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Journal of Bacteriology, March 2004, p. 1811-1817, Vol. 186, No. 6
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.6.1811-1817.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Identification of an Archaeal Type II Isopentenyl Diphosphate Isomerase in Methanothermobacter thermautotrophicus

Sam J. Barkley, Rita M. Cornish, and C. Dale Poulter*

Department of Chemistry, University of Utah, Salt Lake City, Utah 84112

Received 19 June 2003/ Accepted 1 October 2003


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Isopentenyl diphosphate (IPP):dimethylallyl diphosphate isomerase catalyzes the interconversion of the fundamental five-carbon homoallylic and allylic diphosphate building blocks required for biosynthesis of isoprenoid compounds. Two different isomerases have been reported. The type I enzyme, first characterized in the late 1950s, is widely distributed in eukaryota and eubacteria. The type II enzyme was recently discovered in Streptomyces sp. strain CL190. Open reading frame 48 (ORF48) in the archaeon Methanothermobacter thermautotrophicus encodes a putative type II IPP isomerase. A plasmid-encoded copy of the ORF complemented IPP isomerase activity in vivo in Salmonella enterica serovar Typhimurium strain RMC29, which contains chromosomal knockouts in the genes for type I IPP isomerase (idi) and 1-deoxy-D-xylulose 5-phosphate (dxs). The dxs gene was interrupted with a synthetic operon containing the Saccharomyces cerevisiae genes erg8, erg12, and erg19 allowing for the conversion of mevalonic acid to IPP by the mevalonate pathway. His6-tagged M. thermautotrophicus type II IPP isomerase was produced in Escherichia coli and purified by Ni2+ chromatography. The purified protein was characterized by matrix-assisted laser desorption ionization mass spectrometry. The enzyme has optimal activity at 70°C and pH 6.5. NADPH, flavin mononucleotide, and Mg2+ are required cofactors. The steady-state kinetic constants for the archaeal type II IPP isomerase from M. thermautotrophicus are as follows: Km, 64 µM; specific activity, 0.476 µmol mg-1 min-1; and kcat, 1.6 s-1.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The isoprenoid biosynthetic pathway is ubiquitous across all three kingdoms of life. Over 36,000 natural products derived from the fundamental five-carbon isoprenoid building blocks isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) have now been identified (3). Many of these molecules have important biological functions, including glycoprotein synthesis (dolichols), inter- and intracellular signaling (prenylated proteins and steroidal hormones), membrane structure (steroids), electron carriers during redox reactions (ubiquinones), photoprotection (carotenoids), photosynthesis (chlorophyll), and defense against predators (sesquiterpenes and pyrethrins).

There are two independent biosynthetic routes to IPP and DMAPP (Fig. 1). In the mevalonate (MVA) pathway, first discovered in the late 1950s (4, 6), IPP is synthesized from mevalonic acid by the consecutive action of mevalonate kinase (MVA kinase), phosphomevalonate kinase (PMVA kinase), and mevalonate diphosphate decarboxylase (DPMVA decarboxylase). The isomerization of IPP to DMAPP is a mandatory step needed to create the electrophilic allylic diphosphates needed for subsequent prenyl transfer reactions. IPP isomerase is an essential enzyme in archaea, eukaryota, and some gram-positive eubacteria, where IPP is synthesized by the MVA pathway. More recently, it was discovered that IPP and DMAPP are synthesized from D-glyceraldehyde phosphate and pyruvate by the methyl erythritol phosphate (MEP) pathway found in many eubacteria, cyanobacteria, and plant chloroplasts (18, 28). In the final step of the MEP pathway, the ispH gene product synthesizes IPP and DMAPP from 4-hydroxydimethylallyl diphosphate (Fig. 1) (26). In organisms that utilize the MEP pathway, idi is not essential or does not exist (10).



