Studies on the nonmevalonate pathway: Formation of 2-C-methyl-D- erythritol 2,4-cyclodiphosphate from 2-phospho-4-(cytidine 5'-diphospho)-2-C- methyl-D-erythritol

Article abstract of DOI:10.1016/S0040-4039(00)00375-0

2-Phospho-4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol was transformed to 2-C-methyl-D-erythritol 2,4-cyclodiphosphate by a novel Escherichia coli enzyme involved in the nonmevalonate pathway. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4039(00)00375-0

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 41 (2000) 3395–3398
Studies on the nonmevalonate pathway: formation of
2-C-methyl-D-erythritol 2,4-cyclodiphosphate from
2-phospho-4-(cytidine 50-diphospho)-2-C-methyl-D-erythritol
Motoki Takagi,a,† Tomohisa Kuzuyama,a,† Kazuhide Kaneda,a Hiroyuki Watanabe,a
Tohru Dairi b and Haruo Seto a,∗
aInstitute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo 113-0032, Japan
bBiotechnology Research Center, Toyama Prefectural University, Toyama 939-0398, Japan
Received 7 February 2000; accepted 17 February 2000
Abstract
2-Phospho-4-(cytidine 50-diphospho)-2-C-methyl-D-erythritol was transformed to 2-C-methyl-D-erythritol 2,4-
cyclodiphosphate by a novel Escherichia coli enzyme involved in the nonmevalonate pathway. © 2000 Elsevier
Science Ltd. All rights reserved.
Keywords: isopentenyl diphosphate; 2-phospho-4-(cytidine 50-diphospho)-2-C-methyl-D-erythritol; 2-C-methyl-D-erythritol
2,4-cyclodiphosphate; 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase.
Isopentenyl diphosphate 1, a building block of isoprenoids, is biosynthesized through the nonmeva-
lonate pathway in many eubacteria, green algae and the chloroplasts of higher plants (Fig. 1).1 The
initial step of this pathway is the formation of 1-deoxy-D-xylulose 5-phosphate (DXP) 2 by condensation
of pyruvate and D-glyceraldehyde 3-phosphate catalyzed by DXP synthase.2–4 In the second step 2 is
converted to 2-C-methyl-D-erythritol 4-phosphate (MEP) 3 by DXP reductoisomerase as demonstrated
in our study.5,6 Compound 3 is then cytidylylated by MEP cytidylyltransferase to give 4-(cytidine 50-
diphospho)-2-C-methyl-D-erythritol (CDP-ME) 4,7,8 which is phosphorylated by CDP-ME kinase to
afford 2-phospho-4-(cytidine 50-diphospho)-2-C-methyl-D-erythritol (CDP-ME2P) 5.9,10 We have recen-
tly succeeded in the cloning of MEP cytidylyltransferase and CDP-ME kinase genes from Escherichia
coli and demonstrated unequivocally that these enzymes are essential for the formation of 1 in the
nonmevalonate pathway.8,10 The following reactions leading to 1 from 5, however, remain unknown.
∗
†
Corresponding author. Tel: +81-3-5841-7839; fax: +81-3-5841-8485; e-mail: haseto@iam.u-tokyo.ac.jp (H. Seto)
The first two authors contributed equally to this work.
0040-4039/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)00375-0
tetl 16655

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Fig. 1. The nonmevalonate pathway for IPP biosynthesis
In previous papers8,10 we reported the cloning of several E. coli genes that complemented the defects
of blocked mutants in synthesizing 1 from 3.‡ This paper shows conversion of 5 into 2-C-methyl-
D-erythritol 2,4-cyclodiphosphate (MECDP) 6 by an enzyme encoded by one of these genes, ygbB
(DDBJ/EMBL/GenBank accession number AB038256), which is located at 61.8 min on the chromosome
map of E. coli.
Incubation of the ygbB gene product (2 mg)§ with 5 (50 mg) at 37°C for 2 h in 50 ml of 10 mM
Tris–HCl (pH 8.0) containing 1 mM MgCl2 resulted in the decrease of 5 and appearance of a new
peak identified as cytidine 50-monophosphate (CMP) in HPLC with an Asahipak GS-320 HQ column
(7.8×300 mm, Showa Denko, Tokyo, Japan).¶ This decrease of 5 was proportional to the increase of
CMP formation and occurred only in the presence of the ygbB gene product.
1
The reaction product was purified with the assistance and guidance of H NMR spectroscopy. The
reaction mixture was diluted to 100 ml with H2O, subjected to Dowex 1-X8 (Cl− type, 2×6 cm)
chromatography and eluted with 1% NaCl aq. (100 ml). All fractions showing a methyl proton singlet
at 1.43 ppm were combined, concentrated and chromatographed on a Sephadex G-10 column (1.8×100
cm) and eluted with water only. Fractions showing this proton singlet were combined and freeze-dried to
afford a pure reaction product (1.4 mg).
‡
These mutants were derived from an E. coli transformant with the mevalonate kinase, phosphomevalonate kinase and
pyrophosphomevalonate decarboxylase activities. Thus, this transformant can utilize the partial mevalonate pathway for IPP
biosynthesis only by supplementation of mevalonate. Therefore, these mutants, which can grow only in the presence of
mevalonate, are concluded to be deficient in the nonmevalonate pathway (see Ref. 8).
§
Protein expression and purification of the ygbB gene product: The ygbB gene was cloned into the expression vector pQE30
(Qiagen) to give pQECMEP. Escherichia coli M15 containing pREP4 [neo, lacI] (Qiagen) was used as a host for expression
of the ygbB gene. Escherichia coli M15 (pREP4, pQECMEP) was cultured at 37°C in 100 ml of a Luria–Bertani medium
containing 50 µg/ml kanamycin and 200 µg/ml ampicillin for 5 h with 0.1 mM isopropyl-β-D-thiogalactopyranoside upon
reaching an optical density at 660 nm of 0.8. Cells were harvested by centrifugation and resuspended in 100 mM Tris–HCl
(pH 8.0). After brief sonication, the lysate was centrifuged at 10 000×g for 20 min and the supernatant was collected. A 50%
slurry of Ni-nitrilotriacetic acid (NTA) agarose resin (Qiagen) was added into the supernatant and stirred on ice for 60 min.
The resin was washed with 50 mM imidazole in 100 mM Tris–HCl (pH 8.0) and then the protein which bound to the Ni-NTA
agarose resin was eluted with 200 mM imidazole in 100 mM Tris–HCl (pH 8.0). The eluate was used as the purified ygbB
gene product in the subsequent experiments. The purified enzyme afforded a homogeneous protein band on sodium dodecyl
sulfate-polyacrylamide gel electrophoresis with a subunit size of 22 kDa.
¶
Decrease of 5 was monitored at 280 nm with an Asahipak GS-320 HQ column, eluted with 10 mM KH2PO4 (pH 2.5) at the
flow rate of 1.0 ml/min at 30°C. In this HPLC condition, CMP and 5 ware eluted at 9.6 and 11.6 min, respectively.

