lipids

1. THE JOUIWAL OF BIOLOGICAL CHEMIBTRY
Vol. 246, No. 16, Issue of August 25, pp. 502.55030, 1971
Printedin U.S.A.
Biohydrogenation of Unsaturated Fatty Acids
VI. SOURCE OF HYDROGEN AND STEREOSPECIFICITY OF REDUCTION*
(Received for publicat,ion, March 9, 1971)
1. S. ROSENFELI) ANU S. B. TOVE~.
From the Department oj Biochemistry, North Camha State Uwiuersity, Raleigh, North Carolina 2760?’
SUMMARY
The biohydrogenation of either linoleic acid or cis-9, trans-
11 ,cis-13-octadecatrienoic acid (punicic acid) by Butyriuibrio
jbrisolvens results in the formation of trans-11-octadecenoic
acid. Incubation of whole cells with tritiated formate, triti-
ated succinate, and glucose labeled with tritium in various
positions failed to result in the labeling of the monoenoic acid
product. In contrast, experiments performed in DzO indi-
cated that deuterium was incorporated at the cis double
bond(s) reduced by the microorganism. This reduction,
which takes place stereospecifically, was found to occur by
cis addition to the D side of cis-9, tram-1 1-octadecadienoic
acid, an intermediate in the biohydrogenation of linoleic acid.
The distribution of deuterium at the reduced carbon atoms
shows an isotope effect and leads to the speculation that re-
duction occurs by addition of a proton and hydride ion medi-
ated by an unknown carrier.
The pathway of biohydrogen:rtion of linoleic acid by the
anaerobic rumen bacterium, Butyrivibrio Jibrisolvens, consists of
at least two reactions: (a) an initial isomerization to L-9,
trans-ll-octadecadienoic acid and (b) the subsequent hydrogena-
tion of this compound to trans-ll-octadecenoic acid (1, 2).
Partial purification of linoleic acid isomerase, the enzyme that
cntalyzes the isomerization, has been achieved and some of its
properties have been investigated. It shows marked specificity
for a free carboxyl group and a cis-Q,cis-12 pentadiene system
(3). These studies were greatly facilitated by the finding that
this reaction takes place under aerobic conditions. In contrast
to this, t,he hydrogenation reaction appears to be obligately an-
aerobic, and active cell-free preparations have been difficult to
* This work is a contribution from the Department of Bio-
chemistry, School of Agriculture and Life Sciences and School of
Physical and Mathematical Sciences. It is Paper 3421 of the Jour-
nal Series of the, North Carolina State University Agricultural
Experiment Station, Raleigh, North Carolina. This work was
sunnorted in oart bv Public Health Service Research Grant AM-
02483 from &e Naiional Institute of Arthritis and Metabolic
Disecxs. High resolution mass spectrometry was done at the
Research Triangle Institute Cenier for Mass Spectrometry under
Grant PR 330 from the Biotechnology Resources Branch of the
National Institutes of Health.
t To whom correspondence should be addressed.
prepare. This report deals with the hydrogenation reaction with
intact cells in which the source of hydrogen and stereospecificity
of the reduction of the double bond were investigated.
EXPERIMENTAL PROCEDURE
Bacterial Culture
B. fibrisolvens strain A-38 was grown and maintained as pre-
viously described (2) except that the oxidation-reduction poten-
tial dye, resazurin, was not included and the media was gassed
with an atmosphere of oxygen-free 95% COZ and 50/O H, for 2
hours prior to inoculation. The cells were harvested by centri-
fugation in 250~ml capped polypropylene bottles in a Sorvall
GSA rotor at 14,600O X g for 15 min.
Chlorella vulgaris was grown and maintained as described by
Harris and James (4).
Substrates
Linoleic and a-eleostearic acids were obtained from the Hormel
Institute. The tritiated substrate cis-9, trans-ll-[Q, lo-3H]
octadecadienoic acid was prepared by reduction of octadec-Q-
yn, trans-ll-enoic acid with tritium gas and was the generous
gift of Dr. L. J. Morris, Unilever, Shambrook, Bedford, Eng-
land.
