Tài liệu Báo cáo khoa học: Biochemical characterization of Bacillus subtilis type II isopentenyl diphosphate isomerase, and phylogenetic distribution of isoprenoid biosynthesis pathways doc

Biochemical characterization of
Bacillus subtilis
type II isopentenyl
diphosphate isomerase, and phylogenetic distribution of isoprenoid
biosynthesis pathways
Ralf Laupitz
1
, Stefan Hecht
1
, Sabine Amslinger
1
, Ferdinand Zepeck
1
, Johannes Kaiser
1
, Gerald Richter
1
,
Nicholas Schramek
1
, Stefan Steinbacher
2
, Robert Huber
3
, Duilio Arigoni
4
, Adelbert Bacher
1
,
Wolfgang Eisenreich
1

and Felix Rohdich
1
1
Lehrstuhl fu
¨
r Organische Chemie und Biochemie, Technische Universita
¨
tMu
¨
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 Ca
2+
. 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 b
2
and
L
-lactate dehydrogenase and was shown to catalyse the
formation of [2,3-
13
C

2
]lactate from [2,3-
13
C
2
]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
LipitorÒ and ZocorÒ 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: and

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) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04194.x
1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate catalysed
by IspH protein affords both IPP and DMAPP [22–26].
Accordingly, the participation of an IPP isomerase is in
principle not required in this pathway. Nevertheless,
numerous prokaryotes endowed with these genes display
IPP isomerases which may act as salvage enzymes in order
to adjust the ratio of DMAPP and IPP to the specific
requirements of the downstream terpenoid metabolism [27].
A recently discovered IPP isomerase (designated type II)
from Streptomyces sp. CL190 [28] is devoid of sequence
similarity to the previously known IPP isomerases of yeast
and animal origin which are now designated type I.

Whereas type I isomerases only require divalent cations
for catalytic activity, the type II isomerase of Streptomyces
sp. CL190 has been reported to require FMN and NADPH
as well as divalent metals [28]. The structure of a type II IPP
isomerase from Bacillus subtilis has been elucidated by
X-ray crystallography [29]. This paper reports on the
biochemical properties of the recombinant enzyme from
B. subtilis. Phylogenetic patterns of IPP isomerases in the
archaeal and eubacterial kingdoms with respect to the two
IPP/DMAPP biosynthesis pathways were analysed by
bioinformatic methods.
Experimental procedures
Materials
IPP, DMAPP and [3,4,5-
13
C
3
]DMAPP were prepared
by published procedures [30,31]. [U-
13
C
3
]acetone and
[2,3-
13
C
2
]pyruvate were obtained from Isotec (Miamisburg,
OH, USA). Restriction enzymes were purchased from New
England Biolabs (Frankfurt, Germany). Oligonucleotides

were custom synthesized by MWG Biotech (Ebersberg,
Germany). NADPH and NADH were purchased from
Biomol (Hamburg, Germany). FMN was obtained from
Sigma (Steinheim, Germany).
Cloning and expression of the
idi-2
gene from
B. subtilis
A DNA segment extending from bp position 184 997–
186 043 of the B. subtilis chromosome was amplified by
PCR using chromosomal B. subtilis DNA as template and
the oligonucleotides 5¢-TTGGTG
GGATCCGTGACTCG
AGCAGAACGAAAAAGAC-3¢ and 5¢-GGCTTT
GTCG
ACTTATCGCACACTATAGCTTGATG-3¢ as primers
(restriction sites are underlined and start- and stop-codons
are in bold type). The amplificate was purified, treated with
the restriction enzymes BamHI and SalI, and ligated into
the His-tag-encoding expression vector pQE30 (Qiagen,
Hilden, Germany) which had been treated with the same
enzymes. The resulting plasmid pQEidi2 was electrotrans-
formed into Escherichia coli strains XL1-Blue (Stratagene
[32]) and M15 (pREP4) [33] affording the recombinant
strains XL1-pQEidi2 and M15-pQEidi2.
Preparation of the recombinant Idi-2 protein
The recombinant E. coli strain M15-pQEidi2 was grown in
Luria-Bertani broth containing ampicillin (180 mgÆL
)1
)and

kanamycin (50 mgÆL
)1
). Cultures were incubated at 37 °C
with shaking. At an optical density of 0.7 (600 nm),
isopropyl thio-b-
D
-galactoside was added to a final concen-
tration of 2 m
M
, and the culture was incubated for 5 h. The
cells were harvested by centrifugation, washed with 0.9%
(w/v) sodium chloride, and stored at )20 °C under anaer-
obic conditions.
The following steps were carried out under anaerobic
conditions. Frozen cell mass (4 g) was thawed in 38 mL of
100 m
M
Tris hydrochloride, pH 8.0, containing 0.5
M
sodium chloride and 20 m
M
imidazole hydrochloride. The
suspension was passed through a French press and was then
centrifuged. To the supernatant (60 mL), 40 mL of water
were added, and the mixture was applied to a column
of Ni-chelating Sepharose FF (column volume, 11 mL;
Amersham Pharmacia Biotech) which had been equili-
brated with 100 m
M
Tris hydrochloride, pH 8.0, containing