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The structure of the purified product was deduced by spectroscopic methods. The molecular formula
1
was determined to be C5H10O9P2Na2 by HR-FABMS [m/z 344.9481 (M+Na)+, ∆-1.2 mmu]. The H,
13C and 31P NMR spectra of the purified product unequivocally established its structure as 2-C-methyl-
D-erythritol 2,4-cyclodiphosphate 6 [1H NMR (500 MHz): δ 4.21 (m, H-4a), 4.13 (m, H-4b), 4.13 (m,
H-3), 3.77 (d, J=12.5 Hz, H-1a), 3.62 (d, J=12.5 Hz, H-1b), 1.43 (s, 2-Me); 13C NMR (125 MHz): δ
84.3 (d, J=8.5 Hz, C-2), 68.9 (d, J=<3.0 Hz, C-3), 67.4 (J=4.5 Hz, C-1), 66.2 (d, J=6.5, C-4), 16.8
(J=5.5 Hz, 2-Me); 31P NMR (202 MHz): δ −10.9 (d, J=23.2 Hz), −14.9 (d, J=23.2 Hz)].k Thus, the 2-
C-methylerythritol skeleton in 6 was proved to be present as in 5 by 1H and 13C NMR spectral analysis.
The large 13C–31P splittings observed with C-2 and C-4 signals indicated that these positions were
phosphorylated. The characteristic high field 31P signals at −10.9 and −14.9 ppm showing a 31P–31P
coupling with each other proved the presence of a diphosphate diester function in 6. It is interesting to
note that 6 had been isolated from the fermentation broth of a Gram-negative anaerobe, Desulfovibrio
desulfuricans.11 The NMR spectral data of 6 are in complete agreement with the reported values.
These results clearly demonstrate that the elimination of the CMP moiety from the CDP-ME2P
molecule is concomitant with the formation of the cyclic diphosphate 6 (Fig. 2). We propose to designate
this enzyme 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (MECDP synthase).
Fig. 2. MECDP synthase reaction
Although an accumulation of 6 was also found in some bacteria including Corynebacterium ammonia-
genes under oxidative stress conditions caused by treatment of benzyl viologen,12 no reports have been
available to date on the role of 6 in the nonmevalonate pathway. We have clearly showed in this study
that 6 is an intermediate in the nonmevalonate pathway for IPP biosynthesis.
The ygbB gene product showed significant homologies to hypothetical proteins with unknown func-
tions present in Aquifex aeolicus, Thermotoga maritima, Synechocystis, Bacillus subtilis, Mycobacterium
tuberculosis, Haemophilus influenzae, Helicobacter pylori, Treponema pallidum, Chlamydia trachomatis
and Chlamydia pneumoniae which are known to utilize the nonmevalonate pathway for IPP biosynthesis.
Similar distribution patterns in eubacteria are observed with DXP synthase, DXP reductoisomerase, MEP
cytidylyltransferase and CDP-ME kinase, which are involved in the nonmevalonate pathway. These data
corroborate that the ygbB gene is essential for this pathway.
Acknowledgements
We thank Dr. K. Furihata and Dr. H. Kobayashi for collecting NMR and MS spectra, respectively. This
work was supported in part by a grant, Research for the Future Program (RFTF), from The Japan Society
for the Promotion of Science (JSPS) JSPS-RFTF96I00301 to H.S., by a Grant-in-Aid for Scientific
k
All NMR data were collected with an A500 NMR spectrometer (JEOL, Tokyo, Japan) in D2O. The 31P chemical shifts are
relative to phosphoric acid as an external standard at 0 ppm.

3398
Research (B) from The Ministry of Education, Science, Sports and Culture, Japan, 10460047 to H.S.,
by a Grant-in-Aid for Encouragement of Young Scientists from JSPS, 11760086 to T.K., and by a grant
from the Uehara Memorial Foundation to T.K.
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