Punicic acid (c&Q, trans-11 ,cis-13-octadccatrienoic acid) was
isolated from the seed oil of Punica granatum (pomegranate)
purchased in a local market. The outer covers of the pome-
granates were removed and the fruit was allowed to soak in water
for 1 to 2 days. The fruit was then squeezed by hand to remove
the fleshy coating; and the small, hard, white seeds were dried
in a vacuum desiccator over PZOS. The seeds were ground
in a Wiley Mill and extracted under nitrogen with petro-
leum ether (b.p. 40-60”) in a Soxhlet apparatus for 24 hours.
The acid was isolated by low temperature crystallization as de-
scribed by Crombie and Jacklin (5). The white crystalline
product melted at 43” (lit. m.p. 40-42’) (5) and gave the ex-
pected ultraviolet spectrum with maxima at 264, 274 and 285
nm.
The alcohol derivative of punicic acid was prepared from the
methyl ester by treatment with LiAIHt (6). The alcohol gave
the same absorption spectrum as punicic acid and migrated as
a single spot on thin layer chromatoplates of silica gel with
heptane-isopropyl ether-acetic acid (6 :4 :0.3). The infrared ;spec-
trum exhibited characteristic peaks at 3600.0 cm-1 (OH) and
at 981.4 aud 932.0cm-’ (cis, truns-conjugateddoublebondsys-
tern). No peaks were observed in t,he carbonyl region.
5025
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2. 5026 Source of Hydrogen and Stereospecificity of Reduction Vol. 246, No. 16
c1a320
Nuclidic mass Calculated: 264.2453
Found : 264.2448
The ci.s-9, trans-11 ,cis-13-octadecatriene was prepared by
LiAIHd reduction of the mesylate ester of the alcohol (7, 8). The
hydrocarbon had an absorption spectrum identical with that of
punicic acid and gave a single spot when chromatographed on
silica gel plates with hexane as the solvent. When subjected to
gas-liquid chromatography, a single peak was observed. The
infrared spectrum showed no peaks in the carbonyl region, but
the same doublet, characteristic of the cis-trans double bond sys-
tem, was observed.
Nuclidic mass Calculated: 248.2504
Found : 248.2509
Deuterium oxide was supplied by Stohler Isotope Chemicals
and the acid hydrolysate of algae cells grown on D20 was obtained
from Merck.
Tritiated sodium formate, 2, 3-3H-succinic acid, and 5-3H-
glucose were obtained from Amersham-Searle. Glucose labeled
with tritium in positions 1, 2, 3, and 6 was obtained from New
England Nuclear.
The standard paraffins, 9-nonadecene and %heptadecene, were
obtained from the Chemical Samples Company.
Methods
Incubations-A solution of 5 mg of the fatty acid or derivative
in benzene was added to a 125-ml Erlenmeyer flask and the
solvent was removed with a stream of nitrogen. After the
benzene had evaporated, 12 ml of 0.05 M potassium phosphate
buffer, pH 6.6, containing 0.48 g of bovine serum albumin (Frac-
tion V) was added. Twelve milliliters of a bacterial suspension
in 0.1 M phosphate buffer, pH 6.6, were added and the flask was
stoppered with a rubber stopper equipped with two short glass
tubes, on which were placed 2-inch pieces of thin walled rubber
tubing. The flasks were placed in an ice bath and flushed with
hydrogen for 20 min, after which the rubber tubes were closed
with a pinch clamp. Incubation was carried out with gentle
agitation for 4 hours at 37”.
Undue exposure to air was avoided during the preparation of
the bacterial suspension. Following centrifugation, the bacterial
pellet was suspended in 13 ml of 0.1 1~phosphate buffer, pH 6.6,
that had been thoroughly flushed with hydrogen. The tube
containing the cells was flushed with hydrogen for 5 min, stop-
pered, and shaken to disperse the bacteria. The suspension was
diluted with thoroughly gassed buffer such that a 1: 100 dilution
gave an absorbance of 1 at 420 nm.
When the tritium-labeled substrates were used, 100 PCi were
added as an aqueous solution to the buffered albumin. When
cis-9, truns-11[9, 10-3H]octadecadienoic acid was incubated, vol-
umes one-third the usual size were used.
