Differential flux through the quinate and shikimate pathways. Implications for the channelling hypothesis.
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ABSTRACT:
SUBMITTER: Lamb HK
PROVIDER: S-EPMC1132714 | biostudies-other | 1992 May
REPOSITORIES: biostudies-other
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Project description:The shikimate pathway leads to the biosynthesis of aromatic amino acids essential for protein biosynthesis and the production of a wide array of plant secondary metabolites. Among them, quinate is an astringent feeding deterrent that can be formed in a single step reaction from 3-dehydroquinate catalyzed by quinate dehydrogenase (QDH). 3-Dehydroquinate is also the substrate for shikimate biosynthesis through the sequential actions of dehydroquinate dehydratase (DQD) and shikimate dehydrogenase (SDH) contained in a single protein in plants. The reaction mechanism of QDH resembles that of SDH. The poplar genome encodes five DQD/SDH-like genes (Poptr1 to Poptr5), which have diverged into two distinct groups based on sequence analysis and protein structure prediction. In vitro biochemical assays proved that Poptr1 and -5 are true DQD/SDHs, whereas Poptr2 and -3 instead have QDH activity with only residual DQD/SDH activity. Poplar DQD/SDHs have distinct expression profiles suggesting separate roles in protein and lignin biosynthesis. Also, the QDH genes are differentially expressed. In summary, quinate (secondary metabolism) and shikimate (primary metabolism) metabolic activities are encoded by distinct members of the same gene family, each having different physiological functions.
| S-EPMC4156088 | biostudies-literature
Project description:The quinic acid ulitization (qut) pathway in Aspergillus nidulans is a dispensable carbon utilization pathway that catabolizes quinate to protocatechuate via dehydroquinate and dehydroshikimate(DHS). At the usual in vitro growth pH of 6.5, quinate enters the mycelium by means of a specific permease and is converted into PCA by the sequential action of the enzymes quinate dehydrogenase, 3-dehydroquinase and DHS dehydratase. The extent of control on metabolic flux exerted by the permease and the three pathway enzymes was investigated by applying the techniques of Metabolic Control Analysis. The flux control coefficients for each of the three quinate pathway enzymes were determined empirically, and the flux control coefficient of the quinate permease was inferred by use of the summation theorem. There measurements implied that, under the standard growth conditions used, the values for the flux control coefficients of the components of the quinate pathway were: quinate permease, 0.43; quinate dehydrogenase, 0.36; dehydroquinase, 0.18; DHS dehydratase, <0,03. Attempts to partially decouple quinate permease from the control over flux by measuring flux at pH 3.5 (when a significant percentage of the soluble quinate is protonated and able to enter the mycelium without the aid of a permease) led to an increase of approx. 50% in the flux control coefficient for dehydroquinase. Taken together with the fact that A. nidulans has a very efficient pH homeostasis mechanism, these experiments are consistent with the view that quinate permease exerts a high degree of control over pathway flux under the standard laboratory growth conditions at pH 6.5. The enzymes quinate dehydrogenase and 3-dehydroquinase have previously been overproduced in Escherichia coli, and protocols for their purification published. The remaining qut pathway enzyme DHS dehydratase was overproduced in E. coli and a purification protocol established. The purified DHS dehydratase was shown to have a K(m) of 530 microM for its substrate DHS and a requirement for bivalent metal cations that could be fulfilled by Mg(2+), Mn(2+) or Zn(2+). All three qut pathway enzymes were purified in bulk and their elasticity coefficients with respect to the three quinate pathway intermediates were derived over a range of concentrations in a core tricine/NaOH buffer, augmented with necessary cofactors and bivalent cations as appropriate. Using these empirically determined relative values, in conjunction with the connectivity theorem, the relative ratios of the