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  		FIG. 1. MVA (left) and MEP (right) biosynthetic pathways to IPP and DMAPP. Abbreviations: AcCoA, acetyl CoA; AcAcCoA, acetoacetyl-CoA; HMGCoA, 3-hydroxy-3-methylglutaryl-CoA; MVAP, phosphomeualonate; MVAPP, diphosphomenalonate; DXP, deoxyxylulose phosphate; GP, glyceraldehyde phosphate; MEP, methylerythritol phosphate; CDP-MEP, cytidine methylerythritol diphosphate; COP-MEPP, cytidine phosphomethylerythritol diphosphate; cMEPP, cyclomethylerythritol diphosphate; HDMAPP, hydroxydimethylallyl diphosphate.

 
In 1997, Methanothermobacter thermautotrophicus was the first archaeon to have its genome fully sequenced (34). At that time there were no annotations for an open reading frame (ORF) similar to the idi genes previously identified in eukaryota and eubacteria, although Zhang and Poulter had reported activity for IPP isomerase in cell extracts from M. thermautotrophicus (47). Recently, Kaneda et al. identified an unclassified ORF in the MVA pathway gene cluster of Streptomyces sp. strain CL190 that encoded a new form of IPP isomerase (16). All members of the domain Archaea sequenced so far have ORFs for the type II enzyme. In addition, the type II isomerase is found in many pathogenic gram-positive eubacteria, cyanobacteria, and obligate parasitic eubacteria. The type I and type II isomerases have different structures and different cofactor requirements, suggesting that they catalyze isomerizations by different chemical mechanisms. We now report that an idi deletion in a mevalonate-dependent strain of Salmonella enterica serovar Typhimurium is complemented by the putative idi gene from M. thermautotrophicus and biochemical characterization of the encoded type II IPP isomerase.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials. Genomic DNA for M. thermautotrophicus was isolated from frozen cells, provided by Lacy Daniels (University of Iowa), using a genomic DNA isolation kit from Qiagen. [1-14C]isopentenyl diphosphate was purchased from Amersham. L-Arabinose, flavin mononucleotide (FMN), reduced form of NADP (NADPH), and mevalonic acid were purchased from Sigma. Imidazole was purchased from Acros Organics. Ni-nitrilotriacetic acid (NTA) agarose resin was purchased from Qiagen. All restriction endonucleases, Klenow fragment, and T7 DNA polymerase were purchased from New England Biolabs. Taq DNA polymerase was purchased from Promega. Deoxynucleoside triphosphates and T4 DNA ligase were purchased from Pharmacia. pBADA Myc/His and Escherichia coli BL21 DE3/pLysE were purchased from Invitrogen. S. enterica serovar Typhimurium shuttle strain TR6579 was provided by J. R. Roth (University of Utah, Salt Lake City, Utah). Oligonucleotide primers were synthesized by the Protein/DNA Core Facility of the Utah Regional Cancer Center. 2-C-Methyl-D-erythritol was synthesized by the procedure of Duvold et al. (7)

General methods. Minipreparations of plasmid DNA for restriction analysis were obtained by using a Qiagen plasmid miniprep kit. DNA fragments were purified in agarose gels (Bio-Rad) using a gel purification kit from Qiagen. Restriction digestion, ligation, and transformation of competent E. coli and S. enterica serovar Typhimurium cells were conducted as described by Sambrook et al. (29) PCR was performed using the polymerase mix provided in the Advantage PCR kit from Clontech. Radioactivity was measured in Cytoscint scintillation fluid (ICN Biomedicals). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed using the discontinuous buffer system of Laemmli (19). The gels were stained with Coomassie brilliant blue R from Sigma. In-gel tryptic digestion was performed by the procedure of Hellman et al. (13). DNA was sequenced at the Health Sciences Center Sequencing Facility, Eccles Institute of Human Genetics, University of Utah.