In experiments in which DzO was used, the buffer solution
was evaporated to dryness and the buffer salts were dissolved in
the appropriate volume of DZO.
In experiments conducted with the alcohol or paraffin deriva-
tive of punicic acid, the substrate was dispersed by sonic oscil-
lation (Branson) in a small amount of buffer prior to incuba-
tion.
Isolation of Reaction Products-Following incubation, the reac-
tion mixture was extracted according to the method of Dole (9).
The products of the fatty acid substrates were methylated by
diazomethane and the monoenoic acids were isolated by chroma-
tography of their methyl esters on silicic acid-silver nitrate
columns (10, 11). In each case, a single component was ob-
served when examined by gas-liquid chromatography. When
the alcohol or paraffin derivatives of punicic acid were used as
substrates, their hydrogenation products were separated by
chromatography on Florisil (12). The monoene paraffin prod-
uct was indicated by its retention time during gas-liquid chro-
matography with 9-nonadecene and S-heptadecene as standards.
The monoene alcohol was indicated by its cochromatography
with trans-11-octadecenol on silicic acid-silver nitrate thin layer
plates (13).
Stereospecijicity Studies-In these studies cis-9, trans-ll-
[9,10-3H]octsdecadienoic acid was used as the substrate. The
labeled trans-11-octadecenoic acid was isolated, methylated, and
reduced to methyl stearate by hydrazine (14). After saponifica-
tion, l-14C-stearic acid was added and the doubly labeled stearic
acid incubated with a suspension of Chlorella as described by
Morris et al. (15). The algal suspension was then extracted
with chloroform-methanol (2: 1)) and the methyl esters of the
fatty acids were prepared by transmethylation (16). Methyl
oleate and methyl linoleate were isolated by argentation column
chromatography (11). Each gave a single peak upon gas-liquid
chromatography.
To ensure that the tritium label had not moved during the hy-
drogenation of the Ag-bond, l-14C-labeled stearic acid was omitted
from the Chlorella incubation and the tritiated oleic acid was
isolated from the Chlorella suspension as previously described.
Carrier methyl oleate was added and reductive ozonolysis was
accomplished by the method of Edwards (17), except that the
2,4-dinitrophenylhydrazine reagent of Johnson (18) was used.
The dinitrophenylhydrazone derivatives of the aldehyde and
aldehydo-ester fragments were separated by chromatography on
alumina (19). The purity was established by the single spot
obtained for each fragment when chromatographed on thin layer
plates of Microcel-T38 (20). To determine the tritium in each
fragment, the nonanal-dinitrophenylhydrazone and the methyl-
9-oxononanoate dinitrophenylhydrazone were completely oxi-
dized (21) and the tritiated water was absorbed in 20 ml of a
solution of 30% methanol in toluene that contained 6 g of Omni-
fluor (New England Nuclear) per liter.
Oxidative cleavage of the 3H-labeled methyl oleate to nonanoic
acid and monomethyl azelaic acid was accomplished according
to the procedure of Castle and Ackman (22). The nonanoic
acid was isolated by steam distillation and the monomethyl
azelaic acid was isolated by thin layer chromatography on
silica gel plates withheptane-isopropyl ether-acetic acid (6:4 :0.3).
The monocarboxylic acid was extracted with ether and trans-
ferred to a counting vial. The spot corresponding to the mono-
methyl azelaic acid was scraped off and the product was eluted
with methanol and counted.
Mass Spectrometry-Following extraction, methylation, and
isolation of the product of either a deuterated substrate or fatty
acid substrate incubated in DzO, mass spectra were obtained by
means of a AEI-12 mass spectrometer.
