Calculation of '?C-incorporation ratios in !?C-labeled methyl pheophorbide a The signal of the methoxyl carbon, which was derived from CH;0H used in the transesterification reaction to t
Trang 1Evaluation of two biosynthetic pathways to 6-aminolevulinic acid
in Euglena gracilis
Katsumi lida, Ippei Mimura and Masahiro Kajiwara
Department of Medicinal Chemistry, Meiji Pharmaceutical University, Kiyose-shi, Tokyo, Japan
6-Aminolevulinic acid (ALA), which is an intermediate in
the biosynthesis of chlorophyll a, can be biosynthesized via
the C5 pathway and the Shemin pathway in Euglena gracilis
Analysis of the '*C-NMR spectrum of '*C-labeled methyl
pheophorbide a, derived from ‘C-labeled chlorophyll a
biosynthesized from p-[1-'°C]glucose by E gracilis, provid-
ed evidence suggesting that ALA incorporated in the
C-labeled chlorophyll a was synthesized via both the C5
pathway and the Shemin pathway in a ratio of between 1.5
and 1.7 to one The methoxyl carbon of the methoxycar- bonyl group at C-13° of chlorophyll a was labeled with '°C The phytyl moiety of chlorophyll a was labeled on C-P2, C-P3', C-P4, C-P6, C-P7', C-P8, C-P10, C-P11', C-P12, C-P14, C-PI5' and C-P16
Keywords: 6-aminolevulinic acid; C5 pathway; Shemin pathway; Euglena gracilis, "C-NMR
6-Aminolevulinic acid (ALA) (Fig 1, 3), which is an
intermediate in the biosynthesis of tetrapyrrole compounds
such as chlorophyll a (1), vitamin B; and heme, can be
biosynthesized via two pathways, the Shemin pathway (C4
pathway) [1-7] and the C5 pathway [8-14] (Fig 1) In
the Shemin pathway, ALA (3) is biosynthesized by the
condensation of glycine (4) and succinyl CoA (5) In the C5
pathway, ALA (3) is derived from all the carbons of
L-glutamate (L-glutamic acid; 6)
Mayer et al reported that ALA (3) is biosynthesized via
the C5 pathway in Euglena gracilis [12] Beale et al reported
that £ gracilis contains ALA synthase [15], implying that
ALA (3) may also be synthesized via the Shemin pathway
Weinstein e¢ al [16] reported that the C5 pathway in the
chloroplast and ALA synthase probably in the mitochond-
rion of E gracilis operate simultaneously to biosynthesize
ALA They also showed that [2-'*C]glycine was incorpo-
rated specifically into the nontetrapyrrole portion of chlo-
rophyll a (1) by E gracilis Okazaki et al [17] found that
2-'” C]glycine was not incorporated in the tetrapyrrole
portion of chlorophyll a (1) via ALA (3), but was incorpo-
rated into the methoxyl carbon of the methoxycarbonyl
group at C-137 of chlorophyll a (1) by E gracilis Oh-hama
et al [18] and Porra et al [19] reported similar results for
incorporation of isotope-labeled glycine into chlorophyll a
(1) by Scenedesmus obliquus and maize leaves Thus, the
involvement of the Shemin pathway could not be assessed in
terms of labeling in the tetrapyrrole portion of chlorophyll a
Correspondence to K lida, Department of Medicinal Chemistry, Meiji
Pharmaceutical University, 2-522-1 Noshio, Kiyose-shi, Tokyo
204-8588, Japan Fax: + 81 424 95 8612; Tel.: + 81 424 95 8611,
E-mail: iida@my-pharm.ac.jp
Abbreviations: ALA, 6-aminolevulinic acid; DMBI, 5,6-dimethyl-
benzimidazole; GSA, glutamate 1-semialdehyde; MPLC, medium-
pressure liquid chromatography; ODS, octadecyl silica; TCA,
tricarboxylic acid
(Received 31 August 2001, revised 1 November 2001, accepted 2
