Báo cáo Y học: Evaluation of two biosynthetic pathways to d-aminolevulinic acid in Euglena gracilis potx

Evaluation of two biosynthetic pathways to d-aminolevulinic acid
in
Euglena gracilis
Katsumi Iida, Ippei Mimura and Masahiro Kajiwara
Department of Medicinal Chemistry, Meiji Pharmaceutical University, Kiyose-shi, Tokyo, Japan
d-Aminolevulinic acid (ALA), which is an intermediate in
the b iosynthesis o f c hlorophyll a, c an be biosynthesized via
the C5 pathway and the Shemin pathway in Euglena gracilis.
Analysis of the
13
C-NMR spectrum of
13
C-labeled methyl
pheophorbide a, derived from
13
C-labeled chlorophyll a
biosynthesized from
D
-[1-
13
C]glucose by E. gracilis,provid-
ed evidence suggesting that ALA incorporated in the
13
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
2
of chlorophyll a was labeled with
13
C.

The phytyl moiety of chlorophyll a was labeled on C-P2,
C-P3
1
,C-P4,C-P6,C-P7
1
, C-P8, C-P10, C-P11
1
,C-P12,
C-P14, C-P15
1
and C-P16.
Keywords: d-aminolevulinic acid; C5 pathway; Shemin
pathway; Euglena gracilis;
13
C-NMR.
d-Aminolevulinic acid (ALA) (Fig. 1, 3), which is an
intermediate in the biosynthesis of tetrapyrrole compounds
such as chlorophyll a (1), vitamin B
12
and heme, can be
biosynthesized via two pathways, the Shemin pathway (C4
pathway) [1±7] and t he C5 pathway [8±14] (Fig. 1). In
the Shemin pathway, ALA (3) is biosynthesized b y 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 p athway in Euglena gracilis [12]. B eale et al. reported
that E. gracilis contains ALA synthase [15], implying that
ALA (3) may also be synthesized via the Shemin pathway.
Weinstein et al. [16] reported that the C5 pathway in t he
chloroplast and ALA synthase probably in the mitochond-
rion of E. gracilis operate simultaneously to biosynthesize
ALA. They also showed that [2-
14
C]glycine was incorpo-
rated speci®cally into the nontetrapyrrole portion of chlo-
rophyll a (1)byE. gracilis . Okazaki et al. [17] found that
[2-
13
C]glycine was not incorporated in the tetrapyrrole
portion of chlorophyll a (1)viaALA(3), but was incorpo-
rated into the methoxyl carbon of the methoxycarbonyl
group at C-13
2
of chlorophyll a (1)byE. gracilis. Oh-hama
et al. [18] and Porra et al. [19] reported similar results for
incorporation o f isotope-labeled glycine into chlorophyll a
(1)byScenedesmus obliquus and maize leaves. Thus, the
involvement of the Shemin pathway co uld not be assessed in
terms of labeling i n the tetrapyrrole portion of chlorophyll a
(1) from isotope-labeled glycine fed to the organism. Porra
et al. concluded that t he C5 pathway is the predominant
biosynthetic pathway to ALA utilized in chlorophyll a (1),
as shown from feeding experiments with
D
,

L
-[1-
13
C]- and
[5-
13
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 S hemin pathway, based
on feeding experiments with sodium[1-
14
C]- and[5-
14
C]
a-ket oglutarate [2 0].
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
13
C-NMR s pectrum o f
13
C-labeled vitamin B
12
biosynthe-
sized from
D
-[1-

13
C]glucose by P. shermanii provided
evidence that ALA (3) incorporated in the
13
C-labeled
vitamin B
12
may have been synthesized via both the Shemin
pathway and the C5 p athway [21]. We therefore conducted
similar feeding experiments with
D
-[1-
13
C]glucose in E. grac-
ilis, and used
13
C-NMR spectroscopy to examine the
13
C-enrichment ratios of the carbon atoms of
13
C-labeled
chlorophyll a or its derivative,
13
C-labeled methyl pheo-
phorbide a (Fig. 1 ). Our results indicate that the C5 and
Shemin pathways both operate in E. gracilis, an d provide
information about the biosynthetic pathways leading to the
methoxyl carbon of the methoxycarbonyl group at C-13
2
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.
D
-[1-
13
C]Glucose
(90 a tom %
13
C) was purchased from Cambridge Isotope
Laboratories. All other chemicals were of analytical grade.
Correspondence to K. Iida, Department of Medicinal Chemistry, Meiji
Pharmaceutical University, 2-522-1 Noshio, Kiyose-shi, Tokyo
204-8588, Japan. Fa x: + 81 424 95 8612; Tel.: + 81 424 95 8611,
E-mail:
Abbreviations:ALA,d-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)
Eur. J. Biochem. 269, 291±297 (2002) Ó FEBS 2002
Instruments
All
1
H-NMR (400 MHz) and
13