The 11,12-dimethoxy methyl octadecanoate derivative of the
methyl truns-11-octadecenoate obtained from the incubation of
linoleic acid with B. jibrisolvens in DzO was prepared and isolated
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3. Issue of August 25, 1971 I. X. Rosenfeld and S. B. Tove
as described by Neihaus and Ryhage (23). The monoene frac-
tion from the incubation of punicic acid in D20 was reduced to
the paraffin via the alcohol and mesylate ester (7,8), as previously
described and oxidatively cleaved by a modified method of
Scheuerbrandt and Block (24). Since the paraffin was insoluble
in their reaction mixture, the solvent was removed and the
the following solutions were added per 5 mg of unsaturated hy-
drocarbon: 0.8 ml of t-butyl alcohol, 0.3 ml of a mixture of 0.02
M Khln04 and 0.19 M NaI04, 0.12 ml of 0.04 M K&Ox, and
finally 0.6 ml of water. The flask was sealed and stirred for 2
hours at room temperature and the acid fragments were isolated
(24). Mass spectra of their methyl esters were obtained by
using the gas chromatographic inlet system of a model 9000
LKB mass spectrometer. A four-foot column of ethylene glycol
succinate-HaPOd was used with temperature programming.
Several scans were obtained for all samples and the peaks of
int’erest were corrected for natural abundance.
Gus-Liquid Chromatography--The methyl esters of the acids
obtained from incubations with linoleic acid, cr-eleostearic acid,
and the c&runs-conjugated acid mixture were analyzed by
gas-liquid chromatography. The paraffins isolated from incu-
bation of B. jibrisolvens with cis-9, truns-11 ,cis-13-octadecatriene
were also subjected to gas-liquid chromatography. An F and M
model 700 flame ionization instrument equipped with four-foot
columns of 10% diethylene glycol succinate on Chromosorb W
was used.
Other Analytical Procedures-Ester groups were determined by
the procedure of Snyder and Stephens (25).
Radioactivity was measured in a Packard Tri-Carb liquid
scintillation spectrometer by usin, 0‘ a scintillation solution of
Omnifluor (New England Nuclear) in toluene (4 g per liter).
Infrared spectra were measured in a Beckman IR-8 in carbon
disulfide solution.
Jloleculnr formulas were determined by accurate mass meas-
urement on a MS-902 mass spectrometer.
RESULTS
Hydrogenation of Punicic Acid---Linoleic acid isomerase, the
enzyme that catalyzes the first reaction in the biohydrogenation
pathway, has marked substrate specificity requirements (3).
Since B. fibrisolvens was able to hydrogenate a mixture of cis-
frans conjugated dienes (A9~11,A1’J,1z,A8~10)(l), it appeared that the
specificity properties for the hydrogenation reaction were likely
to be less stringent. Accordingly, the naturally occurring con-
jugated octadecatrienoic acid, punicic acid, with a cis-9, truns-
11 ,&s-13 double bond system seemed likely to serve as a sub-
strate. When punicic acid was incubated with the bacteria,
analysis of the methyl esters of the free fatty acids isolated from
the incubation mixture showed a complete disappearance of the
conjugated triene and the appearance of a peak coincident with
methyl oleate. After isolation of this product by argentation
chromatography, it was subjected to analysis by infrared spec-
troscopy and mass spectrometry. In each case the spectra ob-
tained were identical with those of the trans-ll-octadecenoate
product of the linoleic acid incubation. Moreover, reductive
cleavage of the methyl ester yielded heptaldehyde and methyl-
1l-osoundecanoate, which indicated the position of unsatu-
ration to be at C-11.
In contrast to punicic acid, cis-9, trans-11 , trans-13-octadec-
atrienoic acid (a-eleostearic acid) was not changed during incu-
bation with the bacteria. Thus, it would appear that the
TABLE I
Recovery of aH frqm products of &saturation of doubly labeled
stearic acid by Chlorella vulgaris
Experiments with cis-9, trams-11[9, 10-3Hloctadecadienoic acid
were as described in the text. The biohydrogenation product
was reduced to stearate and incubated with Chlorella. Oleic and
linoleic acids were isolated and counted.
Experiment and acid =H “C SH: “C
apm x 10-z dJ%Pz x NJ-’
1. Substrate 18:O~. . 501.8 149.3 3.35
Product 18: 1. . 35.3 11.2 3.15
Product 18:2.. _. . 6.6 2.0 3.30
2. Substrate 18:O.. . 467.5 40.6 11.50
Product 18: 1. 243.1 23.3 10.40
Product 18:2. . . 28.0 2.5 11.20
a The number to the left of the colon represents the number of
carbon atoms in the chain; the number to the right of the colon
designates the number of double bonds.
presence of the trans bond at C-13 prevented the hydrogenation
of the cis-9 bond.