November 2001)
(1) from isotope-labeled glycine fed to the organism Porra
et al concluded that the C5 pathway is the predominant biosynthetic pathway to ALA utilized in chlorophyll a (1),
as shown from feeding experiments with p,L-[1-C]- and [5-'°C]glutamic acid in maize leaves [19] This is in contrast
to their previous estimation of approximately equal contri- butions of the C5 pathway and the Shemin pathway, based
on feeding experiments with sodium[1-!4C]- and[5-'C] a-ketoglutarate [20]
We were interested in investigating the existence of the Shemin pathway for ALA and the ratio of ALA biosyn- thesis from the Shemin pathway to that from the C5 pathway in E gracilis Shemin and others reported that ALA (3) is biosynthesized via the Shemin pathway in Propionibacterium shermanii [6,7], but our analysis of the C-NMR spectrum of C-labeled vitamin Bịa biosynthe- sized from p-[I-'C]glucose by P shermanii provided evidence that ALA (3) incorporated in the ‘C-labeled
vitamin B,» may have been synthesized via both the Shemin
pathway and the C5 pathway [21] We therefore conducted similar feeding experiments with p-[1-'°C]glucosein E grac- ilis, and used °C-NMR spectroscopy to examine the C-enrichment ratios of the carbon atoms of ‘C-labeled chlorophyll a or its derivative, “C-labeled methyl pheo- phorbide a (Fig 1) Our results indicate that the C5 and Shemin pathways both operate in FE gracilis, and provide information about the biosynthetic pathways leading to the methoxyl carbon of the methoxycarbonyl group at C-13° and the phytyl moiety of chlorophyll a (1)
EXPERIMENTAL PROCEDURES
Organism and chemicals The strain used was E gracilis IME E-3 Chlorophyll a (1) (from Spirulina) was purchased from Wako Pure Chemical Industries, Ltd Methyl pheophorbide a (2) was purchased
from Tama Biochemical Co., Ltd p-[I-C]Glucose
(90 atom % '%C) was purchased from Cambridge Isotope Laboratories All other chemicals were of analytical grade
Trang 26 CH„OH
ALA
1 C=O 4
Pa' P7 P11! P15!
P1 P4 P6 P8 P10 P12 P14 P16
Fig 1 Biosynthetic pathways to chlorophyll a (1) from p-glucose (9) and structure of methyl pheophorbide a (2) Chlorophyll a (1) 1s biosynthesized through 6-aminolevulinic acid (ALA) (3) formed via the CS pathway and the Shemin pathway from b-glucose (9), and methyl pheophorbide a (2) 1s derived from chlorophyll a (1)
Instruments
All "H-NMR (400 MHz) and '°C-NMR (100 MHz) spec-
tra were recorded on a Jeol GSX-400 spectrometer UV
spectra were recorded on a Jasco UVIDEC-610C
spectrometer
Examination of optimum amount of p-[1-'*C]glucose
for E gracilis feeding experiments
E gracilis was cultured as described previously, with some
modifications [17] The cultures were grown under illumi-
nation (2400 Lx) in seed culture medium (10 mL), which
consisted of L-glutamic acid (5 gL), p,t-malic acid
(2 gL), t-methionine (50 mg-L7'), thiamine hydrochlo-
ride (1 mgL7'), cyanocobalamin (5 ug-L'), KH>PO,
(0.4 ¢L7'), MgSO,-7H>O (0.5 gL), CaCO; (0.1 gL")
(2mgL"), CuCl2H,O0 (04mgL”), CoClh-6H,O
(2 mg-L~') and H3BO,:7H>O (80 pg-L~'), in a 60-mL test
tube at 27°C After 7 days, the seed culture medium
(10 mL) was added to fermentation culture medium (1 L) in
a 3-L conical flask This fermentation culture medium
contained 2.5-20 g-L™ of p-glucose (9) added in place of
