Zenk1 1 Biozentrum-Pharmazie, Universita¨t Halle, Halle/Saale, Germany;2Lehrstuhl fu¨r Organische Chemie und Biochemie, Technische Universita¨t Mu¨nchen, Garching, Germany;3Laboratorium
Trang 1Studies on the nonmevalonate pathway of terpene biosynthesis
Monika Fellermeier1, Maja Raschke1, Silvia Sagner1, Juraithip Wungsintaweekul2, Christoph A Schuhr2, Stefan Hecht2, Klaus Kis2, Tanja Radykewicz2, Petra Adam2, Felix Rohdich2, Wolfgang Eisenreich2,
Adelbert Bacher2, Duilio Arigoni3and Meinhart H Zenk1
1 Biozentrum-Pharmazie, Universita¨t Halle, Halle/Saale, Germany;2Lehrstuhl fu¨r Organische Chemie und Biochemie, Technische Universita¨t Mu¨nchen, Garching, Germany;3Laboratorium fu¨r Organische Chemie, Eidgeno¨ssische Technische Hochschule Ho¨nggerberg, Zu¨rich, Switzerland
2C-Methyl-D-erythritol 2,4-cyclodiphosphate was recently
shown to be formed from 2C-methyl-D-erythritol
4-phos-phate by the consecutive action of IspD, IspE, and IspF
proteins in the nonmevalonate pathway of terpenoid
biosynthesis To complement previous work with
radio-labelled precursors, we have now demonstrated that
[U-13C5]2C-methyl-D-erythritol 4-phosphate affords
[U-13C5]2C-methyl-D-erythritol 2,4-cyclodiphosphate in
isolated chromoplasts of Capsicum annuum and Narcissus
pseudonarcissus Moreover, chromoplasts are shown to
efficiently convert 2C-methyl-D-erythritol 4-phosphate as
well as 2C-methyl-D-erythritol 2,4-cyclodiphosphate into
the carotene precursor phytoene The bulk of the kinetic data
collected in competition experiments with radiolabeled
substrates is consistent with the notion that the cyclodipho-sphate is an obligatory intermediate in the nonmevalonate pathway to terpenes Studies with [2,20-13C2
]2C-methyl-D-erythritol 2,4-cyclodiphosphate afforded phytoene characterized by pairs of jointly transferred 13C atoms in the positions 17/1, 18/5, 19/9, and 20/13 and, at a lower abundance, in positions 16/1, 4/5, 8/9, and 12/13 A detailed scheme is presented for correlating the observed partial scrambling of label with the known lack of fidelity of the isopentenyl diphosphate/dimethylethyl diphosphate isomerase
Keywords: nonmevalonate pathway; terpene; chromoplasts; 2C-methyl-D-erythritol 2; 4-cyclodiphosphate
For a period of several decades, the mevalonate pathway
elucidated in animal cells and yeast by the studies of Bloch,
Cornforth and Lynen has been considered as the universal
source of isoprenoid precursors for the biosynthesis of
terpenoids (reviewed in [1 – 4]) In recent years, a second
pathway was discovered in certain eubacteria and plants by
the research groups of Rohmer and Arigoni (reviewed in [5 –
7]) Specifically, the incorporation of 13C-labeled acetate
and glucose in bacteria such as Rhodopseudomonas
palustris [8] and Escherichia coli [9], as well as in plants
[10] suggested a triose and pyruvate as precursors for the
formation of isoprenoids via the alternative pathway
Arigoni and his coworkers found that 1-deoxy-D
-xylu-lose, a known precursor of the vitamins thiamine [11] and
pyridoxol [12], could be incorporated into terpenoids by
E coli [9] as well as by higher plants [7] More specifically,
plants were shown to utilize the mevalonate pathway in the
cytoplasmic compartment and the nonmevalonate pathway
in the plastid compartment [7,10,13,14] More recently, the
origin of a variety of plant terpenoids could be assigned to
this plastid-based nonmevalonate pathway (reviewed in [6])
Recent studies by several research groups identified
1-deoxy-D-xylulose 5-phosphate synthase as the first
enzyme of the alternative terpenoid pathway in certain bacteria [15 – 17] and plants [18,19] The enzyme product is converted into the branched chain polyol,
2C-methyl-D-erythritol 4-phosphate, by a reductoisomerase via a skeletal rearrangement followed by an NADPH-dependent reduction [20 – 23]
We have shown that in E coli 2C-methyl-D-erythritol 4-phosphate can be converted into a cyclic diphosphate by the consecutive action of 4-diphosphocytidyl-2C-methyl-D -erythritol synthase, 4-diphosphocytidyl-2C-methyl-D-erythritol kinase and 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase [24 – 26] (Fig 1) In the meantime, some of these results have been confirmed by other authors [27 – 29]
We have also shown that14C-labelled 2C-methyl-D -ery-thritol 2,4-cyclodiphosphate is incorporated into the lipid fraction of Capsicum annuum chromoplasts [26] 2C-methyl-D-erythritol 2,4-cyclodiphosphate had been isolated earlier as a stress metabolite from bacterial cultures
in high yield [30,31]
In this paper we describe the kinetics of
2C-methyl-D-erythritol 2,4-cyclodiphosphate incorporations into chromoplast preparations of C annuum and Narcissus pseudonarcissus, as well as the incorporation of [U-13C5 ]2-C-methyl-D-erythritol 2,4-cyclodiphosphate into phytoene from chromoplasts of C annuum
E X P E R I M E N T A L P R O C E D U R E S
Materials [1-3H]2C-methyl-D-erythritol 4-phosphate was prepared according to a method described by Kis et al using sodium
Correspondence to W Eisenreich, Lehrstuhl fu¨r Organische Chemie
und Biochemie, Technische Universita¨t Mu¨nchen, Lichtenbergstr 4,
D-85747 Garching, Germany Fax: þ 49 89 289 13363,
Tel.: þ 49 89 289 13043, E-mail: wolfgang.eisenreich@ch.tum.de
(Received 5 July 2001, revised 8 October 2001, accepted
9 October 2001)
Trang 2Fig 1 Biosynthesis of phytoene via the nonmevalonate pathway.