Hydrogenation of Parafin and Alcohol Derivatives of Punicie
Acid--Gas-liquid chromatography of the hydrocarbons isolated
after incubation of B. Jibrisolvens with cis-9, truns-11 ,cis-13-
octadecatriene showed the appearance of a peak not observed in
the hydrocarbon fraction from a zero time control. This peak,
amounting to 21.5% of the hydrocarbon fraction, exhibited a
retention time corresponding to that calculated for an octa-
decene.
The alcohol derivative of punicic acid also appears to be re-
duced, since analysis of the reaction products by argentation
thin layer chromatography showed a spot that corresponded to
truns-11-octadecenol.
Stereospecijcity of Biohydrogenation Reaction-Stereospecific
desaturation of stearic acid by C. vulgaris (15) provided the
rationale by which the stereospecificity of the reduction of the
cis-9 double bond of cis-9, truns-ll-octadecadienoic acid was
studied. In these experiments cis - 9, truns - 11[9,10 - 3H]octa-
decadienoic acid was incubated with B. $brisolvens. The la-
beled truns-11-octadecenoate product was isolated as the methyl
ester and converted to stearic acid. Following the addition of
lJ4C-stearic acid, the doubly labeled stearic acid was incubated
with Chlorella. In each of two experiments, the ratio of 3H:14C
in the oleic and linoleic acids isolated from the algae was the
same as the 3H:14C of the stearic acid substrate (Table I). To
ensure that migration of the labeled hydrogens had not occurred
during incubation with B. Jibrisolvens, stearic acid containing
only the tritium label was incubated with Chlorella. The dini-
trophenylhydrazone derivatives of the aldehyde fragments ob-
tained from reductive ozonolysis of the tritiated methyl oleate
were found to contain almost equal amounts of tritium (Table
II). Another portion of the labeled oleate was oxidatively
cleaved. No radioactivity was observed in either the nonanoic
or monomethyl azelaic acid fragments. These results show that
the tritium in the cis-9, truns-1119, 10-3H]octadecadienoate had
remained at positions 9,lO during incubation with B. fibrisol-
vens.
Source of Hydrogen in Hydrogenation Reaction---Initial at-
tempts to ascertain the source of the reducing hydrogen were
made by incubating B. jibrisolvens with a series of tritiated
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4. 5028 Source of Hydrogen and Stereospecificity of Reduction Vol. 246, No. 16
TABLE II
Trilium. in reductive ozonolysis fragments of methyl oleate isolated
S:orn Zhlorella after incubation with cis-9, trans-11[9,1 O-aH]-
octaclecadienoic acid with Butyrivibrio jibrisolvens
The oeonide of methyl oleate was reduced with 2,4-dinitro-
phenylhydrazine and the dinitrophenylhydrazones of nonanal
and methyl 9-oxononanoate were oxidized and the water from
each was collected and counted. The specific activity of the 9, lo-
di-3H-cis-9, trans-11-octadecadienoic acid was 60 mCi per mmole.
Fragment Tritium
&5m/Jmw1e x 10-a
Nonanal................................. 100.0
Methyl 9-oxononanoate. . . 80.0
TABLE III
Incorporation of 3H from 1 -3H-glucose and VH-glucose into trans-
11 -octadecenoic acid and the saturated fatty acids by
Butyrivibrio jibrisolvens
Incubations were carried out with linoleic acid and the labeled
glucose as described in the text. The methyl esters of the satu-
rated fatty acids and trans-11-octadecenoic acid were isolated by
silicic acid-silver nitrate column chromatography. The frac-
tions emerging from the column first were taken as saturated fatty
esters. Ester concentrations were determined on a portion of
the sample, and another portion was counted in a liquid scintilla-
tion spectrometer. Radioactivity measurements were corrected
for background and quench. The specific activity of each trit-
iated glucose was 100 $Zi per mmole.
Substrate
PH-Glucose
3-3H-Glucose.
