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1.
Two distinct groups of 3-deoxy-d-manno-octulosonate 8-phosphate synthase (KDO8PS), a key enzyme of cell-wall biosynthesis, differ by their requirement for a divalent metal ion for enzymatic activity. The unique difference between these groups is the replacement of the metal-binding Cys by Asn. Substitution of just this Asn for a Cys in metal-independent KDO8PS does not create the obligate metal-ion dependency of natural metal-dependent enzymes. We describe how three or four mutations of the metal-independent KDO8PS from Neisseria meningitidis produce a fully functional, obligately metal-dependent KDO8PS. For the substitutions Asn23Cys, Asp247Glu (this Asp binds to the metal ion in all metal-dependent KDO8PS) and Pro249Ala, and for double and triple combinations, mutant enzymes that contained Cys in place of Asn showed an increase in activity in the presence of divalent metal ions. However, combining these mutations with substitution by Ser of the Cys residue in the conserved 246CysAspGlyPro249 motif of metal-independent KDO8PS created enzymes with obligate metal dependency. The quadruple mutant (Asn23Cys/Cys246Ser/Asp247Glu/Pro249Ala) showed comparable activity to wild-type enzymes only in the presence of metal ions, with maximum activity with Cd2+, the metal ion that is strongly inhibitory at micromolar concentrations for the wild-type enzyme. In the absence of metal ions, activity was barely detectable for this quadruple mutant or for triple mutants bearing both Cys246Ser and Asn23Cys mutations. The structures of NmeKDO8PS and its Asn23Cys/Asp247Glu/Pro249Ala and quadruple mutants at pH 4.6 were characterized at resolutions better than 1.85 Å. Aged crystals of the Asn23Cys/Asp247Glu/Pro249Ala mutant featured a Cys23-Cys246 disulfide linkage, explaining the spectral bleaching observed when this mutant was incubated with Cu2+. Such bleaching was not observed for the quadruple mutant. Reverse evolution to a fully functional obligately metal-dependent KDO8PS has been achieved with just three directed mutations for enzymes that have, at best, 47% identity between metal-dependent and metal-independent pairs. 相似文献
2.
Shulami S Yaniv O Rabkin E Shoham Y Baasov T 《Extremophiles : life under extreme conditions》2003,7(6):471-481
3-Deoxy-d-manno-2-octulosonate-8-phosphate (KDO8P) synthase, catalyzes the aldol-type condensation between phosphoenolpyruvate (PEP) and d-arabinose-5-phosphate (A5P) to produce the unusual 8-carbon sugar KDO8P, and inorganic phosphate. A 15.5-kb segment containing the kdsA gene from the hyperthermophilic bacterium Aquifex pyrophilus was cloned from a genomic library and sequenced. The native kdsA gene lacks a typical ribosome binding site, but contains a conserved U,A-rich sequence upstream to the start codon. The purified kdsA gene product catalyzes the formation of KDO8P from its natural substrates, PEP and A5P, as determined by 1H NMR analysis. KDO8P synthase showed maximum activity at 80 °C and pH 5.5–6.0 at 10-min reaction assay. At temperatures of 70, 80, and 90 °C, the enzyme exhibited half-lives of 8.0, 2.25, and 0.5 h, respectively. The kinetic constants at 60 °C were KmA5P=70 M, KmPEP=290 M, and kcat=4 s–1. The isolated enzyme contained 0.19 and 0.26 mol iron and zinc, respectively, per mole of enzyme subunit. Treatment with metal chelators eliminated enzyme activity, and by the addition of several divalent metal ions, the activity was restored and even exceeded the original activity. These results indicate that A. pyrophilus KDO8P synthase is a metal-dependent enzyme. A C11A mutant of KDO8P synthase from A. pyrophulis retained less than 1% of the wild-type activity and was shown to be incapable of metal binding.Communicated by G. Antranikian 相似文献
3.
