Tài liệu Báo cáo khoa học: Biochemical characterization of Bacillus subtilis type II isopentenyl

Biochemical characterization of Bacillus subtilis type II isopentenyl

diphosphate isomerase, and phylogenetic distribution of isoprenoid

biosynthesis pathways

Ralf Laupitz1

, Stefan Hecht1

, Sabine Amslinger1

, Ferdinand Zepeck1

, Johannes Kaiser1

, Gerald Richter1

,

Nicholas Schramek1

, Stefan Steinbacher2

, Robert Huber3

, Duilio Arigoni4

, Adelbert Bacher1

,

Wolfgang Eisenreich1 and Felix Rohdich1

1

Lehrstuhl fu¨r Organische Chemie und Biochemie, Technische Universita¨t Mu¨nchen, Garching, Germany; 2

Division of Chemistry

and Chemical Engineering, California Institute of Technology, Pasadena, CA, USA; 3

Abteilung fu¨r Strukturforschung,

Max-Planck-Institut fu¨r Biochemie, Martinsried, Germany; 4

Laboratorium fu¨r Organische Chemie, Eidgeno¨ssische Technische

Hochschule Zu¨rich, Switzerland

An open reading frame (Acc. no. P50740) on the Bacillus

subtilis chromosome extending from bp 184 997–186 043

with similarity to the idi-2 gene of Streptomyces sp. CL190

specifying type II isopentenyl diphosphate isomerase was

expressed in a recombinant Escherichia coli strain. The

recombinant protein with a subunit mass of 39 kDa was

purified to apparent homogeneity by column chromatog￾raphy. The protein was shown to catalyse the conversion of

dimethylallyl diphosphate into isopentenyl diphosphate and

vice versa at rates of 0.23 and 0.63 lmolÆmg)1

Æmin)1

,

respectively, as diagnosed by 1

H spectroscopy. FMN and

divalent cations are required for catalytic activity; the highest

rates were found with Ca2+. NADPH is required under

aerobic but not under anaerobic assay conditions. The

enzyme is related to a widespread family of (S)-a–hydroxy￾acid oxidizing enzymes including flavocytochrome b2 and

L-lactate dehydrogenase and was shown to catalyse the

formation of [2,3-13C2]lactate from [2,3-13C2]pyruvate, albeit

at a low rate of 1 nmolÆmg)1

Æmin)1

. Putative genes specifying

type II isopentenyl diphosphate isomerases were found in the

genomes of Archaea and of certain eubacteria but not in

the genomes of fungi, animals and plants. The analysis of the

occurrence of idi-1 and idi-2 genes in conjunction with the

mevalonate and nonmevalonate pathway in 283 completed

and unfinished prokaryotic genomes revealed 10 different

classes. Type II isomerase is essential in some important

human pathogens including Staphylococcus aureus and

Enterococcus faecalis where it may represent a novel target

for anti-infective therapy.

Keywords: isoprenoids, mevalonate, deoxyxylulose, Idi-2,

FMN.

Isoprenoids are one of the largest groups of natural

products comprising more than 35 000 reported com￾pounds [1]. Numerous representatives of the terpenoid

family have important physiological functions such as light

perception (retinal), light protection (carotenoids), energy

transduction (retinal, chlorophyll), signal transduction (ret￾inoic acid, steroids), membrane fluidity modulation (ster￾oids, hopanoids), predator repulsion and pollinator or mate

attraction [1].

Despite their enormous structural and functional com￾plexity, all terpenoids are assembled from two simple

precursors, isopentenyl diphosphate (IPP) and dimethylallyl

diphosphate (DMAPP) (Fig. 1). The biosynthesis of these

universal terpene precursors via the mevalonate pathway

has been studied in considerable detail in yeast and animals.

These classical studies established the formation of IPP

from three acetate moieties via mevalonate (reviewed in

[2–5]). IPP is then converted into DMAPP by an isopentenyl

diphosphate isomerase which is essential in all organisms

using the mevalonate pathway (reviewed in [6,7]).

The elucidation of the mevalonate pathway culminated in

the development of the statin type drugs which inhibit

3-hydroxy-3-methylglutaryl-CoA reductase and reduce car￾diovascular morbidity and mortality by reduction of blood

cholesterol levels and probably also by down-regulation

of inflammatory processes [8,9]. Certain statins such as

Lipitor
and Zocor
are record holders with regard to

current drug sales.

A second isoprenoid biosynthesis pathway starting with

1-deoxy-D-xylulose 5-phosphate has been discovered in the

last decade (reviewed in [10–14]). The linear carbohydrate

precursor is transformed into a branched polyol derivative,

2C-methyl-D-erythritol 4-phosphate [15] which is further

converted into 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphos￾phate by the consecutive action of enzymes specified by

the ispCDEFG genes (Fig. 1) [16–21]. The reduction of

Correspondence to F. Rohdich and W. Eisenreich, Lehrstuhl fu¨r

Organische Chemie und Biochemie, Technische Universita¨t,

Lichtenbergstr. 4, D-85747 Garching, Germany.

Fax: + 49 89 289 13363, Tel.: + 49 89 289 13364 and

+49 89 289 13336, E-mail: [email protected] and

[email protected]

Abbreviations: DMAPP, dimethylallyl diphosphate; IPP, isopentenyl

diphosphate.

(Received 26 March 2004, revised 27 April 2004,

accepted 30 April 2004)

Eur. J. Biochem. 271, 2658–2669 (2004) FEBS 2004 doi:10.1111/j.1432-1033.2004.04194.x