L-glutamic acid (5 g-L7') and p,L-malic acid (2 g-L~') in the
seed culture medium The cultures of E gracilis were
continuously grown photosynthetically (2400 Lx) at 27 °C
with or without bubbling of air After 7 days, the wet cells,
collected by centrifugation of the culture broth for 30 min at
12 300 g, were weighed
Feeding of p-[1-'*C]glucose to £ gracilis The above seed culture medium (10 mL x 2), cultivated for
7 days, was added to fermentation culture medium (1 L x 2), which consisted of p-[1-'°C]glucose (2.5 g-L™) added in place of L-glutamic acid (5 gL~') and D,L-malic acid (2 ø;L”) in the seed culture medium, in a 3-L conical flask The cultures of E gracilis were continuously grown photosynthetically (2400 Lx) at 27°C for 7 days with bubbling of air The cells were collected by centrifugation
of the culture broth for 30 min at 12 300 g
Isolation of '*C-labeled chlorophyll a The isolation of chlorophyll a (1) was carried out by modification of the methods described in our previous paper [17] The growing cultures of E gracilis were washed with 0.9% NaCl, and this suspension was centrifuged again for
30 min at 12 300 g The cells were suspended in CH30H (S50 mL), disrupted with an ultrasonicator at 0 °C for 5 min, and centrifuged for 30 min at 12 300 g The supernatant was evaporated 1n the dark Purification of the residue by medium-pressure liquid chromatography (MPLC) using a prepacked glass column [2.5 (internal diameter) x 30 cm, octadecyl silica (ODS)] with CH30H gave ‘C-labeled chlorophyll a The amount of '°C-labeled chlorophyll a
Trang 3isolated was calculated from the UV absorption spectrum
[22]
Transformation from '*C-labeled chlorophyll 2
to '?C-labeled methyl pheophorbide a
Concentrated H2SO, (0.5 mL) was added dropwise, at 0 °C
under argon, to a solution of C-labeled isolated chloro-
phyll ain dry CH3,0H (9.5 mL), and the mixture was stirred
for 12 h at room temperature in the dark The reaction
mixture was diluted with CH2Cl, (200 mL), and quenched
with saturated NaHCO3 The organic layer was washed
with saturated NaHCOs;3, water and saturated NaCl, dried
over dry MgSOu,, and then evaporated Chromatography of
the crude product on silica gel with CHCl3/CH30OH (25 : 1,
v/v) gave '*C-labeled methyl pheophorbide a The amount
of C-labeled methyl pheophorbide a isolated was calcu-
lated from the UV absorption spectrum [22]
'3C-NMR measurements of chlorophyll a
and methyl pheophorbide a
The '°C-NMR spectra were obtained for solutions of '°C-
labeled chlorophyll a (4.8 mm) and chlorophyll a (1) in
C“HCI;/C”H2OH (79 : 6, v/v), and solutions of '°C-labeled
methyl pheophorbide a (3.8 mm) and methyl pheophorbide
a (2) in CÍHCI: The signal of C°7HCl (77.0 p.p.m.) was
used as an internal standard The spectral width was
24 038.5 Hz with 32 768 data points, which corresponds to
a resolution of 0.73 Hz per point The 10-pulse-width was
4.4 us, the acquisition time was 0.682 s, the pulse delay time
was 2.5 s, and the number of scans was 15 000-18 000 The
assignments of “C-NMR signals of chlorophyll a (1) and
methyl pheophorbide a (2) were made on the basis of
reported data [23-28]
Calculation of '?C-incorporation ratios
in !?C-labeled methyl pheophorbide a
The signal of the methoxyl carbon, which was derived from
CH;0H used in the transesterification reaction to the
