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 differe
Trang 1Biochemical 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 Eisenreich1and Felix Rohdich1
1
Lehrstuhl fu¨r Organische Chemie und Biochemie, Technische Universita¨t Mu¨nchen, Garching, Germany;2Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA, USA;3Abteilung fu¨r Strukturforschung,
Max-Planck-Institut fu¨r Biochemie, Martinsried, Germany;4Laboratorium 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)1Æmin)1,
respectively, as diagnosed by1H 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)1Æmin)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 faecaliswhere 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: felix.rohdich@ch.tum.de and
wolfgang.eisenreich@ch.tum.de
Abbreviations: DMAPP, dimethylallyl diphosphate; IPP, isopentenyl
diphosphate.
(Received 26 March 2004, revised 27 April 2004,
accepted 30 April 2004)
Trang 21-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 [3,4,5-13C3]DMAPP were prepared
by published procedures [30,31] [U-13C3]acetone and
[2,3-13C]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 theidi-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¢-TTGGTGGGATCCGTGACTCG AGCAGAACGAAAAAGAC-3¢ and 5¢-GGCTTTGTCG 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 37C 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 mM, 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 mM Tris hydrochloride, pH 8.0, containing 0.5M sodium chloride and 20 mMimidazole 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 mMTris hydrochloride, pH 8.0, containing 0.5M sodium chloride and 20 mM imidazole (flow rate,
2 mLÆmin)1) The column was washed with 90 mL of
100 mM Tris hydrochloride, pH 8.0, containing 0.5M sodium chloride and 20 mM imidazole and was then developed with a gradient of 20–500 mM imidazole in
150 mL of 100 mMTris hydrochloride, pH 8.0, containing 0.5Msodium chloride Fractions were combined (retention volume of Idi-2 protein, 20 mL), dialyzed overnight against
100 mMTris hydrochloride, pH 8.0 and stored at)80 C Assay of IPP isomerase activity
Unless otherwise specified, assay mixtures contained
100 mMTris hydrochloride, pH 8.0, 10 mMMgCl2, 10 lM FMN, 2 mMsodium acetate, 10.8 mMDMAPP or IPP, and protein The mixtures were incubated at 37C under
Fig 1 Biosynthesis of IPP and DMAPP.
Trang 3anaerobic conditions The reaction was terminated by the
addition of EDTA to a final concentration of 26 mM After
the addition of D2O to a final concentration of 10% (v/v),
the samples were analysed by NMR spectroscopy
Assay of lactate dehydrogenase activity
Assay mixtures containing 100 mM Tris hydrochloride,
pH 8.0, 17 mM NADH, 1.6 mM [2,3-13C2]pyruvate, 10%
(v/v) D2O and 2.0 mg of Idi-2 protein (but without added
FMN) in a total volume of 0.7 mL were incubated at 37C,
and13C 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
1H and13C 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 mM 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 mM Tris hydrochloride,
pH 8.0, 33% (v/v) CH3CN, 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) (http://www.ncbi.nlm.nih
gov) were performed with the programs BLASTP and
TBLASTNusing the gappedBLASTandPSI-BLASTalgorithms
[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 programPCGENE(IntelliGenetics,
Uni-versity of Geneva, Switzerland) Alignments were
construc-ted using the programPILEUP(GCG, Madison, Wisconsin)
Phylogenetic analyses of the aligned amino acid sequences
were performed using the Phylogeny Interference Package
PHYLIP3.57c [37] andPHYLO_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 theidi-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 ofB subtilis X-ray structure analysis of the B subtilis enzyme in the presence of FMN indicated a D4symmetric 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 fromB subtilis The reaction catalysed by the recombinant enzyme could be monitored conveniently by NMR spectroscopy (Table 1) The1H NMR and13C NMR signals of IPP and DMAPP have been assigned previously on the basis of1H13C and
13C13C correlation spectroscopy with13C-labelled samples [22] The 1H 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 correct1H NMR assignments are given in the text of [41], reversed assignments of the
Trang 4methyl 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 a 13 C a J HH J PH J CCb
Isopentenyl diphosphate
Dimethylallyl diphosphate
a
Referenced to external trimethylsilylpropane sulfonate;bobserved with [3,4,5-13C 3 ]DMAPP and [2,3-13C 3 ]IPP.