0.5
M
sodium chloride and 20 m
M
imidazole (flow rate,
2mLÆmin
)1
). The column was washed with 90 mL of
100 m
M
Tris hydrochloride, pH 8.0, containing 0.5
M
sodium chloride and 20 m
M
imidazole and was then
developed with a gradient of 20–500 m
M
imidazole in
150 mL of 100 m
M
Tris hydrochloride, pH 8.0, containing
0.5
M
sodium chloride. Fractions were combined (retention
volume of Idi-2 protein, 20 mL), dialyzed overnight against
100 m
M
Tris hydrochloride, pH 8.0 and stored at )80 °C.
Assay of IPP isomerase activity
Unless otherwise specified, assay mixtures contained

100 m
M
Tris hydrochloride, pH 8.0, 10 m
M
MgCl
2
,10l
M
FMN, 2 m
M
sodium acetate, 10.8 m
M
DMAPP or IPP, and
protein. The mixtures were incubated at 37 °C under
Fig. 1. Biosynthesis of IPP and DMAPP.
Ó FEBS 2004 B. subtilis type II IPP isomerase (Eur. J. Biochem. 271) 2659
anaerobic conditions. The reaction was terminated by the
addition of EDTA to a final concentration of 26 m
M
.After
the addition of D
2
O to a final concentration of 10% (v/v),
the samples were analysed by NMR spectroscopy.
Assay of lactate dehydrogenase activity
Assay mixtures containing 100 m
M
Tris hydrochloride,
pH 8.0, 17 m
M

NADH, 1.6 m
M
[2,3-
13
C
2
]pyruvate, 10%
(v/v) D
2
O and 2.0 mg of Idi-2 protein (but without added
FMN) in a total volume of 0.7 mL were incubated at 37 °C,
and
13
C NMR spectra were recorded at intervals.
Sequence determination
DNA was sequenced by the automated dideoxynucleotide
method using a 377 Prism sequencer from Perkin Elmer,
Norwalk, USA [34]. N-terminal peptide sequences were
obtained by Pulsed-Liquid Mode using a PE Biosystems
Model 492 (Perkin Elmer, Weiterstadt, Germany).
NMR spectroscopy
1
Hand
13
C NMR spectra were recorded with a DRX 500
AVANCE spectrometer from Bruker Instruments, Karls-
ruhe, Germany.
Analytical ultracentrifugation
Hydrodynamic studies were performed with an analytical
ultracentrifuge Optima XL-I (Beckman Instruments, Palo

Alto, CA) equipped with ultraviolet and interference
optics. Experiments were performed with double sector
cells equipped with aluminum centerpieces and sapphire
windows. Partial specific volumes and buffer densities
were estimated according to published procedures [35].
Samples contained 100 m
M
Tris hydrochloride, pH 8.0.
Mass spectrometry
Mass spectra were recorded with a Biflex III MALDI-TOF
mass spectrometer from Bruker Instruments, Karlsruhe,
Germany. Samples contained 25 m
M
Tris hydrochloride,
pH 8.0, 33% (v/v) CH
3
CN, saturated a-cyanohydroxycin-
namic acid, 0.1% (v/v) trifluoracetic acid and 0.7 mg of IPP
isomerase per mL.
Bioinformatics
Similarity searches in the GenBank database of completed
and unfinished prokaryotic genomes (among them not yet
specifically assigned genomes) (.
gov) were performed with the programs
BLASTP
and
TBLASTN
using the gapped
BLAST
and

PSI
-
BLAST
algorithms
[36]. Nucleic acid sequences of unfinished genomes were
downloaded from the GenBank database, and open reading
frames were identified and translated into amino acid
sequences with the program
PCGENE
(IntelliGenetics, Uni-
versity of Geneva, Switzerland). Alignments were construc-
ted using the program
PILEUP
(GCG, Madison, Wisconsin).
Phylogenetic analyses of the aligned amino acid sequences
were performed using the Phylogeny Interference Package
PHYLIP
3.57c [37] and
PHYLO
_
WIN
[38]. Phylogenetic trees
were constructed by the Neighbor-joining method. Dayh-
off’s PAM 001 matrix was used to calculate the distances
between pairs of protein sequences [39]. A bootstrapping
analysis using 1000 iterations was performed [40]. Only
groups with bootstrap probablity values >50% were
retained.
Results
Cloning and expression of the

idi-2
gene from
B. subtilis
An open reading frame (Acc. no. P50740) extending from
bp position 184 997–186 043 on the B. subtilis chromosome
with similarity to the idi-2 gene of Streptomyces sp. CL190
[28] (37% identical amino acid residues; Fig. 2A) was
amplified by PCR and was cloned into the plasmid pQE30
affording the recombinant plasmid pQEidi2 (see Experi-
mental procedures). An E. coli strain carrying this plasmid
produced copious amounts of a 39 kDa protein as judged
by SDS electrophoresis (Fig. 3).
The recombinant protein was purified by affinity chro-
matography on Nickel-chelating sepharose and appeared
homogeneous as judged by SDS/PAGE (Fig. 3). Partial
N-terminal Edman degradation afforded the amino acid
sequence MRGSHHHHHHGSVTRAE in agreement with
the sequence of the recombinant gene. MALDI-TOF mass
spectrometry showed a relative mass of 38 463 Da in good
agreement with the calculated mass of 38 455 Da (data not
shown).
Hydrodynamic studies on Idi-2 protein of
B. subtilis
X-ray structure analysis of the B. subtilis enzyme in the
presence of FMN indicated a D
4
symmetric homooctamer
structure with a relative mass of 309 kDa [29]. In order to
check for the potential influence of substrates and cofactors
on the quaternary structure of the enzyme, we performed

