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

Eur J Biochem 271, 2658–2669 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04194.x 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 Lehrstuhl fuăr Organische Chemie und Biochemie, Technische Universitaăt Muănchen, Garching, Germany; 2Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA, USA; 3Abteilung fu Strukturforschung, ăr Max-Planck-Institut fuăr Biochemie, Martinsried, Germany; 4Laboratorium fuăr Organische Chemie, Eidgenoăssische Technische Hochschule Zu ărich, 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 chromatography 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 lmolỈmg)1Ỉmin)1, respectively, as diagnosed by 1H 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–hydroxyacid oxidizing enzymes including flavocytochrome b2 and dehydrogenase and was shown to catalyse the formation of [2,3-13C2]lactate from [2,3-13C2]pyruvate, albeit at a low rate of nmolỈmg)1Ỉmin)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 Isoprenoids are one of the largest groups of natural products comprising more than 35 000 reported compounds [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 (retinoic acid, steroids), membrane fluidity modulation (steroids, hopanoids), predator repulsion and pollinator or mate attraction [1] Despite their enormous structural and functional complexity, 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 cardiovascular 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-diphosphate 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 fur ă Organische Chemie und Biochemie, Technische Universitat, ă 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) L-lactate Keywords: isoprenoids, mevalonate, deoxyxylulose, Idi-2, FMN Ó FEBS 2004 B subtilis type II IPP isomerase (Eur J Biochem 271) 2659 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¢-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 electrotransformed 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 Fig Biosynthesis of IPP and DMAPP 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 The recombinant E coli strain M15-pQEidi2 was grown in Luria-Bertani broth containing ampicillin (180 mgỈL)1) and kanamycin (50 mgỈL)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 concentration of mM, and the culture was incubated for h The cells were harvested by centrifugation, washed with 0.9% (w/v) sodium chloride, and stored at )20 °C under anaerobic 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 0.5 M sodium chloride and 20 mM 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 equilibrated with 100 mM Tris hydrochloride, pH 8.0, containing 0.5 M sodium chloride and 20 mM imidazole (flow rate, mLỈmin)1) The column was washed with 90 mL of 100 mM Tris hydrochloride, pH 8.0, containing 0.5 M sodium chloride and 20 mM imidazole and was then developed with a gradient of 20–500 mM imidazole in 150 mL of 100 mM 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 mM Tris hydrochloride, pH 8.0 and stored at )80 °C Experimental procedures Assay of IPP isomerase activity Materials IPP, DMAPP and [3,4,5-13C3]DMAPP were prepared by published procedures [30,31] [U-13C3]acetone and [2,3-13C2]pyruvate were obtained from Isotec (Miamisburg, Unless otherwise specified, assay mixtures contained 100 mM Tris hydrochloride, pH 8.0, 10 mM MgCl2, 10 lM FMN, mM sodium acetate, 10.8 mM DMAPP or IPP, and protein The mixtures were incubated at 37 °C under Ó FEBS 2004 2660 R Laupitz et al (Eur J Biochem 271) anaerobic 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 37 °C, and 13C 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 H and 13C NMR spectra were recorded with a DRX 500 AVANCE spectrometer from Bruker Instruments, Karlsruhe, 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 