Absence of substrate channeling between active sites in the Agrobacterium tumefaciens IspDF and IspE enzymes of the methyl erythritol phosphate pathway.

The conversion of 2-C-methyl-d-erythritol 4-phosphate (MEP) to 2-C-methyl-d-erythritol 2,4-cyclodiphosphate (cMEDP) in the MEP entry into the isoprenoid biosynthetic pathway occurs in three consecutive steps catalyzed by the IspD, IspE, and IspF enzymes, respectively. In Agrobacterium tumefaciens the ispD and ispF genes are fused to encode a bifunctional enzyme that catalyzes the first (synthesis of 4-diphosphocytidyl-2-C-methyl d-erythritol) and third (synthesis of 2-C-methyl-d-erythritol 2,4-cyclodiphosphate) steps. Sedimentation velocity experiments indicate that the bifunctional IspDF enzyme and the IspE protein associate in solution, raising the possibility of substrate channeling among the active sites in these two proteins. Kinetic evidence for substrate channeling was sought by measuring the time courses for product formation during incubations of MEP, CTP, and ATP with the IspDF and IspE proteins with and without an excess of the inactive IspE(D152A) mutant in the presence or absence of 30% (v/v) glycerol. The time dependencies indicate that the enzyme-generated intermediates are not transferred from the IspD active site in IspDF to the active site of IspE or from the active site in IspE to the active site of the IspF module of IspDF.

Mechanistic studies with 2-C-methyl-D-erythritol 4-phosphate synthase from Escherichia coli.

The mechanism of the reaction catalyzed by 2-C-methyl-d-erythritol 4-phosphate (MEP) synthase from Escherichia coli has been studied by steady-state and single-turnover kinetic experiments for the 1-deoxy-d-xylulose 5-phosphoric acid (DXP) analogues, 1,1,1-trifluoro-1-deoxy-d-xylulose 5-phosphoric acid (CF(3)-DXP), 1,1-difluoro-1-deoxy-d-xylulose 5-phosphoric acid (CF(2)-DXP), 1-fluoro-1-deoxy-d-xylulose 5-phosphoric acid (CF-DXP), and 1,2-dideoxy-d-hexulose 6-phosphate (Et-DXP). CF(3)-DXP, CF(2)-DXP, and Et-DXP were poor inhibitors, most likely because of the increase in steric bulk at C1 of DXP. The three analogues were also poor substrates for the enzyme. In contrast, CF-DXP was a good substrate (k(cat)(CF)(-)(DXP) = 37 +/- 2 s(-)(1), K(m)(CF)(-)(DXP) = 227 +/- 25 microM) for MEP synthase when compared to DXP (k(cat)(DXP) = 29 +/- 1 s(-)(1), K(m)(DXP) = 45 +/- 4 microM). A primary deuterium isotope effect was observed under single-turnover conditions when CF-DXP was incubated with 4S-[(2)H]NADPH ((H)k/(D)k = 1.34 +/-0.01), whereas no isotope effect was observed upon incubation with DXP and 4S-[(2)H]NADPH ((H)k/(D)k = 1.02 +/- 0.02). The reaction did not exhibit burst kinetics for either substrate, indicating that product release is not rate-limiting. These studies suggest that positive charge does not develop at C2 of DXP during catalysis. In addition, the isotope effect with CF-DXP and 4S-[(2)H]NADPH but not DXP indicates that the rearrangement step, which precedes hydride transfer, is rate-limiting for DXP but becomes partially rate-limiting for CF-DXP. Thus, rearrangement appears to be enhanced by substitution of a hydrogen atom in the methyl group of DXP by fluorine. These observations are consistent with a retro-aldol/aldol mechanism for the rearrangement during conversion of DXP to MEP.

Crystal structure of the C67A mutant of isopentenyl diphosphate isomerase complexed with a mechanism-based irreversible inhibitor.