boundary sedimentation experiments under different experi-
mental conditions. In the absence of substrates and
cofactors, the enzyme sedimented as a single, symmetrical
boundary with an apparent sedimentation coefficient of
10.0 S which is well in line with the published octamer
structure [29]. In the aerobic assay mixture, however, the
enzyme sediments with an apparent rate of 4.0 S which
indicates dissociation under substrate turnover conditions.
Catalytic properties of Idi-2 protein from
B. subtilis
The reaction catalysed by the recombinant enzyme could be
monitored conveniently by NMR spectroscopy (Table 1).
The
1
HNMRand
13
C NMR signals of IPP and DMAPP
have been assigned previously on the basis of
1
H
13
Cand
13
C
13
C correlation spectroscopy with
13
C-labelled samples
[22]. The
1

H NMR assignments of DMAPP shown in
Table 1 were confirmed by two-dimensional NOESY
experiments indicating strong NOE interactions between
the methyl signal at 1.79 p.p.m. (E-methyl group) and the
signal at 5.47 p.p.m. (methine group). It should be noted
that some confusion with respect to these assignments reigns
in the literature. Whereas the correct
1
H NMR assignments
are given in the text of [41], reversed assignments of the
2660 R. Laupitz et al. (Eur. J. Biochem. 271) Ó FEBS 2004
methyl signals are given in footnote 26 of that paper and in
[28]. When the enzyme was incubated with IPP as substrate
under aerobic conditions in the presence of NADPH and
Fig. 2. Amino acid residues essential for
functionality of Idi-2 protein. (A) Amino acid
sequence comparison of Idi-2 proteins.
Sequences included in this analysis were
B. subtilis Idi-2 protein and Idi-2 proteins
from major human pathogens. Residues
absolutely conserved in all Idi-2 amino acid
sequences available in the GenBank database
are labelled by open triangles. Residues
involved in FMN binding (as found in the
crystallographic structure of the B. subtilis
protein, see below) are shown by filled
triangles. (B) Stereo representation of the
FMN-binding site of B. subtilis Idi-2 protein.
The disordered regions between Met256 and
Phe263 and Tyr211 and Arg226, respectively,

are indicated by dotted lines. The latter region
is expected to cover FMN. Conserved residues
are shown in blue.
Fig. 3. SDS/PAGE. (A) Molecular mass markers; (B) cell extract of
recombinant E. coli M15-pQEidi2 hyperexpressing the idi-2 gene from
B. subtilis; C, recombinant Idi-2 protein of B. subtilis after nickel
chelating affinity chromatography.
Table 1. NMR data of isopentenyl diphosphate and dimethylallyl
diphosphate.
Position
Chemical shifts
(p.p.m.)
Coupling
constants (Hz)
1
H
a13
C
a
J
HH
J
PH
J
CC
b
Isopentenyl diphosphate
1 4.10 6.6 6.6
2 2.43 6.7
3 143.4 71, 41

4 4.88 111.3 71, 3
5 1.80 21.4 41, 3
Dimethylallyl diphosphate
1 4.49 6.6 6.6
2 5.48 7.1
3 139.7 41, 42
4(E-methyl) 1.79 24.7 42, 4
5(Z-methyl) 1.75 17.0 41, 4
a
Referenced to external trimethylsilylpropane sulfonate;
b
observed
with [3,4,5-
13
C
3
]DMAPP and [2,3-
13
C
3
]IPP.
Ó FEBS 2004 B. subtilis type II IPP isomerase (Eur. J. Biochem. 271) 2661
FMN, we observed the appearance of the signals of both
methyl groups and of the methine group of the enzymat-
ically formed DMAPP (Fig. 4A). Concomitantly, the
signals of IPP were progressively diminished. Using acetate
as an internal standard, the signal integrals afforded the
concentrations of IPP and DMAPP as a function of time
(Fig. 5).
Figure 4B illustrates the reverse reaction, i.e. the conver-

sion of DMAPP into IPP. The
1
H NMR spectrum observed
at equilibrium was virtually identical with that obtained in
the experiment mentioned above (cf. Figure 4A). Under the
experimental conditions described (10.8 m
M
IPP or
DMAPP, respectively, and 0.2 mg of enzyme per mL), the
conversion of IPP into DMAPP and vice versa approached
a state of equilibrium after a reaction period of about 4 h
(Fig. 5).
The rates based on
1
H NMR analysis for the conversion
of IPP into DMAPP and vice versa with Mg
2+
as cofactor
were 0.63 ± 0.042 and 0.23 ± 0.007 lmolÆmg
)1
Æmin
)1
,
respectively (Table 2). These values agree with the activities
of the E. coli Idi-1 protein reported earlier [27]. Their ratio is
similar to the equilibrium constant for the reversible
reaction reported earlier [41].
The isomerization reaction could also be monitored by
13
C NMR spectroscopy using [3,4,5-

