Escolar Documentos
Profissional Documentos
Cultura Documentos
16, Issue
of August
Printedin
25, pp.
502.55030,
Biohydrogenation
VI. SOURCE
1971
U.S.A.
of Unsaturated
OF HYDROGEN
1. S. ROSENFELI)
ANU
AND
S. B.
Fatty
STEREOSPECIFICITY
Acids
OF REDUCTION*
(Received for publicat,ion,
March
Raleigh,
2760?
9, 1971)
TOVE~.
oj Biochemistry,
North
Camha
Carolina
EXPERIMENTAL
Bacterial
PROCEDURE
Culture
B. fibrisolvens strain A-38 was grown and maintained as previously described (2) except that the oxidation-reduction
potential 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 centrifugation 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-Qyn, trans-ll-enoic
acid with tritium gas and was the generous
gift of Dr. L. J. Morris, Unilever, Shambrook,
Bedford, England.
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 pomegranates 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 petroleum ether (b.p. 40-60) in a Soxhlet apparatus for 24 hours.
The acid was isolated by low temperature
crystallization
as described by Crombie and Jacklin (5). The white crystalline
product melted at 43 (lit. m.p. 40-42) (5) and gave the expected 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 ;spectrum exhibited characteristic peaks at 3600.0 cm-1 (OH) and
at 981.4 aud 932.0 cm- (cis, truns-conjugateddoublebond systern). No peaks were observed in t,he carbonyl region.
5025
The biohydrogenation
of either linoleic acid or cis-9, trans11 ,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, tritiated 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 indicated 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 reduction occurs by addition of a proton and hydride ion mediated by an unknown
carrier.
North
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.
SUMMARY
State Uwiuersity,
5026
Source
Nuclidic
c1a320
Calculated:
mass
Found :
of Hydrogen
and Stereospecificity
264.2453
264.2448
Nuclidic
mass
Calculated:
Found :
248.2504
248.2509
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 (Fraction 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
Following centrifugation,
the bacterial
the bacterial suspension.
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, stoppered, 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, volumes 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 derivative of punicic acid, the substrate was dispersed by sonic oscillation (Branson) in a small amount of buffer prior to incubation.
Vol.
246, No. 16
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 5-3Hglucose 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.
of Reduction
Issue
of August
I. X. Rosenfeld
25, 1971
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 cisfrans conjugated dienes (A9~11,A1J,1z,A8~10)(l), it appeared that the
specificity properties for the hydrogenation
reaction were likely
Accordingly,
the naturally occurring conto be less stringent.
jugated octadecatrienoic
acid, punicic acid, with a cis-9, truns11 ,&s-13 double bond system seemed likely to serve as a substrate.
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 spectroscopy and mass spectrometry.
In each case the spectra obtained 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 methyl1 l-osoundecanoate,
which indicated
the position of unsaturation to be at C-11.
In contrast to punicic acid, cis-9, trans-11 , trans-13-octadecatrienoic acid (a-eleostearic acid) was not changed during incubation with the bacteria.
Thus, it would appear that the
TABLE
and acid
=H
apm
1. Substrate
Product
Product
2. Substrate
Product
Product
a The
18:O~.
18: 1.
18:2..
18:O..
18: 1.
18:2.
number
.
.
_. .
.
.
to the left
x 10-z
501.8
35.3
6.6
467.5
243.1
28.0
of the colon
product
Oleic and
SH: C
dJ%Pz x NJ-
149.3
11.2
2.0
40.6
23.3
2.5
represents
3.35
3.15
3.30
11.50
10.40
11.20
the number
of
carbon atoms in the chain; the number to the right of the colon
designates
the
number
of double
bonds.
as described by Neihaus and Ryhage (23). The monoene fraction 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
hydrocarbon: 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
interest 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 incubation of B. jibrisolvens with cis-9, truns-11 , cis-13-octadecatriene
An F and M
were also subjected to gas-liquid chromatography.
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 measurement on a MS-902 mass spectrometer.
and S. B. Tove
5028
Source of Hydrogen
TABLE
and Stereospecificity
of Reduction
II
Trilium.