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
D
-[1-
13
C]glucose
for
E. gracilis
feeding experiments
E. gracilis was cultured as described previously, with some
modi®cations [17]. The cultures were grown under illumi-
nation (2400 Lx) in seed culture medium (10 mL), which
consisted o f
L
-glutamic a cid (5 gáL
)1
),
D
,
L
-malic acid
(2 gáL
)1
),
L
-methionine (50 mgáL
)1

), thiamine hydrochlo-
ride (1 mgáL
)1
), cyanocobalamin (5 lgáL
)1
), KH
2
PO
4
(0.4 gáL
)1
), MgSO
4
á7H
2
O(0.5gáL
)1
), CaCO
3
(0.1 gáL
)1
)
(NH
4
)PO
4
(0.2 gáL
)1
), EDTA (10 mgáL
)1

), ZnCl
2
(10 mgáL
)1
), FeSO
4
á7H
2
O(4mgáL
)1
), MnCl
2
á4H
2
O
(2 mgáL
)1
), CuCl
2
á2H
2
O(0.4mgáL
)1
), CoCl
2
á6H
2
O
(2 mgáL
)1

)andH
3
BO
4
á7H
2
O(80lgáL
)1
), 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 ¯ask. This fermentation culture medium
contained 2.5±20 gáL
)1
of
D
-glucose (9) added in place of
L
-glutamic acid (5 gáL
)1
)and
D
,
L
-malic acid (2 gáL
)1
)inthe
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
D
-[1-
13
C]glucose to
E. gracilis
The above seed culture medium (10 mL ´ 2), cultivated for
7 d ays, was added to fermentation culture medium
(1 L ´ 2), which consisted of
D
-[1-
13
C]glucose (2.5 gáL
)1
)
added in place of
L
-glutamic acid (5 gáL
)1
)and
D
,
L
-malic
acid (2 gáL
)1
) in the seed culture medium, in a 3-L conical
¯ask. 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
13
C-labeled chlorophyll
a
The isolation of chlorophyll a (1) was carried out by
modi®cation 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 CH
3
OH
(50 m L), disrupted with an ultrasonicator at 0 °Cfor5 min,
and centrifuged for 30 min at 12 300 g. The supernatant
was evaporated in the dark. Pu ri®cation of the residue by
medium-pressure liquid chromatography (MPLC) using a
prepacked glass column [2.5 (internal diameter) ´ 30 cm,
octadecyl silica (ODS)] with C H
3
OH gave
13
C-labeled
chlorophyll a. The amount of
13
C-labeled chlorophyll a
Fig. 1. Biosynthetic pathways to chlorophyll a (1) from
D
-glucose (9) and structure of methyl pheophorbide a (2). Chlorophyll a (1) is biosynthesized

through d-aminolevulinic acid (ALA) (3) formed via the C 5 p athway and the Shemin p athway from
D
-glucose (9), an d methyl pheophorbide a (2)is
derived from chlorophyll a (1).
292 K. Iida et al. (Eur. J. Biochem. 269) Ó FEBS 2002
isolated was calculated from the UV absorption spectrum
[22].
Transformation from
13
C-labeled chlorophyll
a
to
13
C-labeled methyl pheophorbide
a
Concentrated H
2
SO
4
(0.5 mL) was added dropwise, at 0 °C
under argon, to a solution of
13
C-labeled isolated chloro-
phyll a in dry CH
3
OH (9.5 mL), and the mixture was stirred
for 12 h at room temperature in the dark. The reaction
mixture was diluted with CH
2
Cl

