Tài liệu Báo cáo Y học: Studies on the nonmevalonate pathway of terpene biosynthesis The role of

Studies on the nonmevalonate pathway of terpene biosynthesis

The role of 2C-methyl-D-erythritol 2,4-cyclodiphosphate in plants

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 Arigoni3 and Meinhart H. Zenk1

1

Biozentrum-Pharmazie, Universita¨t Halle, Halle/Saale, Germany; 2

Lehrstuhl fu¨r Organische Chemie und Biochemie, Technische

Universita¨t Mu¨nchen, Garching, Germany; 3

Laboratorium 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 that 14C-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.

EXPERIMENTAL PROCEDURES

Materials

[1-3

H]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: [email protected]

(Received 5 July 2001, revised 8 October 2001, accepted

9 October 2001)

Eur. J. Biochem. 268, 6302–6310 (2001) q FEBS 2001