Production ofp-Hydroxyhydrocinnamic Acid from Tyrosine

Vol. 12, No. 2
0095-1 137/80/08-0291/03$02.00/0
Production of p-Hydroxyhydrocinnamic Acid from Tyrosine
by Peptostreptococcus anaerobius
Analytical Bacteriology Branch, Center for Disease Control, Atlanta, Georgia 30333
Peptostreptococcus anaerobius was found to metabolize tyrosine to p-hydroxy,
hydrocinnamic acid [3-(p-hydroxyphenyl)propionic acid]. This acid was detected
in spent growth media by gas-liquid chromatography, and its identity was confirmed by mass spectrometry.
A recent report by Babcock (1) showed that
cultures of Peptostreptococcus anaerobius
could be differentiated from other anaerobic
gram-positive cocci by their abiity to degrade
crystals of L-tyrosine which were dispersed
throughout a modified Schaedler agar medium.
Use of this new medium provides a simple and
specific test for the presumptive identification of
P. anaerobius in the clinical laboratory.
Most bacteria metabolize tyrosine through the
homogentisate (2, 4, 6, 9) or homoprotocatechuate (4, 6, 13) pathways to various hydroxyphenolic compounds. In aerobic species, the benzene ring is further hydroxylated, split, and degraded to short-chain carbon compounds which
can then enter the Krebs cycle or other metabolic pathways (2, 4, 6, 9, 13, 14). However, in
anaerobic bacteria, the benzene ring usually remains intact, and only the aliphatic side chains
of these phenolic compounds are metabolized
(2). This report describes the identification of a
major acid metabolite produced when L-tyrosine
is degraded by P. anaerobius.
Cultures of P. anaerobius 17642 (VPI 4329),
19112, and 17790 were obtained from the stock
culture collection of the Center for Disease Control Anaerobe Section. They were inoculated
into thioglycolate broth (135-C; BBL Microbiology Systems, Cockeysville, Md.) and incubated anaerobically for 24 h at 35°C. These
cultures were used to inoculate Schaedler agar
plates containing 0.3% L-tyrosine (1), Schaedler
broth containing 0.05% L-tyrosine, and Schaedler agar and broth without added tyrosine.
The agar plates were prepared, stored, and
inoculated as described by Babcock (1), except
that only one strain was inoculated per plate,
and the inoculum was spread over a 40-mmdiameter area in the center of the plate. The
broth was prepared according to the manufacturer's directions (BBL Microbiology Systems),
dispensed in 100-ml volumes, and inoculated
with 0.3 ml of the thioglycolate culture. The
inoculated media and uninoculated media controls were incubated under anaerobic conditions
for 48 h at 35°C.
The cultures of P. anaerobius grew well in all
media; disappearance of the tyrosine crystals in
the agar plates beneath the area of growth was
observed as previously described (1). The cells
were removed from the agar plates and discarded. A 0.3-ml amount of 25% H2SO4 was
distributed onto the surface of each plate and
allowed to stand for 15 to 30 min. The agar was
cut into small pieces with a spatula, transferred
to 50-ml screw-capped centrifuge tubes, and
melted in a water bath at 80 to 85°C. After
cooling to room temperature, the agar was extracted with 20 ml of diethyl ether; the ether
layer was transferred to a small beaker, concentrated, and transferred to a screw-capped test
tube. The butyl ester or trifluoroacetyl butyl
esters of the acids were prepared as described
previously (8, 10).
The broth cultures were centrifuged, and 5-ml
volumes of the spent growth medium were acidified to pH 2 with 6 N HCL. The acids were
extracted with two 5-ml volumes of diethyl
ether, and the ether layers were combined in a
small beaker, concentrated, and derivatized (8,
10). The butyl ester or trifluoroacetyl butyl ester
was analyzed with a Perkin-Elmer model 990
gas chromatograph (The Perkin-Elmer Corp.,
Norwalk, Conn.) equipped with a flame ionization detector and a coiled glass column (3.6 m
by 4 mm [inside diameter]) packed with 5% OV1 coated on 80/100-mesh, acid-washed, dimethylchlorosilane-treated Chromosorb W (Analabs, North Haven, Conn.) The instrument conditions and column temperatures were the same
as those used in an earlier report (10). Combined
gas-liquid chromatography-mass spectrometry
was done on a model 21-491B mass spectrometer
(Du Pont Instruments, Wilmington, Del.) interfaced with a Varian 2700 gas chromatograph
(Varian Instruments, Walnut Park, Calif.). The
2700 instrument contained a glass column (2 m valeric acids were produced regardless of the
by 2 mm [inside diameter]) packed with 3% 101 medium used. The identity of these acids was
on 100/200-mesh Gas-Chrom Q. The mass spec- established by gas-liquid chromatography retentrometer was equipped for both electron impact tion time comparison with standards and by
ionization and chemical ionization. The reagent mass spectrometry. The large peak eluting at
17.5 min in both chromatograms was identified
gas for chemical ionization was isobutane.