Isopentenyl diphosphate:dimethylallyl diphosphate (IPP:DMAPP) isomerase is a key enzyme in the biosynthesis of isoprenoids. The mechanism of the isomerization reaction involves protonation of the unactivated carbon-carbon double bond in the substrate. Analysis of the 1.97 A crystal structure of the inactive C67A mutant of E. coli isopentenyl diphosphate:dimethylallyl diphosphate isomerase complexed with the mechanism-based inactivator 3,4-epoxy-3-methyl-1-butyl diphosphate is in agreement with an isomerization mechanism involving Glu 116, Tyr 104, and Cys 67. In particular, the results are consistent with a mechanism where Glu116 is involved in the protonation step and Cys67 in the elimination step.

E. coli MEP synthase: steady-state kinetic analysis and substrate binding.

2-C-Methyl-D-erythritol-4-phosphate synthase (MEP synthase) catalyzes the rearrangement/reduction of 1-D-deoxyxylulose-5-phosphate (DXP) to methylerythritol-4-phosphate (MEP) as the first pathway-specific reaction in the MEP biosynthetic pathway to isoprenoids. Recombinant E. coli MEP was purified by chromatography on DE-52 and phenyl-Sepharose, and its steady-state kinetic constants were determined: k(cat) = 116 +/- 8 s(-1), K(M)(DXP) = 115 +/- 25 microM, and K(M)(NADPH) = 0.5 +/- 0.2 microM. The rearrangement/reduction is reversible; K(eq) = 45 +/- 6 for DXP and MEP at 150 microM NADPH. The mechanism for substrate binding was examined using fosmidomycin and dihydro-NADPH as dead-end inhibitors. Dihydro-NADPH gave a competitive pattern against NADPH and a noncompetitive pattern against DXP. Fosmidomycin was an uncompetitive inhibitor against NADPH and gave a pattern representative of slow, tight-binding competitive inhibition against DXP. These results are consistent with an ordered mechanism where NADPH binds before DXP.

Geranylgeranylglyceryl phosphate synthase. Characterization of the recombinant enzyme from Methanobacterium thermoautotrophicum.

Geranylgeranylglyceryl diphosphate synthase (GGGP synthase) catalyzes alkylation of (S)-glyceryl phosphate [(S)-GP] by geranylgeranyl diphosphate (GGPP) to produce (S)-geranylgeranylglyceryl phosphate [(S)-GGGP]. This reaction is the first committed step in the biosynthesis of ether-linked membrane lipids in Archaea. The gene encoding GGGP synthase from Methanobacterium thermoautotrophicum was cloned using probes designed from the N-terminal sequence determined from the purified enzyme. The open reading frame, which encoded a protein of 245 amino acids, was inserted into a pET expression vector and expressed in Escherichia coli. The recombinant GGGP synthase was purified to homogeneity. The enzyme is active as a homopentamer, as determined by size exclusion chromatography and equilibrium sedimentation experiments. GGGP synthase has optimal activity at 55 degrees C in pH 8.0 buffer containing 1 mM MgCl(2). V(max) = 4.0 +/- 0.1 micromol min(-1) mg(-1) (k(cat) = 0.34 +/- 0.03 s(-1) for pentameric GGGP synthase assuming all subunits are fully active), K(m)((S)-GP) = 13.5 +/- 1.0 microM, and K(m)(GGPP) = 506 +/- 47 nM. These steady-state catalytic constants were identical to those for enzyme isolated from cell extracts of M. thermoautotrophicum [Chen, A., Zhang, D., and Poulter, C. D. (1993) J. Biol. Chem. 268, 21701-21705]. Alignment of seven putative archaeal GGGP synthase sequences revealed a number of highly conserved residues consisting of five aspartate/glutamates, three serine/threonines, two prolines, and five glycines, including a conserved GGG motif.

Synthesis of (S)-isoprenoid thiodiphosphates as substrates and inhibitors.