S:orn
in reductive
ozonolysis
fragments
of methyl
oleate isolated
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-dinitrophenylhydrazine
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, lodi-3H-cis-9,
trans-11-octadecadienoic
acid was 60 mCi per mmole.
Fragment
Deuterium
cgntent
of fragments
11 ,I$-dimethoxu
octadecanoate
octadecenoate
isolated
after
Butyrivibrio
of methyl
end and carboxyl
end of
prepared
from
methyl
trans-illinoleic
acid incubation
with
$brisolvens
Mass spectra
(70 e.v.) were obtained
with an AEI-12
ter and the peaks of interest
were corrected
for natural
spectromeabundance.
Tritium
1 D atom
2Datoms
&5m/Jmw1e x 10-a
Nonanal.................................
Methyl
9-oxononanoate.
100.0
80.0
TABLE
TABLE
III
Incorporation
of 3H from 1 -3H-glucose
and VH-glucose
into
11 -octadecenoic
acid and the saturated fatty
acids by
Butyrivibrio
jibrisolvens
Incubations
were
glucose
as described
carried
in the
trans-
Substrate
Saturated acids
Experiment
cis, &s-18:2
IV
(Agn12)
cis, trans,cis-18:3
(Ag.11v13)
1
2
3
1
2
after
incubation
1D
atom
~-__33
16
40
19
24
10
1 13
14
cis-cis-18:2
(A9.12)
cis-trans-&s-18:3
(As.ll.lr)
40
58
Substrate
at positions
C@&/j.Hde
7279
366
TABLE
Deuterium
Deuterium
Substrate
trans-11.18: 1
cpm//mde
PH-Glucose
3-3H-Glucose.
of deuterium
in trans-li-octadecene
prepared
from
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.
2D
atoms
3D
atoms
4D
atoms
43
60
39
34
36
9
7
3
27
25
0
0
0
13
7
substrates.
No tritium was incorporated
from glucose labeled
in positions 1>2 73 95 9 and 6 or from tritiated succinate or formate.
,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 performed.
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-
deuterium
cis double
tion.
To
was
bond(s)
localize
incorporated
during
the
reduction
incorporated
deuterium,
the
of the
of substitumethyl
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 methoxyl groups, yielding 2 ions (m/e 129 and m/e 229) corresponding
to the methyl end and the carboxyl end of the methyl truns-lloctadecenoate (23). The results (Table V) indicate that the deuterium atoms incorporated
during hydrogenation
of the A-9,
silicic
acid-silver
nitrate
column
chromatography.
The fractions 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 scintillation spectrometer.
Radioactivity
measurements
were corrected
for background
and quench. The specific activity of each tritiated glucose
was 100 $Zi per mmole.
VI
Distribution
methyl
Issue of August
I. X. Rosenfeld
25, 1971
DISCUSSION
The hydrogenation
of linolcic acid initially involves the isomerization
of linoleic acid to a cis-9, truns-1 l-octadecadienoic
acid. Several reports on the natire and characteristics of linoleic acid isomerase, the enzyme that catalyzes this reaction,
have been published (2, 3), but, until now, none of the findings
concerning the reduction of the conjugated intermediate
to truns11.octadecenoic
acid have been reported.
As there is no readily available source of this cis-9,truns-lloctadecadienoic
acid intermediate,
punicic acid, cis-9, truns-ll ,
cis-13.octadecatrienoic
acid represents a unique substrate which
facilitates the investigation
of t,he reductive reaction.
It has
been shown (Table IV) that both cis double bonds are hydrogenated, resulting in the same product as that obtained from
linoleic hydrogenation.
It is interesting
to note that, when
ar-eleostearic acid (cis-9, truns-11 , trans.13.octadecatrienoic
acid)
is used as a substrate, no reaction occurs. The inactive aeleostearic acid is a conjugated triene similar to punicic acid
differing only in that the configuration
of the Al3 bond is truns
instead of cis. It is apparent, therefore, that the configuration
of the conjugated truns double bond system imparts a degree of
alteration to the molecule such that the organism is incapable of
reducing the cis bond of the conjugated triene.
The similarity
of deuterium
distribution
at both cis bonds
indicates that, unlike linoleic acid isomerase, the carboxyl group
is a dispensable feature of the substrate.
