Vitamin B6 Antagonists and Growth of Microorganisms. II. S-Desoxypyridoxal and Related Compounds

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Vitamin B6 Antagonists and Growth of Microorganisms.

II. S-Desoxypyridoxal and Related Compounds


Jesse C. Rabinowitz’ and Esmond E. Sell2
From the Department of Biochemistry, Uniwej,sity of Wisconsin,
Madison, Wisconsin
Received November 10, 1952

Relatively few compounds that act antagonistically toward vitamin


Be in microorganisms have been described. The action of 4-desoxypyri-
doxine has been considered in the preceding paper (1). This investiga-
tion deals with a series of recently described (2) compounds related to
pyridoxal, pyridoxamine, and pyridoxine by the replacement of the
5-hydroxymethyl group by a methyl group. These compounds show
inhibitory properties for microorganisms that are prevented by vitamin
B6, but differ from those shown by 4-desoxypyridoxine and w-methyl-
pyridoxine. Details are presented below.
EXPERIMENTAL
Materials and Methods
The various inhibitors used are listed in Table I and were gifts from Dr. Karl
Folkers. Test organisms, cultural conditions, and general methodology are de-
scribed in the preceding paper (1).

Comparative Activities of Individual Inhibitors for Various Organisms


Each of the 5-desoxy analogs of vitamin Bs inhibited growth of the
yeast, Saccharomyces carlsbergensis, although somewhat less effectively
than 4-desoxypyridoxine and w-methylpyridoxine (Table II). The latter
compound is the most active inhibitor found for this organism. In other
experiments, values as low as 4 were obtained for the inhibition ratio of
i Present address: Division of Plant Biochemistry, University of California,
Berkeley, Calif.
*Present address: Department of Chemistry, University of Texas, Austin,
Texas.
408
VITAMIN B6 ANTAGONISTS. II 409

o-methylpyridoxine to pyridoxamine for this organism. This wide range


of values is in contrast to the constancy of the values observed with
4-desoxypyridoxine. In every case, pyridoxine was the most active of
the three forms of vitamin Bs in overcoming these inhibitions. The

TABLE I
Inhibitory Analogs of Vitamin Be

Formula Substituent Trivial name


I
X Y z
-CHI -CHO ’ -CH, 5-Desoxypyridoxal
-CHa --C&NH2 -CHa 5-Desoxypyridoxamine
-CH, -CHzOH -CHJ 5-Desoxypyridoxine
-CHa -CHz -CHzOH 4-Desoxypyridoxine
-CH&Ha -CHzOH -CHzOH o-Methylpyridoxine”

a In some earlier literature (9, 14, 15) this compound has been referred to as
“ethylpyridoxine”.

TABLE II
The Comparative Inhibitory Potencies of Vitamin Be Antagonists for
Saccharomyces carlsbergensis
Reversing agent”
Inhibitor Pyridoxal Pyridoxamine Pyridoxine
Inhibition ratio*
5-Desoxypyridoxal 440 190 1,499
5-Desoxypyridoxamine 12,099 l-,800 35,999
5-Desoxypyridoxine 430 120 6,509
4-Desoxypyridoxinec 27 ‘28 135
w-Methylpyridoxinec 16 16 43
a Fifty millimicrograms of pyridoxamine dihydrochloride or equimolar
amounts of pyridoxal hydrochloride or pyridoxine hydrochloride per 6 ml. were
added unless otherwise indicated.
* The molar ratio of inhibitor to reversing agent at which growth was de-
pressed to 5Oye of that obtained in the absence of added inhibitor. The inhibition
ratio between 5desoxypyridoxal and pyridoxamine in the presence of 100 and
1009 nmg. of the vitamin was 150 and 60, respectively.
c Determined in presence of 109 mFg. of reversing agent.

inhibitory action of the 5-desoxy analogs was overcome more effectively


by pyridoxal than by pyridoxamine; these two forms of the vitamin are
about equally active in counteracting inhibition by 4-desoxypyridoxine
[see (l)] and w-methylpyridoxine.
410 JESSE C. RABINOWITZ AND ESMOND E. SNELL