Thiolo thiophosphate analogues of isopentenyl diphosphate (IPP), dimethylallyl diphosphate (DMAPP), geranyl diphosphate (GPP), farnesyl diphosphate (FPP), and geranylgeranyl diphosphate (GGPP) were synthesized. Inorganic thiopyrophosphate (SPP(i)) was prepared from trimethyl phosphate in four steps. The tris(tetra-n-butylammonium) salt was then used to convert isopentenyl tosylate to (S)-isopentenyl thiodiphosphate (ISPP). (S)-Dimethylallyl (DMASPP), (S)-geranyl (GSPP), (S)-farnesyl (FSPP), and (S)-geranylgeranyl thiodiphosphate (GGSPP) were prepared from the corresponding bromides in a similar manner. ISPP and GSPP were substrates for avian farnesyl diphosphate synthase (FPPase). Incubation of the enzyme with ISPP and GPP gave FSPP, whereas incubation with IPP and GSPP gave FPP. GSPP was a substantially less reactive than GPP in the chain elongation reaction and was an excellent competitive inhibitor, K(I)(GSPP) = 24.8 microM, of the enzyme. Thus, when ISPP and DMAPP were incubated with FPPase, GSPP accumulated and was only slowly converted to FSPP.

Escherichia coli dimethylallyl diphosphate:tRNA dimethylallyltransferase: site-directed mutagenesis of highly conserved residues.

Dimethylallyl diphosphate:tRNA dimethylallyltransferase (DMAPP-tRNA transferase) catalyzes alkylation of the exocyclic amine of adenosine at position 37 in some tRNAs by the hydrocarbon moiety of dimethylallyl diphosphate (DMAPP). A multiple-sequence alignment of 28 gene sequences encoding DMAPP-tRNA transferases from various organisms revealed considerable homology, including 11 charged, 12 polar, and four aromatic amino acids that are highly conserved or conservatively substituted. Site-directed mutants were constructed for all of these amino acids, and a tripeptide Glu-Glu-Phe alpha-tubulin epitope was appended to the C-terminus of the protein to facilitate separation by immunoaffinity chromatography of overproduced mutant enzymes from coexpressed chromosomally encoded wild-type DMAPP-tRNA transferase. Steady-state kinetic constants were measured for wild-type DMAPP-tRNA transferase and the site-directed mutants using DMAPP and a 17-base RNA oligoribonucleotide corresponding to the stem-loop region of tRNA(Phe) as substrates. Substantial changes in k(cat), K(m)(DMAPP), and/or K(m)(RNA) were seen for several of the mutants, suggesting possible roles for these residues in substrate binding and catalysis.

Chrysanthemyl diphosphate synthase: isolation of the gene and characterization of the recombinant non-head-to-tail monoterpene synthase from Chrysanthemum cinerariaefolium.

Chrysanthemyl diphosphate synthase (CPPase) catalyzes the condensation of two molecules of dimethylallyl diphosphate to produce chrysanthemyl diphosphate (CPP), a monoterpene with a non-head-to-tail or irregular c1'-2-3 linkage between isoprenoid units. Irregular monoterpenes are common in Chrysanthemum cinerariaefolium and related members of the Asteraceae family. In C. cinerariaefolium, CPP is an intermediate in the biosynthesis of the pyrethrin ester insecticides. CPPase was purified from immature chrysanthemum flowers, and the N terminus of the protein was sequenced. A C. cinerariaefolium lambda cDNA library was screened by using degenerate oligonucleotide probes based on the amino acid sequence to identify a CPPase clone that encoded a 45-kDa preprotein. The first 50 aa of the ORF constitute a putative plastidial targeting sequence. Recombinant CPPase bearing an N-terminal polyhistidine affinity tag in place of the targeting sequence was purified to homogeneity from an overproducing Escherichia coli strain by Ni(2+) chromatography. Incubation of recombinant CPPase with dimethylallyl diphosphate produced CPP. The diphosphate ester was hydrolyzed by alkaline phosphatase, and the resulting monoterpene alcohol was analyzed by GC/MS to confirm its structure. The amino acid sequence of CPPase aligns closely with that of the chain elongation prenyltransferase farnesyl diphosphate synthase rather than squalene synthase or phytoene synthase, which catalyze c1'-2-3 cyclopropanation reactions similar to the CPPase reaction.