Bacterial strains and growth conditions. All S. enterica serovar Typhimurium strains are derived from S. enterica serovar Typhimurium strain LT2. A high-frequency transducing mutant of phage P22 (HT105 int) was used to mediate all transductional crosses (31). Procedures for propagation of phage and transductional crosses have been described previously (2). The bacterial strains used in this study are listed in Table 1 and were grown at 37°C in Luria-Bertani (LB) medium supplemented with the following antibiotics and concentrations as necessary: ampicillin (AMP), 50 µg/ml; chloramphenicol (CAM), 34 µg/ml; and tetracycline (TET), 25 µg/ml. When needed, 0.2% L-arabinose (AR), 5 mM mevalonic acid (MEV), 0.3 mg of 2-C-methyl-D-erythritol (ME) per ml, and 3 mM isopropyl-ß-D-thiogalactopyranoside (IPTG) were used.


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  		TABLE 1. Bacterial strains and plasmids used in this study

 
Sequence analysis. Homology searches were performed at the National Center for Biotechnology Information (NCBI) website. Pairwise sequence alignments were performed with Vector NTI (Informax) using the Clustal W (1.74) algorithm.

Plasmid constructions. The plasmids used in this study are listed in Table 1. PCR primers were designed incorporating a 5' BglII site (underlined) and a 3' HindIII site (underlined) complementary to ORF48 from M. thermautotrophicus cDNA as follows: BglII 5'-AGATCTATTATTTCGGATAGGAAACTGGAGC-3' and HindIII 5'-AAGCTTTAATGACCTCCTGGCGTATTTTTTAG-3'. The native Met start codon was mutated to Ile (bold), and the native stop codon was mutated to Leu (bold). The gel-purified 1.1-kb PCR product was A-tailed and ligated into the subcloning vector pGEM-T Easy (Promega) to give pJMSB0335. pJMSB0335 was sequenced to verify that the 1.1-kb insert was identical to the deposited sequence for M. thermautotrophicus ORF48. pJMSB0335 was restriction digested with BglII and HindIII and ligated into the doubly digested expression vector pBAD Myc/His A (Invitrogen) to give the expression plasmid pJMSB0338 containing a C-terminal His6 tag.

Construction of strain RMC29 [S. enterica serovar Typhimurium LT2 dxs::mevalonate operon (araC Pbad erg8 erg12 erg19 Kan) idi(del)::Camr(swap)]. Salmonella cells expressing phage lambda recombination genes were used to facilitate linear transformation as described by Price-Carter et al. (25) and Yu et al. (46). A wild-type Salmonella strain carrying plasmid pTP223, strain TT22236, was used as a transformation recipient (25). Plasmid pTP223, supplied and constructed by Fenton and Poteete (8), expressed the phage lambda genes exo, bet, and gam from a lac promoter (25).

For construction of the idi(del)::Camr(swap) in a wild-type background, PCR primers were designed to amplify the CAM resistance (Camr) gene from pACYC184 while incorporating short flanking regions of homology. The 5' end of primer 1 included 40 bp of Salmonella idi sequence beginning 17 bases upstream of the start codon, followed by 20 bp of homologous sequence adjacent to the promoter of the Camr gene of pACYC184. The 5' end of primer 2 included 40 bp of complementary idi sequence beginning 7 bases downstream of the stop codon, followed by complementary sequence homologous to the region immediately following the Camr gene of pACYC184. The Camr gene of pACYC184 was amplified using these primers and Taq DNA polymerase. After purification, the resulting linear fragment was used to transform the recipient strain, TT22236, to CAM resistance. The resulting recombinants carried a Camr gene in place of the idi gene except for the upstream 23 bp and the downstream 33 bp of coding sequence. The insertion site was verified by PCR using primers flanking the expected insertion region followed by sequencing of the amplified DNA.