Aquifex aeolicus 3-deoxy-D-manno-octulosonate 8-phosphate synthase (KDO8PS) catalyzes the condensation of arabinose 5-phosphate (A5P) and phosphoenolpyruvate (PEP) by favoring the activation of a water molecule coordinated to the active-site metal ion. Cys11, His185, Glu222 and Asp233 are the other metal ligands. Wild-type KDO8PS is purified with Zn(2+) or Fe(2+) in the active site, but maximal activity in vitro is achieved when the endogenous metal is replaced with Cd(2+). The H185G enzyme retains 8% of the wild-type activity. ICP mass spectrometry analysis indicates that loss of His185 decreases the enzyme affinity for Fe(2+), but not for Zn(2+). However, maximal activity is again achieved by substitution of the endogenous metal with Cd(2+). We have determined the X-ray structures of the Cd(2+) H185G enzyme in its substrate-free form, and in complex with PEP, and PEP plus A5P. These structures show a normal amount of Cd(2+) bound, suggesting that coordination by His185 is not essential to retain Cd(2+) in the active site. Nonetheless, there are significant changes in the coordination sphere of Cd(2+) with respect to the wild-type enzyme, as the carboxylate moiety of PEP binds directly to the metal ion and replaces water and His185 as ligands. These observations indicate that the primary function of His185 in A.aeolicus KDO8PS is to orient PEP in the active site of the enzyme in such a way that a water molecule on the sinister (si) side of PEP can be activated by direct coordination to the metal ion. 相似文献
4.
3-Deoxy-d-manno-2-octulosonate-8-phosphate (KDO8P) synthase catalyzes the net condensation of phosphoenolpyruvate and d-arabinose 5-phosphate to form KDO8P and inorganic phosphate (Pi). Two classes of KDO8P synthases have been identified. The Class I KDO8P synthases (e.g. Escherchia coli KDO8P synthase) catalyze the condensation reaction in a metal-independent fashion, whereas the Class II enzymes (e.g. Aquifex aeolicus) require metal ions for catalysis. Helicobacter pylori (H. pylori) KDO8P synthase, a Zn2+-dependent metalloenzyme, has recently been found to be a Class II enzyme and has a high degree of clinical significance since it is an attractive molecular target for the design of novel antibiotic therapy. Although the presence of a divalent metal ion in Class II KDO8P synthases is essential for catalysis, there is a paucity of mechanistic information on the role of the metal ions and functional differences as compared with Class I enzymes. Using H. pylori KDO8P synthase as a prototypical Class II enzyme, a steady-state and transient kinetic approach was undertaken to understand the role of the metal ion in catalysis and define the kinetic reaction pathway. Metal reconstitution experiments examining the reaction kinetics using Zn2+, Cd2+, Cu2+, Co2+, Mn2+, and Ni2+ yielded surprising results in that the Cd2+ enzyme has the greatest activity. Unlike Class-I KDO8P synthases, the Class II metallo-KDO8P synthases containing Zn2+, Cd2+, Cu2+, and Co2+ show cooperativity. This study presents the first detailed kinetic characterization of a metal-dependent Class II KDO8P synthase and offers mechanistic insight for how the divalent metal ions modulate catalysis through effects on chemistry as well as quaternary protein structure. 相似文献
5.
3-Deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyses the first step of the shikimate pathway for the biosynthesis of aromatic amino acids. Allosteric regulation of Thermotoga maritima DAH7PS is mediated by l-Tyr binding to a discrete ACT regulatory domain appended to a core catalytic (β/α)8 barrel. Variants of T. maritima DAH7PS (TmaDAH7PS) were created to probe the role of key residues in inhibitor selection. Substitution Ser31Gly severely reduced inhibition by l-Tyr. In contrast both l-Tyr and l-Phe inhibited the TmaHis29Ala variant, while the variant where Ser31 and His29 were interchanged (His29Ser/Ser31His), was inhibited to a greater extent by l-Phe than l-Tyr. These studies highlight the role and importance of His29 and Ser31 for determining both inhibitory ligand selectivity and the potency of allosteric response by TmaDAH7PS. 相似文献
6.
KDO8PS (3-deoxy-D-manno-2-octulosonate-8-phosphate synthase) and DAH7PS (3-deoxy-D-arabino-2-heptulosonate-7-phosphate synthase) are attractive targets for the development of new anti-infectious agents. Both enzymes appear to proceed via a common mechanism involving the reaction of phosphoenolpyruvate (PEP) with arabinose 5-phosphate or erythrose-4-phosphate, to produce the corresponding ulosonic acids, KDO8P and DAH7P, respectively. The synthesis of new inhibitors closely related to the supposed tetrahedral intermediate substrates for the enzymes is described. The examination of the antibacterial activity of these derivatives is reported. 相似文献
7.