3.57c [37] and PHYLO_WIN [38] Phylogenetic trees were constructed by the Neighbor-joining method Dayhoff’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 PHYLIP 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 Experimental 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 chromatography 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 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-cyanohydroxycinnamic acid, 0.1% (v/v) trifluoracetic acid and 0.7 mg of IPP isomerase per mL X-ray structure analysis of the B subtilis enzyme in the presence of FMN indicated a D4 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 experimental 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 Bioinformatics Catalytic properties of Idi-2 protein from B subtilis 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 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, University of Geneva, Switzerland) Alignments were constructed using the program PILEUP (GCG, Madison, Wisconsin) Phylogenetic analyses of the aligned amino acid sequences were performed using the Phylogeny Interference Package The reaction catalysed by the recombinant enzyme could be monitored conveniently by NMR spectroscopy (Table 1) The 1H NMR and 13C NMR signals of IPP and DMAPP have been assigned previously on the basis of 1H13C and 13 13 C C correlation spectroscopy with 13C-labelled samples [22] The 1H NMR assignments of DMAPP shown in Table 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 1H NMR assignments are given in the text of [41], reversed assignments of the Mass spectrometry Ó FEBS 2004 B subtilis type II IPP isomerase (Eur J Biochem 271) 2661 Fig 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 Table NMR data of isopentenyl diphosphate and dimethylallyl diphosphate Chemical shifts (p.p.m.) Position Coupling constants (Hz) JHH JPH 6.6 6.7 6.6 Ha Isopentenyl diphosphate 4.10 2.43 4.88 1.80 Dimethylallyl diphosphate 4.49 5.48 (E-methyl) 1.79 (Z-methyl) 1.75 13 Ca 143.4 111.3 21.4 71, 41 71, 41, 6.6 7.1 139.7 24.7 17.0 JCCb 6.6 41, 42 42, 41, a Referenced to external trimethylsilylpropane sulfonate; b observed with [3,4,5-13C3]DMAPP and [2,3-13C3]IPP Fig 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 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 Ó FEBS 2004 2662 R Laupitz et al (Eur J Biochem 271) Fig 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 mM Tris hydrochloride, pH 8.0, 10 mM MgCl2, mM dithiothreitol, 2.5 mM NADPH, 10 lM FMN, mM or mM sodium acetate, and 10.8 mM IPP or DMAPP j, formation of DMAPP from IPP; s, formation of IPP from DMAPP Table Catalytic rates of Idi-2 protein under different conditions Reaction mixtures contained MgCl2 and were prepared as described under Experimental procedures Procedure/condition Fig 1H-NMR assay of type II IPP isomerase from B subtilis A, part of the 1H 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 1H 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 mM Tris hydrochloride, pH 8.0, 10 mM MgCl2, mM dithiothreitol, 2.5 mM NADPH, 10 lM FMN, mM sodium acetate, and 10.8 mM IPP and 10.8 mM DMAPP, respectively; *, internal standard (acetate) FMN, we observed the appearance of the signals of both methyl groups and of the methine group of the enzymatically 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 conversion of DMAPP into IPP The 1H NMR spectrum observed at equilibrium was virtually identical with that obtained in the experiment mentioned above (cf Figure 4A) Under the Conversion of Aerobica Anaerobic Conversion of Aerobica Anaerobic Conversion of Aerobic Conversion of Aerobicc Specific activity (lmolỈmin)1Ỉmg)1) IPP into DMAPP 0.63 ± 0.042b 0.62 ± 0.037b DMAPP into IPP 0.23 ± 0.007b 0.19 ± 0.093b 13 13 [3,4,5- C3]DMAPP into [3,4,5- C3]IPP 