qFEBS 2001 Isoprenoid biosynthesis in plants (Eur J Biochem 268) 6303
Trang 3[3H]borohydride as reducing agent [32] [2,20-13C2]-,
[2-14C]2C-methyl-D-erythritol 2,4-cyclodiphosphate, and
[U-13C5]2C-methyl-D-erythritol 4-phosphate were prepared
as described [33,34]
Isolation of chromoplasts fromC annuum
Chromoplasts were isolated by a slight modification of the
method first described by Camara [35,36] Pericarp of red
pepper (500 g) was homogenized at 4 8C in 600 mL of 50 mM
Hepes, pH 8.0, containing 1 mM dithioerythritol, 1 mM
EDTA and 0.4 M sucrose (buffer A) The suspension was
filtered through four layers of nylon cloth (50 mm) and
centrifuged (10 min, 3290 g, GSA rotor) to obtain a pellet of
crude chromoplasts which was homogenized in 400 mL of
buffer A The suspension was centrifuged (10 min, 3290 g,
GSA rotor) The pellet was homogenized and resuspended
in 3 mL of 50 mM Hepes, pH 7.6, containing 1 mM
1,4-dithioerythritol The suspension was filtered through one layer
of nylon cloth (50 mm)
Preparation of a chromoplast extract
A suspension of washed C annuum chromoplasts (5 mL;
protein concentration, 10–15 mg·mL21) was diluted with
50 mM Hepes, pH 7.6, containing 1 mMdithioerythritol to a
final volume of 40 mL The mixture was kept for 10 min at
4 8C and was then centrifuged (60 min, 110 560 g, Ti 50
rotor) The supernatant was applied to a Sephadex G-25 column
(type PD-10, Amersham Pharmacia Biotech) which had been
equilibrated with 50 mM Hepes, pH 7.6, containing 1 mM
dithioerythritol The column was developed with the same
buffer Fractions were combined and concentrated using a
Centriprep-10 concentrator (Amicon) The final protein
concentration was about 1–2 mg·mL21
Isolation of chromoplasts fromN pseudonarcissus
The isolation followed a procedure described by Kleinig &
Beyer [37] Inner coronae of N pseudonarcissus (80 g)
were homogenized in 250 mL of 67 mMTris/HCl, pH 7.5,
containing 5 mM MgCl2, 1 mM dithioerythritol, 1 mM
EDTA, 0.2% (w/v) polyvinylpyrrolidone K90, and 0.74M
sucrose The suspension was filtered (three layers of nylon
cloth, 50 mm) and centrifuged (5 min, 1990 g, GSA rotor)
The supernatant was centrifuged (20 min, 25 400 g, GSA rotor) affording a pellet of crude chromoplasts which was resuspended in 2 mL of 67 mM Tris/HCl, pH 7.5, contain-ing 5 mM MgCl2, 1 mM dithioerythritol and 50% (w/v) sucrose The suspension was filtered through one layer of nylon cloth (50 mm) Aliquots of 2 mL were transferred to centrifuge tubes Equal volumes of 40, 30 and 15% (w/v) sucrose in 67 mM Tris/HCl, pH 7.5, containing 5 mM
MgCl2 and 1 mM dithioerythritol were placed on top of the chromoplast suspension Subsequent to centrifugation (60 min, 64 000 g, SW28 rotor), the fraction of intact chromoplasts at the 40/30% interphase was collected and diluted with 67 mM Tris/HCl containing 5 mM MgCl2and
1 mM dithioerythritol to a final sucrose concentration of 15% (w/v) The suspension was centrifuged (20 min,
25 130 g, SS34 rotor) and the pellet was suspended in
2 mL of 67 mM Tris/HCl, pH 7.5, containing 5 mMMgCl2 and 1 mM dithioerythritol
Incorporation experiments with isotope-labeled substrates
Reaction mixtures contained 100 mMHepes, pH 7.6, 2 mM
MnCl2, 10 mM MgCl2, 5 mM NaF, 2 mM NADPþ, 1 mM
NADPH, 6 mM ATP, 20 mM FAD and chromoplasts or chromoplast extract Isotope-labeled 2C-methyl-D-erythritol 4-phosphate and/or 2C-methyl-D-erythritol 2,4-cyclodiphos-phate were added as indicated in Table 1 The mixtures were incubated at 30 8C The reaction was terminated by ethyl acetate extraction The lipid extract was dried over sodium sulfate In experiments with radiolabeled substrates the residue was analyzed by scintillation counting and/or HPLC The aqueous phase was analyzed by reversed phase ion pair HPLC monitored by scintillation counting HPLC analysis of phosphorylated metabolites Reversed phase ion pair HPLC separations were performed with a Luna C8 column (Phenomenex, 5 mm,
4 £ 250 mm) The column was developed with a linear gradient of 0 – 42% methanol in 10 mM tetrabutylammo-nium sulfate, pH 6.0 (total volume, 60 mL; flow rate, 0.75 mL min21) The retention volumes of
2C-methyl-D-erythritol 4-phosphate and 2C-methyl-D-erythritol 2,4-cyclodiphosphate were 10.0 and 29.0 mL, respectively
Table 1 Competition experiments with [1- 3 H]2C-methyl- D -erythritol 4-phosphate (MEP) and [2- 14 C]2C-methyl- D -erythritol 2,4-cyclodi-phosphate (cMEPP) in a chromoplast system from C annuum In each experiment, the sample volume was 150 mL Conc., concentration; Sp radioact., specific radioactivity.