2
(200 mL), and quenched
with saturated NaHCO
3
. The organic layer was washed
with saturated NaHCO
3
, water a nd saturated NaCl, dried
over dry MgSO
4
, and then evaporated. Chromatography of
the crude product on silica gel with CHCl
3
/CH
3
OH (25 : 1 ,
v/v) gave
13
C-labeled methyl pheophorbide a. The amount
of
13
C-labeled methyl pheophorbide a isolated was calcu-
lated from the UV absorption spectrum [22].
13
C-NMR measurements of chlorophyll
a
and methyl pheophorbide
a
The
13

C-NMR spectra were obtained for solutions of
13
C-
labeled chlorophyll a (4 .8 m
M
) and chlorophyll a (1)in
C
2
HCl
3
/C
2
H
3
OH (79 : 6, v/v), an d solutions of
13
C-labeled
methyl pheophorbide a (3.8 m
M
) and methyl pheophorbide
a (2)inC
2
HCl
3
. The signal of C
2
HCl
3
(77.0 p.p.m.) was
used as an internal standard. The spectral width w as

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 ls, 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
13
C-NMR signals of chlorophyll a (1)and
methyl pheophorbide a (2)weremadeonthebasisof
reported data [23±28].
Calculation of
13
C-incorporation ratios
in
13
C-labeled methyl pheophorbide
a
The signal of the methoxyl carbon, which was derived from
CH
3
OH used in the transesteri®cation reaction to the
methyl ester from the phytyl ester of
13
C-labeled chloro-
phyll a of the methoxycarbonyl group at C-17
2
of
13
C-labeled m ethyl pheophorbide a shows t he natural
abundance of
13

C,andthuscanbeusedasareference
signal. T he
13
C-enrichment ratio for each carbon of
13
C-labeled methyl pheophorbide a was calculated from
comparison of the signal intensities or half widths in the
13
C-NMR spectrum of
13
C-labeled methyl pheophorbide a,
with those of methyl pheophorbide a (2).
RESULTS
Suitable amount of
D
-[1-
13
C]glucose
for feeding experiment to
E. gracilis
Cultures of E. gracilis were grown photosynthetically in
E. gracilis fermentation culture medium containing various
amounts of
D
-glucose (9)inplaceof
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 gáL
)1
of
D
-glucose (9) gave 2.11, 4.01 and 4.72 gáL
)1
of E. gracilis,
respectively, as shown in Table 1. With air bubbling, 2.5, 5,
10 a nd 15 gáL
)1
of
D
-glucose (9) gave 3.64, 3.64, 4.23 and
5.85 gáL
)1
of E. gracilis, respectively. For reasons of
economy, we chose to use two cultures, each containing
2.5 gáL
)1
of
D
-[1-
13
C]glucose, with air bubbling f or the
feeding experiments.
Biosynthesis of
13

C-labeled chlorophyll
a
and
13
C-incorporation in its phytyl moiety
13
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 p resence of
D
-[1-
13
C]glucose. Its purity was judged to be high by
comparison of the
1
H-NMR and UV spectra with those of
authentic chlorophyll a (1). The
13
C-enrichments of c arbons
(C-P2, C-P3
1
,C-P4,C-P6,C-P7
1
, C-P8, C-P10, C-P11
1
,
C-P12, C-P14, C-P15
1
and C-P16) of the phytyl moiety of
13

C-labeled chlorophyll a were higher than those of carbons
of the chlorin ring moiety.
Synthesis of
13
C-labeled methyl pheophorbide
a
and determination of
13
C-incorporation ratios
13
C-Labeled methyl pheophorbide a (1.4 mg) was derived
from
13
C-labeled chlorophyll a (2.6 mg). Its purity was
judged to be high by comparison of the
1
H-NMR and UV
spectra with those o f authentic methyl pheophorbide a (2).
The signal of the methoxyl carbon, derived from CH
3
OH
used in the transesteri®cation reaction to the methyl ester
from the phytyl ester of
13
C-labeled chlorophyll a,ofthe
methoxycarbonyl group at C-17
2
of
13
C-labeled methyl

pheophorbide a showed the natural abundance of
13
C, and
thus was used as a reference signal. Comparison of the
signal intensities or half widths in the
13
C-NMR spectrum of
13
C-labeled methyl pheophorbide a with tho se of methyl
pheophorbide a (2) (Fig. 2) gave the
13
C-enrichment ratio
for each carbon of
13
C-labeled methyl pheophorbide a.The
carbons of methyl pheophorbide a (2) are classi®ed into six
groups according t o their biosynthetic origin [12,16,17], i.e.
from each carbon of ALA (3) and the methyl carbon of
L
-methionine, as summarized in Table 2. The average
13
C-enrichment ratio of carbons (C-13
3
and C-17
3
) derived
from C-1 of ALA (3) was 2.4-fold, that of carbons (C-2
1
,
Table 1. Determination of suitable amount of

