SACCHAROMYCES CEREVISIAE

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1 THE EQUIVALENCE OF THIAMIN AND PYRIDOXINE FOR A STRAIN OF SACCHAROMYCES CEREVISIAE I. EFFECT ON GROWTH RATE AND CARBOXYLASE ACTIVITY' WILLIAM MOSES2 AND M. A. JOSLYN Division of Food Technology, University of California, Berkeley, California The existence of a possible relationship between thiamin (BA) and pyridoxine (B6) is inferred in the various reports in the literature that either vitamin may serve as a growth stimulant for differing strains of brewers' or bakers' yeasts. Schultz et al. (1940) reported that several yeast species could be characterized by their requirement for either B, or B6. Atlin et al. (1949) differentiated cultures of top yeasts from bottom yeasts by demonstrating the stimulatory effect of either B, or Be for top yeasts. Stokes et al. (1943) reported a pyridoxineless mutant of-neurospora which at any given level of pyridoxine nutrition demonstrated a growth response proportional to the available exogenous thiamin, though some growth was achieved in its absence. Several explanations for this apparent equivalence between thiamin and pyridoxine were considered for investigation, the most prominent of which were that: (a) either nutrilite might serve as the precursor of an intermediate from which the other may be synthesized, or otherwise catalyze the synthesis of the other, and so permit reversible interconversion of thiamin and pyridoxine; (b) one might serve as a functional substitute for the other in the synthesis of some vital intermediate produced through the influence of either. In the phase of the investigation described below, a comparative study was made of the quantitative effect of B, and BB on growth rate and on the carboxylase activity of one culture of top yeast, as well as of the general conditions under which a B, - B. equivalence occurred. MATERIALS AND METHODS Source of culture. The strain of yeast investigated was a transplant of the culture designated I Based on data presented in Ph.D. Thesis, University of California, February, Chemist, Wallerstein Company, New York. Received for publication February 16, 1953 as 6C44 in the Wallerstein Laboratories culture collection, and represents one of the five cultures of brewery top yeasts, examined by Atkin et al. (1949). Growth condition. The procedure and media used for growing the culture were essentially those described by Atkin et al. (1949). Pyridoxine assay. The yeast microbiological method for pyridoxine assay of Atkin et al. (1943) was used with the following modifications as recommended by Rabinowitz and Snell (1947) and Hopkins and Pennington (1947): (a) samples wereautoclaved at 25 pounds pressure for 5 hours; (b) tests were conducted in 125 ml Erlenmeyer flasks without shaking; (c) nicotinic acid was incorporated into the medium. Thiamin assay. The thiochrome method of Jansen (1936) as modified by Hennessy and Cerecedo (1939) was used for measuring thiamin concentrations. Since better recoveries were obtained without the absorption of thiamin by Decalso, that part of the procedure was omitted. Carboxylase activity. Carboxylase activity of the cultures was measured manometrically in a Warburg constant volume respirometer in an atmosphere of nitrogen using the decarboxylation of pyruvate as an index of carboxylase apoenzyme or coenzyme activity. The method of Lohmann and Schuster (1937) for the estimation of cocarboxylase was used for comparative study of the cocarboxylase activities of the culture grown under conditions of varying nutrition. Dried cellular preparations were made by washing the yeast, after harvesting, with two 50 ml portions of distilled water and drying at room temperature in a current of air. Micro-Kjeldahl nitrogen determinations showed that the cultures from the different media averaged 6.8 per cent i 0.1 per cent nitrogen. Boiled yeast extracts were prepared by suspending yeast dried as above in 5.0 ml of m/15 197