13
C
3
]DMAPP as
Fig. 4.
1
H-NMR assay of type II IPP isomerase from B. subtilis.
A, part of the
1
H NMR spectrum of the reaction mixture (lower lane)
obtained from IPP (
1
H NMR signals, see upper lane) by the catalytic
action of Idi-2 protein under aerobic conditions. B, part of the
1
H
NMR spectrum of the reaction mixture (lower lane) obtained from
DMAPP (
1
H NMR signals, upper lane) by the catalytic action of Idi-2
protein under aerobic conditions. Assay mixtures contained 100 m
M
Tris hydrochloride, pH 8.0, 10 m
M
MgCl
2
,1m
M
dithiothreitol,
2.5 m

M
NADPH, 10 l
M
FMN, 2 m
M
sodium acetate, and 10.8 m
M
IPP and 10.8 m
M
DMAPP, respectively; *, internal standard (acetate).
Fig. 5. Catalytic rates of the reversible conversion of IPP into DMAPP
catalyzed by Idi-2 protein from B. subtilis under aerobic conditions.
Numerical simulations were performed using the DYNAFIT software
[58]. Assay mixtures contained 100 m
M
Tris hydrochloride, pH 8.0,
10 m
M
MgCl
2
,1m
M
dithiothreitol, 2.5 m
M
NADPH, 10 l
M
FMN,
2m
M
or 3 m

M
sodium acetate, and 10.8 m
M
IPP or DMAPP.
j, formation of DMAPP from IPP; s, formation of IPP from
DMAPP.
Table 2. Catalytic rates of Idi-2 protein under different conditions.
Reaction mixtures contained MgCl
2
and were prepared as described
under Experimental procedures.
Procedure/condition
Specific activity
(lmolÆmin
)1
Æmg
)1
)
Conversion of IPP into DMAPP
Aerobic
a
0.63 ± 0.042
b
Anaerobic 0.62 ± 0.037
b
Conversion of DMAPP into IPP
Aerobic
a
0.23 ± 0.007
b

Anaerobic 0.19 ± 0.093
b
Conversion of [3,4,5-
13
C
3
]DMAPP into [3,4,5-
13
C
3
]IPP
Aerobic 0.08
Conversion of [2,3-
13
C
2
]pyruvate into [2,3-
13
C
2
]lactate
Aerobic
c
0.001
a
Reaction mixtures contained NADPH and dithiothreitol;
b
activities were calculated from rate constants (Fig. 5);
c
reaction

mixtures contained NADH.
2662 R. Laupitz et al. (Eur. J. Biochem. 271) Ó FEBS 2004
substrate. The decrease of the
13
C-coupled signals of the
Z-andE-methyl groups resonating at 17.0 and
24.7 p.p.m., respectively, as well as that of the quaternary
carbon atom resonating at 139.7 p.p.m. was accompanied
by the appearance of three new
13
C-coupled signals at
143.4, 111.3 and 21.4 p.p.m. assigned as the carbon atoms
3, 4, and 5 of IPP, respectively (cf. Table 1 and Fig. 6).
Within the limits of experimental accuracy the catalytic
rates determined with this assay were the same as those
described above.
Whereas NADPH or NADH was required for catalytic
activity under aerobic conditions, the reaction could
proceed without NADPH under anaerobic conditions using
enzyme which had been purified under anaerobic condi-
tions. FMN, however, was required under aerobic as well as
under anaerobic conditions. The reaction rates were similar
under aerobic and anaerobic conditions (Table 2). Photo-
metric analysis gave no evidence for reduction of FMN in
aerobic or anaerobic assay mixtures (data not shown). The
recombinant enzyme has an absolute requirement for a
divalent metal ion for catalytic activity; the highest rates
were found with Ca
2+
(Table 3). A different order for the

catalytic activation by such ions has been reported previ-
ously for Streptomyces type II isomerase [28].
Orthologs of Idi-2 protein
An exhaustive
BLAST
search of 283 completed and unfin-
ished prokaryotic genomes in the GenBank database
recovered 91 genes specifying proteins with close similarity
to Idi-2 protein of B. subtilis. This set characterized by an
expect value < 2e-27 is proposed to comprise all type II
isomerases in the set of 283 prokaryotic genomes analysed
for reasons that will become obvious in the following
paragraphs. Additionally to this set, 10 orthologous
sequence entries of microrganism were found in GenBank
whose genomes were not available in their entirety. All 102
putative Idi-2 orthologs of microrganisms studied here
share a significant degree of sequence similarity. Their
lengths range from 330 to 360 amino acid residues
(Fig. 2A). Twenty-one amino acid residues are absolutely
conserved (marked by triangles in Fig. 2). Notably, all
amino acid residues shown by the X-ray structure to be
involved in the binding of the FMN cofactor [29] are
absolutely conserved (Fig. 2A,B). These residues (marked
by filled triangles in Fig. 2A) are located in four different
segments of the peptide chain. Specifically, the residues
Gly66, Gly258, Gly259 contact the phosphate moiety of
FMN via hydrogen bonds (Fig. 2B). The isoalloxazine ring
is coordinated by residues Thr64 (N5), Ser93 (O4), Asn122
(N3) and Lys184 (N1, O2). The amino acid residues His147,
Asn149, Gln152 and Glu153 (marked by open triangles in