The construction of S. enterica serovar Typhimurium strain RMC26 is described elsewhere (40). RMC26 possesses a synthetic operon for the biosynthesis of IPP from mevalonic acid. The synthetic operon consists of the Saccharomyces cerevisiae genes erg12 (mevalonate kinase), erg8 (phosphomevalonate kinase), and erg19 (diphosphomevalonate decarboxylase). These genes are controlled by an arabinose promoter, preceded by the araC gene and followed by a kanamycin resistance gene. The synthetic operon is inserted into a partially deleted chromosomal copy of dxs, the first gene in the MEP pathway. The resulting strain has an absolute requirement for exogenous supplementation with either MEV and AR or ME for the biosynthesis of essential isoprenoids. Strain RMC29 was constructed by crossing the idi(del)::Camr(swap) into strain RMC26 [S. enterica serovar Typhimurium LT2 dxs::mevalonate operon (araC Pbad erg8 erg12 erg19 Kan)] by standard transduction (40).

Construction of strain JMSB0354 (RMC29/pBADA Myc/His). Electrocompetent TR6579 S. enterica serovar Typhimurium cells, metA22 metE551 trpD2 ilv-452 leu pro (leaky) hsdLT6 hsdSA29 hsdB-strA120 galE, were electroporated with the expression vector pBAD Myc/His A from Invitrogen. Individual colonies from LB/AMP (LB plus AMP) agar plates were picked, and the plasmid DNA was isolated. The plasmid DNA was electroporated into freshly prepared electrocompetent RMC29 cells to create strain JMSB0354.

Construction of strain JMSB0351 (RMC29/pJMSB0338). Electrocompetent TR6579 cells were electroporated with pJMSB0338. Individual colonies from LB/AMP agar plates were picked, and the plasmid DNA was isolated. Strain JMSB0351 was created by electroporating the isolated plasmid DNA into electrocompetent strain RMC29.

Construction of E. coli strain JMSB0338 (BL21 DE3 pLysE/pJMSB0338). Electrocompetent BL21 DE3/pLysE cells were electroporated with pJMSB0338. Individual colonies from LB/AMP/CAM agar plates were picked. The plasmid DNA of these transformed colonies was isolated and sequenced for verification.

Expression of M. thermautotrophicus ORF48 and purification of the encoded protein. LB/AMP/CAM cultures (5 ml) were inoculated with single colonies of JMSB0338 and grown overnight at 37°C. These starter cultures were used to inoculate 500 ml of LB/AMP/CAM. The cultures were grown at 37°C to an A600 of approximately 0.6, L-arabinose was added to a final concentration of 0.2%, and incubation was continued for another 6 h. The cells were harvested by centrifugation.

All steps in the purification were performed at 4°C. Cell paste (~3 g) from strain JMSB0338 was suspended in 20 ml of lysis buffer consisting of 50 mM sodium phosphate (pH 8), 300 mM NaCl, and 10 mM imidazole. The cells were disrupted by sonication, and the resulting homogenate was centrifuged at 10,000 x g to remove cellular debris. Ni-NTA (5 ml, 50% slurry) was added to the cleared supernatant, and the suspension was swirled at 4°C at 100 rpm on a rotary shaker. The Ni-NTA and lysate were poured into a 100-ml fritted glass column, and the flowthrough was collected. The Ni-NTA resin was washed twice with 40 ml of a solution consisting of 20 mM imidazole, 300 mM NaCl, and 50 mM sodium phosphate (pH 8) and twice with 10 ml each of 78, 135, 192, and 250 mM imidazole (300 mM NaCl, 50 mM sodium phosphate [pH 8]). The eluates were collected separately and analyzed by SDS-PAGE. The purest fractions were dialyzed against 50 mM HEPES (pH 6.8). The dialyzed protein was concentrated, yielding 195 µg of >90% purity.

