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Enzymes of meso-inositol catabolism in the yeast Schwanniomyces occidentalis
426
PRELIMINARY NOTES
acid is a 1,3-glycerophosphate polymer containing a number of 2-0-a-(N-acetylgalactosaminyl)-glycerol residues and D-alanine ester groups (unpublished observations); consequently, the cross-reactions of S. albus immune sera with S. aureus teichoic acid suggest some lack in serum specificity. Polysaccharide A and the wall teichoic acid from S. aureus H are both N-acetylglucosaminylribitol phosphate polymers and this structure, or a part of it, is presumably essential for cross-reactions between these substances. Neither the D-alanine in the teich6ic acid nor the mucopeptide components in the polysaccharide A preparation are essential for serological reactivity. At present, quantitative precipitation studies have not been completed and the detailed chemical structure of polysaccharide A has not been examined. For these reasons we have used the term "cross-reaction", although it is likely that the two compounds are identical antigens. The serological reactions studied here on S. aureus strains are apparently different from those described by SANDERSON, JUERGENS AND STROMINGER8, as these authors have shown that their immune serum is specific for a-N-acetylglucosamine residues, whereas one of our teichoic acid preparations which shows a high titer in the ring test contains no detectable a residues. We thank Mr. U. L. RAJBHANDARY AND Dr. R. O. MARTIN for samples of S. aureus teichoic acid.
Department o/Microbiology, The Gade Institute, University o/Bergen (Norway) and Department o/Chemistry, King's College, University o/Durham, Newcastle upon Tyne (Great Britain)
G. H A U K E N E S
D. C. ELLWOOD J. BADDILEY P. 0EDING
1 L. A. JULIANELLE AND C. W. WIEGHARD, Proc. Soc. Exptl. Biol. Med., 31 (I934) 947. 2 L. A. JULIANELLE AND C. W. WIEGHARD, J. Exptl. Med., 62 (1935) I I . C. W. WIEGHARD AND L. A. JULIANELLE, f . Exptl. Med., 62 (1935) 23. 4 p. OEDING, Acta Patl~ol. Microbiol. Scand., Suppl. 93 (1952) 356. K. JENSEN, Acta Patlz.ol. Microbiol. Stand., 44 (1958) 421. 6 j . j . ARMSTRONG, J. BADDILEY, J. G. BUCHANAN, B. CARSS AND G. R. GREENBERG, J. Ctwm. Soc., (1958 ) 4344. 7 j . BADDILEY, J. G. BUCHANAN, F. E. HARDY, R. O. MARTIN, U. L. RAJBHANDARY AND A. R. SANDERSON, Biochim. Biophys. Acta, 52 (1961) 4o6. 8 A. R. SANDERSON, W. G. JUERGENS AND J. L. STROMINGER, p e r s o n a l c o m m u n i c a t i o n .
Received July 24th, 1961 Biochim. Biophys. Acta, 53 (1961) 425-426
Enzymes of meso-inositol catabolism in the yeast Schwanniomycesoccidentalis We have previously reported the presence of an inducible enzyme oxidizing inositol in the yeast, Schwanniomyces occidentalis 1, which is capable of growing on inositol as the only carbon source. The first detectable oxidation product in this enzyme reaction is glucuronate. Enzymic evidence has been obtained for the further catabolic breakdown of inositol, and these data are the subject of this note. The organism, method of cultivation, and procedures for obtaining cell-free Biochim. Biophys. Acta, 53 (I96I) 426-428
PRELIMINARY NOTES
427