Fig. 2) in the direct neighborhood of the FMN binding site
are also absolutely conserved (Fig. 2B).
Type II isomerases are restricted to the archaeal and
eubacterial kingdoms. With the exception of Halobacterium
sp. NRC-1, Mycobacterium marinum and Photorhabdus
luminescens featuring both a putative idi-1 and a putative
idi-2 gene, the distribution of type I and type II isomerases in
the prokaryotic kingdom appears to be mutually exclusive.
Genes specifying type II isomerases were found in 19 of 20
(95%) archaebacterial species. Nanoarchaeum equitans is
devoid of IPP biosynthesis as well as of idi genes. In the
group of 263 eubacterial genomes studied, 35 (13%) carry
an idi-1 gene,72(27%)carryanidi-2 gene, 2 carry both an
idi-1 and idi-2 gene, and 154 (59%) appear to be devoid of
IPP isomerases.
Phylogenetic analyses of 102 type II isomerases were
performed as described under Experimental procedures.
The final consensus phylogenetic tree (majority rule) shows
the major phylogenetic grouping of 76 type II isomerases in
the archaeal and eubacterial kingdoms as illustrated in
Fig. 7. Bacillales and Lactobacillales form a cluster which is
separated from other lineages with statistical relevance
(bootstrap value: 100%). Some actinobacteria (Streptomy-
ces sp. CL190, Kitasatospora griseola and Actinoplanes sp.
A40644) group also within this cluster. The separation of
the archaeabacterial from the eubacterial kingdom was not
found to be statistically relevant (bootstrap values < 50%).
In the eubacterial kingdom, Cyanobacteria with the
exception of Crocosphaera watsonii and Synechocystis sp.
PCC6803 (which group together with the sulfur bacterium

Chloroflexus aurantiacus; bootstrap value of 67), some
actinobacteria (Mycobacterium avium, Mycobacterium
Fig. 6.
13
C NMR signals of DMAPP and IPP. A [3,4,5-
13
C
3
]DMAPP;
B, mixture of [3,4,5-
13
C
3
]DMAPP and [3,4,5-
13
C
3
]IPP obtained from
[3,4,5-
13
C
3
]DMAPP by the catalytic action of Idi-2 protein of B. subtilis
under aerobic conditions. Assay mixtures contained 100 m
M
Tris
hydrochloride, pH 8.0, 10 m
M
MgCl
2

,1m
M
dithiothreitol, 2.5 m
M
NADPH, 10 l
M
FMN, and 5.2 m
M
[3,4,5-
13
C
3
]DMAPP.
Table 3. Activation of Idi-2 protein by divalent cations. Reaction mix-
tures were prepared as described under Experimental Procedures.
Metal ions were added to a final concentration of 10 m
M
.
Metal ion Relative Activity (%)
Ca
2+
100
Mg
2+
65
Mn
2+
17
Zn
2+

0.2
Ni
2+
< 0.005
Cu
2+
< 0.005
Co
2+
< 0.005
Ó FEBS 2004 B. subtilis type II IPP isomerase (Eur. J. Biochem. 271) 2663
marinum and Mycobacterium smegmatis) together with the
Deinococcus-Thermus group, and some proteobacteria
form separate lineages (bootstrap values of 95, 100 and
79%, respectively). The remaining orders among the
eubacterial kingdom did not reveal any statistically suppor-
ted relationship. In the archaeal kingdom, the Methanos-
arcinales together with the Archaeoglobales (bootstrap
value: 69%) and the Thermococcales together with the
Methanobacteriales (bootstrap value: 77%) are grouped
into clusters.
The presently available data suggest that Cyano-
bacteria, Bacillales and Lactobacillales with the exception
of B. halodurans, Geobacillus stearothermophilus and
Pasteuria nishizawae use exclusively type II isomerase
(Table S1). Type II isomerases are also found in the
Actinobacteria group (M. avium, M. marinum and
M. smegmatis), and in the a-subgroup of proteobacteria
(Mesorhizobium loti and Rickettsia spp.). Only very few
representatives from other bacteria groups possess idi-2