2 198 WILLIAMS MOSES AND M. A. JOSLYN phosphate buffer, ph 6.2, boiling for 5 minutes, and then adjusting to 10 ml with additional buffer. Yeast atiozymase was prepared by washing one gram of dried yeast with warm (30 C) m/10 Na2HPO4 solution and distilled water. The final suspension containing the residue was adjusted to a total volume of 10 ml with m/15 phosphate buffer, ph.6.2. Acetoin and diacetyl formation. The acetoin and diacetyl content of Warburg residues from IJ S Xt o CASEIN HYDROLYSATE AS N SOURCE TIME, A. Casein hycdrolyzate a nitrogen 8ource. Whereas Atkin et al. (1949) had found apparently slight differences in the amount of growth attained after a 40 hour growth period, marked differences are evident (figure 1) between growth in media A, B, and C at the 24 hour period at which point growth in medium A is almost three times that attained in media B and C. B. Ammonium sulfate a8 nitrogen 8ource. Differences in the rate as well as in extent of growth after 40 hours supported by media A, B, and C, HOURS Figure 1. Growth curves of top brewers' yeast in complete medium A, thiamin deficient medium B, and pyridoxine deficient medium C, with casein hydrolyzate as N source. The thiamin HCl and pyridoxine HCl were present at levels of 0.5 mg per liter. carboxylase tests were measured spectrophotometrically after treating the residual superatants with creatine and alkaline alphanaphthol according to the technique of Westerfeld (1945). RESUrLT Effect of Vitamins Bi and Be on Growth Rate Representative data on the rate of increase in wet weight of yeast grown in media A (complete), B (vitamin B1 deficient), and C (vitamin Be deficient) with casein hydrolyzate and ammonium sulfate, respectively, are shown in figures 1 and 2. [VOL. 66 respectively, are indicated by the data plotted in figure 2. This disparity of growth in media B and C was investigated further following the subsequent demonstration of thiamin synthesis by the org cultured in a thiamin deficient medium. On the assumption that thiamin synthesis was the limiting factor in the growth rate of the yeast in medium B, and that its biosynthesis proceeds through the production of thiazole and pyrimidine followed by coupling of the two fractions, both moieties were tested to determine their relationship to the nitrogen source present. It was found that pyrimidine stimulated growth in

1953] EQUIVALENCE OF THIAMIN AND PYRIDOXINE 199 the thiamin deficient medium regardless of the type of nitrogen source, whereas thiazole stimulated growth only in the medium containing ammonium

3 1953] EQUIVALENCE OF THIAMIN AND PYRIDOXINE 199 the thiamin deficient medium regardless of the type of nitrogen source, whereas thiazole stimulated growth only in the medium containing ammonium sulfate, as shown by the dotted arrow in figure 2. A slight depression in growth in medium C caused by thiazole also was observed as indicated by the dotted arrow in reverse. varying proportions of thiamin were substituted by pyridoxine, the total molar concentration of both being kept constant. The substitution of pyridoxine by thiamin up to the point where the mole fraction of thiamin in the medium was reduced to about 0.7 had no effect on growth. With further decrease in mole fraction of thiamin, the a: w l TIME, HOURS Figure 2. Growth curves of yeast in complete medium A, thiamin deficient medium B, and pyridoxine medium C, with ammonium sulfate as N source. Comparative Rapons to Varying Levels of Bi or Be The increase in growth after 40 hours in media deficient in either pyridoxine or thiamin upon addition of various levels of thiamin or pyridoxine, respectively, is shown in figure 3. On an equimolar basis, thiamin is decidedly more effective as a growth stimulant than pyridoxine. At low concentrations of either vitamin, represented by the initial linear portions of the curves, the ratio of growth stimulation of thiamin to pyridoxine is approximately 2:1. The maimal effect of thiamin is observed at 2.8 X 10- M, whereas that of pyridoxine extrapolated to the same maximum as that of thiamin is 4.88 X 10 Am per 10 ml. To obtain additional data on the relative effect of substituting of thiamin by pyridoxine, the amount of growth after 40 hours in a medium containing initially 1.75 X 10 pm of thsammn per 10 ml (suboptimal level) was determined when growth decreawsed from an initial level of 8.8 to 6.0 mg moist yeast per ml in a medium free of added thiain. Influence of Thiazole and Pyrimidine The relative effects of adding equimolar suboptimal concentrations of pyrimidine and thiasole individually and collectively as well as of adding thiamin upon growth in a medium deficient in both thiamin and pyridoxine were investigated. The data shown in figure 4 indicate that thiazole does not promote any increase in growth over that obtained in the medium deficient in both thiamin and pyridoxine. Although pyrimidine produced an over-all increase in growth approximately two-thirds of that ascribable to either thiamin or pyrimidine and thiazole collectively, it failed to reduce significantly the lag and stationary phases of growth.