D
-glucose (9) for feeding
experiment. The cultures of E. gracilis were grown photosynthetically
in the fermentation cu lture medium, wh ich contained of 2 .5±20 gáL
)1
of
D
-glucose (9) ad ded in p lace of
L
-glutamic acid and
D
,
L
-malic acid,
without or with bubbling of air. After 7 days, the cells were collected
by centrifugat ion of the culture broth, an d the wet weight was mea-
sured. See Experimental procedures for details.
Amount of
D
-glucose (gáL
)1
)
Yield (gáL
)1
)ofE. gracilis cells
No bubbling of air Bubbling of air
2.5 ± 3.64
5 ± 3.64
10 2.11 4.23
15 4.01 5.85

20 4.72
Ó FEBS 2002 Evaluation of two ALA biosynthetic pathways (Eur. J. Biochem. 269) 293
C-3
2
,C-7
1
,C-8
2
,C-12
1
,C-13
2
,C-17
2
and C-18
1
) derived
from C-2 of ALA (3) was 8.8-fold, that of carbons (C-2,
C-3
1
,C-7,C-8
1
, C-12, C-13
1
,C-17
1
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
13
C-enrichment ratio o f the methoxyl
carbon, which is derived from the methyl carbon of
L
-methionine, of the meth oxycarbonyl group at C-13
2
was
1.8-fold. T he C-1 to C-5 carbons of ALA (3) and the methyl
carbon of
L
-methionine were thus labeled with
13
Cfrom
D
-[1-
13
C] glucose.
DISCUSSION
Biosynthetic pathways leading to ALA
and
L
-methionine in
E. 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 p athway (Fig. 1)
[12,16,17]. As shown in Table 2, the average
13
C-enrichment
ratios of carbons derived from C-1 to C-5 of ALA (3)are
2.4-, 8.8-, 4.1-, 4.1- and 3.7-fold, respectively. The
13
C-enrichment ratio of the methoxyl carbon, which is
derived from the methyl carbon of
L
-methionine, of the
methoxycarbonyl group at C-13
2
is 1.8-fold. These results
demonstrate that the C-1 to C-5 carbons of ALA (3)andthe
methyl carbon of
L
-methionine were labeled with
13
Cfrom
D
-[1-
13
C]glucose.
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
13
C-labeled succinyl CoA (5ii to 5vii)
and

13
C-labeled a-ketoglutaric acid (7i to 7v)viatheC5
Fig. 2.
13
C-NMR spectra of
13
C-labeled methyl pheophorbide a and
methyl pheophorbide a (2). Upper: spectrum of
13
C-labeled methyl
pheophorbide a derived from
13
C-labeled chlorophyll a,whichwas
biosynthesized from
D
-[1-
13
C]glucose in E. gracilis.Lower:spectrum
of methyl pheophorbide a (2).
Table 2.
13
C-Enrichment ratios for c arbon atoms in
13
C-labeled methyl pheophorbide a derived from
13
C-labeled chlorophyll a bio synthes ized from
D
-[1-
13
C]glucose in E. gracilis. The cultures of E. gracilis were grown photosynthetically in fermentation culture medium containing

D
-[1-
13
C]glucose with bubbling of air. The E. gracilis cells collected gave rise to
13
C-labeled chlorophyll a after puri®cation. The
13
C-enrichment
ratios for each c arbon of
13
C-labeled methyl pheophorbide a were obtained by comparison of the
13
C-NMR spectrum of
13
C-labeled methyl
pheophorbide a, which was derived from th e
13
C-labeled chlorophyll a, with those of methyl pheophorbide a (2). For each group shown in t he
table, the ®rst line indicates the carbon positions, the second line gives
13
C-NMR ch emical shift values in p.p.m., and t he third line shows the
13
C-enrichment ratio. For de tails of calculation o f
13
C-incorporation ratio in
13
C-labeled m ethyl pheophorbide a, see Experimental pro ce dures. The
reference carb on (reference signal) was the methoxyl carbon of the metho xycarbonyl group at C-17
2
(51.66 p.p.m.,

