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 Chemistry, Kyoto University, Kyoto 606, Japan Received September 12, 1977 Glycolaldehyde dehydrogenase activity in cell-free extract from Escherichia coli B was repressed to 66%, when 10ƒÊM of pyridoxal were added to the growth medium. Ten micro molar of pyridoxine, pyridoxal, pyridoxine 5 L-phosphate, pyridoxal 5 L-phosphate or pyri doxamine 5 L-phosphate added in the growth medium repressed completely the formation of an isozyme C of the enzyme. The formation of isozyme B was repressed by 10ƒÊM of pyridoxine 5 L-phosphate or pyridoxal 5 L-phosphate. One hundred micromolar of pyridoxine, added in the enzyme assay system, inhibited to 33% the isozyme C non-competitively for glycolate. Ki value was calculated to be 0.33 mm. A certain activation by pyridoxine was observed for isozyme B. Glycolaldehyde dehydrogenase was firstly evidenced to be involved in vitamin B6 biosyn thetic pathway in Escherichia coli B by the authors.1,2) In preceding paper,3) it was also found that E. coli B WG1, the wild type strain, has three glycolaldehyde dehydrogenase iso zymes, A, B and C. Dempsey suggested that vitamin B6 biosyn thesis of E. coli B may be controlled by feedback inhibition4) and repression.5) While, the authors6) reported that Flavobacterium sp. 238-7, which produces a high amount of vita min B6, lacks feedback control mechanism in the vitamin B, biosynthesis, Followingly, Nishio et al. discussed the lack of control mechanism with respect to the forms of vitamin B6 produced by the organism.7) However, there has been no enzymatic study on the control of vitamin B, biosynthesis. In this paper, is described the effect of various vitamin B6 compounds on the activity and the enzyme formation of glycolaldehyde dehydrogenase isozymes of E. coli B. MATERIALS AND METHODS Microorganisms. The organism, E. coli B WG1, t Studies on Vitamin B, Metabolism in Microor ganisms. Part XIV. For Part XIII, see reference 3). * deceased. and the cultivation method with the basal medium con taining casamino acid and glycerol were the same as described previously.3) Enzyme activity. Glycolaldehyde dehydrogenase activity was assayed by the NADPH2 method.3) One unit and specific activity of the dehydrogenase were defined as described previously.1) Protein was deter mined by the method of Lowry et al.8) and by measur ing the optical absorbance at 280 nm. Preparation of isozymes. The isozymes were prepared by the separation on DEAE-cellulose column chromatography from the cell-free extract of E. coli B WG1. RESULTS Effect of vitamin B6 compounds on the formation of glycolaldehyde dehydrogenase E. coli B WG1 was cultivated in 500 ml of the basal medium supplemented with various amounts of pyridoxal. The total activity of glycolaldehyde dehydrogenase was assayed in the cell-free extract from each culture. Figure 1 shows the inhibitory effect of pyri doxal on the total activity. About 40 decrease of the activity was observed when The following abbreviations are used: pyridoxine-p, PNP; pyridoxine 5 L-phosphate: pyridoxamine-p, PMP; pyridoxamine 5 L-phosphate: pyridoxal-p, PLP; pyri doxal 5 L-phosphate: PN; pyridoxine: PM; pyridoxa mine: PL; pyridoxal.
70 H. MORITA, Y. TANI and K. OGATA pyridoxal was added in the medium at the con centration of 10ƒÊM. The repression on the total activity of glyco laldehyde dehydrogenase was further studied with the isozymes. E. coif B WG1 was cul tivated in 2 liters of the basal medium con taining each one of the vitamin Be compounds at the concentration of 10ƒÊM. From each cell-free extract, isozymes were separated by DEAE-cellulose column chromatography, as illustrated in Fig. 2. The total activity of the each isozyme was described in Table I. Al though the decrease of the activities of all isozymes was observed with the addition of each vitamin Be compound, the complete re pression of the isozymes B and C was apparent for the cells grown in the media which were supplemented with the phosphorylated forms of vitamin Be. The isozyme C was not found FIG. 1. Effect of Pyridoxal Supplement in the Me dium on the Total Activity of Glycolaldehyde De hydrogenase. scribed previously.8) Cell-free extract was added in the assay mixture for the enzyme. also in the cells grown in the medium which was supplemented with pyridoxine or pyridoxal. On the other hand, the inhibitory effect of pyridoxamine on the enzyme formation was remarkable on the isozyme A. FIG. 2. DEAE-cellulose Column Chromatography of Glycolaldehyde Dehydrogenase f rom E scherichia coli B WG1 Grown in the Medium Supplemented with (a) Vitamin Be Compound or (b) Phosphorylated Vitamin B6 Compound. The column chromatography and enzyme assay were carried out by the method described pre viously.3) A, B and C indicate the isozyme A, B and C from E. coli 13 WG1, respectively.