genes (Dichelobacter nodosus, Legionella pneumophila and
P. luminescens,allthreec-proteobacteria; and the Spiro-
chete Borrelia burgdorferi). Interestingly, the idi-2 gene of
P. luminescens, whose genome specifies the enzymes of the
deoxyxylulose phosphate pathway together with both the
Fig. 7. Consensus cladogram of Idi-2 proteins
from various microorganisms. The simplified
tree (majority rule) was deduced by Neighbor-
joining analysis based on the alignment of the
amino acid sequences of 76 Idi-2 proteins.
Gaps were removed from the alignment, and
the total number of positions taking into
account was 320. The numbers at the nodes
are the statistical confidence estimates
computed by the bootstrap procedure. Only
groups with bootstrap probablity values
>50% were retained. The bar represents 0.137
PAM distance.
2664 R. Laupitz et al. (Eur. J. Biochem. 271) Ó FEBS 2004
idi-1 as well as the idi-2 genes, is interrupted by a
counterclockwise located transposase gene effectively
knocking it out.
Type I isomerases are found preferably in the Actino-
bacteria group (Corynebacterium sp., Mycobacterium sp.
and Streptomyces sp.), but also in the Bacteroidetes group
(Cytophaga hutchinsonii),andsomeinthea-subgroup
(Rhodobacter sphaeroides and Silicibacter pomeroyi)andin
the c-subgroup (Coxiella burnetii, Erwinia carotovora,
Escherichia sp., Klebsiella pneumoniae, P. luminescens,
Salmonella and Shigella sp.) of the proteobacteria group.

Phylogenetic pattern of Idi proteins
It is now in order to analyse the distribution of the two
isomerase types in relation to the two isoprenoid biosyn-
thetic pathways, i.e. the classical mevalonate pathway and
the more recently discovered nonmevalonate pathway via
deoxyxylulose phosphate (Fig. 1). Of the 16 possible
combinations, 10 were actually found in the group of 283
completed and unfinished sequenced prokaryotic genomes
(Fig. 8). Among 204 prokaryotic genomes studied using
exclusively the deoxyxylulose phosphate pathway, 147 carry
no idi gene, 35 carry idi-1 genes, 20 carry idi-2 genes and 2
carry both an idi-1 and idi-2 gene. The majority of
prokaryotes using exclusively the mevalonate pathway
(total number, 64) possess type II isomerases (total number,
62 including several important human pathogens) (Fig. 8).
As an exception, the genomes of Listeria monocytogenes and
Listeria innocua carry complete sets of genes for both
pathways in conjunction with type II isomerases. The
combination of mevalonate pathway genes together with
type I isomerase is found exclusively in the genome of the
eubacterium Coxiella burnetii. The genomes of obligate
intracellular parasitic Rickettsia spp. (total number, 4) are
devoid of an isoprenoid pathway, but carry idi-2 genes.
With the exception of Mycoplasma gallisepticum R, Myco-
plasma penetrans and Spiroplasma kunkelii, the genomes of
the members of the mollicutes group (total number, 7 out of
10) are devoid of genes for the biosynthesis of IPP and
DMAPP. The same is true for the genome of the archaeon
N. equitans. No putative orthologs of the type II isomerase
were found in any eukaryotic species including plants, fungi

and animals. On the other hand, all completely sequenced
eukaryotic genomes comprise putative orthologs of the type
I isomerase. Highest degrees of similarity were found to
Idi-1 proteins of the c-proteobacteria A. vinelandii and
P. luminescens and to Idi-1 protein of the Bacteroid
C. hutchinsonii (expect values 3e-22, 5e-19 and 8e-20,
respectively).
Paralogs of Idi-2 proteins
Database searches conducted with the
BLASTP
program
retrieved a considerable number of proteins with
substantially lower similarity (Expect value >1e-10) to the
B. subtilis Idi-2 protein which was used as search motif.
Notably, type II isomerase shows weak, but significant
similarity with a family of FMN dependent (S)-a-hydroxy-
acid dehydrogenases (pfam database accession no.
PF01070) including flavocytochrome b
2
from yeast
[EC 1.1.2.3 (FCb2)] [42], long chain hydroxyacid oxidase
from mammals [43], glycolate oxidase from spinach
[EC 1.1.3.15 (GOX)] [44],
L
-lactate dehydrogenases from
bacteria (EC 1.1.1.27) [45] (S)-mandelate dehydrogenase
from P. putida (MD) [46] and inosine 5¢-monophosphate
dehydrogenases (EC 1.1.1.205) [47] over the entire length of
their respective sequences (Fig. 9A) (expect values 0.002,
1.1, 0.083, 2e-06, 0.47 and 0.001, respectively).

The members of this enzyme family display a TIM-barrel
fold. Superposition of Idi-2 with the known structures of
GOX [48], FCb2 [49] and MD [50] demonstrates that Idi-2
protein shares a very similar three-dimensional fold in the
C-terminal half of the protein but with significant deviations
in the N-terminal half (Fig. 9B). This finding is reflected by
rms deviations of 1.3–1.5 A
˚
for only about 200 matching
residues out of 349 when comparing Idi-2 to GOX and
FCb2. The rms deviation is only 1.0 A
˚
for about 300
matching residues when comparing GOX with FCb2 or MD.
In addition, the fraction of identical residues for the
significantly lower number of matching residues is below
30% in the first case compared to above 40% in the second
(Table 4).
In addition to a conserved sequence motif
SNHG[AG]RQ [PROSITE database (asy.
org/prosite)], GOX, FCb2 and MD share a number of
conserved active site residues (Tyr24, Tyr129, Arg124,
His254 and Arg257, corresponding to the amino acid
sequence of GOX) (Fig. 9A). Both the sequence motif and
these active site residues are missing in Idi-2. On the other
hand, Idi-2 proteins encompass a set of conserved amino
acid residues (His147, Asn149, Gln152 and Glu153, corres-
ponding to the amino acid sequence of the B. subtilis
protein) in the direct vicinity of the FMN binding site which
is not present in the other members of the family.