flux control coefficients for the various quinate pathway enzymes, and how this control shifts between them, was determined over a range of possible metabolic concentrations. These calculations, although clearly subject to caveates about the relationswhip between kinetic measurements in vitro and the situation in vivo, were able to successfully predict the hiearchy of control observed under the standard laboratory growth conditions. The calculations imply that the hierarchy of control exerted by the quinate pathway enzymes is stable and relatively insensitive to changing metabolite concentrations in the ranges most likely to correspond to those found in vivo. The effects of substituting the type I 3-dehydroquinases from Salmonella typhi and the A. nidulans AROM protein (a pentadomain protein catalysing the conversion of 3-deoxy-D-arabinoheptulosonic acid 7-phosphate into 5-enolpyruvylshikimate 3 phosphate), and the Mycobacterium tuberculosis type II 3-dehydroquinase, in the quinate pathway were investigated and found to have an effect. In the case of S. typhi and A. nidulans, overproduction of heterologous dehydroquinase led to a diminuation of pathway flux caused by a lowering of in vivo quinate dehydrogenase levels increased above those of the wild type. We speculate that these changes in qu
| S-EPMC1217171 | biostudies-other
Project description:A component of the shikimate biosynthetic pathway, dehydroquinate dehydratase (DHQD) catalyzes the dehydration of 3-dehydroquniate (DHQ) to 3-dehydroshikimate. In the type I DHQD reaction mechanism a lysine forms a Schiff base intermediate with DHQ. The Schiff base acts as an electron sink to facilitate the catalytic dehydration. To address the mechanism of Schiff base formation, we determined structures of the Salmonella enterica wild-type DHQD in complex with the substrate analogue quinate and the product analogue shikimate. In addition, we determined the structure of the K170M mutant (Lys170 being the Schiff base forming residue) in complex with quinate. Combined with nuclear magnetic resonance and isothermal titration calorimetry data that revealed altered binding of the analogue to the K170M mutant, these structures suggest a model of Schiff base formation characterized by the dynamic interplay of opposing forces acting on either side of the substrate. On the side distant from the substrate 3-carbonyl group, closure of the enzyme's β8-α8 loop is proposed to guide DHQ into the proximity of the Schiff base-forming Lys170. On the 3-carbonyl side of the substrate, Lys170 sterically alters the position of DHQ's reactive ketone, aligning it at an angle conducive for nucleophilic attack. This study of a type I DHQD reveals the interplay between the enzyme and substrate required for the correct orientation of a functional group constrained within a cyclic substrate.
| S-EPMC3985847 | biostudies-literature
Project description:An 18-kbp Acinetobacter calcoaceticus chromosomal segment contains the pcaIJFBDKCHG operon, which is required for catabolism of protocatechuate, and pobSRA, genes associated with conversion of p-hydroxybenzoate to protocatechuate. The genetic function of the 6.5 kbp of DNA between pcaG and pobS was unknown. Deletions in this DNA were designed by removal of fragments between restriction sites, and the deletion mutations were introduced into A. calcoaceticus by natural transformation. The mutations prevented growth with either quinate or shikimate, growth substrates that depend upon qui gene function for their catabolism to protocatechuate. The location of quiA, a gene encoding quinate-shikimate dehydrogenase, was indicated by its expression in one of the deletion mutants, and the position of the gene was confirmed by determination of its 2,427-bp nucleotide sequence. The deduced amino acid sequence of QuiA confirmed that it is a member of a family of membrane-associated, pyrrolo-quinoline quinone-dependent dehydrogenases, as had been suggested by earlier biochemical investigations. Catabolism of quinate and skikimate is initiated by NAD(+)-dependent dehydrogenases in other microorganisms, so it is evident that different gene pools were called upon to provide the ancestral enzyme for this metabolic step.