Proffered precursors
Radioactivity incorporation into
[1-3H]cMEPP the lipid-soluble material Experiment
Conc.
(m M )
Sp radioact.
(mCi·mmol 21 )
Conc.
(m M )
Sp radioact.
(mCi·mmol 21 )
14
C :3H (% a )
produced (nmol)
14
C (nmol)
3
H (nmol)
14
C :3H (% a )
Trang 4Isolation of 2C-methyl-D-erythritol 2,4-cyclodiphosphate
The reaction mixture was centrifuged and the supernatant
was applied to a CHROMABONDw SB column (500 mg,
Macherey & Nagel) The column was washed with water
and developed with 0.1 M ammonium bicarbonate The
effluent was passed through a column of DOWEX AG
50 W-X8 (100 – 200 mesh, Hþ-form) and was then
lyophilized The residue was dissolved in water and applied
to a Nucleosil 10SB HPLC-column which was developed
with 100 mMammonium formate in 40% (v/v) methanol at
a flow rate of 1 mL·min21 The effluent was monitored by
scintillation counting The retention time of
2C-methyl-D-erythritol 2,4-cyclodiphosphate was 26 min Fractions
were combined and lyophilized
Isolation of phytoene
Fresh red peppers (107 g) were homogenized and
lyophilized The dry powder was extracted with ethyl
acetate (2.5 L) The solution was brought to dryness under
reduced pressure The residue was dissolved in 10 mL of a
hexane/ethylacetate mixture (1 : 1, v/v) The solution was placed on a column of silica gel 60 (5 £ 40 cm) which was developed with a mixture of a hexane/ethylacetate (1 : 1; v/v) The red-colored fraction (400 – 480 mL) was collected and concentrated under reduced pressure The residue was dissolved in 100 mL of chloroform
Purification of phytoene Aliquots (10 mL) of crude phytoene solution in chloroform were applied to a Hypersil RP18 HPLC column (5 mm, 4.5 £ 250 mm, ThermoQuest Germna GmbH, Egelsbach, Germany) that was developed with a mixture of isopropanol/ acetonitrile/water (50 : 45 : 5, v/v) The effluent was monitored photometrically (280 nm) The retention volumes of b-carotene, phytoene, and xanthophylls were
12, 14, and 20 mL, respectively
NMR spectroscopy NMR spectra were recorded with a DRX 500 spectrometer from Bruker Instruments (Karlsruhe, Germany) equipped with four channels and a pulsed gradient unit Two dimensional homocorrelation and heterocorrelation experi-ments were performed withXWINNMRsoftware from Bruker Instruments Phytoene was measured in CDCl3, and 2C-methyl-D-erythritol 2,4-cyclodiphosphate was measured
in D2O
R E S U L T S
Isolated chromoplasts of C annuum were incubated with mixtures of [1-3H]2C-methyl-D-erythritol 4-phosphate and [2-14C]2C-methyl-D-erythritol 2,4-cyclodiphosphate (Table 1) and were then extracted with ethyl acetate The aqueous phase was analyzed by HPLC in order to monitor the conversion of [1-3H]2C-methyl-D-erythritol 4-phosphate into the corresponding 2,4-cyclodiphosphate The organic phase was analyzed for3H and14C in order to monitor the transformation of the proffered radioactive compounds into lipid-soluble material
The data summarized in Table 1 indicate that [1-3H]2C-methyl-D-erythritol 4-phosphate was diverted effici-ently to the 2C-methyl-D-erythritol 2,4-cyclodiphosphate
Fig 3 13 C NMR signals of 2C-methyl- D -erythritol 2,4-cyclodiphosphate obtained by incubation of [U- 13 C 5 ]2C-methyl- D -erythritol 4-phosphate with a chromoplast extract of C annuum.13C and31P coupling patterns are indicated.
Fig 2 Diversion of radioactivity from [2-14C]2C-methyl- D
-ery-thritol 2,4-cyclodiphosphate into lipid-soluble material of
chromo-plasts from Narcissus pseudonarcissus (A) 2C-methyl- D -erythritol
2,4-cyclodiphosphate; (B) lipid-soluble fraction.
qFEBS 2001 Isoprenoid biosynthesis in plants (Eur J Biochem 268) 6305
Trang 5pool The amount of newly formed [1-3
H]2C-methyl-D-erythritol 2,4-cyclodiphosphate increased with the
con-centration of the proffered [1-3H]2C-methyl-D-erythritol
4-phosphate; the transformation showed saturation
characteristics
[1-3H]2C-methyl-D-erythritol 4-phosphate as well as
[2-14C]2C-methyl-D-erythritol 2,4-cyclodiphosphate were
efficiently converted into lipid-soluble material The amount
of [1-3H]2C-methyl-D-erythritol 4-phosphate converted into
lipid-soluble material increased with the concentration of
the profferred substrate; saturation was reached at a
substrate concentration of less than 1 mM
The transformation of14C-labeled cyclic diphosphate into
lipid-soluble compounds had its maximum efficacy
(approximately 35%) at low concentrations of proffered
[1-3H]2C-methyl-D-erythritol 4-phosphate At high
concen-trations of this compound, the incorporation of 14C-label
from the cyclic diphosphate into the lipid-soluble fraction
was significantly diminished
In a similar experiment, we studied the formation of
lipid-soluble material from [2-14C]2C-methyl-D-erythritol
Fig 4. 13C NMR signals of phytoene obtained from [2,20-13C 2
]2-C-methyl- D -erythritol 2,4-cyclodiphosphate by incubation with
chromoplasts of C annuum 13 C coupling patterns are indicated.