In contrast to 4-desoxypyridoxine and w-methylpyridoxine, 5desoxy-


pyridoxal and 5-desoxypyridoxamine inhibit the growth of vitamin
Bs-dependent strains of lactic acid bacteria, such as Streptococcus faecalis
(Table III). Pyridoxamine is more active than pyridoxal in counter-
acting the effects of each of these inhibitors. In promoting growth of this
organism in the absence of inhibitors, pyridoxamine is equally (3) or
somewhat more (4) active than pyridoxal. Pyridoxine is not a source
of vitamin Be for S. jaecalis (4) and was not tested as a reversing agent.
Significantly, 5-desoxypyridoxine is also inactive as an inhibitor. Pyri-
doxamine phosphate is equally or more active than pyridoxamine in

TABLE III
The Comparative Inhibitory Potencies of Vitamin Be Antagonists for
Streptococcus faecalis
Reversing agent
Pyridoxamine
Inhibitor Pyridoxal P&doxamine phosphate
Inhibition ratio”
5-Desoxypyridoxal 2,700 48,000 4,40@
5-Desoxypyridoxamine 400 5,000 7ob
5-Desoxypyridoxine >200,000 >200,000 >800,000
4-Desoxypyridoxine - >500,000 >80,ooo
w-Methylpyridoxine - >200,000 >75,000
a Defined as in Table II and determined in the presence of 5 mpg. of pyridox-
amine dihydrochloride, 10 mpg. of pyridoxal hydrochloride, or 15 wg. of calcium
pyridoxamine phosphate.
b These represent the average of the values determined in the presence of 10,
20,50, and 100 mpg. of calcium pyridoxamine phosphate. With Sidesoxypyridoxal
as the inhibitor, the individual values were 6700,3400,3100, and 4400, respectively.
With 5-desoxypyridoxamine as the inhibitor the corresponding values were 91,
38, 91, and 60.

promoting growth in the absence of inhibitors (5), but is far less active
than pyridoxamine in reversing the inhibitory effects of these analogs.
Under the conditions of growth used here, synthesis of n-alanine
represents the sole reaction for which an external source of vitamin Be
is required by S. jaecalis, and when this amino acid is supplied preformed,
growth occurs in the absence of this vitamin (6, 7). When the sole
function for which vitamin Ba is required is by-passed in this way,
5desoxypyridoxal and 5-desoxypyridoxamine no longer inhibit growth
of 8. jaecalis.
The effectiveness of these inhibitors for various lactic acid bacteria
VITAMIN Bg ANTAGONISTS. II 411

TABLE IV
The Comparative Inhibitory Potencies of Vitamin Be Antagonists for
Lactobacillus helveticus
Inhibitor Inhibition ratid
5-Desoxypyridoxal ‘19,000
5-Desoxypyridoxsmine >91o,ooo
5-Desoxypyridoxine >910,000
0 Determined in the presence of 10 mg. of calcium pyridoxamine phosphate/
10 ml. Pyridoxal and pyridoxamine are essentially inactive as growth factors
for this organism (8).

4 -DESOXYP(fUCClXIN~ HC I 5- DESOXYPYRIDOXAL
MICROGRAMS PER 6 MILLILITERS
FIG. 1. Inhibition of S. carlsbergensis by 4-desoxypyridoxine, 5-desoxypyri-
doxal, and mixtures of the two. All tubes contained 69 wg. of pyridoxamine di-
hydrochloride and 10 pg. of thiamine chloride in addition to the usual
constituents of the medium (10).
1: Response to 4-desoxypyridoxine.
I: As in 1 plus 2 I.cg.of 5-desoxypyridoxal/b ml.
3: Response to 5-desoxypyridoxsl.
.J: As in 3 plus 0.6 pg. of 4-desoxypyridoxine/b ml.

varies from organism to organism. Thus for Lactobacillus helveticus S,


which requires pyridoxamine phosphate for growth (S), only (i-desoxy-
pyridoxd is an effective inhibitor (Table IV).
412 JESSE C. RABINOWITZ AND ESMOND E. SNELL