Vitamin Be Biosynthesis 71 medium was compared with those from the cells grown in the presence of various forms of vitamin Be at the concentration of 100ƒÊM. Among six forms of vitamin Be tested, pyri doxine, pyridoxine-p and pyridoxal apparent ly inhibited the activity, as shown in Table II. TABLE II. EFFECT OF VITAMIN Be COMPOUNDS ON THE ACTIVITY OF GLYCOLALDEHYDE DEHYDROGENASE scribed in the text. Cell-free extract was added in the assay mixture for the enzyme. TABLE 1. FIG. 2. (b) THE ACTIVITY OF EACH ISOZYME OF GLYCOLALDEHYDE DEHYDROGENASE FROM Escherichia coli B WG1 GROWN IN THE MEDIUM SUPPLEMENTED WITH VITAMIN Be COMPOUNDS described previously.3) To clarify the mechanism of inhibition of glycolaldehyde dehydrogenase by vitamin Be, the activity of each isozyme was assayed in the presence of each of vitamin B6 compounds. One hundred micromolar of one of six forms of vitamin Be were added to the reaction mixture TABLE III. EFFECT OF VITAMIN Be COMPOUNDS ON THE ACTIVITY OF GLYCOLALDEHYDE DEHYDROGENASE ISOZYMES The enzyme activity was assayed in a cuvette con taining 33ƒÊmoles of sodium glycolate, 0.17ƒÊmole of NADPH2, 0.17 mmole of potassium phosphate buffer (ph 6.0), 0.1ƒÊmole of one of the vitamin B6 com pounds and a suitable amount of the isozyme in a total volume of 0.92 ml. Effect of vitamin B6 compounds on the activity of glycolaldehyde dehydrogenase The activity of glycolaldehyde dehydro genase in the cell-free extract prepared from the cells of E. coli B WG1 grown on the basal to assay the activity of each one of the isozymes (Table III). None of vitamin B6 compounds considerably affected the activity of the isozyme
72 H. MORITA, Y. TANI and K. OGATA FIG. 3. Effect of Pyridoxine on the Activity of Iso zyme B. described previously,3) except the addition of pyri doxine in the reaction mixture. A, except that pyridoxal activated it. The iso zyme B was activated by pyridoxine and pyri doxal-p. The isozyme C was appreciably inhibited by pyridoxine. Then the effect of pyridoxine concentration on the activity of isozyme B was investigated. Figure 3 shows that the activity increased markedly around the concentration of 0.1 mm. The rate of activation was decreased by pyri doxine at the concentrations higher than 0.1 mm. For the investigation of the effect of pyri doxine concentration on the activity of the isozyme C, the enzyme activity of isozyme C was assayed in the reaction mixture in the presence of various concentrations of pyri doxine. The activity was decreased propor tionally to the amount of pyridoxine, as shown in Fig. 4. The concentration of pyridoxine which inhibited the enzyme activity to 50 (I50) was estimated to be 0.39 mm. The mode of inhibitory effect of pyridoxine on the isozyme C was examined by Hofstee plot (S/V-S plot). As shown in Fig. 5, the inhi bition was found to be non-competitive with glycolate. Ki value for the pyridoxine was calculated to be 0.33 mm. FIG. 4. Effect of Pyridoxine on the Activity of the Isozyme C. scribed previously,3) except the addition of pyrido xine in the reaction mixture. FIG. 5. Inhibition of the Activity of the Isozyme C by Pyridoxine. described previously,3) except the addition of 0.1 mm of pyridoxine in the reaction mixture in the case indi cated as +PN on this figure. S indicates the concen tration of glycolate. V indicates the specific activity of the enzyme which is expressed as unit. DISCUSSION In this paper, it is suggested that the forma tion and the activity of glycolaldehyde de hydrogenase are controlled by vitamin B6 compounds. Pyridoxal-P is known to be an inhibitor of