Table 2.13C NMR assignments for phytoene.
Position
13
C-Chemical shift (d, p.p.m.) a
J CCb
a
Referenced to external TMS; bfrom the experiment with [2,20-13C 2 ]2-C-methyl- D -erythritol 2,4-cyclodiphosphate; c – e assignments may be interchanged.
Table 3.13C-Labeling pattern of phytoene obtained from chromo-plasts of C annuum incubated with [2,20-13C 2
]2C-methyl-D -erythritol 2,4-cyclodiphosphate ND, not determined, due to signal overlapping.
a
Calculated as the relative13C abundance by comparison of13C NMR signal intensities of the labeled sample with 13 C NMR signal intensities of an unlabeled phytoene sample.bcalculated as the fraction of the13C-coupled satellites in the global 13 C NMR intensity of a given atom c – e assignments may be interchanged.
Trang 62,4-cyclodiphosphate using isolated chromoplasts from
N pseudonarcissus (Fig 2) The incorporation of
radio-actvity into lipid-soluble material was again checked by
solvent extraction of reaction mixtures and the consumption
of 2C-methyl-D-erythritol 2,4-cyclodiphosphate was
monitored by HPLC analysis of the aqueous phase using a
scintillation detector As shown in Fig 2, the radioactive
substrate was virtually completely depleted and up to 94%
of the proffered radioactivity was transformed into
lipid-soluble material
Next, experiments with precursors labeled with stable
isotopes were initiated An extract prepared from isolated
chromoplasts of C annuum was depleted of low molecular
mass compounds by gel filtration and was then incubated
with [U-13C5]2C-methyl-D-erythritol 4-phosphate in
admix-ture of a small amount of [2-14C]2C-methyl-D-erythritol
4-phosphate at 30 8C for 15 h as described under
Experimental procedures A radioactive product was then
isolated from the reaction mixture and was analyzed by13C
NMR spectroscopy (Fig 3)
All 13C NMR signals of the isolated compound were
multiplets due to13C13C coupling Based on chemical shift
values and coupling constants, the compound was identified
as 2C-methyl-D-erythritol 2,4-cyclodiphosphate (see [26]
for NMR data of the authentic compound) The absence of
singlet signals for the carbon atoms 1, 2, 2-Me, 3 and 4 in the
spectrum of the isolated material demonstrates that the
proffered material had not been diluted by significant
amounts of endogenous material with natural 13C
abun-dance It follows that the chromoplast extract used did not
contain significant amounts of endogenous, unlabeled
2C-methyl-D-erythritol 2,4-cyclodiphosphate
Isolated chromoplasts from C annuum were
sub-sequently incubated with 0.7 mM [2,20-13C2
]2C-methyl-D-erythritol 2,4-cyclodiphosphate at 30 8C for 12 h The
suspension was extracted with ethyl acetate, and phytoene
(Fig 1, compound 10) was isolated from the resulting
mixture of lipophilic compounds.13C NMR signals of the
isolated compound are shown in Fig 4
Signal assignments taken from [38] are supported by
1-and 2- dimensional analysis of the 13C-labeled phytoene
sample (Table 2) Eight of the 2013C signals of the labeled
phytoene appeared as singlets, eight signals showed
high-intensity satellites caused by 13C –13C coupling, and four
signals were characterized by13C –13C coupling satellites of
lower intensity (Table 3) The13C connectivity was further
analyzed by a two-dimensional INADEQUATE experiment
showing four pairs of13C atoms (Fig 5)
The terminal moiety of phytoene is biosynthetically derived from dimethylalkyl diphosphate Both methyl groups (i.e C-16 and C-17) of this moiety showed13C–13C coupling satellites, albeit of different intensities (Table 3) The labeling pattern of the reconstructed DMAPP unit is summarized in Fig 6 and the evaluation of the signal intensities indicated a ratio of 10 : 1 for the two isotopomers a and b The13C NMR signals of the methyl groups C-18, C-19, and C-20 of phytoene (biosynthetically equivalent to C-5 of IPP) showed
13C-coupled satellites of high intensity (Table 3) From the signal intensities the molar fraction of the IPP isotopomer c can be calculated (Fig 6)
The signals of C-12 and the coincident signals of carbon atoms 4 and 8 showed one bond13C –13C coupling satellites
of lower intensities that were substantially broadened by comparison with the central signal (Fig 4) When processed for maximum resolution, these satellites appeared as pseudotriplets that could be due to long range coupling involving vicinal isoprenoid moieties Due to the line broadening, the precision of signal integration is substan-tially reduced However, within the experimental limits, it appears that the abundance of IPP isotopomer d is similar to that of DMAPP isotopomer b (Fig 6)
D I S C U S S I O N
The conversion of 2C-methyl-D-erythritol 4-phosphate into the corresponding 2,4-cyclodiphosphate by the consecutive action of three recombinant E coli enzymes (specified by the ispD, ispE and ispF genes) has been described [24 – 29] Orthologs of ispD and ispE from Arabidopsis thaliana and tomato, respectively, have been expressed in recombinant
E coli cells [39,40]
This paper shows that isolated chromoplasts from
C annuum and N pseudonarcissus catalyze the con-version of 2C-methyl-D-erythritol 4-phosphate into the 2,4-cyclodiphosphate in a process that displays saturation kinetics and that the product of this reaction is further processed efficiently into phytoene
The results of the competition experiments summarized in Table 1 demonstrate that the incorporation of radioactivity from [1-14C]2C-methyl- -erythritol 2,4-cyclodiphosphate
Fig 5 Two-dimensional INADEQUATE spectrum of phytoene
obtained from [2,20-13C 2 ]2C-methyl- D -erythritol
2,4-cyclodipho-sphate by incubation with chromoplasts of C annuum.