The Combined Action of Vitamin Be Antagonists


If two antagonists inhibit growth by the same mechanism, their
effects should be roughly additive, whereas, if different mechanisms are
operative, nonadditive effects are to be expected. Experiments of this
nature were conducted with S. carlsbergensis. From curves 1 and 3 of
Fig. 1, it is seen that addition of either 4-desoxypyridoxine or 5-desoxy-
pyridoxal to a medium containing a constant level of pyridoxamine
first stimulates growth, then inhibits it. The reason for growth stimula-

c,yMETHYLPYRlDOXlNE
MICROGRAMS PER 6 ML.
FIG. 2. The inhibition of S. carlsbergensis by w9ethylpyridoxine in the ab-
sence and presence of other inhibitors. Each tube contains thiamine and pyri-
doxamine as in Fig. 1.
f: w-Methylpyridoxine alone.
8: w-Methylpyridoxine plus 0.6 pg. of 4-desoxypyridoxine/6 ml.
3: w-Methylpyridoxine plus 2.0 pg. of 5-desoxypyridoxal/6 ml.

tion at low levels of inhibitor is not known, but such stimulation at


subinhibitory concentrations of a toxic compound is not unusual (9).
The lowest concentrations at which the inhibitory action of 4-desoxy-
pyridoxine or 5-desoxypyridoxal overcomes this stimulatory effect or
begins to depress growth are 0.6 and 2.0 pg./6 ml., respectively. If the
effects of increasing amounts of 4-desoxypyridoxine are now determined
in the presence of 2.0 rg of 5-desoxypyridoxal, inhibition is not observed
until 0.8 pg. of 4-desoxypyridoxine has been added (curve 2, Fig. 1); this
same amount, however, produces the same degree of inhibition in the
absence of 5desoxypyridoxal (curve 1, Fig. 1). The converse experiment,
VITAMIN Be ANTAGONISTS. II 413

in which increasing amounts of 5desoxypyridoxal are added to tubes


containing a constant level (0.6 pg.) of 4-desoxypyridoxine, shows that
within experimental error the same amount of 5-desoxypyridoxal is
required for inhibition in the presence or absence of this low level of
4-desoxypyridoxine. Although the amounts of 5-desoxypyridoxal and
4-desoxypyridoxine added for establishing curves 2 and 4 (Fig. 1)
suffice to overcome the stimulatory effects of these antagonists, their
presence does not prevent a similar stimulation due to the other in-
hibitor. There is thus no evidence for an additive effect of the two
inhibitors; the findings point instead to independent loci for their action.
Entirely similar experiments conducted with 4-desoxypyridoxine and
w-methylpyridoxine show conclusively an additive effect of these in-
hibitors (compare curves 1 and 2, Fig. 2), indicating that these two
substances act at the same locus within the cell. If this is so, then
w-methylpyridoxine and 5desoxypyridoxal should act independently;
an experimental test provided results largely in accord with this expecta-
tion (curves 1 and 3, Fig. 2).

DISCUSSION

Cells of S. faecalis phosphorylate 4-desoxypyridoxine, and the product


thus formed competes with pyridoxal phosphate for the apoenzyme of
the tyrosine decarboxylase of this organism (11). These experiments
indicate a possible mechanism by which this inhibitor may act, although
further investigation of the phenomenon is needed, inasmuch as 4-
desoxypyridoxine is ineffective as a growth inhibitor for S. jaecalis (1).
The present results indicate that 4-desoxypyridoxine and w-methyl-
pyridoxine inhibit growth of S. carlsbergensis in the same way; sig-
nificantly, both compounds have a free hydroxymethyl group in the
5-position that would permit phosphorylation, as postulated for 4-desoxy-
pyridoxine by Umbreit and Waddell (11). In contrast, the 5-desoxy
analogs cannot be phosphorylated in this position, and our results
indicate that 5-desoxypyridoxal functions by a different mechanism
than do 4-desoxypyridoxine and w-methylpyridoxine. Among other pos-
sibilities, the 5-desoxy analogs may inhibit growth by (a) interfering
with absorption of vitamin Bc by the cell or by (5) interfering with the
phosphorylation of vitamin Bg. The failure of pyridoxamine phosphate
to function as a noncompetitive antagonist of the 5desoxy analogs
argues against the latter mechanism as the exclusive mode of action
of these compounds.
414 JESSE C. RABINOWITZ AND ESMOND E. SNELL