enzyme preparations have been described 1, 2. In addition to cell extracts obtained with the cell disintegrator of MERKENSCHLAGERet al. 3, the HUGHES' press 4 also yielded extracts suitable for the demonstration of all enzymes mentioned in this paper except the inositol oxygenase. An enzyme converting D-glucuronate to L-gulonate was demonstrated in both directions, and was found to be specific for the free acids as substrates. The corresponding lactones are attacked only very slowly. NADP and N A D P H , act as hydrogen acceptor and hydrogen donor, respectively. The enzyme activity was determined by measuring the decrease in absorbancy at 34 ° m~ due to the disappearance of N A D P H 2 at pH 7.5 in the case of glucuronate reduction, and by measuring the increase in absorbancy at the same wavelength due to the formation of N A D P H 2 at pH 9.1 in the case of gulonate dehydrogenation. Preliminary measurements indicate that the Km values for gulonate and NADP are similar to those found by YORK et al. 5 for their mammalian enzyme of the same specificity. Fructuronate has been implicated as an intermediate in glucuronate metabolism in certain bacteria 6 and plants 7 and cannot be completely ruled out as an intermediate in our reaction scheme. However, the rate measurements with glucuronate are more in favour of a direct reduction to gulonate. Employing L-gulonate as substrate, a NAD-dependent dehydrogenase s-l° was demonstrated. Under the conditions of this test, an evolution of CO 2 could be observed in the Warburg manometer. The dehydrogenation of L-gulonate was followed spectrophotometrically at pH 9.1 measuring the formation of NADH 2. Since the enzyme preparation was a crude extract, it is not known whether the oxidation of L-gulonate and the decarboxylation are due to a single enzyme; recent evidence in a mammalian system 1° has indicated that these activities are caused by two separate enzymes. The decarboxylation of the presumed intermediate, 3-oxo-L-gulonic acid, will give rise to L-xylulose. COOH OH OH H~/H- ' \ OHH~ H0
>
OH
NADPH2 -. H O - - C - - H
H O - - C]- - H ~" NADP
J
H0--C--H
J
CH2OH
I)-glucuronate
_
L-gulonate
CH~OH
3-oxo-L-gulonat
CH2OH
CH20H
f
P C~O
, H - - Cf- - O H "
r
H--C--0H
HO--C--H
CHO
--CO S
C--0
H--C--OH
HO--C--H
--,
HO--C--H
NAD
J
H--C--OH
COOH
r
H O - - CI - - H ~ NADH~
r
meso-inositol
--
I
HO--C--H
Oi
H H/ N]--i/H
H
COOH
]
NADPH ~~. H O - - C - - H NADP
~
J
NAD
H - - C - - O H ? NADH~
H0--C--H H--C--0H
I
HO--C--H
I
CH2OH L-xylulose
HO--C--H
J
CH2OH Xylitol
C~0
/
CH~0H D-xylulose
Fig. I. Catabolism of inositol.
Biochim. Biophys. Acta, 53 (1961) 426-428
428
PRELIMINARY NOTES
Once again employing spectrophotometric methods to measure transformations of the nicotinamide coenzymes, a NADP-dependent dehydrogenasen, 12 was found which reduces L-xylulose to xylitol. This pentitol could be oxidized also by a NADdependent dehydrogenase giving rise to n-xylulose. With the exception of the inositol oxygenase, all enzymes of inositol catabolism, described in this paper, are found in our organism regardless of whether it has been grown on inositol or glucose as the sole carbon source, and must therefore be considered constitutive. Since in previous work (not yet published) the presence of the enzymes necessary for the functioning of the pentose phosphate cycle has been demonstrated in our organism, the present investigation lends considerable support to the theory that inositol is catabolized in Schwanniomyces occidentalis via n-glucuronate, L-gulonate, L-xylulose, xylito1, and n-xylulose (Fig. I), followed by phosphorylation of n-xylulose and oxidation of o-xylulose 5-phosphate through the pentose phosphate cycle. However, for the complete establishment of this pathway, an investigation with radioisotopically labelled inositol will be necessary. It is of interest that inositol catabolism in Schwanniomyces occidentalis seems to follow the same pathway as has been postulated for mammalian tissue 13. The authors are indebted to Dr. R. G. JANKE (Technische Hochschule, Vienna, Austria) for her invaluable help with the microbiological part of this work, and also to Dr. G. G. ASHWELL (National Institutes of Health, Bethesda, Md.) for a generous gift of several pentoses.