Saturated acids
cpm//mde
7279
366
trans-11.18: 1
C@&/j.Hde
40
58
TABLE IV
Deuterium in methyl trans-11 -octadecenoate isolated after incubation
of linoleic.acid and punicic acid
Ex-
Per cent of parent ions containing
Substrate peri-
ment XoD 1D 2D 3D 4D
atoms atom atoms atoms atoms
~-__-
cis, &s-18:2 (Agn12) 1 15 33 43 9 0
2 15 16 60 7 0
3 9 40 39 3 0
cis, trans,cis-18:3 (Ag.11v13) 1 7 19 34 27 13
2 8 24 36 25 7
substrates. No tritium was incorporated from glucose labeled
in positions 1 2 3 5 and 6 or from tritiated succinate or formate.> 7 9 9
,Evidence that l-3H-glucose and 3-3H-glucose were metabolized
as expected, i.e. provided reducing equivalents for fatty acid
synthesis, comes from the observation that tritium was found in
the fatty acids synthesized by the cell (Table III).
To determine whether or not water provides the hydrogens
for reduction, incubations of I?. jlbrisolvens in DzO were per-
formed. When incubated in D20, a single deuterium atom was
found to be incorporated at C-13 during the isomerization of
linoleic acid to cis-9, truns-11-octadecadienoic acid (2), but the
hydrogenated product was not examined. More recent experi-
TABLE V
Deuterium cgntent of fragments of methyl end and carboxyl end of
11 ,I$-dimethoxu octadecanoate prepared from methyl trans-il-
octadecenoate isolated after linoleic acid incubation with
Butyrivibrio $brisolvens
Mass spectra (70 e.v.) were obtained with an AEI-12 spectrome-
ter and the peaks of interest were corrected for natural abundance.
Per cent of parent ions containing
No D atoms / 1 D atom 1 2Datoms
TABLE VI
Distribution of deuterium in trans-li-octadecene prepared from
methyl trans-11 -octadecenoate product of linoleic and punicic
acid incubations
After isolation, methyl trans-11-octadecenoate was converted
to the paraffin derivative and oxidatively cleaved to heptanoic
and undecanoic acids. The methyl esters of these acids were
subjected to gas-liquid chromatography-mass spectrometer
analyses on a LKB model 9000 spectrometer (70 e.v.). The peaks
of interest were corrected for natural abundance. The positions
refer to the original trans-11-octadecenoic acid, positions 9 and 10
coming from undecanoic acid and positions 13 and 14 coming from
heptanoic acid.
Substrate
Deuterium at positions
1 9 1 10 1 13 / 14
cis-cis-18:2 (A9.12)
cis-trans-&s-18:3 (As.ll.lr)
ments in which approximately 3 ml of bacterial pellet were
suspended in 12 ml of 99% DzO indicate that, during reduction
of the cis-9, trans-11-octadecadienoic acid in DzO, 2 additional
atoms of deuterium were present in the resulting truns-ll-
octadecenoic acid (Table IV). The actual level of deuterium
incorporated reflects not only the specific activity of the water
but the rate of equilibration of deuterium with the active hydro-
gens of the bacterial cell.
Similar experiments were performed with punicic acid as a
substrate. Since punicic acid does not undergo isomerization
prior to its hydrogenation to the truns-11-monoene and since
both cis bonds are reduced, it was expected that 4 deuterium
atoms would be incorporated. The results in Table IV show
this to be the case.
These experiments analyzed by mass spectrometry indicated
that deuterium was incorporated during the reduction of the
cis double bond(s) but did not reveal the positions of substitu-
tion. To localize the incorporated deuterium, the methyl ester
of truns-11-monoenoic acid resulting from linoleic incubation
was converted to the 11,12-dimethoxy derivative and subjected
to mass spectrometry (23). When treated in this manner, the
dimethoxy compound undergoes cleavage between the meth-
oxyl groups, yielding 2 ions (m/e 129 and m/e 229) corresponding
to the methyl end and the carboxyl end of the methyl truns-ll-
octadecenoate (23). The results (Table V) indicate that the deu-
terium atoms incorporated during hydrogenation of the A-9,
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5. trans-11-octadecadienoic acid were located in the carboxyl por- acid were hydrogenated and, thus, support this conjecture.
