Diversity of Glycerol Dehydrogenase in Methylotrophic Yeasts

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1 Agric. Biol. Chem., 51 (9), , Diversity of Glycerol Dehydrogenase in Methylotrophic Yeasts Keiko Yamada and Yoshiki Tani Research Center for Cell and Tissue Culture, Faculty of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606, Japan Received March 19, 1987 Twotypes of glycerol dehydrogenase (GDH) were found on DEAE-cellulose column chromatography of cell-free extracts of methylotrophic yeasts. One type, designated as GDHI, showed only the reductive activity which was detected in the reaction system containing dihydroxyacetone and NADH,at ph 6.0. The other type, designated as GDHII, showed the oxidative activity which was detected in the system containing glycerol and NAD+, at ph 9.0, together with the reductive activity. Candida boidinii No. 2201, which possesses the phosphorylative pathway for glycerol dissimilation, had only GDHI when grown on glycerol or methanol as the carbon source. Hansenula ofunaensis, which has the oxidative pathway, had both GDHI and GDHII when grown on glycerol, but only GDHI when grown on methanol. Hansenula polymorpha DL-1, which has both pathways, had both GDHI and GDHII when grown on glycerol or methanol. The significance of glycerol dehydrogenase (GDH) in glycerol metabolism has been discussed by many workers. NAD+-Linked GDH was reported to be involved in the pathway for glycerol catabolism in Schizosaccharomyces pombe}'2) Klebsiella pneumoniae utilizes glycerol anaerobically by means of NAD+-linked GDH.3) The involvement of NADP+-linked GDHin glycerol metabolism was also reported for Neurosporaerassa.4) Babel and Hofmann suggested that GDH was used for glycerol assimilation by some methylotrophic yeasts.5) In previous papers,6'7) we showed that methylotrophic yeasts exhibited three different patterns, represented by Candida boidinii (Kloeckera sp.) No. 2201, Hansenula ofunaensis and Hansenula polymorphadl-1, with regard to the initial step of glycerol dissimilation; the phosphorylative pathway involving glycerol kinase in C. boidinii No. 2201, the oxidative pathway involving NAD+-linked GDHin H. ofunaensis, and both pathways in H. polymorpha DL-1. The activity level of NAD+-linked GDHin H. polymorpha DL-1 was found to be the same when glycerol and methanol were used as the sole carbon source, and to be induced by transfer from glucose medium to methanol medium. Enzyme activity which preferentially converted dihydroxyacetone to glycerol was also detected in extracts of these strains grownon methanol. From these results, it was suggested that there might be a variety of GDHin the methylotrophic yeasts. In the present paper, the multiformity of GDHin these representative methylotrophic yeasts, i.e., C. boidinii No. 2201, H. ofunaensis and H. polymorpha DL-1, will be demonstrated. MATERIALS AND METHODS Microorganisms. C. boidinii No. 2201, H. ofunaensis and H. polymorpha DL-1 were from our laboratory collection.8* Chemicals. All chemicals were from usual commercial sources and used without further purification. Cultivation. The basal medium consisted of 0.5g NH4C1, 0.1g K2HPO4, 0.1g KH2PO4, 0.05g MgSO4-7H2O. 3mg FeCl3-6H2O, 1mg CaCl2-2H2O, 1mg