4 200 WILLIAMS MOSES AND M. A. JOSLYN [VOL. 66 Vitamin B1 anl Vitamin B. Content Following Yeast Growth in Media Deficient in either or both Vitamins To determine the validity of one possible interpretation for the equivalence of the two vitamins, that one might serve as a precursor of the other, an investigation was made of the vitamin B1 and vitamin Be content of the yeast grown in media deficient in vitamins B1 and B6, respectively. The vitamin B1 and vitamin B, content of yeast grown in a medium deficient in both growth -J 4 This discrepancy is even more pronounced with respect to the thiamin content following growth in the medium deficient in both thiamin and in pyridoxine and in that lacking thiamin. Carboxylase Activity The influence of exogenous pyridoxine on the formation of thiamin pyrophosphate was investigated using enzymatic decarboxylation of pyruvic acid to test activity of carboxylase and co-carboxylae. I THIAMIN OR PYRIDOXINE,,UMOLS X 101 PER 10 ML Figure S. Comparative response of yeast to varying concentrations of thiamin (B1) and pyridoxine (B.), respectively. The upper curve (B1) shows increase in growth obtained in medium supplemented with pyridoxine upon addition of increasing amounts of thiamin; the lower one of increase in growth obtained in medium supplemented with thiamin upon addition of increasing amounts of pyridoxine. factors was determined also. To attain comparable growth in the latter medi required approximately 120 hours, instead of 40 hours. Vitamin B1 asays were carried out by centrifuging the yeast crop from the mother liquor and subsequently treating the yeast as described in the procedure given previously. No detectable amounts of vitamin B1 were found in the supernate. Pyridoxine assays were made on the entire suspensions. As shown in table 1, growth of the yeast in media from which thiamin or pyridoxine or both were omitted resulted in the synthesis of all deficiencies. Quantitative differences, however, are evident between the pyridoxine content of the yeast from the pyridoxine deficient medium and the medium deficient in both nutrilites. Fgure 5 shows the results of a comparative manometric study of the carboxylase activity of cellular suspensions of dried yeast preparations of the cultures obtained from media differing in nutritional adequacy. In these tests the main chamber of the manometric vessel contained 20 mg dried cells and 1.0 ml m/15 phosphate buffer, ph 6.2. To alternate vessels were added also 100 mg of co-carboxylase. The side arm contained 0.5 ml of 0.72 M sodium pyruvate. The total volume in all manometric tests was 3.0 ml. The results indicate that the yeast from the complete medium A has the greatest carboxylase activity, the culture from thiamin deficient medium B has the least, and the culture from pyridoxine deficient medium C is intermediate in activity. Al-

5 19531 EQUIVALENCE OF THIAMIN AND PYRIDOXINE 201 though the addition of co-carboxylase caused but slight increase in carbon dioxide evolution in yeast grown in pyridoxine deficient medium (C), it produced an increase of more than 20 per cent with the yeast from the thiamin deficient medium (B), resulting in a rate of carbon dioxide evolution comparable to that of the yeast from C. 1.0 as TIME, HOURS Figure 4. Growth curves of yeast in media deficient in thiamin and pyridoxine supplemented by equimolar quantities of thiazole, pyrimidine, and thiamin, respectively. P-Thiamin and pyridoxine deficient medium supplemented by pyrimidine (2 methyl-5-ethoxymethyl-6-amino-pyrimidine). T-Thiamin and pyridoxine deficient medium supplemented by thiazole (4-methyl-5-t-hydroxyethyl thiazole). Bl-Medium deficient in pyridoxine. -(B1 + B.)-Medium deficient in pyridoxine and thiamin. Acetoin Formation The function of thiamin pyrophosphate as the coenzyme for the carboligase enzyme of yeast, bacteria, and animal tissues was shown by Silverman and Werkman (1941) and Green et al. (1942). The over-all reaction for acetoin synthesis by yeast is represented by the equation: CH,COCOOH + CH.CHO -a CH3CHOHCOCH, + C02 For a valid interpretation of the results of the carboxylase determination it was essential therefore to estimate the extent to which this reaction influenced carbon dioxide evolution. The data shown in table 2 indicate that no significant TABLE 1 Comparison of thiamin and pyridoxine content of yeast 6C44-Following growth in media deficient in thiamin or pyridoxine or both pm X 10-' B, or B. per 100 mg moist yeast MEDIUM DEFICIZNCY VITAMIN CONTENT Thiamin Pyridoxine Pyridoxine Thiamin Thiamin and pyridoxine (n ab w. J TIME, MINUTES Figure 5. Pyruvate decarboxylation by yeast following growth in a complete medium, A; thiamin deficient medium, B; and a pyridoxine deficient medium, C. Co-100 pg thiamin pyrophosphate. effect is exerted on the latter by the small percentage of total carbon dioxide evolved attributable to the carboligatic enzyme. Carboxylase Acity of Cultures from Thiamin Deficient Media An investigation was made of the relationship between exogenous pyridoxine and carboxylase