Fig 6 Reconstruction of the labeling pattern of IPP (isotopomers a and b) and DMAPP (isotopomers c and d) from the labeling pattern
of the phytoene sample obtained in the experiment with [2,20- 13 C 2 ]2C-methyl- D -erythritol 2,4-cyclodiphosphate Bold lines denote bonds linking adjacent 13C atoms, numbers indicate the percentage molar fraction of the isotopomers.
qFEBS 2001 Isoprenoid biosynthesis in plants (Eur J Biochem 268) 6307
Trang 7into phytoene (the main labeled component of the
lipid-soluble fraction) is systematically diminished by the
addition of increasing amounts of [1-3H]2C-methyl-D
-ery-thritol 4-phosphate Moreover, the data show that even at
saturating concentrations of the tritiated compound, the
relative transfer of14C-label from the cyclodiphosphate pool
is always in excess of the value calculated from the original
molar concentration of the two precursors This requires that
within the nonmevalonate pathway the cyclodiphosphate is
nearer than the 4-phosphate to IPP and DMAPP, the two C5
building blocks from which phytoene is assembled Thus, on
all the available accounts the cyclodiphosphate behaves as
expected for an obligatory intermediate
NMR spectroscopic analysis of the phytoene specimen
obtained from [2,20-13C2]2C-methyl-D-erythritol
2,4-cyclo-diphosphate reveals a partial scrambling of label between
(Z)- and (E)-methyl groups of DMAPP derived units and for
the corresponding IPP-derived internal units A similar
partial scrambling of label between the (Z)- and (E)-methyls
of the starter DMAPP unit matched by a corresponding
scrambling within nonstarter C5-units derived from IPP in
the elongation process has been observed in the mevalonate
independent biosynthesis of carotenoids in cell cultures of Catharanthus roseus [13] as well as for the DMAPP-derived
C5-unit of mevalonoid origin present in several clavine alkaloids [41 – 43]; but for a possible exception [44], a corresponding scrambling within the nonstarter C5units of mevalonoid terpenes seems to have gone undetected, probably because of the inadequacy of the analytical tools employed in earlier work using a14C label In all the cases
in which such a scrambling was observed it was usually ascribed to a lack of fidelity of the isomerase that inter-converts IPP and DMAPP Participation of the isomerase is
of crucial importance in the mevalonate pathway, in which formation of IPP and DMAPP take place in sequential steps;
in contrast, the available evidence indicates that within the new pathway IPP and DMAPP are formed in independent steps from a common and yet unidentified intermediate [45 – 50], but a subsequent partial equilibration of the pre-formed units can nevertheless occur in organisms equipped with the isomerase, as is the case in higher plants in which the two metabolic pathways are known to coexist
The isomerization of IPP to DMAPP is an antarafacial process in which a proton is added to the re-re face of the double bond with subsequent or concomitant stereospecific removal of the HB hydrogen at C-2 (Fig 7) from the opposite face of the molecule [51]; in the specific case of a recombinant yeast enzyme, the catalytic groups have been identified as Cys139, respectively, Glu207 [52] In refinement and extension of previous observations by other authors [53], the Poulter group has carried out a thorough investigation on the lack of fidelity of this isomerase by analyzing the proton exchange that occurs when IPP is incubated with the enzyme in D2O [54]; a rapid exchange was detected for the C-4 hydrogens and one of C-2 hydrogens of IPP as well as for the (E)-methyl group of DMAPP, followed by a slower exchange (2% of the isomerization rate) of the methyl group of IPP and the (Z)-methyl group of DMAPP, and an even slower exchange (0.5% of the isomerization rate) of the olefinic hydrogen of DMAPP corresponding to the HA-hydrogen at C-2 of IPP
It is tempting to correlate this lack of regiochemical and stereochemical fidelity of the enzyme with the bidentate nature of the Glu207 carboxylate group positioned in the ES complex of the reaction as indicated in Fig 7; in this geometric arrangement one of the oxygens is competent for efficient removal of the HB hydrogen from C-2 of the
Fig 7 Model representation for the positioning of the substrate in
the active site of IPP isomerase; (a) and (b) represent alternative
paths for proton abstraction from the substrate by the ambident
carboxylate group of Glu207.
Fig 8 A detailed mechanistic scheme accounting for the known lack of fidelity of IPP isomerase The resulting isotopic scrambling can be visualized by following the fate of the C atom labeled with a black dot in the starting material represented in the squares The alternative reaction paths (a) and (b) correspond to the ones illustrated in Fig 7 Sets A and B illustrate two different binding modes for the substrate.
Trang 8substrate (path a), while the second oxygen is close enough to
the methyl group to catalyze, as an alternative, the occasional
removal of one of its hydrogens (path b) The outcome of the
two competing deprotonation paths is illustrated in Fig 8 for
the predominant ES complex A of a sample of IPP carrying a
13C label in its methyl group; a similar scheme involving a
less stable ES complex B is necessary to account for the
observed very slow exchange of the HA-hydrogen of IPP In
both cases, scrambling of the label takes place within the IPP
pool and the error is then transcribed into the DMAPP pool by
the normal action of the isomerase The validity of the
proposed scheme is rewardingly supported by the observation
that the enzyme is capable to convert the IPP homolog X into
its isomer Y (see Fig 9) in a process which bypasses the
formation of allylic isomers [53]
A C K N O W L E D G E M E N T S
This work was supported by grants from the Fonds der Chemischen
Industrie and the Deutsche Forschungsgemeinschaft (SFB369) to A B.,
W E and M H Z and a fellowship from the
Hans-Fischer-Gesellschaft to T R We thank Katrin Ga¨rtner for skillfull assistence
and Prof B Camara, Strasbourg, for a sample of phytoene Financial
support by Novartis International AG Basel (to D A.) is gratefully
acknowledged.