0.08 [2,3-13C2]pyruvate into [2,3-13C2]lactate 0.001 a Reaction mixtures contained NADPH and dithiothreitol; activities were calculated from rate constants (Fig 5); c reaction mixtures contained NADH b 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 h (Fig 5) The rates based on 1H 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 lmolỈmg)1Ỉmin)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 [3,4,5-13C3]DMAPP as Ó FEBS 2004 B subtilis type II IPP isomerase (Eur J Biochem 271) 2663 Table Activation of Idi-2 protein by divalent cations Reaction mixtures were prepared as described under Experimental Procedures Metal ions were added to a final concentration of 10 mM Metal ion 2+ Ca Mg2+ Mn2+ Zn2+ Ni2+ Cu2+ Co2+ Fig 13C NMR signals of DMAPP and IPP A [3,4,5-13C3]DMAPP; B, mixture of [3,4,5-13C3]DMAPP and [3,4,5-13C3]IPP obtained from [3,4,5-13C3]DMAPP by the catalytic action of Idi-2 protein of B subtilis under aerobic conditions Assay mixtures contained 100 mM Tris hydrochloride, pH 8.0, 10 mM MgCl2, mM dithiothreitol, 2.5 mM NADPH, 10 lM FMN, and 5.2 mM [3,4,5-13C3]DMAPP substrate The decrease of the 13C-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 of IPP, respectively (cf Table 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 conditions FMN, however, was required under aerobic as well as under anaerobic conditions The reaction rates were similar under aerobic and anaerobic conditions (Table 2) Photometric 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 previously for Streptomyces type II isomerase [28] Orthologs of Idi-2 protein An exhaustive BLAST search of 283 completed and unfinished 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 Relative Activity (%) 100 65 17 0.2 < 0.005 < 0.005 < 0.005 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%) carry an idi-2 gene, 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 Bacillales and Lactobacillales form a cluster which is separated from other lineages with statistical relevance (bootstrap value: 100%) Some actinobacteria (Streptomyces 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 2664 R Laupitz et al (Eur J Biochem 271) Ó FEBS 2004 Fig Consensus cladogram of Idi-2 proteins from various microorganisms The simplified tree (majority rule) was deduced by Neighborjoining 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 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 supported relationship In the archaeal kingdom, the Methanosarcinales 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 Cyanobacteria, 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 Spirochete Borrelia burgdorferi) Interestingly, the idi-2 gene of P luminescens, whose genome specifies the enzymes of the deoxyxylulose phosphate pathway together with both the Ó FEBS 2004 B subtilis type II IPP isomerase (Eur J Biochem 271) 2665 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 Actinobacteria 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, Salmonella and Shigella sp.) of the proteobacteria group 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) Phylogenetic pattern of Idi proteins Paralogs of Idi-2 proteins It is now in order to analyse the distribution of the two isomerase types in relation to the two isoprenoid biosynthetic 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 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, Mycoplasma penetrans and Spiroplasma kunkelii, the genomes of the members of the mollicutes group (total number, out of 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-hydroxyacid 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, corresponding 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-hydroxyacidoxidizing 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 Distribution of isoprenoid biosynthesis