Solid-phase synthesis of a radiolabeled, biotinylated, and farnesylated Ca(1)a(2)X peptide substrate for Ras- and a-mating factor converting enzyme.

Eukaryotic proteins with carboxyl-terminal Ca(1)a(2) motifs undergo three posttranslational processing reactions--prenylation, endoproteolysis, and carboxymethylation. Two genes in yeast encoding Ca(1)a(2)X endoproteases, AFC1 and RCE1, have been identified. Rce1p is solely responsible for proteolysis of yeast Ras proteins. When proteolysis is blocked, localization of Ras2p to the outer membrane is impaired. The mislocalization of undermodified Ras in the cell suggests that Rce1p is an attractive target for cancer therapeutics. A biotinylated, farnesylated Ca(1)a(2)X peptide [(1-N-biotinyl-(13-N-succinimidyl-(S-(E,E-farnesyl)-L-cysteinyl)-L-valinyl-L-isoleucinyl-L-alanine))-4,7,10-trioxatridecanediamine] 1 containing a poly(ethylene glycol) linker was prepared by solid-phase synthesis for use in an assay for Ca(1)a(2)X endoprotease activity that relies on the strong affinity of avidin for biotin. The peptide was radiolabeled in the penultimate step of the synthesis by cleavage of the biotinylated, farnesylated Ca(1)a(2) precursor from Kaiser's oxime resin with [(14)C]-L-alanine methyl ester. [(14)C]1 was a good substrate for yRce1p with K(M) = 1.3 +/- 0.3 microM. Analysis of the carboxyl terminal products by reverse phase HPLC confirmed that VIA was the only radioactive fragment released upon incubation of [(14)C]1 with a yeast membrane preparation of recombinant yRce1p. The solid-phase methodology developed using Kaiser's benzophenone oxime resin to synthesize [(14)C]1 should be generally applicable for peptides containing sensitive side chains. In addition, introduction of the radiolabeled unit at the end of the synthesis mostly circumvents problems associated with handling radioactive materials.

Structure-activity relationships for inhibition of farnesyl diphosphate synthase in vitro and inhibition of bone resorption in vivo by nitrogen-containing bisphosphonates.

It has long been known that small changes to the structure of the R(2) side chain of nitrogen-containing bisphosphonates can dramatically affect their potency for inhibiting bone resorption in vitro and in vivo, although the reason for these differences in antiresorptive potency have not been explained at the level of a pharmacological target. Recently, several nitrogen-containing bisphosphonates were found to inhibit osteoclast-mediated bone resorption in vitro by inhibiting farnesyl diphosphate synthase, thereby preventing protein prenylation in osteoclasts. In this study, we examined the potency of a wider range of nitrogen-containing bisphosphonates, including the highly potent, heterocycle-containing zoledronic acid and minodronate (YM-529). We found a clear correlation between the ability to inhibit farnesyl diphosphate synthase in vitro, to inhibit protein prenylation in cell-free extracts and in purified osteoclasts in vitro, and to inhibit bone resorption in vivo. The activity of recombinant human farnesyl diphosphate synthase was inhibited at concentrations > or = 1 nM zoledronic acid or minodronate, the order of potency (zoledronic acid approximately equal to minodronate > risedronate > ibandronate > incadronate > alendronate > pamidronate) closely matching the order of antiresorptive potency. Furthermore, minor changes to the structure of the R(2) side chain of heterocycle-containing bisphosphonates, giving rise to less potent inhibitors of bone resorption in vivo, also caused a reduction in potency up to approximately 300-fold for inhibition of farnesyl diphosphate synthase in vitro. These data indicate that farnesyl diphosphate synthase is the major pharmacological target of these drugs in vivo, and that small changes to the structure of the R(2) side chain alter antiresorptive potency by affecting the ability to inhibit farnesyl diphosphate synthase.

