and Amano, E.: Cloning and prokaryotic expression of a biologically active human placental aldose reductase. DNA and Cell Biol., 9, 149-157 (1990). Carper, D., Nishimura, C., Shinohara, T., Dietschold, B., Wiitow, G., Craft, C., Kador, P., and Kinoshita, J. H.: Aldose reductase and r-crystallin belong to the same protein superfamily as aldehyde reductase. FEBS Lett., 220, 209-213 (1987). Schade, S. Z., Early, S. L., Williams, T. R., Kbzdy, F. J., Heinrlkson, R. L., Grlmshaw, C. E., and Doughty, C. C.: Sequence analysis of bovine lens aldose reductase. J. Biol. Chem., 265, 3628-3635 (1990). Garcia-Perez, A., Martin, B., Murphy, H. R., Uchida, S., Murer, H., Benjamin, D., Cowley, J., Handler, J. S., and Burg, M. B.: Molecular cloning of cDNA coding for kidney aldose reductase. J. Biol. Chem., 264, 16815-16821 (1989). Pailhoux, E. A., Martinez, A., Veyssiere, G. M., and Jean, C. G.: Androgen-dependent protein from mouse vas deferens. cDNA cloning and protein homology with the aldo-keto reductase superfamily. J.~ Biol. Chem., 265, 19932-19936 (1990). Verduvn. C., van-Diikea. J. P.. and Scheffers. W. A.: Multiule forms-of xyiose redictasd in Pkhysolen tann~philw CBS4&. FEMS Microbial. Lett., 30, 313-317 (1985). MaehovB, E.: Induction of aldose reductase and polyol dehydrogenase activities in Aureobasidium pullulans by Dxylose, L-arabinose and o-garactose. Appl. Microbial. Biotechnol., 37, 374-377 (1992). Suzuki, T. and Onishi, H.: Purification and properties of polyol: NADP oxidoreductase from Pichia quercuum. Agr.

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