13
C-Enrichment ratio of 1.0).
Carbons of m ethy l p heophorbi de a are c lassi®ed in to six groups accordin g t o their biosynthetic origin: C-1 to C -5 ind icate carb ons o f A LA (3), and
methyl indicates the methyl carbon of
L
-methionine.
C-1
a
13
3
169.56
2.4
17
3
173.34
2.4
C-2
b
2
1
3
2
7
1
8
2
12
1
13
2

17
2
18
1
12.05 122.83 11.26 17.42 12.10 64.70 29.84 23.06
9.2 9.5 8.1 8.2 9.2 8.6 8.4 8.8
C-3
c
23
1
78
1
12 13
1
17
1
18
131.88 128.93 136.21 19.48 129.06 189.62 31.01 50.09
4.4 4.6 3.7 3.9 4.6 4.0 3.4 4.2
C-4
d
1 3 6 8 11 13 17 19
142.07 136.32 155.70 145.26 137.94 129.00 51.08 172.19
4.4 4.0 4.6 3.2 3.4 4.6 4.4 4.2
C-5
e
4 5 91014151620
136.53 97.58 151.01 104.47 149.67 105.18 161.19 93.13
3.4 4.1 3.8 3.8 3.2 3.5 3.7 4.4
Methyl Methoxyl carbon of the methoxycarbonyl group at C-13

2
52.85
1.8
a
Average
13
C-enrichment ratio for C-1 of ALA (3) is 2.4.
b
Average
13
C-enrichment ratio for C-2 of ALA (3) is 8.8.
c
Average
13
C-
enrichment ratio for C-3 of ALA (3) is 4.1.
d
Average
13
C-enrichment ratio for C-4 of ALA (3) is 4.1.
e
Average
13
C-enrichment ratio for C-5
of ALA (3) is 3.7.
294 K. Iida et al. (Eur. J. Biochem. 269) Ó FEBS 2002
pathway and the Shemin pathway. As shown in Figs 1 and
3, the C-2 to C-5 carbons of ALA (3) generated via the C5
pathway are labeled with
13

Cfrom
D
-[1-
13
C]glucose. The
C-1 carbon of ALA (3) formed via the C 5 pathway is not
labeled with
13
Cfrom
D
-[1-
13
C]glucose, as this carbon is
derived from C-1, whose carbon is not labeled with
13
Cfrom
D
-[1-
13
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
13
Cfrom
D
-[1-
13
C]glucose. The
C-5 carbon of ALA (3) formed via the Shemin pathway is
not labeled with

13
Cfrom
D
-[1-
13
C]glucose, as this carbon is
derived from C-2, whose carbon is not labeled with
13
Cfrom
D
-[1-
13
C]glucose, of glycine (4) derived from
L
-[3-
13
C]serine,
which is generated from
D
-[1-
13
C]glucose via [2-
13
C]acetyl
CoA and [3-
13
C]pyruvic acid. Thus, ALA (3) labeled with
13
C on C-1 appears via the Shemin pathway, never via the
C5 pathway, and ALA (3) labeled with

13
C on C-5 appears
via the C5 pathway, never via the Shemin pathway.
Therefore, the observed
13
C-enrichment at carbons of
13
C-
labeled methyl pheophorbide a derived from C-1 and C-5 of
ALA (3) suggests that both pathways to ALA (3)operatein
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
13
ConC-1andC-5canberationalizedas
follows. Succinyl CoA, which is formed in the s econd cycle
of the tricarboxylic acid (TCA) cycle, is labeled with
13
Con
C-1atthe®rstentryof[2-
13
C]acetyl CoA (8i) into the TCA
cycle and transformed to succinic acid. At this time, succinic
acid molecules labeled with
13
C on C-4 and C-1 appear in
equal quantity. Succinic acid labeled with
13
ConC-4and