1-Deoxy-D-xylulose 5-phosphate synthase, the gene product of open reading frame (ORF) 2816 and ORF 2895 in Rhodobacter capsulatus.

In eubacteria, green algae, and plant chloroplasts, isopentenyl diphosphate, a key intermediate in the biosynthesis of isoprenoids, is synthesized by the methylerythritol phosphate pathway. The five carbons of the basic isoprenoid unit are assembled by joining pyruvate and D-glyceraldehyde 3-phosphate. The reaction is catalyzed by the thiamine diphosphate-dependent enzyme 1-deoxy-D-xylulose 5-phosphate synthase. In Rhodobacter capsulatus, two open reading frames (ORFs) carry the genes that encode 1-deoxy-D-xylulose 5-phosphate synthase. ORF 2816 is located in the photosynthesis-related gene cluster, along with most of the genes required for synthesis of the photosynthetic machinery of the bacterium, whereas ORF 2895 is located elsewhere in the genome. The proteins encoded by ORF 2816 and ORF 2895, 1-deoxy-D-xylulose 5-phosphate synthase A and B, containing a His(6) tag, were synthesized in Escherichia coli and purified to greater than 95% homogeneity in two steps. 1-Deoxy-D-xylulose 5-phosphate synthase A appears to be a homodimer with 68 kDa subunits. A new assay was developed, and the following steady-state kinetic constants were determined for 1-deoxy-D-xylulose 5-phosphate synthase A and B: K(m)(pyruvate) = 0.61 and 3.0 mM, K(m)(D-glyceraldehyde 3-phosphate) = 150 and 120 microM, and V(max) = 1.9 and 1.4 micromol/min/mg in 200 mM sodium citrate (pH 7.4). The ORF encoding 1-deoxy-D-xylulose 5-phosphate synthase B complemented the disrupted essential dxs gene in E. coli strain FH11.

Farnesyl diphosphate synthase. Altering the catalytic site to select for geranyl diphosphate activity.

Farnesyl diphosphate synthase (FPPase) catalyzes chain elongation of the C(5) substrate dimethylallyl diphosphate (DMAPP) to the C(15) product farnesyl diphosphate (FPP) by addition of two molecules of isopentenyl diphosphate (IPP). The synthesis of FPP proceeds in two steps, where the C(10) product of the first addition, geranyl diphosphate (GPP), is the substrate for the second addition. The product selectivity of avian FPPase was altered to favor synthesis of GPP by site-directed mutagenesis of residues that form the binding pocket for the hydrocarbon residue of the allylic substrate. Amino acid substitutions that reduced the size of the binding pocket were identified by molecular modeling. FPPase mutants containing seven promising modifications were constructed. Initial screens using DMAPP and GPP as substrates indicated that two of the substitutions, A116W and N144'W, strongly discriminated against binding of GPP to the allylic site. These observations were confirmed by an analysis of the products from reactions with DMAPP in the presence of excess IPP and by comparing the steady-state kinetic constants for the wild-type enzyme and the A116W and N114W mutants.

Solid-phase synthesis of a farnesylated CaaX peptide library: inhibitors of the Ras CaaX endoprotease.

A solid-phase method, based on Kaiser's p-benzophenone oxime resin, was developed for the synthesis of a series of N-acetyl-S-(E, E-farnesylated) Ca(1)a(2)X tetrapeptides as potential inhibitors of recombinant Ras and a-factor converting enzyme (RCE). N-Acetyl-S-(E, E-farnesyl)-L-cysteine was coupled to resin-bound a(1)a(2) dipeptide using HOBt/DCC activation in conjunction with N-BOC chemistry. The protected farnesylated tripeptide was cleaved from the resin with simultaneous addition of the X residue by treating the resin-bound farnesylated Ca(1)a(2) tripeptide with L-amino acid benzyl ester tosylates under mildly acidic conditions. The benzyl ester was saponified, and the resulting carboxylate precipitated by ether to afford a library of tetrapeptides as a mixture of diastereomers at the cysteine center. The peptides were evaluated as inhibitors of recombinant yeast RCE endoprotease (yRCE) to obtain information about the affinity of the enzyme for the a(1)a(2)X portion of the Ca(1)a(2)X moiety.

