Regulatory Tasks of the Phosphoenolpyruvate-Phosphotransferase System of Pseudomonas putida in Central Carbon Metabolism

  1. Víctor de Lorenzoa
  1. Systems Biology Program, Centro Nacional de Biotecnología (CNB-CSIC), Cantoblanco-Madrid, Spain,a and
  2. Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerlandb
  1. Address correspondence to: Víctor de Lorenzo, vdlorenzo{at}cnb.csic.es.

  1. Editor E. Peter Greenberg, University of Washington

  1. FIG 1
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    FIG 1

    Organization and interplay of the fructose-related and N-related branches of the P. putida PTS. (Top left) Components of PTSFru, which includes the membrane-bound permease FruA, consisting of a fusion of EIIBFru and EIICFru domains. It is generally believed that P-EIIB phosphorylates the carbohydrate bound to EIIC. FruA thus transports extracellular fructose through a phosphorylation-dependent process to yield fructose 1-P (F1P), which is then channeled towards the central metabolism following the action of FruK to yield fructose 1,6-bisphosphate (FBP). The second component of the PTSFru is FruB, a fusion of EIIAFru, HPr, and EI modules into a single polypeptide, the last domain of which is responsible for conveying high-energy phosphates from phosphoenolpyruvate (PEP) into the system. The flow of such phosphate through each of the constituents of the system is indicated. (Bottom left) PTSNtr, which is composed of the proteins PtsN (EIIANtr), PtsO (NPr), and PtsP (EINtr). The transfer of phosphate from PEP among them is shown, although EIIANtr~P lacks any plausible EIIBC counterpart. The arrows between the two PTS branches represent the known cross talk between them (21). Note also that PEP is the shared ultimate donor of phosphate for the two systems.

  2. FIG 2
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    FIG 2

    Glucose and fructose metabolism in P. putida. The scheme summarizes the network of reactions in cells growing on either sugar as the sole carbon source. The upper metabolic domain comprises the Entner-Doudoroff (ED) pathway, the Embden-Meyerhof-Parnas (EMP) glycolytic route and the pentose phosphate pathway (PPP, represented as a cycle). The lower metabolic domain encompasses the phosphoenolpyruvate-pyruvate-oxaloacetate (PEP-Pyr-OAA) node, including the pyruvate shunt and the TCA cycle. The metabolites involved in each of the corresponding transformations are indicated: G6P, glucose 6-phosphate; F1P, fructose 1-phosphate; FBP, fructose 1,6-bisphosphate; 6PG, 6-phosphogluconate; 2K6PG, 2-keto-6-phosphogluconate; 2K3D6P, 2-keto-3-deoxy-6-phosphogluconate; GA3P, glyceraldehyde 3-phosphate; DHAP, dihydroxyacetone phosphate; PEP, phosphoenolpyruvate; Pyr, pyruvate; AcCoA, acetyl-CoA; OAA, oxaloacetate; 2-OX, 2-oxoglutarate. The enzymes that catalyze each of the transformations are shown with the corresponding ORF code from the P. putida genome: FruBA, permease/phosphotransferase system for fructose (23); Pgi, glucose-6-phosphate isomerase (PP_1808); Fbp, fructose 1,6-bisphosphate phosphatase (PP_5040); Fda, fructose 1,6-bisphosphate aldolase (PP_4960); Zwf-1, glucose 6-phosphate 1-dehydrogenase (PP_1022); Edd, phosphogluconate dehydratase (PP_1010); Eda, keto-hydroxyglutarate-aldolase (PP_1024); TpiA, triosephosphate isomerase (PP_4715); Gap, glyceraldehyde-3-phosphate dehydrogenase (PP_1009); Pgk, phosphoglycerate kinase (PP_4963); Pgm, phosphoglyceromutase (PP_5056); Eno, phosphopyruvate hydratase (PP_1612); PykA, pyruvate kinase (PP_1362); AceE, pyruvate dehydrogenase subunit E1 (PP_0339); AceF, dihydrolipoamide acetyltransferase (PP_0338); LpdG, dihydrolipoamide dehydrogenase (PP_4187); GltA, citrate synthase (PP_4194); AcnA, aconitate hydratase 1 (PP_2112); AcnB, aconitate hydratase 2 (PP_2339); Icd, isocitrate dehydrogenase (PP_4011); Kgd, alpha-ketoglutarate decarboxylase (PP_4189); KgdB, dihydrolipoamide acetyltransferase (PP_4188); SucA, 2-oxoglutarate dehydrogenase E1 component (PP_4189); SucC, succinyl-CoA synthetase beta subunit (PP_4186); SucD, succinyl-CoA synthetase (PP_4185); SdhA, succinate dehydrogenase flavoprotein subunit (PP_4191); SdhB, succinate dehydrogenase iron sulfur subunit (PP_4190); SdhC, succinate dehydrogenase, cytochrome b556 subunit (PP_4193); SdhD, succinate dehydrogenase, hydrophobic membrane anchor protein (PP_4192); Fum, fumarate hydratase (PP_0897); Mdh, malate dehydrogenase (PP_0654); OadA, pyruvate carboxylase subunit B (PP_5346); AccC-2, pyruvate carboxylase subunit A (PP_5347); MaeB, malic enzyme (PP_5085); Ppc, phosphoenolpyruvate carboxylase (PP_1505). The 16 sites of the network where metabolic fluxes could be calculated as explained in the text are indicated.