2 2402 K. Yamada and Y. Tani MnSO4-3H2O, 1mg ZnSO4-7H2O, 0.2mg thiamin- HC1 and 2/ig biotin, in 100ml of tap water, ph 6.0. Glycerol, methanol or glucose as the carbon source was added to the basal medium at the concentration of 1%(w/v). Cultivation of each yeast was carried out in 500ml of the medium in a 2-1 shaking flask at 28 C under reciprocal shaking at 120rpm until the late logarithmic phase. The inoculum was 5ml of a seed culture with the same medium. Enzymepreparation and assay. All operations for the enzyme preparation were performed at 0 ~ 5 C. Potassium phosphate buffer, ph 7.0, containing 0.5 mmdithiothreitol was used throughout, unless otherwise noted. Cells from 500-ml and 5-1 cultures were suspended in about 30 ml and 300 ml of0.01 m buffer, respectively. A cell-free extract was prepared by ultrasonic oscillation followed by centrifugation as described previously.6) GDHactivities were determined as follows: the oxidative activity was determined by measuring the reduction of NAD+in a system containing the enzyme solution, 100mM NH4C1-NH4OH, ph 9.0, 100mM glycerol and 0.17mM NAD+,in a total volume of3ml. As a reference, the reaction mixture without glycerol was used. The reductive activity was determined by measuring the oxidation of NADH in a system containing the enzyme solution, 100mM K2HPO4-KH2PO4, ph 6.0, 100mM dihydroxyacetone and 0.17mM NADH, in a total volume of 3 ml. As a reference, the reaction mixture without dihydroxyacetone was used. The enzyme unit was defined as described previously.7) The concentration of protein was determined by measuring the absorbance at 280nm and with a BIO-RADprotein assay kit. The conductivity of the eluate from the chromatographic column was monitored with a CD-35MII conductivity meter (M&SInstruments Inc.) at 10 C. RESULTS Determination of two types of GDH A cell-free extract prepared from glycerolgrowncells of H. ofunaensis wasapplied to a DEAE-cellulose column. Figure 1 shows the elution pattern with a gradient system of 0 to 1m KC1 in 0.03m buffer. The GDHactivity appeared in two fractions. The first fraction was eluted with 0.03m buffer containing m KC1, corresponding to a conductivity of around lommho, and represented the reductive activity. The second fraction was eluted with 0.03m buffer containing 0.24m KG1, corresponding to a conductivity of around 40 mmho,and represented both the reductive and oxidative activities. The recovery of activity in the eluate from the column was calculated to be 124% for the reductive activity and 1 10% for the oxidative activity, respectively. These recoveries showed that there was no dispersion of the activity during the prepara- I 1 I.2.0- : \ E : \ : i -^ oo : I\"à" : "" jc HL «1.0- : I V...à"à"à"K.> /' -J, -53 J ^g5^ ^bcoccccccjo Jo Fraction No. (10 ml/tube) Fig. 1. Separation of GDHby DEAE-Cellulose Column Chromatography with a Gradient System. A cell-free extract of H. ofunaensis prepared from a 5-1 culture in the glycerol mediumwas applied on a DEAE-cellulose column (5 x 22 cm), which had been equilibrated with 0.01 m buffer. The column was washed with 0.03m buffer and then the absorbed enzyme was eluted with a gradient ofkc1, from 0 to 1 m, in 0.03m buffer at a flow rate of 75 ml/hr. The eluate was collected in 10 ml fractions. The oxidative (#) and reductive (O) activities of GDHin each fraction were determined as described under Materials and Methods.

3 Glycerol Dehydrogenase in Methylotrophic Yeasts 2403 tion procedure and the appearance of all the GDH in the cell-free extract in the chromatogram. Fromthe results, we concluded that there were two types of GDHin the cell-free extract of H. ofunaensis, which were detected with the assay system and separated into two fractions with the elution system employed: One type had only the reductive activity, and was designated as GDHI. The other type had both the reductive and oxidative activities, and was designated as GDH II. Thus, GDHin methylotrophic yeasts might be concerned in not only the oxidation of glycerol as the first step of the oxidative pathway for glycerol dissimilation but also in the reduction of dihydroxyacetone. Consequently, the GDHpatterns of C. boidinii No. 2201, H. ofunaensis and H. polymorpha DL-1 were compared in the following experiments, using cells grown on glycerol, methanol or glucose as the carbon source. GDH of C. boidinii No.2201 The specific activity of GDHin the cell-free extract prepared from C. boidinii No grownon glycerol, methanol or glucose was determined (Table I). The oxidative activity Table I. Activities of GDHin Cell-free Extracts of C. boidinii NO. 2201, H. ofunaensis and H. polymorpha DL-1 When Grown on Glycerol, Methanol or Glucose The oxidative and reductive activities of GDHin the cell-free extracts were determined as described under Materials and Methods. Carbon source.,. Glycerol Methanol Glucose Strain Specific activity (units/mg) Reductive Oxidative Reductive Oxidative Reductive Oxidative C. boidinii No Trace 0.80 Trace Trace H. ofunaensis Trace Trace 0 H. polymorpha.dl Glycerol Methanol O Glucose 2.o- 0.03M 0.07 M 0.03 M 0.07 M 0.03 M 0.07 M -4.o^ I : o O i I Q - -20I :, A I C> ^ S Fraction No. (5 ml/tube) Fig. 2. DEAE-Cellulose Column Chromatography of GDHof C. boidinii No A cell-free extract prepared from glycerol-, methanol- or glucose-grown cells from a 500-ml culture was applied on a DEAE-cellulose column (1.8 x 20 cm), which had been equilibrated with 0.01 m buffer. Elution was performed stepwisely with 0.03m, 0.07m and 0.1 m buffer, 0.1 m buffer containing 0.2m KC1 (0.3m) and finally 0.1 m buffer containing 0.5m KC1 (0.6m) at a flow rate of 75ml/hr. The eluate was collected in 5-ml fractions. The oxidative (#) and reductive (O) activities of GDHwere determined as described under Materials and Methods.