Assays. Type II IPP isomerase activity was assayed by a modified version of the acid lability assay (30) described by Kaneda et al. (16). The assay cocktail consisted of 50 mM HEPES buffer (pH 6.8), 200 µM IPP (2 µCi/µmol), 20 µM FMN, 10 mM NADPH, 50 mM MgCl2, and 50 µM dithiothreitol (DTT). The reaction was initiated by adding 10 µl of sample to 40 µl of the assay cocktail. The reaction mixture was incubated at 70°C for 10 min, quenched with 200 µl of methanol-HCl (4:1), and incubated for an additional 10 min at 37°C. Ligroine (1 ml), with a boiling point of 90 to 110°C, was added to the quenched reaction mixture and vortexed. A 500-µl portion of the organic layer was added to 10 ml of Cytosint scintillation fluid and counted by liquid scintillation spectrometry.

Purified type II IPP isomerase from M. thermautotrophicus was incubated in polybuffer with a pH range from 4.0 to 9.0, in 0.5 pH increments, for 30 min at room temperature and assayed as described above. The temperature dependence of the activity was determined by performing incubation at temperatures ranging from 22 to 95°C. FMN concentration was varied from 0 to 250 µM. NADPH concentration was varied from 0 to 20 mM. Divalent magnesium concentration was varied from 0 to 100 mM.

Identification of expressed M. thermautotrophicus ORF48. SDS-PAGE was performed on the Ni-NTA-purified protein from strain JMSB0338. A band corresponding to 50 kDa was cut from the denaturing 10% polyacrylamide gel. An in-gel tryptic digestion was performed on the excised band (13). The peptides generated from the proteolytic digestion were subjected to matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. The mass-to-charge ratios of the isolated peptides were queried against the database using MS-Fit (5).

Product studies. A 500-µl sample containing 8 mM IPP or DMAPP, 65 nM isomerase, 10 mM NADPH, 20 µM FMN, 50 mM MgCl2, and 1 mM DTT in 50 mM phosphate buffer (pH 7.0) was incubated for 15 h at 70°C. The sample was lyophilized, dissolved in D2O, and analyzed by nuclear magnetic resonance (NMR) spectroscopy at 500 MHz.

Kinetic parameters. Km and Vmax for IPP were determined at various IPP concentrations between 10 and 800 µM [1-14C]IPP (2 µCi/µmol). Assays of the initial velocities were performed in triplicate under optimal conditions as described above. Conversion of IPP to DMAPP was limited to 10% or less. Km and Vmax for IPP were obtained by fitting to the appropriate form of the Michaelis-Menten equation.

Sedimentation equilibrium. Sedimentation equilibrium experiments were conducted in a Beckman Optima XL-A analytical ultracentrifuge, with a Ti60 rotor at 20°C, using six-channel, 12-mm-thick charcoal-Epon centerpieces. The three sample channels in each cell contained three different loading concentrations of protein in 50 mM Tris, pH 7.5, with 150 mM NaCl and 25 µM DTT, while reference channels contained the dialysate. Loading concentrations varied between 1.12 and 4.47 µM. Cells were scanned radially in continuous mode, with data resulting from 10 absorbance readings taken at 0.001-cm intervals. Equilibrium was confirmed by no change in scans taken at 4 hourly intervals. Values of v-bar and the extinction coefficients for each protein were calculated from the amino acid sequence by the method of Laue et al. (21). Various models describing the concentration distribution were fit to final absorbance versus radius data using nonlinear least-squares techniques and the analysis program NONLIN (15, 45).


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Archaeal genomes contain a gene for type II IPP isomerase. Database searches were conducted for homology to the amino acid sequence of type II IPP isomerase from Streptomyces sp. strain CL190. Type II IPP isomerase ORFs were found in all of the archaeal genomes examined, including Archaeoglobus fulgidus, Aeropyrum pernix, Methanothermobacter thermautotrophicus, Methanococcus jannaschii, Pyrococcus abyssi, Pyrococcus horikoshii, Sulfolobus solfutaricus, Thermoplasma acidophilum, Pyrobaculum aerophilum, Pyrococcus furiosus, Sulfolobus tokodaii, Thermoplasma volcanium, and Halobacterium sp. strain NRC-1. The archaeal IPP isomerases varied in length from 345 amino acids in A. fulgidus to 394 amino acids in P. furiosus.