2022 WILLIAMS MOSES AND M. A. JOSLYN [VOL. 66 activity. The carboxylase activities of cultures from thiamin deficient media containing 4.88 X 108 and 14.

6 2022 WILLIAMS MOSES AND M. A. JOSLYN [VOL. 66 activity. The carboxylase activities of cultures from thiamin deficient media containing 4.88 X 108 and X 10-l pm of pyridoxine, respectively, are shown in figure 6. In these tests the main compartment of the Warburg vessel contained 20 mg of dried cells and 100 ml m/15 phosphate buffer, ph 6.2. The side arm contained 0.5 ml 0.72 M pyruvate. The nitrogen content of TABLE 2 Carbon dioxide evolved due to acetoin* formation PEtR CNT (ujl "ACE. YEAST CEARACTERISTIC TOIN 1C1OO AL TOTAL CCh) "Normal" (A) Thiamin deficient (B) Pyridoxine deficient (C) *This also may include diacetyl. -I a ojm 0 I) is i 21 TIME, MINUTTS Figure 6. Pyruvate decarboxylation following growth of yeast in thiamin deficient media containing respectively 4.88 X 10-' and X lor1 asm of pyridoxine per 10 ml. the two cultures varied by 40.5 per cent. The reslts obtained (figure 6) show that the most rapid rate of pyruvate decarboxylation occurs in the culture from the medium containing the greater concentration of pyridoxine. The reproducibility of quantitative determination of carboxylase activity of yeast grown in duplicate cultures and dried separately was checked by determining the total C02 evolved from sodium pyruvate under our conditions on media containing 5 and 15 pg, respectively, of pyridoxine per 50 ml. The total C02 evolved at the lower level was 395 and 434,&L, respectively, averaging 415; at the higher level it was 546 and 575 pl, respectively, averaging 561. DISCUSSION The data presented indicate the indispensability of both thiamin and pyridoine for the attainment of a maxmal growth rate. The retarded growth rate of the yeast in a medium lacking both thiamin and pyridoxine but supplemented by pyrimidine is indicative of a metabolic impairment in the organism. This impairment is characterized by a slow rate of synthesis of the pyrimidine moiety of thiamin and to a much lesser extent the thiazole component. A relationship between thiazole synthesis and the source of exogenous nitrogen is suggested by the stimulatory effect of thiasole on the growth of the culture using ammonium sulfate as a nitrogen source. Although Bonner and Buchman (1938) reported that thiawole synthesis in isolated pea roots proceeds via ring formation from chloroacetylpropyl alcohol and thioforma& mide, Tatum and Bell (1946) failed to confirm this observation with Neurospora strains. Based on the observations reported here, it is not inconceivable that thiazole formation may proceed in culture 6C44 by way of one or more of the amino acid components of the casein hydrolyzate employed in these tests. In addition, since growth rate is influenced by exogenous thiazole, the rate of synthesis of thiazole apparently is the limiting factor in cell synthesis. The data on carboxylae formation confirm the observation that thiamin synthesis occurs in the organism when cultured in a thiaminle medium. They demonstrate that the organism grown in the absence of added thiamin has synthesized the carboxylase apoenzyme in excess of its available coenzyme. Leijnse and Terpstra (1951) recently reported that carboxylase formation based on CO2 production from Na pyruvate by living bakers' yeast was optimum when glucose was present with thiamin and ammonilm sufate or pyrimidyl plus ammonium sulfate. They found that in the absence of glucose, carboxylase formation in living bakers' yeast was higher in thiamin or pyrimidyl than in the presence of amon sulfate alone, and suggest that glucose and ammonium sulfate may be involved in formation of apocarboxylase. The influence of the environmental content of pyridoxine upon the corre-

1953] EQUIVALENCE OF THIAMIN AND PYRIDOXINE 203 sponding carboxylase and co-carboxylase activity suggest strongly that thiamin and, consequently, thiamin pyrophosphate synthesis depend on the