Organisch-Chemisches Institut der Universiti~t, Vienna (Austria)
A. SIVAK* O. HOFFMANN-OSTENHOF
l C. JUNGWlRTH, A. SIVAK, O. HOFFMANN-OSTENHOF AND R. G. JANKE, Mona~sh. Chem., 92 (1961) 72. 2 R. G. JANKE, C. JUNGWIRTH, I. B. DWAID AND O. HOFFMANN-OSTENHOF, Monatsh. Chem., 9 ° (1959) 382. a M. MERKENSCHLAOER, K. SCHLOSSMANN AND "vV. KURZ, Biochem. Z., 329 (1957) 332. 4 D. E. HUGHES, Brit. J. Exptl. Pathol., 32 (1951) 97. 5 j. L. YORK, A. P. GROLLMAN ANn C. BUBLITZ, Biochim. Biophys. Acta, 47 (1961) 298R. A. MCRoRIE, A. K. WILLIAMS AND W. J. PAYNE, J. Bacteriol., 77 (1959) 212. 7 F. A. L o E w u s AND S. KELLY, Biochem. Biophys. Research Communs. i (1959) 143. s C. BOBLITZ, Biochim. Biophys. Acta, 48 (1961) 61. 9 C. BOBLITZ AND J. C. YORK, Biochim. Biophys. Acta, 48 (1961) 56. 10 j. D. SMILEY AND G. G. ASHWELL, J. Biol. Chem., 236 (1961) 35711 C. CHIAN~ AND S. G. KNIGHT, Biochem. Biophys. Research Communs., 3 (196o) 554. 12 S. HOLLMANN, Z. physiol. Chem. Hoppe-Seyler's, 317 (1959) 193. ta j. j . BURNS, •. TROUSOF, C. EVANS, N. PAPADOPOULOS AND B. ~V. AGRANOFF, Biochim. Biophys. Acla, 33 (1959) 215.
Received July 2ist, 1961 * U.S. Public Health Service Postdoctoral Fellow. Present address: A r t h u r D. Little, Inc., Cambridge, Mass. (U.S.A.).
Biochim. Biophys. Acta, 53 (I96~) 426-428
PRELIMINARY NOTES
acid is a 1,3-glycerophosphate polymer containing a number of 2-0-a-(N-acetylgalactosaminyl)-glycerol residues and D-alanine ester groups (unpublished observations); consequently, the cross-reactions of S. albus immune sera with S. aureus teichoic acid suggest some lack in serum specificity. Polysaccharide A and the wall teichoic acid from S. aureus H are both N-acetylglucosaminylribitol phosphate polymers and this structure, or a part of it, is presumably essential for cross-reactions between these substances. Neither the D-alanine in the teich6ic acid nor the mucopeptide components in the polysaccharide A preparation are essential for serological reactivity. At present, quantitative precipitation studies have not been completed and the detailed chemical structure of polysaccharide A has not been examined. For these reasons we have used the term "cross-reaction", although it is likely that the two compounds are identical antigens. The serological reactions studied here on S. aureus strains are apparently different from those described by SANDERSON, JUERGENS AND STROMINGER8, as these authors have shown that their immune serum is specific for a-N-acetylglucosamine residues, whereas one of our teichoic acid preparations which shows a high titer in the ring test contains no detectable a residues. We thank Mr. U. L. RAJBHANDARY AND Dr. R. O. MARTIN for samples of S. aureus teichoic acid.