Escherichia coli dimethylallyl diphosphate:tRNA dimethylallyltransferase: pre-steady-state kinetic studies.

Escherichia coli dimethylallyl diphosphate:tRNA dimethylallyltransferase (DMAPP-tRNA transferase) catalyzes the first step in the biosynthesis of the hypermodified A37 residue in tRNAs that read codons beginning with uridine. The mechanism of the enzyme-catalyzed reaction was studied by isotope trapping, pre-steady-state rapid quench, and single turnover experiments. Isotope trapping indicated that the enzyme.tRNA complex is catalytically competent, whereas the enzyme.DMAPP complex is not. The results are consistent with an ordered sequential mechanism for substrate binding where tRNA binds first. The association and dissociation rate constants for the enzyme.tRNA binary complex are 1. 15+/-0.33x10(7) M(-1) s(-1) and 0.06+/-0.01 s(-1), respectively. Addition of DMAPP gives an enzyme.tRNA.DMAPP ternary complex in rapid equilibrium with the binary complex and DMAPP. Rapid quench studies yielded a linear profile (k(cat)=0.36+/-0.01 s(-1)) with no evidence for buildup of enzyme-bound product. Product release from DMAPP-tRNA transferase is therefore not rate-limiting. The Michaelis constant for tRNA and the equilibrium dissociation constant for DMAPP calculated from the individual rate constants determined here are consistent with values obtained from a steady-state kinetic analysis.

Synthesis of geranyl S-thiolodiphosphate. A new alternative substrate/inhibitor for prenyltransferases.

The tris(tetra-n-butylammonium) salt of thiopyrophosphate 5 was prepared from trimethyl phosphate in four steps. Treatment of geranyl bromide with 5 gave an 80% yield of geranyl S-thiolodiphosphate (6). Thiolodiphosphate 6 is substantially less reactive than geranyl diphosphate (7) in the prenyl transfer reaction catalyzed by farnesyl diphosphate synthase and is a good inhibitor of the enzyme.

Escherichia coli dimethylallyl diphosphate:tRNA dimethylallyltransferase: essential elements for recognition of tRNA substrates within the anticodon stem-loop.

Escherichia coli dimethylallyl diphosphate:tRNA dimethylallyltransferase (DMAPP-tRNA transferase) catalyzes the alkylation of the exocyclic amine of A37 by a dimethylallyl unit in tRNAs with an adenosine in the third anticodon position (position 36). By use of purified recombinant enzyme, steady- state kinetic studies were conducted with chemically synthesized RNA oligoribonucleotides to determine the essential elements within the tRNA anticodon stem-loop structure required for recognition by the enzyme. A 17-base oligoribonucleotide corresponding to the anticodon stem-loop of E. coli tRNA(Phe) formed a stem-loop minihelix (minihelix(Phe)) when annealed rapidly on ice, while the same molecule formed a duplex structure with a central loop when annealed slowly at higher concentrations. Both the minihelix and duplex structures gave k(cat)s similar to that for the normal substrate (full-length tRNA(Phe) unmodified at A37), although the K(m) for minihelix(Phe) was approximately 180-fold higher than full-length tRNA. The A36-A37-A38 motif, which is completely conserved in tRNAs modified by the enzyme, was found to be important for modification. Changing A36 to G in the minihelix resulted in a 260-fold reduction in k(cat) compared to minihelix(Phe) and a 13-fold increase in K(m). An A38G variant was modified with a 9-fold reduction in k(cat) and a 5-fold increase in K(m). A random coil 17-base oligoribonucleotide in which the loop sequence of E. coli tRNA(Phe) was preserved, but the 5 base pair helix stem was completely disrupted and showed no measurable activity, indicating that a helix-loop structure is essential for recognition. Finally, altering the identity of several base pairs in the helical stem did not have a major effect on catalytic efficiency, suggesting that the enzyme does not make base-specific contacts important for binding or catalysis in this region.