A multiple-sequence alignment of the archaeal proteins revealed several conserved amino acids and regions of potential significance. Type II IPP isomerase is classified in the NCBI database as a member of the 1304 cluster of orthologous groups (COG) (39) that includes L-lactate dehydrogenase (FMN dependent) and related alpha-hydroxy acid dehydrogenases. The inclusion of the type II IPP isomerases into COG 1304 is based on the strong specific alignment with the conserved domain for FMN-dependent dehydrogenases. There are several conserved glycine-rich regions that are likely part of the binding motifs for FMN and NADPH (42). Apart from the archaeal type II IPP isomerases, we identified additional homologous genes in the NCBI database using the M. thermautotrophicus sequence as a probe.

M. thermautotrophicus ORF48 complements IPP isomerase activity in vivo. S. enterica serovar Typhimurium strain RMC29 differs from RMC26 by the deletion or insertion of a Camr cassette into the chromosomal copy of idi. Both RMC26 and RMC29 are viable when supplemented with ME through use of the MEP pathway genes downstream of ispC which biosynthesize IPP and DMAPP without the necessity of IPP isomerase activity. ME complements disruptions in dxs and dxr (9, 38). The alcohol is imported and phosphorylated before it enters the MEP pathway. Strain RMC26 is also viable in the presence of MEV, through utilization of both the synthetic operon, containing the yeast genes for the conversion of MEV to IPP, as well as the endogenous isomerase which converts IPP to DMAPP. However, RMC29 is not viable when supplemented with MEV due to the absence of endogenous isomerase activity; DMAPP is not produced in its absence. JMSB0351, JMSB0354, and RMC29 grew on LB/CAM/ME, demonstrating that all three strains do not require a functional IPP isomerase to synthesize DMAPP when utilizing the MEP pathway (Fig. 2) (27). Strains JMSB0354 and RMC29, which do not have a functional copy of idi, did not grow on LB/AMP/CAM/MEV/AR (Fig. 2). The disruption of chromosomal idi in JMSB0351 was complemented by a plasmid-encoded copy of ORF48 from M. thermautotrophicus. The S. enterica serovar Typhimurium strain grew on LB/AMP/CAM/MEV/AR by utilizing the MVA pathway to synthesize IPP and the archaeal type II isomerase to convert IPP to DMAPP (Fig. 2).



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  		FIG. 2. Growth of S. enterica serovar Typhimurium strains JMSB0351, JMSB0354, and RMC29 on selective media.

 
M. thermautotrophicus type II IPP isomerase. Type II IPP isomerase from M. thermautotrophicus was purified to >90% homogeneity in one step using Ni2+ agarose affinity chromatography. The expected molecular mass of the protein containing the C-terminal Myc/His tag from the pBADA Myc/His expression vector is 40,886 Da. A single band corresponding to ~48 kDa was cut from a SDS-polyacrylamide gel, and in-gel tryptic digestion was performed on the excised band (13). The peptides generated from the proteolytic digestion were subjected to MALDI-TOF mass spectrometry. The mass-to-charge ratios of the isolated peptides were queried against the database using MS-Fit (5). The resulting matched peptides corresponded to an excellent fit of the encoded protein from M. thermautotrophicus ORF48, with 39% of the amino acids covered, including major peaks in the mass spectrum at m/z 1148.614 for peptide KIDISLDFLGRE and m/z 1808.036 for peptide KSPVIITGHTGEWLNQRG.

Type II IPP isomerase activity was measured by the acid lability technique with the modifications offered by Kaneda et al. (16). The archaeal type II IPP isomerase from M. thermautotrophicus required the combined presence of FMN, NADPH, and Mg2+ for activity. The enzyme was maximally active at 70°C (Fig. 3).



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  		FIG. 3. Dependence of type II IPP isomerase activity from M. thermautotrophicus on temperature (A), pH (B), [MgCl2] (C), [FMN] (D), and [NADPH] (E).