R E F E R E N C E S
1 Qureshi, N & Porter, J.W (1981) Biosynthesis of mevalonic acid
from acetyl-CoA In Biosynthesis of Isoprenoid Compounds
(Porter, J.W & Spurgeon, S.L., eds), Vol 1, pp 47 – 94 John
Wiley, New York, USA.
2 Bloch, K (1992) Sterol molecule: structure, biosynthesis and
function Steroids 57, 378 – 382.
3 Bach, T.J (1995) Some new aspects of isoprenoid biosynthesis in
plants – a review Lipids 30, 191 – 202.
4 Bochar, D.A., Friesen, J.A., Stauffacher, C.V & Rodwell, V.W.
(1999) Biosynthesis of mevalonic acid from acetyl-CoA In
Comprehensive Natural Product Chemistry (Cane, D., ed.),Vol 2,
pp 15 – 44 Pergamon, Oxford, UK
5 Rohmer, M (1999) A mevalonate-independent route to isopentenyl
diphosphate In Comprehensive Natural Product Chemistry (Cane,
D., ed.), Vol 2, pp 45 – 68 Pergamon, Oxford, UK.
6 Eisenreich, W., Schwarz, M., Cartayrade, A., Arigoni, D., Zenk, M.
& Bacher, A (1998) The deoxyxylulose phosphate pathway of
terpenoid biosynthesis in plants and microorganisms Chem Biol.
5, R221 – R233.
7 Schwarz, M & Arigoni, D (1999) Ginkgolide biosynthesis In
Comprehensive Natural Product Chemistry (Cane, D., ed), Vol 2,
pp 367 – 399 Pergamon, Oxford, UK.
8 Rohmer, M., Knani, M., Simonin, P., Sutter, B & Sahm, H (1993)
Isoprenoid biosynthesis in bacteria: a novel pathway for the early
steps leading to isopentenyl diphosphate Biochem J 295,
517 – 524.
9 Broers, S.T.J (1994) U ¨ ber die fru¨hen Stufen der Biosynthese von Isoprenoiden in Escherichia coli PhD Thesis, ETH Zu¨rich, Switzerland.
10 Schwarz, M.K (1994) Terpen-Biosynthese in Ginkgo biloba: Eine u¨berraschende Geschichte PhD Thesis, ETH Zu¨rich, Switzerland.
11 Spenser, I.D & White, R.L (1997) Biosynthesis of vitamin B 1
(thiamin): an instance of biochemical diversity Angew Chem Int.
Ed 36, 1032 – 1046.
12 Hill, R.E., Himmeldirk, K., Kennedy, I.A., Panloski, R.M., Sayer, B.G., Wolf, E & Spenser, I.D (1996) The biogenetic anatomy of vitamin B 6 A 13C NMR investigation of the biosynthesis of pyridoxol in Escherichia coli J Biol Chem 271, 30426– 30435.
13 Arigoni, D., Sagner, S., Latzel, C., Eisenreich, W., Bacher, A & Zenk, M.H (1997) Terpene biosynthesis from 1-deoxy- D -xylulose
in higher plants by intramolecular skeletal rearrangement Proc Natl Acad Sci USA 94, 10600– 10605.
14 Lichtenthaler, H.K (1999) The 1-deoxy- D -xylulose 5-phosphate pathway of isoprenoid biosynthesis in plants Annu Rev Plant Phys Plant Mol Biol 50, 47 – 65.
15 Sprenger, G.A., Scho¨rken, U., Wiegert, T., Grolle, S., deGraaf, A.A., Taylor, S.V., Begley, T.P., Bringer-Meyer, S & Sahm, H (1997) Identification of a thiamin-dependent synthase in Escher-ichia coli required for the formation of the 1-deoxy- D -xylulose 5-phosphate precursor to isoprenoids, thiamin, and pyridoxol Proc Natl Acad Sci USA 94, 12857– 12862.
16 Lois, L.M., Campos, N., Putra, S.R., Danielsen, K., Rohmer, M & Boronat, A (1998) Cloning and characterization of a gene from Escherichia coli encoding a transketolase-like enzyme that catalyzes the synthesis of D -1-deoxyxylulose 5-phosphate, a common precursur for isoprenoid, thiamin, and pyridoxol biosynthesis Proc Natl Acad Sci USA 95, 2105 – 2110.
17 Kuzuyama, T., Takagi, M., Takahashi, S & Seto, H (2000) Cloning and characterization of 1-deoxy- D -xylulose 5-phosphate synthase from Streptomyces sp Strain CL190, which uses both the mevalonate and nonmevalonate pathways for isopentenyl dipho-sphate biosynthesis J Bacteriol 182, 891 – 897.
18 Lange, B.M., Wildung, M.R., McCaskill, D & Croteau, R (1998)
A family of transketolases that directs isoprenoid biosynthesis via a mevalonate-independent pathway Proc Natl Acad Sci USA 95,
2100 – 2104.
19 Bouvier, F., d’Harlingue, A., Suire, C., Backhaus, R.A & Camara,
B (1998) Dedicated roles of plastid transketolases during the early onset of isoprenoid biogenesis in pepper fruits Plant Physiol 117,
1423 – 1431.