7 1953] EQUIVALENCE OF THIAMIN AND PYRIDOXINE 203 sponding carboxylase and co-carboxylase activity suggest strongly that thiamin and, consequently, thiamin pyrophosphate synthesis depend on the exogenous pyridoxine content. SUMMARY An investigation was made of the conditions governing the curious relationship between thiamin and pyridoxine in one strain of a brewers' top yeast, which required an exogenous supply of either nutrilite for the attainment of a rapid rate of growth. The indispensability of both vitamins for a maximal growth rate was found. The culture was characterized by its inability to synthesize pyrimidine at a rate required to support optimum growth and, to a lesser extent, by its retarded rate of synthesis of thiazole. The depression in the rate of growth of the orgamin a medium containing ammonium sulfate as the nitrogen source and the acceleration of its growth rate by thiazole indicated a relationship between thiazole synthesis and the source of exogenous nitrogen. The rate of synthesis of thiazole may be the limiting factor in cell production when the organism utilizes am imonium sulfate as its nitrogen source. Thiamin or pyridoxine served as functional substituents for each other since in the absence of either the organism synthesized the deficiency. A functional impairment in carboxylase activity, largely eliminated by an addition of the coenzyme, was demonstrated by the culture from a pyridoxine deficient medium. The synthesis of thiamin may be a function of the environmental pyridoine content. REFERENCES ATKIN, L., GRAY, P. P., MOSES, W., AND FEIN- STEIN, M Growth and fermentation factors for different brewery yeasts. European Brewery Convention Congress-Lucerne. Elsevier Publishing Co., New York. ATIN, L., ScHnuvz, A. S., WILAMs, W. L., AND FRiY, C. N Yeast microbiological methods for determination of vitamins: Pyridoxine. Ind. Eng. Chem., 15, BoNNER, J., AND BucHmN, E. R Syntheses carried out in vivo by isolated pea roots. Proc. Natl. Acad. Sci. U. S., 24, GREEN, D. C., WESTmRnLD, W. W., VENNESLAND, B., AND KNOX, W. E Carboxylases of animal tissues. J. Biol. Chem., 145, HENNESsY, T. J., AND CERECEDO, L. R The determination of free and phosphorylated thiamine by a modified thiochrome assay. J. Am. Chem. Soc., 61, HoPKiNs, R. H., AND PENNINGTON, R. J The asay of the vitamin Bs complex. Biochem. J., 41, JANSEN, B. C. P Chemical determination of aneurin by the thiochrome reaction. Rec. trav. chim., 55, LEuNsE, B., AND TERPsTA, W Formation of carboxylase and thiamine pyrophosphate in living bakers' yeast. Biochim. et Biophys. Acta, 7, LOHmANN, K., AND ScHUSTER, P Untersuchungen fiber die cocarboxylase. Biochem. Z., 294, RAEINOWITZ, J. C., AND SNELL, E. E An improved method for assay of vitamin B, with Streptococcus faecalis. J. Biol. Chem., 169, ScHUTZ, A. B., ATEIN, L., AND FREY, C. N The biochemical classification of yeast strains. J. Bact., 40, SILVERMAN, M., AND WE1RwAN, C. H The formation of acetylmethylcarbinol from pyruvic acid by a bacterial enzyme preparation. J. Biol. Chem., 161, SToKEs, J. L., FosTzR, J. W., AND WOODWARD, C. R Synthesis of pyridoxine by a "pyridoxineless" x-ray mutant of Neurospora 8itophila. Arch. Biochem., 2, TATum, E. L., AND BELL, T. T Biosynthesis of thiamine. Am. J. Botany, 33, WESTERFELD, W. W A colorimetric determination of blood acetoin. 161, J. Biol. Chem.,

possibilities occurs. It has been found that the organism acquires addition of vitamin B1 to cells of P. pentosaceum which had

possibilities occurs. It has been found that the organism acquires addition of vitamin B1 to cells of P. pentosaceum which had ADAPTATION OF THE PROPIONIC-ACID BACTERIA TO VITAMIN B1 SYNTHESIS INCLUDING A METHOD OF ASSAY M. SILVERMAN AND C. H. WERKMAN Bacteriology Section, Industrial Science Research Institute, Iowa State College,