methyl ester from the phytyl ester of ‘C-labeled chloro-
phyll a of the methoxycarbonyl group at C-17° of
C-labeled methyl pheophorbide a shows the natural
abundance of °C, and thus can be used as a reference
signal The 'C-enrichment ratio for each carbon of
C-labeled methyl pheophorbide a was calculated from
comparison of the signal intensities or half widths in the
'3C-NMR spectrum of '°C-labeled methyl pheophorbide a,
with those of methyl pheophorbide a (2)
RESULTS
Suitable amount of p-[1-'?C]glucose
for feeding experiment to F gracilis
Cultures of E gracilis were grown photosynthetically in
E gracilis fermentation culture medium containing various
amounts of p-glucose (9) in place of L-glutamic acid and
D,L-malic acid, which are the carbon sources of chlorophyll a
(1), without or with bubbling of air After 7 days, the culture
broth was centrifuged for 30 min at 12 300 g, and the cells
were weighed Without air bubbling, 10, 15 and 20 g:L™ of
p-glucose (9) gave 2.11, 4.01 and 4.72 gL! of E gracilis,
respectively, as shown in Table 1 With air bubbling, 2.5, 5,
10 and 15 gL! of p-glucose (9) gave 3.64, 3.64, 4.23 and 5.85¢L7' of E gracilis, respectively For reasons of economy, we chose to use two cultures, each containing 2.5¢L' of pfl-C]glucose, with air bubbling for the feeding experiments
Biosynthesis of '*C-labeled chlorophyll a and '3C-incorporation in its phytyl moiety C-Labeled chlorophyll a (2.6 mg) was isolated from growing cultures (6.7 g) of E gracilis cultivated in two 1-L fermentation culture medium in the presence of D-[I-'C]glucose Its purity was judged to be high by comparison of the ‘H-NMR and UV spectra with those of authentic chlorophyll a (1) The '*C-enrichments of carbons (C-P2, C-P3', C-P4, C-P6, C-P7', C-P8, C-P10, C-P11’, C-P12, C-P14, C-P15' and C-P16) of the phytyl moiety of C-labeled chlorophyll a were higher than those of carbons
of the chlorin ring moiety
Synthesis of '*C-labeled methyl pheophorbide a and determination of '2C-incorporation ratios
!%C-Labeled methyl pheophorbide a (1.4 mg) was derived from C-labeled chlorophyll a (2.6 mg) Its purity was judged to be high by comparison of the 'H-NMR and UV spectra with those of authentic methyl pheophorbide a (2) The signal of the methoxyl carbon, derived from CH;0H used in the transesterification reaction to the methyl ester from the phytyl ester of '*C-labeled chlorophyll a, of the methoxycarbonyl group at C-17? of “C-labeled methyl pheophorbide a showed the natural abundance of ‘°C, and thus was used as a reference signal Comparison of the signal intensities or half widths in the "C-NMR spectrum of C-labeled methyl pheophorbide a with those of methyl pheophorbide a (2) (Fig 2) gave the '*C-enrichment ratio for each carbon of C-labeled methyl pheophorbide a The carbons of methyl pheophorbide a (2) are classified into six groups according to their biosynthetic origin [12,16,17], 1-e from each carbon of ALA (3) and the methyl carbon of L-methionine, as summarized in Table 2 The average C-enrichment ratio of carbons (C-13° and C-17°) derived from C-1 of ALA (3) was 2.4-fold, that of carbons (C-2',
Table 1 Determination of suitable amount of p-glucose (9) for feeding experiment The cultures of E gracilis were grown photosynthetically
in the fermentation culture medium, which contained of 2.5-20 ¢L7!