| S-EPMC197224 | biostudies-other
Project description:Lignin-based aromatics are attractive raw materials to derive medium-chain length poly(3-hydroxyalkanoates) (mcl-PHAs), biodegradable polymers of commercial value. So far, this conversion has exclusively used the ortho-cleavage route of Pseudomonas putida KT2440, which results in the secretion of toxic intermediates and limited performance. Pseudomonas putida H exhibits the ortho- and the meta-cleavage pathways where the latter appears promising because it stoichiometrically yields higher levels of acetyl-CoA. Here, we created a double-mutant H-ΔcatAΔA2 that utilizes the meta route exclusively and synthesized 30% more PHA on benzoate than the parental strain but suffered from catechol accumulation. The single deletion of the catA2 gene in the H strain provoked a slight attenuation on the enzymatic capacity of the ortho route (25%) and activation of the meta route by nearly 8-fold, producing twice as much mcl-PHAs compared to the wild type. Inline, the mutant H-ΔcatA2 showed a 2-fold increase in the intracellular malonyl-CoA abundance - the main precursor for mcl-PHAs synthesis. As inferred from flux simulation and enzyme activity assays, the superior performance of H-ΔcatA2 benefited from reduced flux through the TCA cycle and malic enzyme and diminished by-product formation. In a benzoate-based fed-batch, P. putida H-ΔcatA2 achieved a PHA titre of 6.1 g l-1 and a volumetric productivity of 1.8 g l-1 day-1 . Using Kraft lignin hydrolysate as feedstock, the engineered strain formed 1.4 g l- 1 PHA. The balancing of carbon flux between the parallel catechol-degrading routes emerges as an important strategy to prevent intermediate accumulation and elevate mcl-PHA production in P. putida H and, as shown here, sets the next level to derive this sustainable biopolymer from lignin hydrolysates and aromatics.
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Project description:Understanding substrate binding and product release in cytochrome P450 (CYP) enzymes is important for explaining their key role in drug metabolism, toxicity, xenobiotic degradation and biosynthesis. Here, molecular simulations of substrate and product exit from the buried active site of a mammalian P450, the microsomal CYP2C5, identified a dominant exit channel, termed pathway (pw) 2c. Previous simulations with soluble bacterial P450s showed a different dominant egress channel, pw2a. Combining these, we propose two mechanisms in CYP2C5: (i) a one-way route by which lipophilic substrates access the enzyme from the membrane by pw2a and hydroxylated products egress along pw2c; and (ii) a two-way route for access and egress, along pw2c, for soluble compounds. The proposed differences in substrate access and product egress routes between membrane-bound mammalian P450s and soluble bacterial P450s highlight the adaptability of the P450 fold to the requirements of differing cellular locations and substrate specificity profiles.
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Project description:Chlorogenic acids (CGAs) are a group of soluble phenolic compounds that are produced by a variety of plants, including Coffea canephora (robusta coffee). The last step in CGA biosynthesis is generally catalysed by a specific hydroxycinnamoyl-CoA quinate hydroxycinnamoyltransferase (HQT), but it can also be catalysed by the more widely distributed hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyltransferase (HCT). Here, the cloning and overexpression of HCT from C. canephora in Escherichia coli as well as its purification and crystallization are presented. Crystals were obtained by the sitting-drop technique at 293?K and X-ray diffraction data were collected on the microfocus beamline ID23-2 at the ESRF. The HCT crystals diffracted to better than 3.0?Å resolution, belonged to space group P4(2)2(1)2 with unit-cell parameters a = b = 116.1, c = 158.9?Å and contained two molecules in the asymmetric unit. The structure was solved by molecular replacement and is currently under refinement. Such structural data are needed to decipher the molecular basis of the substrate specifities of this key enzyme, which belongs to the large plant acyl-CoA-dependent BAHD acyltransferase superfamily.
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Project description:Autophagy has a key role in metabolism and impacts on tumorigenesis. Our previous study found that halofuginone (HF) exerts anticancer activity in colorectal cancer (CRC) by downregulating Akt/mTORC1 (mechanistic target of rapamycin complex 1) signaling pathway. But whether and how HF regulates autophagy and metabolism to inhibit cancer growth remains an open question. Here, we unveil that HF activates ULK1 by downregulation of its phosphorylation site at Ser757 through Akt/mTORC1 signaling pathway, resulting in induction of autophagic flux under nutrient-rich condition. On the other hand, HF inactivates ULK1 by downregulation of its phosphorylation sites at Ser317 and Ser777 through LKB1/AMPK signaling pathway, resulting in autophagic inhibition under nutrient-poor condition. Furthermore, Atg7-dependent autophagosome formation is also induced under nutrient-rich condition or blocked in nutrient-poor environment, respectively, upon HF treatment. More interestingly, we also found that HF inhibits glycolysis under nutrient-rich condition, whereas inhibits gluconeogenesis under nutrient-poor condition in an Atg7-dependent manner, suggesting that autophagy has a pivotal role of glucose metabolism upon HF treatment. Subsequent studies showed that HF treatment retarded tumor growth in xenograft mice fed with either standard chow diet or caloric restriction through dual regulation of autophagy in vivo. Together, HF has a dual role in autophagic modulation depending on nutritional conditions for anti-CRC.