pathways and IPP isomerases in 283 completed and unfinished prokaryotic genomes MEV, mevalonate pathway genes; DXP, deoxyxylulose pathway genes 2666 R Laupitz et al (Eur J Biochem 271) Ó FEBS 2004 Fig Structural relationship of Idi-2 with a-hydroxyacid dehydrogenases A, structure based sequence alignment of glycolate oxidase (gox), flavocytochrome b2 (cy), S-mandelate dehydrogenase (md; Acc no P20932) and Idi-2 (idi) The sequence of L-lactate dehydrogenase 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 conserved in IDI proteins located close to FMN in green The similarity between the a-hydroxyacid dehydrogenase and Idi-2 proteins is most pronounced in the C-terminal half which harbours the standard phosphate binding site (SPB) B, stereo-view of the superposition of Idi-2 (N-terminal extension in yellow, TIMbarrel 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) Secondary 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 displays a bound active site inhibitor [4-carboxy5-(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 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 b2), 1p5b (S-mandelate dehydrogenase) and 1pno (Idi-2) GOX Idi-2 GOX FCb2 FCb2 MD 1.5, 201, 27.9% 1.3, 203, 27.1% 1.0, 303, 42.9% 1.5, 202, 24.3 1.0, 306, 41.2% 1.1, 307, 34.2% 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 prompted 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 nmolỈmg)1Ỉmin)1 by 13C NMR spectroscopy (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 dehydrogenase activity displayed by Idi-2 protein did not result from E coli wildtype background activities caused by protein impurities 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, 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 conditions, in the range of 0.6 lmolỈmg)1Ỉmin)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 Sulfolobus 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 recombinant 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 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 B subtilis type II IPP isomerase (Eur J Biochem 271) 2667 microorganisms have either retained Idi-1 or Idi-2, or of a lateral gene transfer similar to that reported for 3-hydroxy3-methylglutaryl coenzyme A reductase [55,56] It is interesting 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 Actinobacteria) observed here (cf Figure 7) may be explained by a loss of evolutionary constraints due to nonessential functions 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 isomerization 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 available 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 Gartner ă for skillful assistance, and Angelika Werner for expert help with the preparation of the manuscript References Sacchettini, J.C & Poulter, C.D (1997) Creating isoprenoid diversity Science 277, 1788–1789 Bach, T.J (1995) Some new aspects of isoprenoid biosynthesis in plants – a review Lipids 30, 191–202 Bloch, K (1992) Sterol molecule: structure, biosynthesis, and function Steroids 57, 378–382 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 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 Poulter, C.D & Rilling, H (1981) Conversion of farnesyl pyrophosphate to squalene In Biosynthesis of Isoprenoid Compounds (Porter, J.W & Spurgeon, S.L., eds), Vol 1, pp 162–209 John Wiley, New York Ó FEBS 2004 2668 R Laupitz et al (Eur J Biochem 271) Koyama, T & Ogura, K (1999) Isopentenyl diphosphate, isomerase and prenyltransferases In Comprehensive Natural Product Chemistry (Cane, D., ed.), Vol 2, pp 69–96 Pergamon, Oxford Slater, E.E & MacDonald, J.S (1988) Mechanism of action and biological profile of HMG CoA reductase inhibitors A new therapeutic alternative Drugs 36, 72–82 Stancu, C & Sima, A (2001) Statins: mechanism of action