of p-glucose (9) added in place of L-glutamic acid and D,L-malic acid, without or with bubbling of air After 7 days, the cells were collected
by centrifugation of the culture broth, and the wet weight was mea- sured See Experimental procedures for details
Yield (g:-L~') of E gracilis cells
Amount of b-glucose (g-L~!) No bubbling of air Bubbling of air
Trang 4
Lu | Uh
I I I I Ỉ | Ỉ Ỉ Ỉ | | 1 I Ỉ Ỉ |
|
Fig 2 '°C-NMR spectra of ‘C-labeled methyl pheophorbide a and
methyl pheophorbide a (2) Upper: spectrum of '*C-labeled methyl
pheophorbide a derived from '*C-labeled chlorophyll a, which was
biosynthesized from p-[1-'*C]glucose in E gracilis Lower: spectrum
of methyl pheophorbide a (2)
C-3°, C-7', C-87, C-12', C-13°, C-17° and C-18') derived
from C-2 of ALA (3) was 8.8-fold, that of carbons (C-2,
C-3', C-7, C-8', C-12, C-13', C-17' and C-18) derived from
C-3 of ALA (3) was 4.1-fold, that of carbons (C-1, C-3, C-6,
C-8, C-11, C-13, C-17 and C-19) derived from C-4 of ALA
(3) was 4.1-fold, and that of carbons (C-4, C-5, C-9, C-10,
C-14, C-15, C-16 and C-20) derived from C-5 of ALA (3)
was 3.7-fold The '°C-enrichment ratio of the methoxyl carbon, which is derived from the methyl carbon of L-methionine, of the methoxycarbonyl group at C-13° was 1.8-fold The C-1 to C-5 carbons of ALA (3) and the methyl carbon of L-methionine were thus labeled with '°C from p-[1-'°C] glucose
DISCUSSION
Biosynthetic pathways leading to ALA and :-methionine In £ gracilis The chlorin ring moiety of methyl pheophorbide a (2), in addition to the methyl carbon of L-methionine, is derived from the carbons of ALA (3), which may in principle be formed via the C5 pathway or the Shemin pathway (Fig 1) [12,16,17] As shown in Table 2, the average '°C-enrichment ratios of carbons derived from C-1 to C-5 of ALA (3) are
2.4-, 88-, 4.1-, 41- and 3.7-fold, respectively The
'C-enrichment ratio of the methoxyl carbon, which is derived from the methyl carbon of L-methionine, of the methoxycarbonyl group at C-13* is 1.8-fold These results demonstrate that the C-1 to C-5 carbons of ALA (3) and the methyl carbon of L-methionine were labeled with !3C from D-[I-!3C]ølucose
Figure 3 shows the positions that are predicted to be labeled in ALA (3ii-5ii to 3vii-5vii and 3i-7i to 3v-7v) biosynthesized from '°C-labeled succinyl CoA (5ii to 5vii) and '°C-labeled a-ketoglutaric acid (7i to 7v) via the C5
Table 2 '°C-Enrichment ratios for carbon atoms in '°C-labeled methyl pheophorbide a derived from '*C-labeled chlorophyll a biosynthesized from p-[1-'°C]glucose in E gracilis The cultures of E gracilis were grown photosynthetically in fermentation culture medium containing p-[1-'°C]glucose with bubbling of air The E gracilis cells collected gave rise to '*C-labeled chlorophyll a after purification The '*C-enrichment ratios for each carbon of '°C-labeled methyl pheophorbide a were obtained by comparison of the '°C-NMR spectrum of ‘C-labeled methyl pheophorbide a, which was derived from the '°C-labeled chlorophyll a, with those of methyl pheophorbide a (2) For each group shown in the table, the first line indicates the carbon positions, the second line gives '*C-NMR chemical shift values in p.p.m., and the third line shows the '°C-enrichment ratio For details of calculation of '*C-incorporation ratio in '*C-labeled methyl pheophorbide a, see Experimental procedures The reference carbon (reference signal) was the methoxyl carbon of the methoxycarbonyl group at C-177 (51.66 p.p.m., '°C-Enrichment ratio of 1.0) Carbons of methyl pheophorbide a are classified into six groups according to their biosynthetic origin: C-1 to C-5 indicate carbons of ALA (3), and methyl indicates the methyl carbon of L-methionine
169.56 173.34
52.85
1.8
4 Average '°C-enrichment ratio for C-1 of ALA (3) is 2.4 ° Average '°C-enrichment ratio for C-2 of ALA (3) is 8.8 © Average '°C- enrichment ratio for C-3 of ALA (3) is 4.1 Average '°C-enrichment ratio for C-4 of ALA (3) is 4.1 ° Average '°C-enrichment ratio for C-5
of ALA (3) is 3.7.