Preliminary
experiments showed that the alcohol and paraffin derivatives of punicic
5029
trans-11-octadecadienoic
acid were located in the carboxyl portion of the molecule.
The distinct positions of substitution
were obtained by reducing the deutcrated truns-11-octadecenoic
acid from the punicic
acid and linoltic acid incubations
to the trans-11-octadecene.
Oxidative cleavage and mass spectrometry
of the methyl esters
of the heptanoic acid and undecanoic acid fragments allowed
the use of the McLafferty
rearrangement
to determine
the
location of deuterium in the original truns-1 l-octadecenoic
acid.
The major peak of methyl esters longer than Cs is due to a
rearranged ion of m/e 74. This ion contains 3 hydrogen atoms:
2 from the a-carbon and 1 from the y-carbon of the fatty acid
methyl ester (26). In this case, the two hydrogens bonded to
the a-carbon of the methyl heptanoate fragment represent the
hydrogen atoms at C-13 of the truns-11-octadecenoic
acid.
Those bonded at C-10 of the trans-11-octadecenoic
acid would
correspond to a-hydrogens of the methyl undecanoate fragment.
The appearance of a large peak at m/e 75 in the spectrum of
each of the monocarboxylic
methyl esters from both substrates
indicates that hydrogen from HZ0 is incorporated
at C-10 of
linoleic acid and at C-10 and C-13 of punicic acid.
From the ratio of the m/e 74 ion to the m/e 75 ion and the
assumption that all of the deuterium incorporated
was bonded
to the carbons of the cis double bond(s), the distribution
of
deuterium at each of the positions of the double bond could be
calculated.
The results (Table VI) show that the carbons adjacent to the truns double bond contain less deuterium than those
From the mass peaks associated with
distal to the truns bond.
the parent ions, it may be calculated that 3056 of the hy-drogen
atoms at C-13 and C-14 and 28T1 of the hydrogen at C-9 and
These results, together with
C-10 were replaced by deuterium.
the similarity in distribution,
suggest that both of the cis bonds
of punicic acid were hydrogenated by the same system.
and S. B. Tove
5030
Source of Hydrogen
and SkreospeciJicity
expected to show one-half of the tritium label. The results (Table 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 biohydrogenation
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
is
involves cis addition to the L side. However, B. fibrisolvens
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 biohyParticular effort is being directed
drogenation
are in progress.
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
is extended
trometry.
derivatives
and further
to Dr.
L. J. Morris
appreciation
21. PETERSON,
J. I., Anal.
Biochem.,
31, 189 (1969).
22. CASTLE,
J. D., AND ACKMAN,
R. G., Can. J. Chem., 45, 1405
(1967).
23. NEIHAUS,
W. J., JR., AND RYHAGE,
R., Anal.
Chem., 40, 1840
(1968).
24. SCHEUERBRANDT,
G., AND BLOCK. , K.. , J. Biol.
Chem.,
237,
2064 (1962).
25. SNYDER,
F., AND STEPHENS, N., Biochim.
Biophys.
Acta, 34,
244 (1959).
26. RYHAGE,
R., AND STENHAGEN,
E., in F. W. MCLAFFERTY
(Editor),
Mass spectroscopy
of organic ions, Academic
Press,
New York,
1963, p. 400.
27. SHORLAND,
F. B., WEENINK,
It. O., JOHNS, A. T., AND McDONALD,
I. R. C., Biochem.
J., 67, 328 (1957).
28. MOIZTENSON,
L. E., VALENTINE,
R. C., AND CARNHAN,
J. E.,
Biochem.
Biophys.
Res. Commun.,
7, 448 (1962).
29. MORRIS,
L. J., Biochem.
J., 118, 681 (1970).
30. SCHROEPFER,
G. J., JR., J. Biol. Chem., 241, 5441 (1966).
31. KEPLER,
C. R., TUCKER, W. P., AND TOVE, S. B., J. Biol.
Chem., 246, 2765 (1971).
32. POLAN, C. E., MCNEILL,
J. J., AND TOVE, S. B., J. Bacterial.,
88, 1056 (1964).
7. BAUMANN,
2,4-dinitrophenylhydrazone
of Reduction