Peng Tao H. Bernhard Schlegel Domenico L. Gatti 《Journal of inorganic biochemistry》2010,104(12):1267-1275
The three-dimensional structures of metal and non-metal enzymes that catalyze the same reaction are often quite different, a clear indication of convergent evolution. However, there are interesting cases in which the same scaffold supports both a metal and a non-metal catalyzed reaction. One of these is 3-deoxy-D-manno-octulosonate 8-phosphate (KDO8P) synthase (KDO8PS), a bacterial enzyme that catalyzes the synthesis of KDO8P and inorganic phosphate (Pi) from phosphoenolpyruvate (PEP), arabinose 5-phosphate (A5P), and water. This reaction is one of the key steps in the biosynthesis of bacterial endotoxins. The evolutionary tree of KDO8PS is evenly divided between metal and non-metal forms, both having essentially identical structures. Mutagenesis and crystallographic studies suggest that one or two residues at most determine whether or not KDO8PS requires a metal for function, a clear example of “minimalist evolution”. Quantum mechanical/molecular mechanical (QM/MM) simulations of both the enzymatic and non-enzymatic synthesis of KDO8P have revealed the mechanism underlying the switch between metal and non-metal dependent catalysis. The principle emerging from these studies is that this conversion is possible in KDO8PS because the metal is not involved in an activation process, but primarily contributes to orienting properly the reactants to lower the activation energy, an action easily mimicked by amino acid side-chains. 相似文献
8.
Nicholas J. Harmer 《Journal of molecular biology》2010,400(3):379-11313
Heptoses are found in the surface polysaccharides of most bacteria, contributing to structures that are essential for virulence and antibiotic resistance. Consequently, the biosynthetic enzymes for these sugars are attractive targets for novel antibiotics. The best characterized biosynthetic enzyme is GmhA, which catalyzes the conversion of sedoheptulose-7-phosphate into d-glycero-d-manno-heptopyranose-7-phosphate, the first step in the biosynthesis of heptose. Here, the structure of GmhA from Burkholderia pseudomallei is reported. This enzyme contains a zinc ion at the heart of its active site: this ion stabilizes the active, closed form of the enzyme and presents coordinating side chains as a potential acid and base to drive catalysis. A complex with the product demonstrates that the enzyme retains activity in the crystal and thus suggests that the closed conformation is catalytically relevant and is an excellent target for the development of therapeutics. A revised mechanism for the action of GmhA is postulated on the basis of this structure and the activity of B. pseudomallei GmhA mutants. 相似文献
9.
Bianca Derrer Gabriela Guédez Rodríguez Martin Gengenbacher Wolf D. Lehmann Irmgard Sinning Barbara Kappes 《FEBS letters》2010,584(19):4169-4174
Most organisms synthesise the B6 vitamer pyridoxal 5-phosphate (PLP) via the glutamine amidotransferase PLP synthase, a large enzyme complex of 12 Pdx1 synthase subunits with up to 12 Pdx2 glutaminase subunits attached. Deletion analysis revealed that the C-terminus has four distinct functionalities: assembly of the Pdx1 monomers, binding of the pentose substrate (ribose 5-phosphate), formation of the reaction intermediate I320, and finally PLP synthesis. Deletions of distinct C-terminal regions distinguish between these individual functions. PLP formation is the only function that is conferred to the enzyme by the C-terminus acting in trans, explaining the cooperative nature of the complex.
Structured summary
MINT-7994448: PfPdx1 (uniprotkb:C6KT50) and PfPdx1 (uniprotkb:C6KT50) bind (MI:0407) by molecular sieving (MI:0071)MINT-7994425, MINT-7994413, MINT-7994435: PfPdx1 (uniprotkb:C6KT50) and PfPdx1 (uniprotkb:C6KT50) bind (MI:0407) by cosedimentation in solution (MI:0028). 相似文献10.
