Tài liệu Báo cáo khoa học: Isoprenoid biosynthesis via the methylerythritol phosphate pathway

Isoprenoid biosynthesis via the methylerythritol phosphate pathway

Mechanistic investigations of the 1-deoxy-D-xylulose 5-phosphate reductoisomerase

Jean-Franc¸ois Hoeffler, Denis Tritsch, Catherine Grosdemange-Billiard and Michel Rohmer

Universite´ Louis Pasteur/CNRS, Institut Le Bel, Strasbourg, France

The 1-deoxyxylulose 5-phosphate reductoisomerase (DXR,

EC 1.1.1.267)catalyzes the conversion of 1-deoxy-D-xylulose

5-phosphate (DXP)into 2-C-methyl-D-erythritol 4-phos￾phate (MEP). This transformation is a two-step process

involving a rearrangement of DXP into the putative inter￾mediate 2-C-methyl-D-erythrose 4-phosphate followed by a

NADPH-dependent reduction of the latter aldehyde. By

using [1-13C]DXP as a substrate, the rearrangement of DXP

into [5-13C]2-C-methyl-D-erythrose 4-phosphate was shown

to be NADPH dependent, although it does not involve

a reduction step. The putative aldehyde intermediate,

obtained by chemical synthesis, was converted into MEP by

the DXR in the presence of NADPH and into DXP in the

presence of NADP+, indicating the reversibility of the

reaction catalyzed by the DXR. This reversibility was con￾firmed by the conversion of MEP into DXP in the presence

of NADP+. The equilibrium was, however, largely dis￾placed in favour of the formation of MEP. The reduction

step required the presence of a divalent cation such as Mg2+

or Mn2+.

Keywords: isoprenoid, 2-C-methyl-D-erythritol 4-phosphate,

1-deoxyxylulose 5-phosphate reductoisomerase, 2-C-methyl￾D-erythrose4-phosphate.

Many bacteria, the unicellular green algae and the chlo￾roplasts from phototrophic organisms synthesize their

isoprenoids via the mevalonate-independent 2-C-methyl￾D-erythritol phosphate 5 (MEP)pathway (Fig. 1)[1–3].

The initial step of this route is the formation of 1-deoxy￾D-xylulose 5-phosphate 3 (DXP)by the condensation of

(hydroxyethyl)thiamine resulting from pyruvate 1 decarb￾oxylation on glyceraldehyde 3-phosphate 2 catalyzed by

the thiamine diphosphate-dependent DXP synthase (DXS)

[4–6]. The second enzyme of this biosynthetic pathway,

the DXP reductoisomerase (DXR), catalyzes the trans￾formation of DXP into MEP 5 in two steps. DXR is a

class B dehydrogenase [7,8]. The corresponding gene has

now been cloned from Escherichia coli [9], Zymomonas

mobilis [10], Mentha x piperita [11], Arabidopsis thaliana

[12], Synechocystis sp. [7], Streptomyces coelicolor [13] and

Pseudomonas aeruginosa [14]. In the postulated mechanism

of the reaction catalyzed by the DXR, DXP 3 is first

rearranged into 2-C-methyl-erythrose-4-phosphate 4 [15],

which is subsequently reduced by NADPH to yield MEP

5. The latter aldehyde intermediate 4 was, however, never

characterized, neither directly, nor indirectly. It is appar￾ently not released from the enzyme active site during the

catalysis [16,17]. Three reactions are successively per￾formed on the MEP framework, yielding three additional

intermediates of the MEP pathway: conversion of MEP 5

into 4-diphosphocytidyl-2-C-methyl-D-erythritol 6 [18,19],

phosphorylation of the C-2 hydroxyl group of 6 yielding 7

[20,21] and conversion of 7 into 2-C-methyl-D-erythritol

2,4-cyclodiphosphate 8 [22,23]. The two last steps of the

pathway were identified by a combination of genetic and

biochemical methods. An E . coli strain engineered for the

utilization of exogenous mevalonate accumulated tritium￾labelled 2-C-methyl-D-erythritol 2,4-cyclodiphosphate upon

incubation of [1-3

H]-2-C-methyl-D-erythritol and after

disruption of the gcpE gene, suggesting that 2-C-methyl￾D-erythritol 2,4-cyclodiphosphate 8 is the substrate of the

GcpE protein [24]. Incubation of [3-14C]-2-C-methyl￾D-erythritol 2,4-cyclodiphosphate 8 with a crude cell-free

system from an E . coli strain overexpressing gcpE resulted

in the formation of 4-hydroxy-3-methylbut-2-enyl diphos￾phate 9 [25,26]. Deletion of the lytB gene in a similarly

engineered E . coli strain, resulted in the accumulation of

the same diol diphosphate 9 [27]. In addition, feeding

with uniformly labelled [U-13C5]-1-deoxy-D-xylulose E . coli

strains overexpressing the gene of the xylulose kinase

(responsible for the phosphorylation of free 1-deoxy-D￾xylulose)as well as of all genes of the enzymes down￾stream of gcpE or lytB resulted in the accumulation of

uniformly labelled 4-hydroxy-3-methylbut-2-enyl diphos￾phate 9 or of isopentenyl diphosphate (IPP) 10 and

dimethylallyl diphosphate 11, respectively [28,29]. The

nature of the cofactors required for the conversion of

2-C-methyl-D-erythritol 2,4-cyclodiphosphate 8 into IPP 10

and dimethylallyl diphosphate 11 is still a matter of

investigation (Fig. 1).

This paper focuses on the two intriguing consecutive

steps catalyzed by the DXR from E . coli. Recently,

Correspondence to M. Rohmer, Universite´ Louis Pasteur/CNRS,

Institut Le Bel, 4 rue Blaise Pascal, 67070 Strasbourg Cedex, France.

Fax: +33 3 90241345, E-mail: [email protected]

Abbreviations: AHIR, acetohydroxy acid isomeroreductase;

H2-NADPH, dihydro-NADPH; DXP, 1-deoxy-D-xylulose

5-phosphate; DXR, 1-deoxy-D-xylulose 5-phosphate reducto￾isomerase; DXS, 1-deoxy-D-xylulose 5-phosphate synthase;

H-DXR, His-tagged DXR; IPP, isopentenyl diphosphate;

MEP, 2-C-methyl-D-erythritol 4-phosphate.

Enzymes: acetohydroxy acid isomeroreductase (EC 1.1.1.86),

1-deoxy-D-xylulose 5-phosphate reductoisomerase (EC 1.1.1.267),

1-deoxy-D-xylulose 5-phosphate synthase (EC 4.1.3.7),

NADP-dependent alcohol dehydrogenase (EC 1.1.1.2).

(Received 12 June 2002, accepted 24 July 2002)

Eur. J. Biochem. 269, 4446–4457 (2002)
FEBS 2002 doi:10.1046/j.1432-1033.2002.03150.x