| S-EPMC5520722 | biostudies-literature
Project description:Although we understand many aspects of how small proteins (number of residues less than about hundred) fold, it is a major challenge to quantitatively describe how large proteins self-assemble. To partially overcome this challenge, we performed simulations using the self-organized polymer model with side chains (SOP-SC) in guanidinium chloride (GdmCl), using the molecular transfer model (MTM), to describe the folding of the 110-residue PDZ3 domain. The simulations reproduce the folding thermodynamics accurately including the melting temperature (Tm), the stability of the folded state with respect to the unfolded state. We show that the calculated dependence of ln kobs (kobs is the relaxation rate) has the characteristic chevron shape. The slopes of the chevron plots are in good agreement with experiments. We show that PDZ3 folds by four major pathways populating two metastable intermediates, in accord with the kinetic partitioning mechanism. The structure of one of the intermediates, populated after polypeptide chain collapse, is structurally similar to an equilibrium intermediate. Surprisingly, the connectivities between the intermediates and hence, the fluxes through the pathways depend on the concentration of GdmCl. The results are used to predict possible outcomes for unfolding of PDZ domain subject to mechanical forces. Our study demonstrates that, irrespective of the size or topology, simulations based on MTM and SOP-SC offer a theoretical framework for describing the folding of proteins, mimicking precisely the conditions used in experiments.
| S-EPMC5893999 | biostudies-literature
Project description:BackgroundThe most commonly occurring cancer mutations, including oncogenes such as MYC, Ras and PIK3C, are found in signal transductions pathways feeding into the translational machinery. A broad range of translation initiation factors are also commonly found to be either amplified or mis-regulated in tumours, including eIF4E (elongation initiation factor 4E). eIF4E is a subunit of the eIF4F protein initiation complex and required for its recruitment. Here we measure the formation of the eIF4F complex through interactions of eIF4E and eIF4G subunits, and the effect of oncogenic signalling pathways on complex formation.ResultsWe developed a protein fragment complementation (PCA) assay that can accurately measure the status of the eIF4E-eIF4G interaction in cells and quantify the signalling flux through the RAS/ERK and PI3K/AKT pathways regulating eIF4F assembly. Complex disruption induced by inhibition of either pathway was shown to be a function of the phosphorylation status of 4EBP1, a key mediator of eIF4F assembly that interacts directly with eIF4E, confirming 4EBP1's ability to integrate multiple signals affecting cap-dependent translation. Maximal measured disruption of the eIF4F complex occurred under combined mTORC1 and mTORC2 inhibition, whilst combined inhibition of both RAS/ERK and PI3K/AKT pathways in parallel resulted in greater inhibition of eIF4F formation than individually. v-Myc-mediated resistance to dual mTORC/PI3K inhibition was also principally demonstrated to depend on the lack of competent 4EBP1 available in the cell to bind eIF4E.ConclusionsWe show that 4EBP1 is a critical regulator of the mitogen responsive RAS/ERK and PI3K/AKT pathways and a key transducer of resistance mechanisms that affect small molecule inhibition of these pathways, principally by attenuating their effects on cap-dependent translation. These findings highlight the importance of highly efficacious direct inhibitors of eIF4E and eIF4F assembly, which could potentially target a wide spectrum of tumours containing differing mutations that effect these pathways and which confer chemo-resistance.
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