The CaaX proteases, Afc1p and Rce1p, have overlapping but distinct substrate specificities.

Many proteins that contain a carboxyl-terminal CaaX sequence motif, including Ras and yeast a-factor, undergo a series of sequential posttranslational processing steps. Following the initial prenylation of the cysteine, the three C-terminal amino acids are proteolytically removed, and the newly formed prenylcysteine is carboxymethylated. The specific amino acids that comprise the CaaX sequence influence whether the protein can be prenylated and proteolyzed. In this study, we evaluated processing of a-factor variants with all possible single amino acid substitutions at either the a(1), the a(2), or the X position of the a-factor Ca(1)a(2)X sequence, CVIA. The substrate specificity of the two known yeast CaaX proteases, Afc1p and Rce1p, was investigated in vivo. Both Afc1p and Rce1p were able to proteolyze a-factor with A, V, L, I, C, or M at the a(1) position, V, L, I, C, or M at the a(2) position, or any amino acid at the X position that was acceptable for prenylation of the cysteine. Eight additional a-factor variants with a(1) substitutions were proteolyzed by Rce1p but not by Afc1p. In contrast, Afc1p was able to proteolyze additional a-factor variants that Rce1p may not be able to proteolyze. In vitro assays indicated that farnesylation was compromised or undetectable for 11 a-factor variants that produced no detectable halo in the wild-type AFC1 RCE1 strain. The isolation of mutations in RCE1 that improved proteolysis of a-factor-CAMQ, indicated that amino acid substitutions E139K, F189L, and Q201R in Rce1p affected its substrate specificity.

Synthesis of 2-C-methyl-D-erythritol 4-phosphate: the first pathway-specific intermediate in the methylerythritol phosphate route to isoprenoids.

[reaction: see text] 2-C-methyl-D-erythritol 4-phosphate (4), formed from 1-deoxy-D-xylulose 5-phosphate (3), is the first pathway-specific intermediate in the methylerythritol phosphate route for the biosynthesis of isoprenoid compounds in bacteria, algae, and plant chloroplasts. In this report, 4 was synthesized from 1,2-propanediol (7) in seven steps with an overall yield of 32% and in an enantiomeric excess of 78%.

Studies with recombinant Saccharomyces cerevisiae CaaX prenyl protease Rce1p.

Eukaryotic proteins with carboxyl-terminal CaaX motifs undergo three post-translational processing reactions-protein prenylation, endoproteolysis, and carboxymethylation. Two genes in yeast encoding CaaX endoproteases, AFC1 and RCE1, have been identified. Rce1p is solely responsible for proteolysis of yeast Ras proteins. When proteolysis is blocked, plasma membrane localization of Ras2p is impaired. The mislocalization of undermodified Ras in the cell suggests that Rce1p is an attractive target for cancer therapeutics. Homologous expression of plasmid-encoded Saccharomyces cerevisiae RCE1 under the control of the GAL1 promoter gave a 370-fold increase in endoprotease activity over an uninduced control. Yeast Rce1p was detected by Western blotting with a yRce1p antibody or with an anti-myc antibody to Rce1p bearing a C-terminal myc-epitope. Membrane preparations were examined for their sensitivity to a variety of protease inhibitors, metal ion chelators, and heavy metals. The enzyme was sensitive to cysteine protease inhibitors, Zn(2+), and Ni(2+). The substrate selectivity of yRce1p was determined for a variety of prenylated CaaX peptides including farnesylated and geranylgeranylated forms of human Ha-Ras, Ki-Ras, N-Ras, and yeast Ras2p, a-mating factor, and Rho2p. Six site-directed mutants of conserved polar and ionic amino acids in yRce1p were prepared. Four of the mutants, H194A, E156A, C251A, and H248A, were inactive. Results from the protease inhibition studies and the site-directed mutagenesis suggest that Rce1p is a cysteine protease.