4 2404 K. Yamada and Y. Tani could hardly be detected in any of the cell-free extracts. The reductive activity was found in all cell-free extracts, that in the extract from cells grown on methanol being the highest. Each cell-free extract was applied to a DEAE-cellulose column. The absorbed enzyme was eluted in a stepwise manner, on the basis of the data obtained with a gradient elution system. GDHactivity was detected in fractions eluted with 0.03 m and 0.07 m buffer. Figure 2 shows the elution pattern of each fraction of a DEAE-cellulose column. GDH activity of cell-free extracts of glycerol- and methanol-grown cells was found in the 0.03 m and 0.07m fractions. The activity in that of glucose-grown cells appeared only in the 0.07 m fraction. Each fraction showed the reductive activity but not the oxidative activity, and thus corresponded to GDHI of H. ofunaensis. No detectable activity of GDHII was found in any fractions of the eluate for cell-free extracts of glycerol-, methanol- and glucosegrown cells. To compare the activities of GDH I in glycerol-, methanol- and glucose-grown cells, the total activity of GDHI in each fraction was calculated with the following formula; total units of the reductive activity in each fraction x 100/mg of protein in the cell-free extract with each carbon source. As shown in Table II. Total GDHActivity in Each Fraction of Eluates on DEAE-Cellulose ColumnChromatography The total reductive activity in each fraction obtained on DEAE-cellulose column chromatography was calculated as described in the text. DEAEc.. Cellulose Carbon source Strain _ frac tion eluted with Glycerol Methanol Glucose C. boidinii 0.03 m No m H. ofunaensis 0.07 m m 92 Trace 0 H. polymorpha 0.03 m DL m Table II, the total activity in the 0.03 m fraction of the cell-free extract of methanol-grown cells was 3.6-fold that of glycerol-grown cells. The total activity of the 0.07m fraction of methanol-grown cells was about 1 1-fold those of glycerol- and glucose-grown cells. From these results, it seems that C. boidinii No. 2201, which dissimilates glycerol through the phosphorylative parhway, which involves glycerol kinase, has GDH I, which plays a significant role in methanol metabolism rather than in glycerol metabolism. GDHof H. ofunaensis The GDHactivity of H. ofunaensis, which dissimilates glycerol through the oxidative pathway, was estimated as the specific activity of oxidation and reduction when grown on glucose, glycerol or methanol. As shown in Table I, cells of H. ofunaensis exhibited the high oxidative activity in the glycerol medium, but little or no activity in the glucose or methanol medium. This coincided with the occurrence ofcatabolite repression of GDHby glucose in the yeast, as previously reported.7) The reductive activity was found in cell-free extracts from both glycerol- and methanolgrown cells, and that of glycerol-grown cells was 3.9-fold that of methanol-grown cells. The reductive activity was hardly found in glucosegrowncells. Subsequently, the cell-free extract of cells grown on glycerol, methanol or glucose was applied to a DEAE-cellulose column in the same manner as in the case of C. boidinii No (Fig. 3). GDH activity was detected in fractions eluted with 0.07m buffer and 0.1 m buffer containing 0.2m KC1 (0.3m). GDH of glycerol-grown cells appeared in the 0.07m fraction as GDHI and in the 0.3m fraction as GDHII. GDHof methanol-grown cells was found only in the 0.07m fraction as GDHI. Neither GDHI nor GDHII was detected in the eluate of the cell-free extract of glucosegrown cells. The total activity of GDHI eluted in the 0.07m fraction was somewhat higher in the cell-free extract of methanol-growncells than in that of glycerol-grown cells, as shown

