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 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
,

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).

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

), 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

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

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

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

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

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

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

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,

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

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.,

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

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
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

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

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-

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

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

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

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

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