Trang 5a |
8i cccCc — [2-'SC]ALA a
the TCA
cycle
XA Ả han
cCc6c ŠŠ [2,4-!3C„]ALA ; 2 ccC©c —› [2,3- 4, 12,3-13 ”C2]ALA the 7ii | ii-7ii 7i — 3ii-7li | second cycle of
c©cC > [1,3-'3C,JALA CcG©c = [2,4-'SC,JALA cGCc > [2,3-'°CaJALA cCGc > [2,3-'SCaJALA |
5iv 3iv-5iv 5v 3v-5V 5vi 3vi-5vi 5vii 3vii-5vii
cGcCc = [2,4- “CoJALA CcGCc = [2,3,5- “C3]ALA
Tiv 3iv-7iv 7V 3V-7V the third
cycle of the TCA cycle
Fig 3 Positions of ‘°C in products derived from p-[1-'°C]glucose Changes of '°C-label position during the biosynthesis of ALA (3ii-5ii to 3vii-Svii and 3i-7i to 3v-7v), through the C5 pathway or the Shemin pathway via the TCA cycle from [2-'°C]acetyl CoA (8i to 8iii) derived from p- [1-'*C]glucose (ccccc) represents «-ketoglutaric acid (7i to 7v), (cccc) represents succinyl CoA (5ii to 5vii), and (cc) represents acetyl CoA (8i to 8iii) (c) is unlabeled carbon, (C) is '*C-carbon from first entry of [2-'°C]acetyl CoA (8i) into the TCA cycle, (CG) 1s 'SC-carbon from the second entry of [2-'°C]acetyl CoA (8ii) into the TCA cycle, and (C) is '°C-carbon from the third entry of [2-'°C]acetyl CoA (S8iii) into the TCA cycle '*C-Labeled positions of succinyl CoA (cccc) (Sii to 5vii) are those of the product formed by reversion from succinic acid Numbers shown under (ccccc) (cccc) and (cc) are the carbon numbers of the compounds '*C-Labeled positions of ALA (3ii-5ii to 3vii-Svii and 3i-7i to 3v-7v) formed via the C5 pathway from each '*C-labeled (ccccc) and via the Shemin pathway from each '*C-labeled (cccc) are shown at the side (a) and (b) on arrows ( > ) show the
CS pathway and the Shemin pathway, respectively
pathway and the Shemin pathway As shown in Figs | and
3, the C-2 to C-5 carbons of ALA (3) generated via the C5
pathway are labeled with '°C from p-[1-'°C]glucose The
C-1 carbon of ALA (3) formed via the C5 pathway 1s not
labeled with '°C from p-[1-’°C]glucose, as this carbon is
derived from C-1, whose carbon is not labeled with °C from
p-[1-'°C]glucose, of acetyl CoA (8) On the other hand, the
C-1 to C-4 carbons of ALA (3) produced via the Shemin
pathway are labeled with '°C from p-[1-'°C]glucose The
C-5 carbon of ALA (3) formed via the Shemin pathway 1s
not labeled with '°C from p-[1-'°C]glucose, as this carbon is
derived from C-2, whose carbon is not labeled with '°C from
p-[1-'°C]glucose, of glycine (4) derived from L-[3-'*C]serine,
which is generated from p-[1-'°C]glucose via [2-'°C]acetyl
CoA and [3-'°C]pyruvic acid Thus, ALA (3) labeled with
'°C on C-1 appears via the Shemin pathway, never via the
C5 pathway, and ALA (3) labeled with ‘°C on C-5 appears
via the CS pathway, never via the Shemin pathway
Therefore, the observed '°C-enrichment at carbons of °C-
labeled methyl pheophorbide a derived from C-1 and C-5 of
ALA (3) suggests that both pathways to ALA (3) operate in
E gracilis
As shown in Fig 3 and discussed in our previous report
[21], the biosynthesis of ALA molecules (3iv-5iv and 3v-7v)
labeled with ‘°C on C-1 and C-5 can be rationalized as
follows Succinyl CoA, which is formed in the second cycle
of the tricarboxylic acid (TCA) cycle, is labeled with ‘°C on
C-1 at the first entry of [2-'°C]acetyl CoA (8i) into the TCA
cycle and transformed to succinic acid At this time, succinic
acid molecules labeled with '*C on C-4 and C-1 appear in
equal quantity Succinic acid labeled with 'ÌC on C-4 and
C-1 can revert to succinyl CoA (5Siv and 5y), giving rise to
succinyl CoA (Siv and 5v) labeled with '°C on C-4 and C-1