Chromatograms of the esterified acids pro- by gas-liquid chromatography-mass spectromeduced by P. anaerobius in Schaedler broth with try as hydrocinnamic acid or 3-phenylpropionic
and without added tyrosine are shown in Fig. 1 acid. The electron impact ionization spectrum of
and 2, respectively. Large to moderate amounts the butyl ester showed a molecular ion (M+) of
of acetic, isocaproic, butyric, and benzoic acids 206, major ions at m/e 91 and m/e 104, and
and small amounts of isobutyric, lactic, and iso- relatively large fragment ions at m/e 151 (M+55) and m/e 133 (M+-73). The chemical ionization spectrum showed a large M+1 ion at m/e
207. These data are identical to those reported
previously for the butyl ester derivative of a
standard of hydrocinnamic acid (3).
The peak at retention time of 19.6 min in Fig.
1 was identified asp-hydroxyhydrocinnamic acid
or 3-(p-hydroxyphenyl)propionic acid. The mass
spectrum of the butyl ester showed an M+ at 222
in the electron impact mode and an M+1 ion at
223 in the chemical mode. In the electron impact
ionization spectrum, the base peak ion was at
m/e 107, and characteristic fragment ions were
at m/e 120 (M+-102), m/e 149 (M+-73), and m/e
166 (M+-56). Identical mass spectra and retention time data were observed for a standard of
FIG. 1. Gas-liquid chromatogram of esterified 3-(p-hydroxyphenyl)propionic acid (Aldrich
short-chain acids produced by P. anaerobius 17642 Chemical Co., Milwaukee, Wis.) derivatized and
after growth in Schaedler broth containing 0.05% L
analyzed under the same conditions. When the
tyrosine. Abbreviations: C2, acetic acid; R, reagent; butyl ester derivatives of the culture extracts or
iC4, isobutyric acid; C4, butyric acid; L, lactic acid; thep-hydroxyhydrocinnamic acid standard were
iC5, isovaleric acid; iCe,, isocaproic acid; B> benzoic treated with trifluoroacetic anhydride (Pierce
acid; HCn, hydrocinnamic acid; and OH-HCn, hydroxyhydrocinnamic acid. Analysis was made on a Chemical Co., Rockford, Ill.), the retention time
decreased from 23.1 to 19.6 min, indicating that
5% OV-I column.
the hydroxyl group on the benzene ring had
reacted to form a more volatile compound. The
electron impact ionization mass spectrum of the
trifluoroacetyl butyl ester derivative had an M+
at 318, confirming the addition of -COCF3 to
the butyl ester.
A small amount of hydroxyhydrocinnamic
was produced by all cultures grown in
Schaedler medium without added tyrosine (Fig.
2), indicating that low concentrations of this
amino acid were present in unsupplemented met4
dia. The substantial increase in the amount of
this acid produced by all cultures of P. anaerobius grown in the tyrosine-enriched medium
(Fig. 1) clearly shows that hydroxyhydrocinnamic acid is a major product when tyrosine is
metabolized by this species.
These data indicate that the homogentisate
FIG. 2. Gas-liquid chromatogram of esterified
homoprotocatechuate pathways are not inshort-chain acids produced by P. anaerobius 17642
after growth in Schaedler broth. Analysis was made volved in the degradation of tyrosine by P. anaerobius since the expected intermediates of
on a 5% OV-I column. Abbreviations are as in the
legend to Fig. 1.
these pathways (p-hydroxyphenylpyruvic,p-hy-
VOL. 12, 1980
droxyphenyllactic, or p-hydroxyphenylacetic
acid) were not detected (2, 4, 9, 13). This organism may metabolize tyrosine by initial deamination to p-hydroxycinnamic acid and subsequent reduction of this acid to p-hydroxyhydrocinnamic acid. The deamination reaction has
been demonstrated in several fungi (4) and is
catalyzed by either a tyrosine or phenylalanine
ammonia-lyase (EC The latter enzymatic reaction could be similar to that reported
for Lactobacillus pastorianus subsp. quinicus
with various phenolic compounds (14). Similar
reactions may have been observed previously in
Clostridium sporogenes when phenylalanine, an
aromatic amino acid structurally related to tyrosine, was first metabolized to cinnamic acid
and then to hydrocinnamic acid (11). An analogous enzyme system may be responsible for the
degradation of tyrosine and phenylalanine in P.
anaerobius because p-hydroxyhydrocinnamic
and hydrocinnamic acids were both major metabolites of this organism. Obviously, other studies are needed to firmly establish the metabolic
pathway(s) involved in these reactions.
Other microorganisms, including the pathogenic aerobic actinomycetes (5), nonfermentative gram-negative bacilli (7), and members of
the Enterobacteriaceae (12), can also actively
degrade L-tyrosine. To our knowledge, the metabolites produced from tyrosine by these organisms have not been determined.
We thank Ann Y. Armfield, Anaerobe Section, Center for
Disease Control for providing the cultures used in this study.
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