20 Takahashi, S., Kuzuyama, T., Watanabe, H & Seto, H (1998) A 1-deoxy- D -xylulose 5-phosphate reductoisomerase catalyzing the formation of 2-C-methyl- D -erythritol 4-phosphate in an alternative nonmevalonate pathway for terpenoid biosynthesis Proc Natl Acad Sci USA 95, 9879 – 9884.
21 Lange, B.M & Croteau, R (1999) Isoprenoid biosynthesis via a mevalonate-independent pathway in plants: cloning and heterologous expression of 1-deoxy- D -xylulose-5-phosphate reductoisomerase from peppermint Arch Biochem Biophys 365,
170 – 174.
22 Schwender, J., Mu¨ller, C., Zeidler, J & Lichtenthaler, H.K (1999) Cloning and heterologous expression of a cDNA encoding 1-deoxy- D -xylulose-5-phosphate reductoisomerase of Arabidopsis thaliana FEBS Lett 455, 140 – 144.
23 Jomaa, H., Wiesner, J., Sanderbrand, S., Altinicicek, B., Weidemeyer, C., Hintz, M., Tu¨rbachova, I., Eberl, M., Zeidler, J., Lichtenthaler, H.K., Soldati, D & Beck, E (1999) Inhibitors of the non-mevalonate pathway of isoprenoid biosynthesis as antimalarial drugs Science 285, 1573 – 1576.
24 Rohdich, F., Wungsintaweekul, J., Fellermeier, M., Sagner, S., Herz, S., Kis, K., Eisenreich, W., Bacher, A & Zenk, M.H (1999) Cytidine
50-triphosphate biosynthesis of isoprenoids: YgbP protein of
Fig 9 The anomalous reaction of the IPP homolog X catalyzed by
the IPP isomerase acting along path (b) of Fig 7.
qFEBS 2001 Isoprenoid biosynthesis in plants (Eur J Biochem 268) 6309
Trang 9Escherichia coli catalyzes the formation of
4-diphosphocytidyl-2C methylerythritol Proc Natl Acad Sci USA 96, 11758 – 11763.
25 Lu¨ttgen, H., Rohdich, F., Herz, S., Wungsintaweekul, J., Hecht, S.,
Schuhr, C.A., Fellermeier, M., Sagner, S., Zenk, M.H., Bacher, A.
& Eisenreich, W (2000) Biosynthesis of terpenoids: YchB protein
of Escherichia coli phosphorylates the 2-hydroxy group of
4-diphosphocytidyl-2C-methyl- D -erythritol Proc Natl Acad Sci.
USA 97, 1062 – 1067.
26 Herz, S., Wungsintaweekul, J., Schuhr, C.A., Hecht, S., Lu¨ttgen,
H., Sagner, S., Fellermeier, M., Eisenreich, W., Zenk, M.H.,
Bacher, A & Rohdich, F (2000) Biosynthesis of terpenoids: YgbB
protein converts 4-diphosphocytidyl-2C-methyl- D -erythritol
2-phosphate to 2C-methyl- D -erythritol 2,4-cyclodiphosphate.
Proc Natl Acad Sci USA 97, 2486 – 2490.
27 Kuzuyama, T., Takagi, M., Kaneda, K., Dairi, T & Seto, H (2000)
Formation of 4-(cytidine 5 0 -diphospho)-2-C-methyl- D -erythritol
from 2-C-methyl- D -erythritol 4-phosphate by 2-C-methyl- D
-ery-thritol 4-phosphate cytidyltransferase, a new enzyme in the
nonmevalonate pathway Tetrahedron Lett 41, 703 – 706.
28 Kuzuyama, T., Takagi, M., Kaneda, K., Watanabe, H., Dairi, T &
Seto, H (2000) Studies on the nonmevalonate pathway: conversion
of 4-(cytidine 50-diphospho)-2-C-methyl- D -erythritol to its
2-phos-pho derivative by 4-(cytidine 50-diphospho)-2-C-methyl- D
-erythri-tol kinase Tetrahedron Lett 41, 2925 – 2928.
29 Takagi, M., Kuzuyama, T., Kaneda, K., Watanabe, H., Dairi, T &
Seto, H (2000) 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 Tetrahedron
Lett 41, 3395 – 3398.
30 Turner, D., Santos, H., Fareleira, P., Pacheco, I., LeGall, Y &
Xavier, A.V (1992) Structure determination of a novel cyclic
phosphocompound isolated from Desulfovibrio desulfuricans.
Biochem J 285, 387 – 390.
31 Ostrovsky, D., Kharatian, E., Dubrovsky, T., Ogrel, O., Shipanova,
I & Sibeldina, L (1992) The ability of bacteria to synthesize a new
cyclodiphosphate correlates with their tolerance to redox-cycling
drugs: on a crossroad of chemotherapy, environmental toxicology
and immunobiochemical problems Biofactors 4, 63 – 68.
32 Kis, K., Wungsintaweekul, J., Eisenreich, W., Zenk, M.H & Bacher,
A (2000) An efficient preparation of 2-C-methyl- D -erythritol
4-phosphoric acid and its derivatives J Org Chem 65, 587 – 592.
33 Schuhr, C.A., Hecht, S., Eisenreich, W., Wungsintaweekul, J.,
Bacher, A & Rohdich, F (2001) Studies on the non-mevalonate
pathway – preparation and properties of isotope-labeled
2C-methyl- D -erythritol 2,4-cyclodiphosphate Eur J Org Chem.,
3221 – 3226.