in equal quantity Part of succinyl CoA (iv and 5y) labeled with ‘°C on C-4 and C-1 goes into the Shemin pathway, and condenses with glycine (4) ALA (3iv-Siv) labeled with '°C
on C-1 1s biosynthesized from succinyl CoA (iv) labeled with '°C on C-4, and gives rise to a 2.4-fold '*C-enrichment
in '°C-labeled methyl pheophorbide a ALA (3v-5v) labeled with '°C on C-4 is concomitantly biosynthesized from succinyl CoA (5v) labeled with ‘°C on C-1 The rest of succinyl CoA (Siv and 5v) labeled with '°C on C-4 and C-1 re-enters the TCA cycle, and generates ‘C-labeled a-ketoglutaric acid (7iv and 7v) via '°C-labeled succinic
acid, '°C-labeled oxaloacetic acid, '°C-labeled citric acid and
other ‘C-labeled intermediates '°C-Labeled 1-glutamic acid, which is formed from '°C-labeled œ-ketoglutaric acid (Jiv and 7v), goes into the C5 pathway, and generates '°C- labeled ALA (3iv-7iv and 3v-7v) Namely, succinyl CoA (5yv) labeled with '°C on C-1 generates ALA (3v-7v) labeled with 'C on C-5 via o-ketoglutaric acid (7v) labeled with '°C on C-1, and '°C on C-4 of succinyl CoA (Siv) labeled at the first entry of [2-'Clacetyl CoA (8i) into the TCA cycle disappears from ‘'°C-labeled ALA (3iv-7iv) The '°C-
enrichment ratio of C-5 of '°C-labeled ALA (3v-7¥) is
decreased in comparison with that of C-1 of '°C-labeled succinyl CoA (5v) that re-entered the TCA cycle owing to the many pathways leaving from the pathway between succinyl CoA (5) and L-glutamic acid (6), and ALA (3v-7v) labeled with '°C on C-5 gives rise to at least a 3.7-fold '°C- enrichment in '°C-labeled methyl pheophorbide a Further, the '°C-enrichment ratio of C-5 of '°C-labeled ALA (3v-7v) generated from [2-'°C]acetyl CoA (8i) via only the C5 pathway 1n the third cycle of the TCA cycle can not be larger
Trang 6than the average '°C-enrichment ratio (4.1-fold), which is
mainly due to ALA (3ii-7ii and 3v-5v) labeled with ‘°C on
C-4 generated from [2-'*C]acetyl CoA (8i) via both the C5
pathway and the Shemin pathway in the second cycle of the
TCA cycle, of carbons of '°C-labeled methyl pheophorbide
a derived from C-4 of ALA (3) Thus, the '°C-enrichment
ratio of C-5 of '°C-labeled ALA (3v-7v) takes the value of
between 3.7- and 4.1-fold
On the basis of relation of the biosynthetic pathways of
ALA (3ivy-Siv and 3v-7v) labeled with '°C on C-1 and C-5,
the '°C-enrichment ratio (2.4-fold) of carbons of '*C-labeled
methyl pheophorbide a derived from C-1 of ALA (3) should
reflect the ratio of ALA biosynthesis from the Shemin
pathway, and the '°C-enrichment ratio (between 3.7-fold
and 4.1-fold) of carbons of ‘C-labeled methyl pheophor-
bide a derived from C-5 of ALA (3) should reflect the ratio
of ALA biosynthesis from the C5 pathway Thus, on the
assumption that substantial scrambling of the label does not
occur, we can estimate the relative contributions of the C5
pathway and the Shemin pathway to ALA biosynthesis in a
ratio of between 1.5 (1.e 3.7/2.4 ) and 1.7 (i.e 4.1/2.4) to
one E gracilis also biosynthesizes ALA from the conden-
sation of glycine (4) and succinic acid [15,29,30] However,
simultaneous biosynthesis of ALA from succinyl CoA and
succinic acid would not influence the estimation of the ratio
of ALA biosynthesis via the C5 pathway to that via the
Shemin pathway
It is crucial to evaluate the extent of scrambling of the
label due to possible alternative or competing biosynthetic
pathways or degradative reactions, particularly as a culture
period of 7 days was employed Although we cannot assess
the importance of all the possible reactions, we can assess