Thus, under structural aspects, Idi-2 appears as a fairly
distant relative of the FMN-dependent a-hydroxyacid-
oxidizing enzymes. However, the sequence similarity in
conjunction with the TIM barrel fold and the conserved
FMN binding site leave no doubt about the evolutionary
relatedness of Idi-2 with the dehydrogenase superfamily.
Fig. 8. Distribution of isoprenoid biosynthesis pathways and IPP
isomerases in 283 completed and unfinished prokaryotic genomes. MEV,
mevalonate pathway genes; DXP, deoxyxylulose pathway genes.
Ó FEBS 2004 B. subtilis type II IPP isomerase (Eur. J. Biochem. 271) 2665
L
-Lactate dehydrogenase activity of Idi-2 protein
from
B. subtilis
Partial sequence similarity of the Idi-2 gene and the
paralogous lldD gene specifying
L
-lactate dehydrogenase
together with similarities in the TIM-barrel fold and
FMN binding site of the two respective proteins promp-
ted a search for the presence of a residual redox activity
in type II isomerase. In order to obtain maximum
sensitivity in combination with maximum selectivity, we
used [2,3-
13
C
2
]pyruvate as substrate. Using NADH as
cofactor, we observed the formation of [2,3-
13

C
2
]lactate
at a rate of 1 nmolÆmg
)1
Æmin
)1
by
13
C NMR spectro-
scopy (Table 2). The addition of FMN did not increase
the catalytic activity. A protein sample prepared from an
E. coli strain harbouring the expression vector without
insert and eluted from the Nickel affinity column with
the same volume as compared to the recombinant Idi-2
protein did not show any lactate dehydrogenase activity.
This result clearly indicated that the lactate dehydroge-
nase activity displayed by Idi-2 protein did not result
from E. coli wildtype background activities caused by
protein impurities.
Fig. 9. Structural relationship of Idi-2 with
a-hydroxyacid dehydrogenases. A, structure
based sequence alignment of glycolate oxidase
(gox), flavocytochrome b
2
(cy), S-mandelate
dehydrogenase (md; Acc. no. P20932) and
Idi-2 (idi). The sequence of
L
-lactate dehydro-

genase was added based on a sequence
alignment to glycolate oxidase. Secondary
structures and sequence numbers refer to gox
(top lines) and idi (bottom lines), respectively.
The eight ba modules of the TIM-barrel
domainarecoloredyellowandnamedS1toS8
and H1 to H8, respectively. Active site residues
of the a-hydroxyacid dehydrogenase family are
shown in red, the characteristic
NHG[GA]RQL-motif is boxed (note that it is
not conserved in IDI proteins). FMN-binding
residues are coloured blue and residues con-
served in IDI proteins located close to FMN in
green. The similarity between the a-hydroxy-
acid dehydrogenase and Idi-2 proteins is most
pronounced in the C-terminal half which har-
bours the standard phosphate binding site
(SPB). B, stereo-view of the superposition of
Idi-2 (N-terminal extension in yellow, TIM-
barrel in grey, C-terminal extension in green)
and glycolate dehydrogenase (N-terminal
extensionindarkblue,TIM-barrelinlight
blue, C-terminal extension in purple). Secon-
dary structure elements of the TIM-barrel
superimpose very well, especially for modules
b7/a7andb8/a8 which harbor the standard
phosphate binding site (SPB). Deviations are
found for the N- and C-terminal extensions.
FMNisdepictedinorange(Idi-2)andgreen
(GOX). In addition, the GOX structure dis-

plays a bound active site inhibitor [4-carboxy-
5-(1-pentenyl)hexylsulfanyl-1,2,3-triazole
(TACA)] in pink. His254 and Arg257 of the
signature motif NHG[AG]RQL of GOX are
depicted as ball and stick.
Table 4. Structure superimposion of Idi-2 protein with (S)-a hydroxy-
acid dehydrogenases. Rmsd in A
˚
, # of matching residues, % identity for
matching residues. The structures have been superimposed with
TOP3D using the PDB entries 1al8 (glycolate dehydrogenase), 1ltd
(flavocytochrome b
2
), 1p5b (S-mandelate dehydrogenase) and 1pno
(Idi-2).
GOX FCb2 MD
Idi-2 1.5, 201, 27.9% 1.3, 203, 27.1% 1.5, 202, 24.3
GOX 1.0, 303, 42.9% 1.0, 306, 41.2%
FCb2 1.1, 307, 34.2%
2666 R. Laupitz et al. (Eur. J. Biochem. 271) Ó FEBS 2004
Discussion
The type I IPP isomerases which have been known for a
long time require only divalent metal cations for activity
[6,7]. On the other hand, the type II isomerases of
Streptomyces sp. CL190 and Staphylococcus aureus have
been reported to require FMN and NADPH in addition to
divalent metal cations for activity under aerobic as well as
under anaerobic conditions [28]. Under aerobic conditions,
thetypeIIIPPisomeraseofB. subtilis requires NADPH
as well as FMN for activity in close similarity with the