tion of the molecule. Some naturally occurring compounds that contain a truns-con-
The distinct positions of substitution were obtained by reduc- jugated double bond system, such as the carotenes, escape
ing the deutcrated truns-11-octadecenoic acid from the punicic hydrogenation in the rumen (27). The findings with cu-eleo-
acid and linoltic acid incubations to the trans-11-octadecene. stearic acid and the punicic acid derivatives suggest that it is
Oxidative cleavage and mass spectrometry of the methyl esters the presence of the truns configuration rather than the absence
of the heptanoic acid and undecanoic acid fragments allowed of a carboxyl group which accounts for their lack of hydrogena-
the use of the McLafferty rearrangement to determine the tion.
location of deuterium in the original truns-1 l-octadecenoic acid. Experiments with tritiated glucose (labeled in positions 1, 2, 3,
The major peak of methyl esters longer than Cs is due to a 5, and 6) showed that the hydrogen that reduces the double bond
rearranged ion of m/e 74. This ion contains 3 hydrogen atoms: did not come directly from glucose. Similarly, absence of tritium
2 from the a-carbon and 1 from the y-carbon of the fatty acid incorporation in truns-11-octadecenoic acid from labeled formate
methyl ester (26). In this case, the two hydrogens bonded to and succinate, as well as the absence of deuterium incorporation
the a-carbon of the methyl heptanoate fragment represent the from a totally deuterated algal hydrolysate, indicated that the
hydrogen atoms at C-13 of the truns-11-octadecenoic acid. direct addition of hydrogen from an organic substrate was un-
Those bonded at C-10 of the trans-11-octadecenoic acid would likely. In contrast, the fact that 2 deuterium atoms were incor-
correspond to a-hydrogens of the methyl undecanoate fragment. porated from D,O during the hydrogenation of cis-9, truns-ll-
The appearance of a large peak at m/e 75 in the spectrum of octadecadienoic acid and 4 deuterium atoms from DzO were
each of the monocarboxylic methyl esters from both substrates incorporated in the biohydrogenation of punicic acid indicates
indicates that hydrogen from HZ0 is incorporated at C-10 of that water is the immediate source of hydrogens used to reduce
linoleic acid and at C-10 and C-13 of punicic acid. the cis bond(s). These results, however, do not preclude the
From the ratio of the m/e 74 ion to the m/e 75 ion and the direct reduction of a carrier by an organic substrate if the hydro-
assumption that all of the deuterium incorporated was bonded gen carrier can undergo rapid exchange with water.
to the carbons of the cis double bond(s), the distribution of Examination of the isotope distribution in the reduced products
deuterium at each of the positions of the double bond could be showed that the position(s) adjacent to the truns double bond
calculated. The results (Table VI) show that the carbons adja- contains less deuterium than the distal position(s). This dis-
cent to the truns double bond contain less deuterium than those tribution indicates that discrimination against deuterium had oc-
distal to the truns bond. From the mass peaks associated with curred at C-10 of cis-9,truns-ll-octadecadienoic acid, and at (‘-10
the parent ions, it may be calculated that 3056 of the hy-drogen and C-13 of punicic acid. Therefore, the hydrogens added at
atoms at C-13 and C-14 and 28T1 of the hydrogen at C-9 and C-10 or C-13 must have experienced at least one more bond-
C-10 were replaced by deuterium. These results, together with breaking event than those added at C-9 or C-14. These results
the similarity in distribution, suggest that both of the cis bonds lead to the suggestion that the mechanism of biohydrogenation
of punicic acid were hydrogenated by the same system. involves the addition of a proton to the cis bond at the position
distal to the truns bond and that reduction of the double bond is
DISCUSSION
finally completed by a hydride ion provided by an unknown car-
The hydrogenation of linolcic acid initially involves the isom- rier.
erization of linoleic acid to a cis-9, truns-1 l-octadecadienoic Since ferredoxin occurs commonly in anaerobic organisms, one
acid. Several reports on the natire and characteristics of lino- might expect this electron carrier to be involved in biohydrogena-
leic acid isomerase, the enzyme that catalyzes this reaction, tion. However, we were unable to observe a ferredoxin band on
have been published (2, 3), but, until now, none of the findings a DEAE-cellulose column following chromatography (28) of cell
concerning the reduction of the conjugated intermediate to truns- extracts of B. $brisolvens.