the contribution of the second passage through the TCA
cycle, which is likely to be one of the major contributors to
label scrambling That is, there is a contribution to the
biosynthesis of ALA, which would be labeled with '°C on
C-1 and C-5, from [2-'°C]acetyl CoA (8ii) generated in the
second cycle of the TCA cycle (shown as cG) As this results
in the synthesis of ALA (3iii-7iii, 3vi-5vi and 3vii-5vii) with
adjacent labeled carbons at C-2 and C-3 (Fig 3), we can
estimate the contribution of [2-'°C]acetyl CoA (8ii) from the
second turn of the TCA cycle from the ratio of doublet and
singlet signals in the '*C-NMR spectrum; the average was
~ 10% This suggests that extensive scrambling of the label
does not occur, and that this approach to evaluate the
contributions of the two pathways is reasonable It 1s worth
noting that the contributions of more complex scrambling
pathways would tend to be diluted out
A comment is necessary regarding the enrichment ratio
(1.8-fold) of the methoxyl carbon of the methoxycarbonyl
group at C-13° of '°C-labeled methyl pheophorbide a
During the exchange of the phytyl ester to the methyl
ester in the transformation of ‘°C-labeled chlorophyll a to
C-labeled methyl pheophorbide a in CH;OH and con-
centrated H»SQOg,, it is possible that some exchange of the
methoxyl carbon of the methoxycarbonyl group at C-137
with the carbon of CH3OH also occurs, though the
reactivities of the phytyl and methyl esters are likely to be
different Thus, all we can say about the '°C-enrichment of
the methoxyl carbon of the methoxycarbonyl group at
C-137 of chlorophyll a, is that the observed value of 1.8-fold
in methyl pheophorbide a represents a minimum value
With regard to the source of the methoxyl carbon of the
methoxycarbonyl group at C-137 of '°C-labeled methyl pheophorbide a, [2-'°C]acetyl CoA, which would be formed from p-[1-'°C]glucose by glycolysis, is transformed to L-[3-'°C]serine via [3-'°C]pyruvic acid The 1-[3-'°C]serine
is transformed to glycine (4) in the presence of tetrahydrof- olic acid, and N°,N'°-['°C]methylenetetrahydrofolic acid is derived from the '*C-carbon of L-[3-'°C]serine and tetra- hydrofolic acid N°,N'°-['°C]Methylenetetrahydrofolic acid gives rise to L-[methyl-'°C}methionine Therefore, the methoxyl carbon of the methoxycarbonyl group at C-137
of ‘C-labeled methyl pheophorbide a is labeled with
°C from p-[1-'°C]glucose, as this carbon is derived from the methyl carbon of L-methionine [17—19]
CONCLUSION
Our results suggest that ALA (3) is synthesized via both the C5 pathway and the Shemin pathway from the TCA cycle in
E gracilis, with the relative contributions being 1n a ratio of between 1.5 and 1.7 to one The extent of label scrambling could not be quantitatively determined, but the effect of second passage through the TCA cycle (likely to be a major contributor) was estimated to be only 10% We also found that the phytyl moiety of chlorophyll a (1) 1s synthesized via the condensation of ‘C-labeled isoprene ({1,2-methyl, 3-'°C]2-methyl-1,3-butadiene) generated from p-[1-'°C]- glucose via [2-'°C]acetyl CoA The methoxyl carbon of the methoxycarbonyl group at C-137 of chlorophyll a (1) was derived from the '°C-labeled methyl carbon of L-[methyl-'°C]methionine generated from p-[1-'*C]glucose via [2-'°C]acetyl CoA and L-[3-'°C]serine
ACKNOWLEDGEMENT
We thank Prof R Timkovich (University of Alabama, AL, USA) for advice on ALA biosynthetic pathways
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