Vitamin B6 Biosynthesis 73 some enzymes which contain lysine residues in their active site.9) As shown in Table III, pyridoxine, but not pyridoxal nor pyridoxal-p, inhibited the isozyme C. This fact shows that the inhibition cannot be ascribed to Schiff base formation between vitamin B6 compounds and lysine residues in the active site of the enzyme. The inhibitory mechanism should be specific to the feedback mechanism on the possible first enzyme of vitamin B6 biosynthetic pathway. The forms of produced vitamin B6 were discussed using E. coli7,10) or Flavo bacterium sp. 238-7,7) in which vitamin B6 biosynthesis is controlled or non-controlled by vitamin B6, respectively. The present result will give further information and speculation on this problem. Activation of enzyme by pyridoxal has already been observed in horse liver alcohol dehydrogenase,11) but the activation by pyridoxine and pyridoxal-p has not been re ported yet. Glycolaldehyde dehydrogenase isozyme B is firstly found to be activated by pyridoxine and pyridoxal-p as shown in Table III and Fig. 3. However, the physiological significance of the activation is not clear yet. In the present work, all the phosphorylated vitamin B6 compounds repressed the formation of the glycolaldehyde dehydrogenase, as shown in Fig. 2, when E. coli B was grown in the pho sphate rich medium. These results suggest intact incorporation of phosphorylated vitamin B6 compounds into E. coli like human red blood cells.12) Dempsey observed that 0.6ƒÊ M of pyridoxine-p, pyridoxamine or pyrido xamine-p added in the medium did not re press the synthesis of vitamin B6 in E. coli B.4) Yamada et al. reported that E. coli KG980 (vitamin B6 auxotroph derived from K-12 strain) could not utilize the phosphorylated vitamin B6 compounds when it was grown in a phosphate rich medium.13) The relation of these results should be made clear by complete elucidation about the biosynthetic pathway and the incorporation mechanism of vitamin B6. Acknowledgement. The authors wish to thank Prof. H. Yamada, Kyoto University, for his interest and help ful discussion. REFERENCES 1) Y. Tani, H. Morita and K. Ogata, Agric. Biol. Chem., 38, 2057 (1974). 2) Y. Tani, H. Morita and K. Ogata, ibid., 41, 1749 (1977). 3) Y. Tani, H. Morita, H. Nishise and K. Ogata, ibid., 42, 63 (1978). 4) W. B. Dempsey, J. Bacteriol., 90, 431 (1965). 5) W. B. Dempsey, ibid., 108, 415 (1971). 6) Y. Tani, T. Nakamatsu, Y. Izumi and K. Ogata, Agric. Biol. Chem., 36, 189 (1972). 7) N. Nishio, T. Suzuki, M. Hayashi and T. Kami kubo, J. Ferment. Technol., 55, 277 (1977). 8) O. H. Lowry, N. J. Rosebrough, A. L. Farr and R. T. Randall, J. Biol. Chem., 193, 265 (1951). 9) S. Ronchi, M. C. Zapponi and G. Ferri, Eur. J. Biochem., 8, 325 (1969). 10) W. B. Dempsey and L. J. Arcement, J. Bacteriol., 107, 580 (1971). 11) D. C. Sogin and B. V. Plapp, J. Biol. Chem., 250, 205 (1975). 12) R. Suzue and M. Tachibana, J. Vitaminol.,16, 16,164 (1970). 13) R. Yamada, T. Tsuji and Y. Nose, J. Nutr. Sci. Vitaminol., 23, 7 (1977).