and effects J Cell Mol Med 5, 378–387 10 Schwarz, M & Arigoni, D (1999) Gingkolide biosynthesis In Comprehensive Natural Product Chemistry (Cane, D., ed.), Vol 2, pp 367–399 Pergamon, Oxford 11 Rohmer, M (1999) A mevalonate-independent route to isopentenyl diphosphate In Comprehensive Natural Product Chemistry (Cane, D., ed.), Vol 2, pp 45–68 Pergamon, Oxford 12 Eisenreich, W., Schwarz, M., Cartayrade, A., Arigoni, D., Zenk, M.H & Bacher, A (1998) The deoxyxylulose phosphate pathway of terpenoid biosynthesis in plants and microorganisms Chem Biol 5, R221–R233 13 Eisenreich, W., Rohdich, F & Bacher, A (2001) Deoxyxylulose phosphate pathway to terpenoids Trends Plant Sci 6, 78–84 14 Rohdich, F., Hecht, S., Bacher, A & Eisenreich, W (2003) Deoxyxylulose phosphate pathway of isoprenoid biosynthesis Discovery and function of ispDEFGH genes and their cognate enzymes Pure Appl Chem 75, 393–405 15 Takahashi, S., Kuzuyama, T., Watanabe, H & Seto, H (1998) A 1-deoxy-D-xylulose 5-phosphate reductoisomerase catalyzing the formation of 2-C-methyl-D-erythritol 4-phosphate in an alternative nonmevalonate pathway for terpenoid biosynthesis Proc Natl Acad Sci USA 95, 9879–9884 16 Rohdich, F., Wungsintaweekul, J., Fellermeier, M., Sagner, S., Herz, S., Kis, K., Eisenreich, W., Bacher, A & Zenk, M.H (1999) Cytidine 5¢-triphosphate-dependent biosynthesis of isoprenoids: YgbP protein of Escherichia coli catalyzes the formation of 4-diphosphocytidyl-2-C-methylerythritol Proc Natl Acad Sci USA 96, 11758–11763 17 Luttgen, H., Rohdich, F., Herz, S., Wungsintaweekul, J., Hecht, ă S., Schuhr, C.A., Fellermeier, M., Sagner, S., Zenk, M.H., Bacher, A & Eisenreich, W (2000) Biosynthesis of terpenoids: YchB protein of Escherichia coli phosphorylates the 2-hydroxy group of 4-diphosphocytidyl-2C-methyl-D-erythritol Proc Natl Acad Sci USA 97, 1062–1067 18 Herz, S., Wungsintaweekul, J., Schuhr, C.A., Hecht, S., Luttgen, ¨ H., Sagner, S., Fellermeier, M., Eisenreich, W., Zenk, M.H., Bacher, A & Rohdich, F (2000) Biosynthesis of terpenoids: YgbB protein converts 4-diphosphocytidyl-2C-methyl-D-erythritol 2-phosphate to 2C-methyl-D-erythritol 2,4-cyclodiphosphate Proc Natl Acad Sci USA 97, 2486–2490 19 Hecht, S., Eisenreich, W., Adam, P., Amslinger, S., Kis, K., Bacher, A., Arigoni, D & Rohdich, F (2001) Studies on the nonmevalonate pathway to terpenes: the role of the GcpE (IspG) protein Proc Natl Acad Sci USA 98, 14837–14842 20 Kollas, A.K., Duin, E.C., Eberl, M., Altincicek, B., Hintz, M., Reichenberg, A., Henschker, D., Henne, A., Steinbrecher, I., Ostrovsky, D.N., Hedderich, R., Beck, F., Jomaa, H & Wiesner, J (2002) Functional characterization of GcpE, an essential enzyme of the non-mevalonate pathway of isoprenoid biosynthesis FEBS Lett 532, 432–436 21 Wolff, M., Seemann, M., Grosdemange-Billiard, C., Tritsch, D., Campos, N., Rodriguez-Concepcion, M., Boronat, A & Rohmer, M (2002) Isoprenoid biosynthesis via the methylerythritol phosphate pathway (E)-4-Hydroxy-3-methylbut-2-enyl diphosphate: chemical synthesis and formation from methylerythritol cyclodiphosphate by a cell-free system from Escherichia coli Tetrahedron Lett 43, 2555–2559 22 Rohdich, F., Hecht, S., Gartner, K., Adam, P., Krieger, C., ¨ Amslinger, S., Arigoni, D., Bacher, A & Eisenreich, W (2002) 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 Studies on the nonmevalonate terpene biosynthetic pathway: metabolic role of IspH (LytB) protein Proc Natl Acad Sci USA 99, 1158–1163 Adam, P., Hecht, S., Eisenreich, W., Kaiser, J., Grawert, T., ă Arigoni, D., Bacher, A & Rohdich, F (2002) Biosynthesis of terpenes: studies on 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase Proc Natl Acad Sci USA 99, 12108– 12113 Altincicek, B., Duin, E.C., Reichenberg, A., Hedderich, R., Kollas, A.K., Hintz, M., Wagner, S., Wiesner, J., Beck, E & Jomaa, H (2002) LytB protein catalyzes the terminal step of the 2-C-methyl-D-erythritol-4-phosphate pathway of isoprenoid biosynthesis FEBS Lett 532, 437–440 Wolff, M., Seemann, M., Tse Sum Bui, B., Frapart, Y., Tritsch, D., Estrabot, A.G., Rodriguez-Concepcion, M., Boronat, A., Marquet, A & Rohmer, M (2002) Isoprenoid biosynthesis via the methylerythritol phosphate pathway: the (E)-4-hydroxy-3methylbut-2-enyl diphosphate reductase (LytB/IspH) from Escherichia coli is a [4Fe-4S] protein FEBS Lett 541, 115–120 Rohdich, F., Zepeck, F., Adam, P., Hecht, S., Kaiser, J., Laupitz, R., Grawert, T., Amslinger, S., Eisenreich, W., Bacher, A & ă Arigoni, D (2003) The deoxyxylulose phosphate pathway of isoprenoid biosynthesis: studies on the mechanisms of the reactions catalyzed by IspG and IspH protein Proc Natl Acad Sci USA 100, 1586–1591 Hahn, F.M., Hurlburt, A.P & Poulter, C.D (1999) Escherichia coli open reading frame 696 is idi, a nonessential gene encoding isopentenyl diphosphate isomerase J Bacteriol 181, 4499–4504 Kaneda, K., Kuzuyama, T., Takagi, M., Hayakawa, Y & Seto, H (2001) An unusual isopentenyl diphosphate isomerase found in the mevalonate pathway gene cluster from Streptomyces sp strain CL190 Proc Natl Acad Sci USA 98, 932–937 Steinbacher, S., Kaiser, J., Gerhardt, S., Eisenreich, W., Huber, R., Bacher, A & Rohdich, F (2003) Crystal structure of the type II isopentenyl diphosphate: dimethylallyl diphosphate isomerase from Bacillus subtilis J Mol Biol 329, 973–982 Davisson, V., Jo, Woodside, A.B., Neal, T.R., Stremler, K.E., Muhlbacher, M & Poulter, C.D (1986) Phosphorylation of isoă prenoid alcohols J Org Chem 51, 4768–4779 Christensen, D.J & Poulter, C.D (1994) Enzymatic synthesis of isotopically labeled isoprenoid diphosphates Bioorg Med Chem 2, 631–637 Bullock, W.O., Fernandez, J.M & Short, J.M (1987) XL1-Blue: a high efficiency plasmid transforming recA Escherichia coli strain with beta-galactosidase selection Biotechniques 5, 376–379 Zamenhof, P.J & Villarejo, M (1972) Constructions and properties of Escherichia coli strains exhibiting a-complementation of b-galactosidase fragments in vivo J Bacteriol 110, 171–178 Sanger, F., Nicklen, S & Coulson, A.R (1977) DNA sequencing with chain-terminating inhibitors Proc Acad Natl Sci USA 74, 5463–5468 Laue, T.M., Shah, B.D., Ridgeway, T.M & Pelletier, S.L (1992) Computer-aided interpretation of analytical sedimentation data for proteins In Analytical Ultracentrifugation in Biochemistry and Polymer Science (Harding, S.E., Rowe, A.J & Horton, J.C., eds), pp 90–125 Royal Society of Chemistry, Cambridge, UK Altschul, S.F., Madden, T.L., Schaer, A.A., Zhang, J., Zhang, ă Z., Miller, W & Lipman, J (1997) Gapped BLAST and PSIBLAST: a new geneeration of protein database search programs Nucleic Acids Res 25, 3389–3402 Felsenstein, J (1995) phylip (Phylogeny Interference Package), Version 3.57c (Distributed by the Author) Department of Genetics University of Washington, Seattle, MA Ó FEBS 2004 38 Galtier, N., Gouy, M & Gautier, C (1996) SEAVIEW and PHYLO_WIN: two graphic tools for sequence alignment and molecular phylogeny Comput Applic Biosci 12, 543–548 39 Dayhoff, M.O (1979) Atlas of Protein Sequence and Structure Vol5 (Suppl 3) National Biomedical Research Foundation, Washington, D.C 40 Felsenstein, J (1985) Confidence limits on phylogenies: an approach using the bootstrap Evolution 39, 783–791 41 Street, I.P., Christensen, D.J & Poulter, C.D (1990) Hydrogen exchange during the enzyme-catalyzed isomerization of isopentenyl diphosphate and dimethylallyl diphosphate J Am Chem Soc 112, 8577–8578 42 Lederer, F (1991) Flavocytochrom b2 In Chemistry and Biochemistry of the Flavoenzymes (Muller, F., ed.), Vol 2, ă pp 153242 CRC Press, Boca Raton, FL 43 