Department o/Microbiology, The Gade Institute, University o/Bergen (Norway) and Department o/Chemistry, King's College, University o/Durham, Newcastle upon Tyne (Great Britain)
G. H A U K E N E S
D. C. ELLWOOD J. BADDILEY P. 0EDING
1 L. A. JULIANELLE AND C. W. WIEGHARD, Proc. Soc. Exptl. Biol. Med., 31 (I934) 947. 2 L. A. JULIANELLE AND C. W. WIEGHARD, J. Exptl. Med., 62 (1935) I I . C. W. WIEGHARD AND L. A. JULIANELLE, f . Exptl. Med., 62 (1935) 23. 4 p. OEDING, Acta Patl~ol. Microbiol. Scand., Suppl. 93 (1952) 356. K. JENSEN, Acta Patlz.ol. Microbiol. Stand., 44 (1958) 421. 6 j . j . ARMSTRONG, J. BADDILEY, J. G. BUCHANAN, B. CARSS AND G. R. GREENBERG, J. Ctwm. Soc., (1958 ) 4344. 7 j . BADDILEY, J. G. BUCHANAN, F. E. HARDY, R. O. MARTIN, U. L. RAJBHANDARY AND A. R. SANDERSON, Biochim. Biophys. Acta, 52 (1961) 4o6. 8 A. R. SANDERSON, W. G. JUERGENS AND J. L. STROMINGER, p e r s o n a l c o m m u n i c a t i o n .
Received July 24th, 1961 Biochim. Biophys. Acta, 53 (1961) 425-426
Enzymes of meso-inositol catabolism in the yeast Schwanniomycesoccidentalis We have previously reported the presence of an inducible enzyme oxidizing inositol in the yeast, Schwanniomyces occidentalis 1, which is capable of growing on inositol as the only carbon source. The first detectable oxidation product in this enzyme reaction is glucuronate. Enzymic evidence has been obtained for the further catabolic breakdown of inositol, and these data are the subject of this note. The organism, method of cultivation, and procedures for obtaining cell-free Biochim. Biophys. Acta, 53 (I96I) 426-428
PRELIMINARY NOTES
427
enzyme preparations have been described 1, 2. In addition to cell extracts obtained with the cell disintegrator of MERKENSCHLAGERet al. 3, the HUGHES' press 4 also yielded extracts suitable for the demonstration of all enzymes mentioned in this paper except the inositol oxygenase. An enzyme converting D-glucuronate to L-gulonate was demonstrated in both directions, and was found to be specific for the free acids as substrates. The corresponding lactones are attacked only very slowly. NADP and N A D P H , act as hydrogen acceptor and hydrogen donor, respectively. The enzyme activity was determined by measuring the decrease in absorbancy at 34 ° m~ due to the disappearance of N A D P H 2 at pH 7.5 in the case of glucuronate reduction, and by measuring the increase in absorbancy at the same wavelength due to the formation of N A D P H 2 at pH 9.1 in the case of gulonate dehydrogenation. Preliminary measurements indicate that the Km values for gulonate and NADP are similar to those found by YORK et al. 5 for their mammalian enzyme of the same specificity. Fructuronate has been implicated as an intermediate in glucuronate metabolism in certain bacteria 6 and plants 7 and cannot be completely ruled out as an intermediate in our reaction scheme. However, the rate measurements with glucuronate are more in favour of a direct reduction to gulonate. Employing L-gulonate as substrate, a NAD-dependent dehydrogenase s-l° was demonstrated. Under the conditions of this test, an evolution of CO 2 could be observed in the Warburg manometer. The dehydrogenation of L-gulonate was followed spectrophotometrically at pH 9.1 measuring the formation of NADH 2. Since the enzyme preparation was a crude extract, it is not known whether the oxidation of L-gulonate and the decarboxylation are due to a single enzyme; recent evidence in a mammalian system 1° has indicated that these activities are caused by two separate enzymes. The decarboxylation of the presumed intermediate, 3-oxo-L-gulonic acid, will give rise to L-xylulose. COOH OH OH H~/H- ' \ OHH~ H0
>
OH
NADPH2 -. H O - - C - - H
H O - - C]- - H ~" NADP
J
H0--C--H
J
CH2OH
I)-glucuronate
_
L-gulonate
CH~OH
3-oxo-L-gulonat
CH2OH
CH20H
f
P C~O
, H - - Cf- - O H "
r
H--C--0H
HO--C--H
CHO
--CO S
C--0
H--C--OH
HO--C--H
--,
HO--C--H
NAD
J
H--C--OH
COOH
r
H O - - CI - - H ~ NADH~
r
meso-inositol
--
I
HO--C--H
Oi
H H/ N]--i/H
H
COOH
]
NADPH ~~. H O - - C - - H NADP
~
J
NAD
H - - C - - O H ? NADH~
H0--C--H H--C--0H
I
HO--C--H
I
CH2OH L-xylulose
HO--C--H
J
CH2OH Xylitol
C~0
/
CH~0H D-xylulose
Fig. I. Catabolism of inositol.