The results emphasize once more the great differences in sensitivity


of different organisms to single inhibitors [cf. (12, 13)]. Thus the lactic
acid bacteria are readily inhibited by appropriate 5-desoxy analogs but
are insensitive under the same conditions to 4-desoxypyridoxine and
w-methylpyridoxine. The latter two inhibitors, however, are considerably
more effective than the 5-desoxy analogs in inhibiting yeast growth.
w-Methylpyridoxine, the most effective against yeast of the antagonists
tested, was early shown to replace pyridoxine in promoting growth of
excised tomato roots (14), although it inhibited growth of Ceratostomella
ulmi (15).

SUMMARY

Three 5-desoxy analogs of vitamin Bs were compared with 4-desoxy-


pyridoxine and w-methylpyridoxine as antagonists of vitamin Bs.
Except for minor va.riations, the order of decreasing effectiveness
as antagonists of vitamin Be for Saccharomyces carlsbergensis was:
w-methylpyridoxine > 4-desoxypyridoxine > 5-desoxypyridoxine 2
5-desoxypyridoxal > 5-desoxypyridoxamine. Pyridoxine was more effec-
tive than pyridoxal in preventing the inhibitory effects of these antago-
nists; pyridoxal in turn was more effective than pyridoxamine.
For Streptococcus faecalis, 5-desoxypyridoxamine was an effective
antagonist of vitamin Be; 5-desoxypyridoxal was somewhat less active;
the remaining compounds were inactive. Pyridoxamine was more effec-
tive than either pyridoxal or pyridoxamine phosphate in preventing
growth inhibition by these antagonists. The antagonists did not inhibit
growth when vitamin Bc was made nonessential by the addition of
n-alanine to the medium. For Lactobacillus helveticus, only 5-desoxy-
pyridoxal proved effective as an antagonist of vitamin Be.
The inhibitory effects on Saccharomyces carlsbergensis of 4-desoxy-
pyridoxine and w-methylpyridoxine were additive, indicating that these
two compounds inhibited the same reaction in this organism. In contrast,
the effects of 4-desoxypyridoxine (or of w-methylpyridoxine) and of
5-desoxypyridoxal were exerted independently of one another; these
inhibitors, therefore, appear to act by independent mechanisms.

REFERENCES
1. RABINOWITZ, J. C., AND SNELL, E. E., Arch. Biochem. and Biophye. 43, 399
(1953).
2. HEYL. D., HARRIS, S. A., AND FOLKERS, K., J. Am. Chem. Sot. in press.
VITAMIN B,y ANTAGONISTS. II 415

3. RABINOWITZ, J. C., AND SNELL, E. E., J. Biol. Chem. 169, 631 (1947).
4. SNELL, E. E., AND RANNEFELD, A. N., J. Biol. Chem. 167, 475 (1945).
5. RABINOWITZ, J. C., AND SNELL, E. E., J. Biol. Chem. 189, 643 (1947).
6. HOLDEN, J. T., AND SNELL, E. E., J. Biol. Chem. 178, 799 (1949).
7. SNELL, E. E., Symposium sur le Metabolism Microbien, p. 47, IIe Congr&s
International de Biochimie, Paris, 1952.
8. MCNUTT, W. S., AND SNELL, E. E., J. Biol. Chem. 173,801 (1948).
9. WOOLLEY, D. W., A Study of Antimetabolites. John Wiley and Sons, New
York, 1952.
10. RABINOWITZ, J. C., AND SNELL, E. E., Anal. Chem. 19, 277 (1947).
11. UMBREIT, W. W., AND WADDELL, J. W., Proc. Sot. Exptl. Biol. Med. ‘IO, 293
(1949).
12. SNELL, E. E., AND SHIVE, W., J. Biol. Chem. 168,551 (1945).
13. SHIVE, W., AND SNELL, E. E,, Science 162, 401 (1945).
14. ROBBINS, W. J., Am. J. Botany 29, 241 (1942).
15. ROBBINS, W. J., AND MA, R., Bull. Torrey Botan. Club 69, 342 (1942).

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