5 Glycerol Dehydrogenase in Methylotrophic Yeasts 2405 Glycerol q Methanol j Glucose 0.07M I0.3M A 0.07M \0.3M à" ; 0.07M 03M å # I! - -. I I - -I J i l l! If w10- :: -2.o... :; à" I ^ ^ ^ o Fraction No. (5ml/tube) Fig. 3. DEAE-Cellulose Column Chromatography of GDHof H. ofunaensis. The experimental conditions and symbols were the same as in Fig. 2. Glycerol 0.03M Methanol 0.03M.,0.07M 0.07M rnkrr" *&;å Glucose 0.03M 0.07M - J0 Fraction 50 0 No. 50 (5 0 ml/tube) Fig. 4. DEAE-Cellulose Column Chromatography of GDHof H. polymorpha DL-1. The experimental conditions and symbols were the same as in Fig. 2. in Table II. These results show that GDH I might be significantly involved in methanol metabolism in H. ofunaensis. GDHII should be involved in the oxidative pathway for the glycerol dissimilation. GDHof H. polymorpha DL-1 It was shown that H. polymorpha DL-1 possessed the oxidative pathway together with the phoshorylative pathway for glycerol dissimilation, and its GDHwas subject to catabolite repression by glucose.6'7) The oxidative activity was found in the cell-free extract of glycerol-, methanol- or glucose-grown cells, as shown in Table I. The activities in glycerol- and methanol-grown cells were 8.1-fold and 9.5-fold that in glucose-grown cells, respectively. Cells grown on glycerol or methanol showed 5.1-fold and 5.7-fold the reductive activity of those grown on glucose, respectively. GDHin the cell-free extract of cells grown on glycerol, methanol or glucose was separateraphy, by DEAE-cellulose column chromatog- as described above. The activity appeared in the 0.03m and 0.07m fractions (Fig. 4). The activity in the 0.03m fraction was low but was found in eluates of cell-free extracts with all carbon sources. The fraction showed only the reductive activity and thus corresponded to 0.07m fraction GDHI of H. ofunaensis. The GDHwas also found in cells grownon all was high in carbon sources, and the content glycerol- and methanol-grown cells. These three 0.07 m fractions showed both

6 2406 K. Yamada and Y. Tani the oxidative and reductive activities, and thus corresponded to GDHII of H. ofunaensis. Table II shows that the 0.03m fraction GDHI was induced by methanol, up to two-fold the activity in glycerol- and glucose-grown cells. The level ofgdhi in cells grown on glycerol was the same as that in those grown on glucose. The total activities of GDHII in the 0.07m fractions from cells grown on glycerol and methanol were 6.7-fold and 9.0-fold that in the case of glucose medium, respectively. These results show that GDH I might be involved in methanol metabolism in H. polymorpha DL-1, and that it is formed rather constitutively compared to those of C. boidinii No and H. ofunaensis. The high activity of GDHII in methanol-grown cells showed its possible involvement in methanol metabolism as well as glycerol dissimilation. DISCUSSION On the basis of the activity ofnad+-linked GDHin methylotrophic yeasts, the enzyme was separated into two types, GDHI having reductive activity and GDHII having both reductive and oxidative activities. In previous papers,6'7) it was demonstrated that C. boidinii No had glycerol kinase activity, which was induced by glycerol. H. ofunaensis exhibited little glycerol kinase activity but had GDHactivity which was induced by glycerol. H. polymorpha DL-1 had glycerol kinase activity, which was induced by glycerol, and Methanol / HCHO GDHactivity, which was induced by glycerol and methanol. It was demonstrated in the present study that C. boidinii No. 2201, H. ofunaensis and H. polymorpha DL-1 had GDH I, which was inducibly formed in methanolgrown cells. GDHII was found in H. ofunaensis and H. polymorpha DL-1, which was inducibly formed in glycerol-grown cells, and glycerol- and methanol-grown cells, respectively. From these results, the relation ship between the dissimilation of glycerol and that of methanol can be summarized as shown in Fig. 5. The activity of NAD+-linked GDHwas demonstrated in Candida methylica, although the strain cannot grow on glycerol.5) It was also shown that methylotrophic yeasts were capable of transforming dihydorxyacetone phosphate enzymatically into methylglyoxal,9) which is known to be a toxic compound.10) As reported in the previous paper,7) high dihydroxyacetone kinase activity was found in extracts of C. boidinii No grown on methanol, and of H. ofunaensis and H. polymorpha DL-1 grown on methanol or glycerol. Thus, methylotrophic yeasts when grown on methanol might be able to control the cellular level of dihydroxyacetone by some means, by chance, using GDHI and possibly GDH II. Although the physiological role of GDHI in glycerol-grown cells is uncertain, it is likely to be inducibly formed by dihydroxyacetone formed from glycerol by GDHII. On the other hand, it was shown that glyc- X5P H. Qo^ oftnaensis I ^^^^- GL Glyceraldehyde3-P^ t aw' * > DH/ DHA ^..* ^H.polymoipteDL-1 O bgww Na 2201 G3P DHAP Fig. 5. Relation Ship between the Dissimilation of Glycerol and That of Methanol. GL, glycerol; DHA, dihydroxyacetone; G3P, glycerol 3-phosphate; DHAP, dihydroxyacetone phosphate; X5P, xylulose 5-phosphate. G3P