Biochim. Biophys. Acta, 53 (1961) 426-428
428
PRELIMINARY NOTES
Once again employing spectrophotometric methods to measure transformations of the nicotinamide coenzymes, a NADP-dependent dehydrogenasen, 12 was found which reduces L-xylulose to xylitol. This pentitol could be oxidized also by a NADdependent dehydrogenase giving rise to n-xylulose. With the exception of the inositol oxygenase, all enzymes of inositol catabolism, described in this paper, are found in our organism regardless of whether it has been grown on inositol or glucose as the sole carbon source, and must therefore be considered constitutive. Since in previous work (not yet published) the presence of the enzymes necessary for the functioning of the pentose phosphate cycle has been demonstrated in our organism, the present investigation lends considerable support to the theory that inositol is catabolized in Schwanniomyces occidentalis via n-glucuronate, L-gulonate, L-xylulose, xylito1, and n-xylulose (Fig. I), followed by phosphorylation of n-xylulose and oxidation of o-xylulose 5-phosphate through the pentose phosphate cycle. However, for the complete establishment of this pathway, an investigation with radioisotopically labelled inositol will be necessary. It is of interest that inositol catabolism in Schwanniomyces occidentalis seems to follow the same pathway as has been postulated for mammalian tissue 13. The authors are indebted to Dr. R. G. JANKE (Technische Hochschule, Vienna, Austria) for her invaluable help with the microbiological part of this work, and also to Dr. G. G. ASHWELL (National Institutes of Health, Bethesda, Md.) for a generous gift of several pentoses.
Organisch-Chemisches Institut der Universiti~t, Vienna (Austria)
A. SIVAK* O. HOFFMANN-OSTENHOF
l C. JUNGWlRTH, A. SIVAK, O. HOFFMANN-OSTENHOF AND R. G. JANKE, Mona~sh. Chem., 92 (1961) 72. 2 R. G. JANKE, C. JUNGWIRTH, I. B. DWAID AND O. HOFFMANN-OSTENHOF, Monatsh. Chem., 9 ° (1959) 382. a M. MERKENSCHLAOER, K. SCHLOSSMANN AND "vV. KURZ, Biochem. Z., 329 (1957) 332. 4 D. E. HUGHES, Brit. J. Exptl. Pathol., 32 (1951) 97. 5 j. L. YORK, A. P. GROLLMAN ANn C. BUBLITZ, Biochim. Biophys. Acta, 47 (1961) 298R. A. MCRoRIE, A. K. WILLIAMS AND W. J. PAYNE, J. Bacteriol., 77 (1959) 212. 7 F. A. L o E w u s AND S. KELLY, Biochem. Biophys. Research Communs. i (1959) 143. s C. BOBLITZ, Biochim. Biophys. Acta, 48 (1961) 61. 9 C. BOBLITZ AND J. C. YORK, Biochim. Biophys. Acta, 48 (1961) 56. 10 j. D. SMILEY AND G. G. ASHWELL, J. Biol. Chem., 236 (1961) 35711 C. CHIAN~ AND S. G. KNIGHT, Biochem. Biophys. Research Communs., 3 (196o) 554. 12 S. HOLLMANN, Z. physiol. Chem. Hoppe-Seyler's, 317 (1959) 193. ta j. j . BURNS, •. TROUSOF, C. EVANS, N. PAPADOPOULOS AND B. ~V. AGRANOFF, Biochim. Biophys. Acla, 33 (1959) 215.
Received July 2ist, 1961 * U.S. Public Health Service Postdoctoral Fellow. Present address: A r t h u r D. Little, Inc., Cambridge, Mass. (U.S.A.).
Biochim. Biophys. Acta, 53 (I96~) 426-428