Hak Suk Chung Eun Gyeong Yang Dohyeon Hwang Ji Eun Lee Ziqiang Guan Christian R.H. Raetz 《Biochemical and biophysical research communications》2014
The lipopolysaccharide (LPS) isolated from certain important Gram-negative pathogens including a human pathogen Yersinia pestis and opportunistic pathogens Burkholderia mallei and Burkholderia pseudomallei contains d-glycero-d-talo-oct-2-ulosonic acid (Ko), an isosteric analog of 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo). Kdo 3-hydroxylase (KdoO), a Fe2+/α-KG/O2 dependent dioxygenase from Burkholderia ambifaria and Yersinia pestis is responsible for Ko formation with Kdo2-lipid A as a substrate, but in which stage KdoO functions during the LPS biosynthesis has not been established. Here we purify KdoO from B. ambifaria (BaKdoO) to homogeneity for the first time and characterize its substrates. BaKdoO utilizes Kdo2-lipid IVA or Kdo2-lipid A as a substrate, but not Kdo-lipid IVAin vivo as well as in vitro and Kdo-(Hep)kdo-lipid A in vitro. These data suggest that KdoO is an inner core assembly enzyme that functions after the Kdo-transferase KdtA but before the heptosyl-transferase WaaC enzyme during the Ko-containing LPS biosynthesis. 相似文献
11.
Robert C. Seid Herman Schneider Sophia Bondarew Robert A. Boykins 《Analytical biochemistry》1982,124(2):320-326
A partition chromatographic procedure utilizing a cationic exchange resin column in the Li+ form and 90% ethanol as the mobile phase was employed to quantify 3-deoxy-d-manno-octulosonic acid (KDO) and l-glycero-d-manno-heptose in the lipopolysaccharides (LPS) of Re and RdP? rough mutants of Salmonella minnesota. In a standard mixture of monosaccharides, KDO eluted shortly after the void volume and heptose eluted after the neutral hexoses. Mild acid treatment of either the Re or RdP? LPS with 0.16 n methanesulfonic acid in the presence of Dowex 50-X8 resin (H+ form) released more than 80% of the KDO residues within 15 min. The heptose of the RdP? LPS, first detected after 90 min of hydrolysis, increased gradually to a maximum level at 12 h. A secondary gradual increase in KDO became apparent during the heptose release. The weight contents of these two monosaccharides based upon aheir maximum values detected during hydrolysis were 20.3 ± 0.6% KDO, for the Re LPS, and 13.8 ± 0.4% KDO and 12.0 ± 0.4% heptose, for the RdP? LPS. The relationship between the kinetics of release of KDO and heptose and the nature of the linkages involving these two monosaccharides are discussed. 相似文献
12.
The tautomeric composition of a solution of ammonium 3-deoxy-d-manno-octulosonate (KDO, 1a) in D2O at 28° was assessed by means of 13 C-F.t.-n.m.r. spectroscopy. The results revealed the presence of 6?0 and 11 % of the α and β anomers of the pyranose, and 20 and 9 % of the two furanoses, and suggested, but did not unequivocally prove, that the major furanose form is the α anomer. To facilitate interpretation of the spectral results for 1, ammonium 3,5-dideoxy-d-arabino(or ribo)-octulosonate (3a) was prepared by the reaction of 5-deoxy-d-erythro-pentose with sodium oxalacetate at pH 11. A chromatographically homogeneous, noncrystalline sample of 3 was obtained by lyophilization, and characterized as its (4-nitrophenyl)hydrazone (m.p. 162-163°). The 13C-n.m.r. spectrum of a solution of 3a in D2O revealed it to be substantially all in the α-pyranose form. No signals were obtained for the possible 1,4-lactone of 3. As the 1,5-lactone and furanose forms are impossible for 3, it exhibited no signals analogous to those attributed to furanoid 1. On the basis of these results for 3, the two lactone forms of 1 were excluded from consideration, and the three pairs of 13C-n.m.r. signals observed at ≈45, 86, and 104 p.p.m. were assigned to the furanose forms of 1. 相似文献
13.
David B. Hicks 《BBA》2010,1797(8):1362-1377
This review focuses on the ATP synthases of alkaliphilic bacteria and, in particular, those that successfully overcome the bioenergetic challenges of achieving robust H+-coupled ATP synthesis at external pH values > 10. At such pH values the protonmotive force, which is posited to provide the energetic driving force for ATP synthesis, is too low to account for the ATP synthesis observed. The protonmotive force is lowered at a very high pH by the need to maintain a cytoplasmic pH well below the pH outside, which results in an energetically adverse pH gradient. Several anticipated solutions to this bioenergetic conundrum have been ruled out. Although the transmembrane sodium motive force is high under alkaline conditions, respiratory alkaliphilic bacteria do not use Na+- instead of H+-coupled ATP synthases. Nor do they offset the adverse pH gradient with a compensatory increase in the transmembrane electrical potential component of the protonmotive force. Moreover, studies of ATP synthase rotors indicate that alkaliphiles cannot fully resolve the energetic problem by using an ATP synthase with a large number of c-subunits in the synthase rotor ring. Increased attention now focuses on delocalized gradients near the membrane surface and H+ transfers to ATP synthases via membrane-associated microcircuits between the H+ pumping complexes and synthases. Microcircuits likely depend upon proximity of pumps and synthases, specific membrane properties and specific adaptations of the participating enzyme complexes. ATP synthesis in alkaliphiles depends upon alkaliphile-specific adaptations of the ATP synthase and there is also evidence for alkaliphile-specific adaptations of respiratory chain components. 相似文献
14.