34 Hecht, S., Wungsintaweekul, J., Rohdich, F., Kis, K., Radykewicz,
T., Schuhr, C.A., Eisenreich, W., Richter, G & Bacher, A (2001)
Biosynthesis of terpenoids: Efficient multistep biotransformation
procedures affording isotope-labeled 2C-methyl- D -erythritol
4-phosphate using recombinant 2C-methyl- D -erythritol
4-phos-phate synthase J Org Chem., in press.
35 Camara, B (1985) Carotene synthesis in Capsicum chromoplasts.
Methods Enzymol 110, 244 – 253.
36 Camara, B (1993) Plant phytoene synthase complex: component
enzymes, immunology, and biogenesis Methods Enzymol 214,
352 – 365.
37 Kleinig, H & Beyer, P (1985) Carotene synthesis in spinach
(Spinacia oleracea L.) chloroplasts and daffodil (Narcissus
pseudonarcissus L.) chromoplasts Methods Enzymol 110,
267 – 273.
38 Clough, J.M & Pattenden, G.J (1983) Stereochemical assignment
of prolycopene and other poly-Z-isomeric carotenoids in fruits of
the tangerine tomato Lycopersicon esculentum var ‘Tangella’.
J Chem Soc Perkin Trans 1, 3011 – 3018.
39 Rohdich, F., Wungsintaweekul, J., Eisenreich, W., Richter, G., Schuhr, C.A., Hecht, S., Zenk, M.H & Bacher, A (2000) Biosynthesis of terpenoids: 4-diphosphocytidyl-2C-methyl- D -ery-thritol synthase of Arabidopsis thaliana Proc Natl Acad Sci USA
97, 6451 – 6456.
40 Rohdich, F., Wungsintaweekul, J., Lu¨ttgen, H., Fischer, M., Eisenreich, W., Schuhr, C.A., Fellermeier, M., Schramek, N., Zenk, M.H & Bacher, A (2000) Biosynthesis of terpenoids: 4-dipho-sphocytidyl-2-C-methyl- D -erythritol kinase from tomato Proc Natl Acad Sci USA 97, 8251 – 8251.
41 Fehr, T., Acklin, W & Arigoni, D (1966) The role of the chanoclavines in the biosynthesis of ergot alkaloids J Chem Soc Chem Commun 801 – 802
42 Pachlatko, P., Tabacik, C., Acklin, W & Arigoni, D (1975) Natural and unnatural precursors in the biosynthesis of ergot alkaloids Chimia 29, 526.
43 Shibuya, M., Chou, H.-M., Fountoulakis, M., Hassam, S., Kim, S.-U., Kobayashi, K., Otsuka, H., Rogalska, E., Cassady, J.M & Floss, H.G (1990) Stereochemistry of the isoprenylation of tryptophan catalyzed by 4-(g,g-dimethylallyl) tryptophan synthase from Claviceps, the first pathway-specific enzyme in ergot alkaloid biosynthesis J Am Chem Soc 112, 297 – 304.
44 Croteau, R & Loomis, W.D (1972) Biosynthesis of mono- and sesquiterpenes in peppermint from mevalonate 2- 14 C Phytochem-istry 11, 1055 – 1066.
45 Giner, J.-L., Jaun, B & Arigoni, D (1998) Biosynthesis of isoprenoids in Escherichia coli The fate of the 3-H and 4-H atoms
1857 – 1858
46 Leyes, A.E., Baker, J.A., Hahn, F.M & Poulter, C.D (1999) Biosynthesis of isoprenoids in Escherichia coli: stereochemistry of the reaction catalyzed by isopentenyl diphosphate: dimethylallyl diphosphate isomerase J Chem Soc., Chem Commun., 717 – 718
47 Leyes, A.E., Baker, J.A & Poulter, C.D (1999) Biosynthesis of isoprenoids in Escherichia coli Stereochemistry of the reaction catalyzed by farnesyl diphosphate synthase Org Lett 1,
1071 – 1073.
48 Hahn, F.M., Hurlburt, A.P & Poulter, C.D (1999) Escherichia coli open reading frame 696 is idi, a nonessential gene encoding isopentenyl diphosphate isomerase J Bacteriol 181, 4499 – 4504.
49 Rodriguez-Concepcion, M., Campos, N., Maria Lois, L., Maldonado, C., Hoeffler, J.F., Grosdemange-Billiard, C., Rohmer,
M & Boronat, A (2000) Genetic evidence of branching in the isoprenoid pathway for the production of isopentenyl diphosphate and dimethylallyl diphosphate in Escherichia coli FEBS Lett 473,
328 – 332.
50 Rieder, C.H., Jaun, B & Arigoni, D (2000) On the early steps of cineol biosynthesis in Eucalyptus globulus Helv Chim Acta 83,
2504 – 2513.
51 Poulter, C.D & Rilling, H.C (1981) Prenyl transferases and isomerase Biosynthesis of Isoprenoids Compounds (Porter, J.W & Spurgeon, S.L., eds), Vol 1, pp 161 – 224 John Wiley & Sons, New York, USA.
52 Street, I.P., Coffman, H.R., Baker, J.A & Poulter, C.D (1994) Identification of Cys139 and Glu207 as catalytically important groups in the active site of isopentenyl diphosphate: dimethylallyl diphosphate isomerase Biochemistry 33, 4212 – 4217.
53 Koyama, T., Ogura, K & Seto, S (1973) Studies on isopentenyl pyrophosphate isomerase with artificial substrates J Biol Chem.
248, 8043 – 8051.
54 Street, I.P., Christiansen, D.J & Poulter, C.D (1990) Hydrogen exchange during the enzyme-catalyzed isomerization of isopente-nyl diphosphate and dimethylallyl diphosphate J Am Chem Soc.
112, 8577 – 8578.