Trang 5FMN, 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 The1H 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 mM 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 on1H NMR analysis for the conversion
of IPP into DMAPP and vice versa with Mg2+as cofactor were 0.63 ± 0.042 and 0.23 ± 0.007 lmolÆmg)1Æmin)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
13C NMR spectroscopy using [3,4,5-13C]DMAPP as
Fig 4.1H-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 (1H 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 (1H 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 , 1 m 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 , 1 m M dithiothreitol, 2.5 m M NADPH, 10 l M FMN,
2 m 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)1Æmg)1) Conversion of IPP into DMAPP
Conversion of DMAPP into IPP
Conversion of [3,4,5-13C 3 ]DMAPP into [3,4,5-13C 3 ]IPP
Conversion of [2,3- 13 C 2 ]pyruvate into [2,3- 13 C 2 ]lactate
a
Reaction mixtures contained NADPH and dithiothreitol;
b activities were calculated from rate constants (Fig 5); c reaction mixtures contained NADH.
Trang 6substrate The decrease of the13C-coupled signals of the
Z- and E-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 13C-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 Ca2+(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 exhaustiveBLASTsearch 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 luminescensfeaturing both a putative idi-1 and a putative idi-2gene, 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%) carry an idi-2 gene, 2 carry both an idi-1and 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-cessp 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 6.13C NMR signals of DMAPP and IPP A [3,4,5-13C 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 , 1 m 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
Trang 7marinumand 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, all three c-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.
Trang 8idi-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), and some in the a-subgroup
(Rhodobacter sphaeroides and Silicibacter pomeroyi) and in
the c-subgroup (Coxiella burnetii, Erwinia carotovora,
Escherichia sp., Klebsiella pneumoniae, P luminescens,
Salmonellaand 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 penetransand 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 b2 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 (http://www.expasy 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.
Trang 9L-Lactate dehydrogenase activity of Idi-2 protein
fromB subtilis
Partial sequence similarity of the Idi-2 gene and the
paralogous lldD gene specifying -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 [2,3-13C2]pyruvate as substrate Using NADH as cofactor, we observed the formation of [2,3-13C2]lactate
at a rate of 1 nmolÆmg)1Æmin)1 by 13C 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 relationshipof 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 domain are colored yellow and named S1 to S8 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 extension in dark blue, TIM-barrel in light blue, C-terminal extension in purple) Secon-dary structure elements of the TIM-barrel superimpose very well, especially for modules b7/a7 and b8/a8 which harbor the standard phosphate binding site (SPB) Deviations are found for the N- and C-terminal extensions FMN is depicted in orange (Idi-2) and green (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).
Idi-2 1.5, 201, 27.9% 1.3, 203, 27.1% 1.5, 202, 24.3
Trang 10The 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,
the type II IPP isomerase of B 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)1Æmin)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] and the two Archaea
Methanothermobacter thermoautothrophicus[53] and
Sulf-olobus shibatae [54] These enzymes were found to be
dependent on NADPH and Mg2+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 Ca2+ 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 aureusunder 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
Enterococciand 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.
References
1 Sacchettini, J.C & Poulter, C.D (1997) Creating isoprenoid diversity Science 277, 1788–1789.
2 Bach, T.J (1995) Some new aspects of isoprenoid biosynthesis in plants –a review Lipids 30, 191–202.
3 Bloch, K (1992) Sterol molecule: structure, biosynthesis, and function Steroids 57, 378–382.
4 Bochar, D.A., Friesen, J.A., Stauffacher, C.V & Rodwell, V.W (1999) Biosynthesis of mevalonic acid from acteyl-CoA.
In Comprehensive Natural Product Chemistry (Cane, D., ed.), Vol 2, pp 15–44 Pergamon, Oxford.
5 Qureshi, N & Porter, J.W (1981) Conversion of acetyl-coenzyme
A to isopentyl pyrophosphate In Biosynthesis of Isoprenoid Compounds (Porter, J.W & Spurgeon, S.L., eds), Vol 1, pp 47–
94 John Wiley, New York.
6 Poulter, C.D & Rilling, H (1981) Conversion of farnesyl pyro-phosphate to squalene In Biosynthesis of Isoprenoid Compounds (Porter, J.W & Spurgeon, S.L., eds), Vol 1, pp 162–209 John Wiley, New York.