Belmouden, A., Le, K.H., Lederer, F & Garchon, H.J (1993) Molecular cloning and nucleotide sequence of cDNA encoding rat kidney long-chain L-2-hydroxyacid oxidase Expression of the catalytically active recombinant protein as a chimaera Eur J Biochem 214, 17–25 44 Lindqvist, Y & Branden, C.I., (1989) The active site of spinach glycolate oxidase J Biol Chem 264, 3624–3628 45 Garvie, E.I (1980) Bacterial lactate dehydrogenases Microbiol Rev 44, 106–139 46 Sukumar, N., Xu, Y., Gatti, D.L., Mitra, B & Mathews, F.S (2001) Structure of an active soluble mutant of the membraneassociated (S)-mandelate dehydrogenase Biochemistry 40, 9870– 9878 47 Whitby, F.G., Luecke, H., Kuhn, P., Somoza, J.R., Huete-Perez, J.A., Phillips, J.D., Fletterick, R.J & Wang, C.C (1997) Crystal structure of Tritrichomonas foetus inosine-5¢-monophosphate dehydrogenase and the enzyme-product complex Biochemistry 36, 10666–10674 48 Stenberg, K & Lindqvist, Y (1997) Three-dimensional structures of glycolate oxidase with bound active-site inhibitors Protein Sci 6, 1009–1015 49 Tegoni, M & Cambillau, C (1994) The 2.6-A refined structure of the Escherichia coli recombinant Saccharomyces cerevisiae flavocytochrome b2-sulfite complex Protein Sci 3, 303–313 50 Sukumar, N., Dewanti, A.R., Mitra, B & Mathews, F.S (2004) High resolution structures of an oxidized and reduced flavoprotein: The water switch in a soluble form of (S)-mandelate dehydrogenase J Biol Chem 279, 3749–3757 B subtilis type II IPP isomerase (Eur J Biochem 271) 2669 51 Wouters, J., Oudjama, Y., Barkley, S.J., Tricot, C., Stalon, V., Droogmans, L & Poulter, C.D (2003) Catalytic mechanism of Escherichia coli isopentenyl diphosphate isomerase involves Cys67, Glu-116, and Tyr-104 as suggested by crystal structures of complexes with transition state analogues and irreversible inhibitors J Biol Chem 278, 11903–11908 52 Takagi, M., Kaneda, K., Shimizu, T., Hayakawa, Y., Seto, H & Kuzuyama, T (2004) Bacillus subtilis ypgA gene is fni, a nonessential gene encoding type isopentenyl diphosphate isomerase Biosci Biotechnol Biochem 68, 132–137 53 Barkley, S.J., Cornish, R.M & Poulter, C.D (2004) Identification of an archaeal type II isopentenyl diphosphate isomerase in Methanothermobacter thermoautotrophicus J Bacteriol 186, 1811–1817 54 Yamashita, S., Hemmi, H., Ikeda, Y., Nakayama, T & Nishino, T (2004) Type isopentenyl diphosphate isomerase from a thermoacidiphilic archaeon Sulfolobus shibita Eur J Biochem 271, 1087–1093 55 Boucher, Y & Doolittle, W.F (2000) The role of lateral gene transfer in the evolution of isoprenoid biosynthesis pathways Mol Microbiol 37, 703–716 56 Boucher, Y., Huber, H., L’Haridon, S., Stetter, K.O & Doolittle, W.F (2001) Bacterial origin for the isoprenoid biosynthesis enzyme HMG-CoA reductase of the archaeal Thermoplasmatales and Archaeoglobales Mol Biol Evol 18, 1378–1386 57 Weigel, L.M., Clewell, D.B., Gill, S.R., Clark, N.C., McDougal, L.K., Flannagan, S.E., Kolonay, J.F., Shetty, J., Killgore, G.E & Tenover, F.C (2003) Genetic analysis of a high-level vancomycinresistant isolate of Staphylococcus aureus Science 302, 1569– 1571 58 Kuzmic, P (1996) Program DYNAFIT for the analysis of enzyme kinetic data: application to HIV proteinase Anal Biochem 237, 260–273 Supplementary material The following material is available from http://www.black wellpublishing.com/products/journals/suppmat/EJB/EJB4194/ EJB4194sm.htm Table S1 Isoprenoid biosynthesis in completed and unfinished prokaryotic genomes  ... 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... 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... acetate, and 10.8 mM IPP and 10.8 mM DMAPP, respectively; *, internal standard (acetate) FMN, we observed the appearance of the signals of both methyl groups and of the methine group of the enzymatically