 
The state of aggregation in solution for type II IPP isomerase from M. thermautotrophicus was examined using sedimentation equilibrium. In each case, all data were initially inspected for light redistribution at the base of the cell, and affected data were not included in the fitting process. The final fit used data from three different concentrations of protein. The fitting strategy involved fitting the various models of association to the concentration distributions using the program NONLIN. After final convergence, the baseline offset terms were included in the fit using a starting value measured by overspeeding the sample at the conclusion of the experiment. The best fit was obtained for a tetramer-octamer equilibrium, with a KD of 17 µM. The data were well described by this model, as shown by the randomly distributed residuals (Fig. 4). At the concentration in the assays (29 nM), the protein exists in a homotetrameric state.



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  		FIG. 4. Sedimentation equilibrium data for type II IPP isomerase from M. thermautotrophicus. The lower panel shows experimental data points for three different loading concentrations ({square}, 4.47 µM; {triangleup}, 2.24 µM; {circ}, 1.12 µM) of the protein. The upper panels show the residuals for fits of experimental data to the tetrameric model. The small and random deviations indicate a good fit corresponding to a KD of 17 µM.

 
The products of the isomerization were determined by 1H NMR spectroscopy. The enzyme, NADPH, and FMN were incubated with IPP or DMAPP. The sample was lyophilized, dissolved in D2O, and analyzed. The conversion of IPP to DMAPP was confirmed by the appearance of peaks at 5.45 (H at C-2), 1.77 (E-methyl group), and 1.72 ppm (Z-methyl group) for IPP and a corresponding reduction in the intensity of peaks at 2.40 (H at C-2) and 1.78 ppm (methyl group) (35). Peaks for the protons at C-1 of both diphosphates and the C-4 olefinic protons in IPP were obscured.

The steady-state kinetic constants for M. thermautotrophicus type II IPP isomerase were as follows: Km of 64 µM and kcat of 1.6 s-1. The catalytic efficiency (kcat/Km) of the enzyme was 2.5 x 104 M-1 s-1. The kinetic parameters for the type II isomerases from M. thermautotrophicus, Streptomyces sp. strain CL190, and Staphylococcus aureus (16) and the type I isomerases from E. coli (10), S. cerevisiae (1), and Homo sapiens (11) are compared with type I isomerase from M. thermautotrophicus in Table 2. Except for the Streptomyces enzyme, the catalytic efficiencies of the type I and type II IPP isomerases from widely divergent organisms are similar.


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  		TABLE 2. Kinetic parameters for isomerases across the three kingdoms

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Isoprenoid molecules are required to support life, and all organisms must synthesize isoprenoid molecules de novo to survive. The Archaea have a unique, signature requirement for isoprenoids during the biosynthesis of their cellular membranes (41). The membrane lipids of M. thermautotrophicus consist of C40 and C80 glyceryl ethers where the alkyl moieties are methyl branched isoprenoid chains (23). The basic membrane lipid is a C40 diether consisting of two C20 phytanyl chains ether linked to C-2 and C-3 of glycerol. The tetraethers are macrocyclic molecules formed by cross-linking the ends of the phytanyl chains of two diether molecules (12, 23). The proportion of tetraethers in membrane lipids increases with increases in temperature (14) and contributes to the robust nature of archaeal membranes designed to function in extreme environments (17).

Labeling studies indicate that archaeal membrane ethers are synthesized from acetyl coenzyme A (acetyl-CoA) by the MVA pathway (24). Thus far, putative genes for acetoacetyl-CoA thiolase, 3-hydroxy-3-methylglutaryl-CoA synthase, 3-hydroxy-3-methylglutaryl-CoA reductase, and MVA kinase have been identified in the M. thermautotrophicus chromosome (20). Interestingly, homologs for PMVA kinase and DPMVA decarboxylase have not yet been found in the DNA database for the archaeon.