  3. FIG 3
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    FIG 3

    Channeling of glucose and fructose through each of the upstream sugar-catabolic pathways. The activities on the y axes represent the net fluxes of carbon through each of the routes calculated using metabolic flux analysis of P. putida MAD2 grown on the compound indicated in each case (see the text and Fig. S1). Note that P. putida degrades glucose mainly through the Entner-Doudoroff (ED) pathway (~96%). Fructose is also catabolized mostly by the ED route (52%) but with an important contribution from standard glycolysis (the Embden-Meyerhof-Parnas [EMP] pathway), which accounts for ~34% of the corresponding flux.

  4. FIG 4
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    FIG 4

    The origin of the components of the phosphoenolpyruvate-pyruvate-oxaloacetate (PEP-Pyr-OAA) node. (a) Major biochemical reactions that connect the ED and EMP routes with the malate and oxaloacetate components of the Krebs cycle. The routes necessary for the conversion of glucose and fructose into metabolic currency (Pyr and PEP) are illustrated on top along with the enzymes that belonging to the ED or the EMP pathways. The bottom highlights the reactions at the boundary between the upper and the lower metabolic domains, including the PEP-Pyr-OAA node and the Pyr shunt. (b) A breakdown of the route of key metabolites (PEP, OAA, and pyruvate) through each of the connecting reactions of the upper and lower metabolic boundaries for either glucose or fructose. The percentage of each precursor compound or pathway is indicated in every case: PEP derived from the EMP pathway, from the ED pathway, or from OAA (via phosphoenolpyruvate carboxylase [Ppc]); OAA directly from malate (via malate dehydrogenase [Mdh]) or from pyruvate (through pyruvate carboxylase [AccC-2/OadA]); and pyruvate derived from malate (via malic enzyme [MaeB]), directly from the ED pathway (Eda enzyme) or from PEP (via the pathway GA3P→PEP→Pyr).

  5. FIG 5
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    FIG 5

    Activity of the enzymes of the pyruvate shunt in extracts of wild-type P. putida MAD2 and ptsN mutants. (a and b) Activity of pyruvate carboxylase in P. putida MAD2 (wt) and its ptsN and ptsN ptsO variants in cells grown on glucose (a) and on fructose (b). (c and d) Activity of the malic enzyme in P. putida MAD2 (wt) and ptsN, ptsN ptsO, and ptsN(H68A) mutants grown on glucose (c) and on fructose (d). The levels of pyruvate carboxylase and malic enzyme were measured in cell extracts from bacteria grown on the sugar indicated as the sole carbon source, as explained in Materials and Methods. The values of the corresponding enzymatic activities are shown in units per milligram of protein (U/mg protein). The bars represent the means for ≥6 independent biological replicates. Note that enzymatic activities were systematically higher in the ptsN and ptsN ptsO strains than in the wild type.

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    May/June 2017, volume 8 issue 3
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