7 Glycerol Dehydrogenase in Methylotrophic Yeasts 2407 erol was produced through a reversible sequence dependent on an NADP+-linked GDH in Dunaliella.11] Ben-Amotz and Avron introduced the NADP-linked GDH of Dunaliella as NADP-linked dihydroxyacetone reductase though it catalyzed both the oxidative and reductive reactions.12) Further, an NADPH-dependent oxidoreductase was isolated from a fungus, Mucorjavanicus.13) From the facts that dihydroxyacetone was isolated from the mycelial extract and that it proved to be a highly active substrate, the enzyme was regarded as dihydroxyacetone reductase. Methylotrophic yeasts assimilate methanol through the xylulose monophosphate pathway, in which dihydroxyacetone is formed by means of fixation of formaldehyde to xylulose 5-phosphate.14) The reduction of dihydroxyacetone to glycerol might have some physiological role in the yeast though it is not involved in the regeneration system for xylulose 5-phosphate. Thus, it may be proper that the GDHI in the present study can be called an NAD-linked dihydroxyacetone reductase. The significance of the enzyme in methanol metabolism remains to be clarified in a further study. REFERENCES 1) J. W. MayandJ. Sloan, J. Gen. Microbiol., 123, 183 (1981). 2) C. Gancedo, A. Llobell, J.-C. Ribas and F. Luchi, Eur. J. Biochem., 159, 171 (1986). 3) E. C. C. Lin, Annu. Rev. Microbiol., 30, 535 (1976). 4) M. Viswanath-Reddy, S. N. Bennett and H. B. Howe, Mol. Gen. Genet., 153, 29 (1977). 5) W. Babel and K. H. Hofmann, Arch. Microbiol., 132, 179 (1982). 6) Y. Tani and K. Yamada, FEMS Microbiol. Let., 40, 151 (1987). 7) Y. Tani andk. Yamada, Agric. Biol. Chem., 51, 1927 (1987). 8) T. Yonehara (1986). and Y. Tani, Agric. Biol. Chem., 50, 899 9) W. Babel and K. H. Hofmann, FEMS Microbiol. Lett., 10, 133 (1981). 10) N. Krymkiewicz, E. Dieguez, U. D. Rekarte and N. Zwaig, J. Bacteriol., 10H, 1338 (1971). ll) A. D. Brown and L. J. Borowitzka, "Biochemistry and Physiology of Protozoa," Vol. 1, ed. by M. Levandowsky and S. H. Hunter, Academic Press, New York, 1979, p ) A. Ben-Amotz and M. Avron, FEBS Lett., 29, 153 (1973). 13) E. Hochuli, K. E. Taylor and H. Dutler, Eur. J. Biochem., 75, 433 (1977). 14) Y. Tani, "Methylotrophs: Microbiology, Biochemistry and Genetics," ed. by C.-T. Hou, CRC Press, Florida, 1984, p. 55.

Control of Glycolaldehyde Dehydrogenase in Vitamin B6 Biosynthesis. in Escherichia coli B õ. Hiroshi MORITA, Yoshiki TANI and Koichi OGATA*

Agric. Biol. Chem., 42 (1), 69 `73, 1978 Control of Glycolaldehyde Dehydrogenase in Vitamin B6 Biosynthesis in Escherichia coli B õ Hiroshi MORITA, Yoshiki TANI and Koichi OGATA* Department of Agricultural