IPP isomerase activity is essential to convert IPP to DMAPP for isoprenoids synthesized by the MVA pathway. The type I enzyme fulfills this function in eukaryota and some members of the domain Bacteria. Type I IPP isomerase is monomeric and requires Mg2+ or Mn2+ for activity. Affinity labeling (37) and site-directed mutagenesis (36) experiments reveal that the enzyme has essential active- site glutamic acid and cysteine residues. These amino acids are thought to be involved in the addition and abstraction of protons during the isomerization of IPP to DMAPP by a proton addition-elimination mechanism (36). Recent crystal structures of type I IPP isomerase from E. coli (43, 44) indicate that the active enzyme has two metals in the active site. One is presumably magnesium, which stabilizes interactions between the diphosphate residue and the protein. The other metal is coordinated by three histidines and two glutamates, one of which was previously implicated in catalysis by the affinity labeling and site-directed mutagenesis.

Homologs for type II IPP isomerase have been found in all archaeal chromosomes sequenced so far and in some eubacteria (mostly proteobacteria, cyanobacteria, and gram-positive eubacteria). In contrast to the type I enzyme, type II IPP isomerase exists in a homotetrameric state in solution, requiring FMN, NADPH, and Mg2+ as cofactors. Although the chemical mechanism for the isomerization catalyzed by the type II enzyme is not known, it is undoubtedly different than the protonation or deprotonation sequence employed by type I IPP isomerase.

IPP isomerase activity was first detected in the cellular lysate of M. thermautotrophicus in 1993 (47). The genome of the archaeon was sequenced in 1997 before the discovery of the type II enzyme, and it was somewhat surprising that no homologue of the known type I IPP isomerase was found. After bioinformatic approaches failed to find an idi gene in Archaea, it was suggested that archaeal IPP isomerase might be a novel enzyme (33). Following the discovery of type II IPP isomerase in Streptomyces sp. strain CL190 (16), several ORFs that encoded proteins homologous proteins were found in Archaea, including ORF48 in M. thermautotrophicus.

We have now established that ORF48 encodes a type II IPP isomerase. The archaeal gene complements an IPP isomerase deletion in a strain of S. enterica serovar Typhimurium specifically engineered to use either methyl erythritol or mevalonate as the sole precursor for isoprenoid biosynthesis. When complemented with ME, isomerase activity is not required for growth. This observation is consistent with the recently established ability of the ispH gene product to synthesize both IPP and DMAPP in the last step of the MEP pathway (27). In contrast, a functional IPP isomerase is essential when the strain is grown on media supplemented with only mevalonate. Interestingly, the M. thermautotrophicus enzyme was sufficiently active at 37°C to complement the disruption of the idi gene in S. enterica serovar Typhimurium. M. thermautotrophicus is a moderate thermophile, and the optimal temperature for the enzyme is ~70°C (32). The activity of the enzyme is substantially lower at 37°C.

In summary, ORF48 from M. thermautotrophicus encodes type II IPP isomerase. The recombinant protein was characterized by MALDI mass spectrometry of peptide fragments generated by in-gel tryptic digestion. ORF48 complemented the disruption of idi in a S. enterica serovar Typhimurium mutant strain engineered to synthesize IPP from MEV. IPP isomerase activity for the archaeal enzyme was also established biochemically. IPP isomerases have now been established in organisms across the three kingdoms of life.


   ACKNOWLEDGMENTS
 
We thank Lisa Joss for assistance with the sedimentation equilibrium experiments, Cindy Chepanoske for assistance with the MALDI mass spectrometry experiments, and J. R. Roth for providing Salmonella strains.

This work was supported by NIH grant GM25521.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Chemistry, University of Utah, 315 South 1400 East, Room 2020, Salt Lake City, UT 84112. Phone: (801) 581-6685. Fax: (801) 581-4391. E-mail: poulter{at}chem.utah.edu. Back


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Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Journal of Bacteriology, March 2004, p. 1811-1817, Vol. 186, No. 6
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.6.1811-1817.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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