Streptomyces enzyme. However, when the enzyme is
purified and assayed under anaerobic conditions, NADPH
is not required. The catalytic activities observed with IPP as
substrate are similar under aerobic and anaerobic condi-
tions, in the range of 0.6 lmolÆmg
)1
Æmin
)1
. No evidence was
obtained for redox cycling of FMN. The amino acid
residues involved in the FMN binding site are absolutely
conserved throughout a large number of orthologs
(Figs 2,9). This suggests an essential role for FMN despite
the low affinity of the enzyme for that cofactor and the
apparent absence of a redox process as part of the catalytic
cycle. The substrate binding site of the type II enzyme
remains veiled. However, a patch of absolutely conserved
amino acid residues comprising the polar amino acid
residues H147, N149, Q152 and E153 in close proximity
to the FMN binding site suggests that the substrates could
be bound in close proximity to the isoalloxazine moiety. In
the absence of direct evidence for the involvement of a redox
process it is tempting to postulate that the cofactor might act
as a dipole stabilizing a cationic intermediate or transition
state of the reaction. A similar role has been postulated for
tryptophan 121 in the case of E. coli Idi-1 [51].
During the preparation of this manuscript three groups
reported the catalytic properties of recombinant type II IPP
isomerases from B. subtilis [52]andthetwoArchaea
Methanothermobacter thermoautothrophicus [53] and Sulf-

olobus shibatae [54]. These enzymes were found to be
dependent on NADPH and Mg
2+
as cofactors. Anaerobic
conditions were not tested in these studies. In contrast to
these findings [52–54], the catalytic activity of the recom-
binant enzyme studied here was maximal with Ca
2+
.In
addition, we show for the first time, that, under anaerobic
conditions, the enzyme did not require NADPH. However,
unchanging of NADPH-dependency was claimed earlier for
the respective enzymes of Streptomyces sp. CL190 and
Staphylococcus aureus under anaerobic conditions [28].
Idi-2 protein is clearly a member of a superfamily of (S)-
a-hydroxyacid dehydrogenases, and the coenzyme pattern
of Idi-2 protein, where the roles of FMN and of NADPH
required under aerobic conditions are at present not
understood, may ultimately find an explanation by the
evolutionary relationship with oxidoreductases.
Numerically, bacteria using the deoxyxylulose phosphate
pathway without any isomerase (147 out of 283) and
bacteria using the mevalonate pathway in conjunction with
type II isomerase (62 out of 283) constitute the largest sets
within the prokaroytic kingdom (Fig. 8). The combination
of the deoxyxylulose phosphate pathway with type I
isomerase (35 out of 263) and with type II isomerase (20
out of 263) occur with lower frequency. This situation could
be the result of a differential gene loss, in which some
microorganisms have either retained Idi-1 or Idi-2, or of a

lateral gene transfer similar to that reported for 3-hydroxy-
3-methylglutaryl coenzyme A reductase [55,56]. It is inter-
esting in this context that both types of isomerases are found
in the Actinobacteria group. The anomalous positions for
some eubacterial species (e.g. Cyanobacteria and Actino-
bacteria) observed here (cf. Figure 7) may be explained by a
loss of evolutionary constraints due to nonessential func-
tions of Idi-2 proteins in bacteria using the deoxyxylulose
phosphate pathway.
With regard to the complex distribution of the two
different terpenoid pathways and of the two different
isomerase types in the eubacterial kingdom, it is relevant to
emphasize that certain highly pathogenic Gram-positive
cocci including Enterococcus and Staphylococcus species use
type II isomerases in conjunction with the mevalonate
pathway which has an absolute requirement for isomeriza-
tion of IPP in order to generate DMAPP. Hence, the type II
isomerase is an essential enzyme in this group of human
pathogens.
Enterococci and Staphylococci have a dramatic history of
resistance development against virtually all currently avail-
able antibiotics. Most notably, many strains are multidrug
resistant, and the rapidly spreading resistance against
vancomycin and methicillin constitutes a life-threatening
problem in affected patients [57]. Clearly, there is an urgent
requirement for novel therapeutic strategies directed at
these microorganisms. As the human type I IPP isomerase
and the type II isomerase of the microorganisms mentioned
have no detectable similarity, it should be possible to
develop inhibitors for the bacterial enzyme which have little

or no significant cross-inhibitory activity for the human
enzyme.
Acknowledgements
We thank the Deutsche Forschungsgemeinschaft, the Fonds der
Chemischen Industrie, and the Hans Fischer Gesellschaft for support.
Financial support by Novartis International AG, Basel (to D. A.) is
gratefully acknowledged. We thank Fritz Wendling and Katrin Ga
¨
rtner
for skillful assistance, and Angelika Werner for expert help with the
preparation of the manuscript.
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Supplementary material
The following material is available from ck
wellpublishing.com/products/journals/suppmat/EJB/EJB4194/
EJB4194sm.htm
Table S1. Isoprenoid biosynthesis in completed and unfin-
ished prokaryotic genomes.
Ó FEBS 2004 B. subtilis type II IPP isomerase (Eur. J. Biochem. 271) 2669