Macarena Marín 《Biochemical and biophysical research communications》2009,390(4):1345-1348
Pseudomonasreinekei MT1 is capable of growing on 4- and 5-chlorosalicylate as the sole carbon source involving a pathway with trans-dienelactone hydrolase as the key enzyme. This enzyme transforms 4-chloromuconolactone to maleylacetate and thereby avoids the spontaneous formation of toxic protoanemonin. trans-Dienelactone hydrolase is a Zn2+-dependent hydrolase where activity can be modulated by the exchange of Zn2+ by Mn2+ in at least two of the three metal-binding sites. Site directed variants of conserved residues of the Q101XXXQ105XD107XXXH111 motif and of H281 and E294 exhibit a two order of magnitude decrease in activity and a strong decrease in metal-binding capability. As none of the variants exhibited a change in secondary structure, the analyzed amino acid residues can be assumed to be involved in metal binding, forming a novel trinuclear metal-binding motif. 相似文献
15.
Peter Biely Mária MastihubováVladimír Puchart 《Biochimica et Biophysica Acta (BBA)/General Subjects》2007
Positional specificity of NodB-like domain of a multidomain xylanase U from Clostridium thermocellum (CtAxe) was investigated. Of three monoacetates of 4-nitrophenyl β-d-xylopyranoside the acetylxylan esterase domain showed a clear preference for the 2-acetate. Moreover, the enzyme was significantly activated by Co2+. Acetylated methyl β-d-xylopyranosides were deacetylated slightly better at position 3 than at position 2, suggesting that the enzyme binds the substrate with the small methyl aglycone also in the opposite orientation. Nevertheless, both positions 2 and 3 of methyl β-d-xylopyranoside were deacetylated much faster in the presence of the activating metal ion. In contrast, replacement of the hydroxyl group at either of these positions with fluorine or hydrogen, as well as acetylation of both positions, abolished the enzyme activity, regardless the absence or the presence of Co2+. Thus, the presence of the free vicinal hydroxyl group seems to be a prerequisite not only for an efficient deacetylation of position 2 or 3, but also for the activation of the enzyme with cobalt ion. The demonstrated involvement of the vicinal hydroxyl groups in the mechanism of deacetylation is in accord with 3-D structures of CtAxe as well as other CE4 metal-dependent deacetylases. 相似文献
16.
Background
Malaria is a devastating disease and Plasmodium falciparum is the most lethal parasite infecting humans. Understanding the biology of this parasite is vital in identifying potential novel drug targets. During every 48-hour intra-erythrocytic asexual replication cycle, a single parasite can produce up to 32 progeny. This extensive proliferation implies that parasites require substantial amounts of lipid precursors for membrane biogenesis. Glycerol kinase is a highly conserved enzyme that functions at the interface of lipid synthesis and carbohydrate metabolism. P. falciparum glycerol kinase catalyzes the ATP-dependent phosphorylation of glycerol to glycerol-3-phosphate, a major phospholipid precursor.Methods
The P. falciparum glycerol kinase gene was disrupted using double crossover homologous DNA recombination to generate a knockout parasite line. Southern hybridization and mRNA analysis were used to verify gene disruption. Parasite growth rates were monitored by flow cytometry. Radiolabelling studies were used to assess incorporation of glycerol into parasite phospholipids.Results
Disruption of the P. falciparum glycerol kinase gene produced viable parasites, but their growth was significantly reduced to 56.5 ± 1.8% when compared to wild type parasites. 14C-glycerol incorporation into the major phospholipids of the parasite membrane, phosphatidylcholine and phosphatidylethanolamine, was 48.4 ± 10.8% and 53.1 ± 5.7% relative to an equivalent number of wild type parasites.Conclusions
P. falciparum glycerol kinase is required for optimal intra-erythrocytic asexual parasite development. Exogenous glycerol may be used as an alternative carbon source for P. falciparum phospholipid biogenesis, despite the lack of glycerol kinase to generate glycerol-3-phosphate.General significance
These studies provide new insight into glycerolipid metabolism in P. falciparum. 相似文献17.