C-1 can revert to succinyl CoA (5iv and 5v), giving rise to
succinyl CoA (5iv and 5v)labeledwith
13
ConC-4andC-1
in equal quantity. Part of succinyl CoA (5iv and 5v) labeled
with
13
C on C-4 and C-1 goes into the Shemin pathway, and
condenses with glycine ( 4). ALA (3iv-5iv) labeled with
13
C
on C-1 is biosynthesized from succinyl CoA (5iv) labeled
with
13
C on C-4, and gives rise to a 2.4-fold
13
C-enrichment
in
13
C-labeled methyl pheophorbide a.ALA(3v-5v) labeled
with
13
C on C-4 is concomitantly biosynthesized from
succinyl CoA (5v) labeled with
13
C on C-1. The rest of
succinyl CoA (5iv and 5v)labeledwith
13
ConC-4andC-1
re-enters the TCA cycle, and generates

13
C-labeled
a-ket oglutaric acid ( 7iv and 7v)via
13
C-labeled succinic
acid,
13
C-labeled oxaloacetic acid,
13
C-labeled citric acid and
other
13
C-labeled intermediates.
13
C-Labeled
L
-glutamic
acid, which is formed from
13
C-labeled a-ketoglutaric acid
(7iv and 7v), goes into the C5 pathway, and generates
13
C-
labeled ALA (3iv-7iv and 3v-7v). Namely, succinyl C oA (5v)
labeled with
13
C on C-1 generates ALA (3v-7v) labeled w ith
13
ConC-5viaa-ketoglutaric acid (7v) labeled with
13

Con
C-1, and
13
C on C-4 of succinyl CoA (5iv) labeled at the ®rst
entry of [2-
13
C]acetyl CoA (8i) into the TCA cycle
disappears from
13
C-labeled ALA (3iv-7iv). The
13
C-
enrichment ratio of C-5 of
13
C-labeled ALA (3v-7v)is
decreased in c omparison with that of C-1 of
13
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
13
C on C-5 gives rise to at least a 3.7-fold
13
C-
enrichment in
13

C-labeled methyl pheophorbide a.Further,
the
13
C-enrichment ratio of C-5 of
13
C-labeled ALA (3v-7v )
generated from [2-
13
C]acetyl CoA (8i) v ia only t he C5
pathway in the third cycle of the TCA cycle can not be larger
Fig. 3. Positions of
13
C in products derived from
D
-[1-
13
C]glucose. Changes of
13
C-label position d uring the biosynthesis of ALA (3ii-5ii to 3vii-5vii
and 3i-7i to 3v-7v), through the C5 pathway or the Shemin pathway via the TCA cycle f rom [2-
13
C]acetyl CoA (8i to 8iii) derived from
D
-
[1-
13
C]glucose. (ccc cc) r epresents a-ket oglutaric acid (7i to 7v ), (cccc ) r epresen ts suc cinyl C oA ( 5ii to 5vii), and (cc) represents acetyl CoA (8i to 8iii ).
(c) is unlabeled carbon, (C)is
13
C-carbon from ®rst entry of [2-

13
C]acetyl CoA (8i)intotheTCAcycle,( )is
13
C-carbon from the second entry of
[2-
13
C]acetyl CoA (8ii) into the TCA cycle, and (C) is
13
C-carbon from the third entry of [2-
13
C]acetyl CoA (8iii)intotheTCAcycle.
13
C-Labeled
positions of succinyl CoA (cccc) (5ii to 5vii) are those of the product formed by reversion from succinic acid. Numbers shown under (ccccc) (cccc)
and (cc) a re the carbon numbers of th e com pounds.
13
C-Labeled positions of ALA ( 3ii-5ii to 3vii-5vii and 3i-7i to 3v-7v) formed via the C 5 p athway
from each
13
C-labeled ( cccc c) a nd via the Shemin pathway from ea ch
13
C-labeled (cccc) a re s hown a t the s ide. (a ) an d (b) o n arro ws ( ® ) show the
C5 pathway and the Shemin p athway, respe ctively.
Ó FEBS 2002 Evaluation of two ALA biosynthetic pathways (Eur. J. Biochem. 269) 295
than the average
13
C-enrichment ratio (4.1-fold), which is
mainly due to ALA (3ii-7ii and 3v-5v)labeledwith
13
Con