11.octadecenoic acid have been reported. Upon biohydrogenation and reduction of the monoenoic acid to
As there is no readily available source of this cis-9,truns-ll- stearic acid, it is possible, with the stearic acid as a substrate for
octadecadienoic acid intermediate, punicic acid, cis-9, truns-ll , Chlorellu, to determine the stereochemistry of hydrogen addition
cis-13.octadecatrienoic acid represents a unique substrate which by B. jibrisolvens. Morris has used this approach to study the
facilitates the investigation of t,he reductive reaction. It has stereospecificity of the biohydrogenation of oleic and elaidic acids
been shown (Table IV) that both cis double bonds are hydro- by mixed rumen flora (29), and Schroepfer, using Corynebucterium
genated, resulting in the same product as that obtained from diphtheriue instead of ChZoreZZu,determined the stereospecificity
linoleic hydrogenation. It is interesting to note that, when of the hydroxylation of oleic acid (30). As reported in the pre-
ar-eleostearic acid (cis-9, truns-11 ,trans.13.octadecatrienoic acid) vious paper of this series, the same approach was used to show
is used as a substrate, no reaction occurs. The inactive a- the stereospecificity of hydrogen addition at C-13 of linoleic acid
eleostearic acid is a conjugated triene similar to punicic acid during its isomerization (31).
differing only in that the configuration of the Al3 bond is truns If the biohydrogenation of cis-9, truns-11-octadecadienoic acid
instead of cis. It is apparent, therefore, that the configuration occurs by cis addition, then either DD or LL-9, 10-di-aH-truns-ll-
of the conjugated truns double bond system imparts a degree of octadecenoic acid would result. Desaturation by Chore&x of the
alteration to the molecule such that the organism is incapable of stearic acid derived from the truns-ll-octadecenoic acid would
reducing the cis bond of the conjugated triene. yield oleic and linoleic acids showing either complete recovery of
The similarity of deuterium distribution at both cis bonds the tritium for the D-labeled enantiomer or complete loss of trit-
indicates that, unlike linoleic acid isomerase, the carboxyl group ium for the L-labeled enantiomer. The truns addition of hydro-
is a dispensable feature of the substrate. Preliminary experi- gen by B. fibrisolvens would yield threo-di-aH-truns-ll-octudccc-
ments showed that the alcohol and paraffin derivatives of punicic noic acid, and the oleic acid isolated from Chlorellu would be
Issue of August 25, 1971 I. X. Rosenfeld and S. B. Tove 5029
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6. 5030 Source of Hydrogen and SkreospeciJicity of Reduction Vol. 246, No. 16
expected to show one-half of the tritium label. The results (Ta-
ble I) showed complete recovery, and reductive and oxidative
ozonolysis of the oleic acid showed that the tritium label had not
moved during incubation. We conclude, therefore, that the bio-
hydrogenation of L-9, trns-1 I-octadecadienoic acid by B.
fibrisolvens occurs by cis addition to the D side of carbons 9 and 10.
Morris (29) has shown that the biohydrogenation of oleic acid
involves cis addition to the L side. However, B. fibrisolvens is
unable to hydrogenate oleic acid (32). Consequently, although
the biohydrogenation of oleic and &s-9, trans-11-octadecadienoic
acids is similar in that both involve cis addition to a cis double
bond, it is clear that, the two systems are different.
Studies with a cell-free system capable of carrying out biohy-
drogenation are in progress. Particular effort is being directed
toward the elucidation of the nature of the electron donor and
carrier.
Acknowledgments-We wish to thank Dr. Marion Miles of the
Department of Chemistry for some of the mass spectrometric
analyses. We also thank Drs. D. P. Schwartz and 0. W. Parks
of the USDA, Washington, D. C., for helping us separate the
2,4-dinitrophenylhydrazone derivatives and further appreciation
is extended to Dr. Parks for his help in gas-liquid mass spec-
trometry. We are also indebted to Dr. L. J. Morris for his many
helpful comments and discussions.
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