Phosphoribosyl pyrophosphate (PRPP) synthetase catalyzes the transfer of the pyrophosphate group from ATP to ribose-5-phosphate (R5P) yielding PRPP and AMP. PRPP is an essential metabolite that plays a central role in cellular metabolism. The enzyme from a thermophilic archaeon Thermoplasma volcanium (Tv) was expressed in Escherichia coli, crystallized, and its X-ray molecular structure was determined in a complex with its substrate R5P and with substrate analogs β,γ-methylene ATP and ADP in two monoclinic crystal forms, P21. The β,γ-methylene ATP- and the ADP-bound binary structures were determined from crystals grown from ammonium sulfate solutions; these crystals diffracted to 1.8 Å and 1.5 Å resolutions, respectively. Crystals of the ternary complex with ADP-Mg2+ and R5P were grown from a polyethylene glycol solution in the absence of sulfate ions, and they diffracted to 1.8 Å resolution; the unit cell is approximately double the size of the unit cell of the crystals grown in the presence of sulfate. The Tv PRPP synthetase adopts two conformations, open and closed, at different stages in the catalytic cycle. The binding of substrates, R5P and ATP, occurs with PRPP synthetase in the open conformation, whereas catalysis presumably takes place with PRPP synthetase in the closed conformation. The Tv PRPP synthetase forms a biological dimer in contrast to the tetrameric or hexameric quaternary structures of the Methanocaldococcus jannaschii and Bacillus subtilis PRPP synthetases, respectively. 相似文献
18.
A glyphosate (N-[phosphonomethyl]glycine)-insensitive 5-enolpyruvylshikimic acid-3-phosphate (EPSP) synthase has been purified from a strain of Klebsiella pneumoniae which is resistant to this herbicide [(1984) Arch. Microbiol. 137, 121-123] and its properties compared with those of the glyphosate-sensitive EPSP synthase of the parent strain. The apparent Km values of the insensitive enzyme for phosphoenolpyruvate (PEP) and shikimate 3-phosphate (S-3-P) were increased 15.6- and 4.3-fold, respectively, as compared to those of the sensitive enzyme, and significant differences were found for the optimal pH and temperature, as well as the isoelectric points of the two enzymes. While PEP protected both enzymes against inactivation by N-ethylmaleimide, 3-bromopyruvate, and phenylglyoxal, glyphosate protected only the sensitive enzyme. 相似文献
19.
Eric S. Sharps 《Analytical biochemistry》1984,140(1):183-189
A new assay for 5-enolpyruvylshikimate-3-phosphate synthase is described. This enzyme of the shikimate pathway of aromatic amino acid biosynthesis generates 5-enolpyruvylshikimate 3-phosphate and orthophosphate from phosphoenolpyruvate and shikimate 3-phosphate. The shikimate pathway is present in bacteria and plants but not in mammals. The assay employs a paper-chromatographic separation of radiolabeled substrate from product. The method is specific, is sensitive to 50 pmol of product, and is suitable for use in crude extracts of bacteria. This enzyme appears to be the primary target site of the commercial herbicide glyphosate (N-phosphonomethyl glycine). A procedure for the enzymatic synthesis of [14C]shikimate 3-phosphate from the commercially available precursor [14C]shikimic acid is also described. 相似文献
20.
The methylerythritol phosphate (MEP) pathway of Plasmodium falciparum (P. falciparum) has become an attractive target for anti-malarial drug discovery. This study describes a kinetic model of this pathway, its use in validating 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) as drug target from the systemic perspective, and additional target identification, using metabolic control analysis and in silico inhibition studies. In addition to DXR, 1-deoxy-d-xylulose 5-phosphate synthase (DXS) can be targeted because it is the first enzyme of the pathway and has the highest flux control coefficient followed by that of DXR. In silico inhibition of both enzymes caused large decrement in the pathway flux. An added advantage of targeting DXS is its influence on vitamin B1 and B6 biosynthesis. Two more potential targets, 2-C-methyl-d-erythritol 2,4-cyclodiphosphate synthase and 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase, were also identified. Their inhibition caused large accumulation of their substrates causing instability of the system. 相似文献