C-4 generated from [2-
13
C]acetyl CoA (8i) via both the C5
pathway and the Shemin pathway in the second cycle of the
TCA cycle, of carbons of
13
C-labeled methyl pheop horbide
a derived from C-4 of ALA (3). Thus, the
13
C-enrichment
ratio of C-5 of
13
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 (3iv-5iv and 3v-7v) labeled with
13
ConC-1andC-5,
the
13
C-enrichment ratio (2.4-fold) of carbons of
13
C-labeled
methyl pheophorbide a derived from C-1 of ALA (3) should
re¯ect the ratio of ALA biosynthesis from t he Shemin
pathway, and the
13
C-enrichment ratio ( between 3.7-fold
and 4.1-fold) of carbons of
13

C-labeled methyl pheophor-
bide a derived from C-5 of ALA (3) should re¯ect the ratio
of ALA biosynthesis from the C5 pathway. Thus, on the
assumption that substantial scrambling o f the label does not
occur, we can estimate the relative contributions of the C5
pathway and the Shemin path way to ALA biosynthesis in a
ratio of between 1.5 (i.e. 3.7/2.4 ) and 1.7 (i.e. 4.1/2.4) to
one. E. gracilis also biosyn thesizes 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 in¯uence the estimation of the ratio
of ALA biosynthesis via the C5 p athway to that via t he
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 a ll 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
13
Con
C-1 and C-5, from [2-
13
C]acetyl CoA (8ii) generated in the
second cycle of the TCA cycle (shown as c
). As this results
in the synthesis of AL A (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-
13
C]acetyl CoA (8ii)fromthe
second turn of the TCA cycle from the ratio of doublet and
singlet signals in the
13
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 is worth
noting that the contributions of more complex scrambling
pathways would tend to be diluted out.
A comment is necessary re garding the enrichment ratio
(1.8-fold) of the methoxyl carbon of the methoxycarbonyl
group at C-13
2
of
13
C-labeled methyl pheophorbide a.
During the exchange of the phytyl ester to the m ethyl
ester i n the transformation of
13
C-labeled chlorophyll a to
13
C-labeled methyl pheophorbide a in CH
3
OH and con-
centrated H
2

SO
4
, it is possible that some exchange of the
methoxyl carbon of the methoxycarbonyl group at C-13
2
with the carbon of CH
3
OH also occurs, though the
reactivities of the phytyl and methyl esters are likely to b e
different. Thus, all we can say about the
13
C-enrichment of
the methoxyl car bon of the methoxycarbonyl group at
C-13
2
of chlorophyll a, is that the observed value of 1.8-fold
in m ethyl pheophorbide a represents a minimum value.
With regard to the source of the methoxyl carbon of the
methoxycarbonyl group at C-13
2
of
13
C-labeled methyl
pheophorbide a,[2-
13
C]acetyl CoA, which would be formed
from
D
-[1-
13

C]glucose by glycolysis, is transformed to
L
-[3-
13
C]serin e via [3-
13
C]pyruvic acid. The
L
-[3-
13
C]serine
is transformed to glycine (4) in the presence of tetrahydrof-
olic acid , and N
5
,N
10
-[
13
C]methylenetetrahydrofolic acid is
derived f rom t he
13
C-carbon of
L
-[3-
13
C]serine and tetra-
hydrofolic acid. N
5
,N
10

-[
13
C]Methylenetetrahydrofolic acid
gives rise to
L
-[methyl-
13
C]methionine. Therefore, the
methoxyl carbon of the methoxycarbonyl group at C-13
2
of
13
C-labeled methyl pheophorbide a is labeled with
13
Cfrom
D
-[1-
13
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 in 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) is synthesized via
the condensation of
13
C-labeled isoprene ([1,2-methyl,
3-
13
C
3
]2-methyl-1,3-butadiene) generated from
D
-[1-
13
C]-
glucose via [2-
13
C]acetyl CoA. The methoxyl carbon of
the methoxycarbonyl group at C-13
2
of chlorophyll a (1)
was derived from the
13
C-labeled methyl carbon of
L
-[methyl-
13
C]methionine generated from
D
-[1-
13
C]glucose

via [2-
13
C]acetyl CoA and
L
-[3-
13
C]serine.
ACKNOWLEDGEMENT
We thank Prof. R. Timkovich (University of Alabama, AL, USA) for
advice on ALA biosynthetic pathways.
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