Erythritol Feeds the Pentose Phosphate Pathway Via Three New Isomerases Leading to D-Erythrose-4- Phosphate in Brucella

Erythritol feeds the pentose phosphate pathway via three new isomerases leading to D-erythrose-4- phosphate in Brucella

Thibault Barbiera,1, François Collardb,1, Amaia Zúñiga-Ripac, Ignacio Moriyónc, Thibault Godardd, Judith Beckere, Christoph Wittmanne, Emile Van Schaftingenb,2,3, and Jean-Jacques Letessona,2,3

aResearch Unit in Microorganisms Biology, University of Namur, B-5000 Namur, Belgium; bWelbio and de Duve Institute, Université Catholique de Louvain, B-1200 Brussels, Belgium; cDepartamento de Microbiología e Instituto de Salud Tropical, Universidad de Navarra, 31008 Pamplona, Spain; dInstitute of Biochemical Engineering, Technische Universität Braunschweig, 38106 Braunschweig, Germany; and eInstitute of Systems Biotechnology, Saarland University, 66123 Saarbrücken, Germany

Edited by Hans Leo Kornberg, Boston University, Boston, MA, and approved November 3, 2014 (received for review July 31, 2014) Erythritol is an important nutrient for several α-2 Proteobacteria, injections of erythritol increase the numbers of Brucella in the spleens including N2-fixing plant endosymbionts and Brucella, a worldwide of intraperitoneally infected calves (3, 7). For these reasons, erythritol pathogen that finds this four-carbon polyol in genital tissues. has been considered a major factor in Brucella pathogenesis. Erythritol metabolism involves phosphorylation to L-erythritol- Erythritol metabolism (Fig. 1A, accepted pathway) starts with 4-phosphate by the kinase EryA and oxidation of the latter to its phosphorylation by EryA to yield L-erythritol-4-P (10, 11), L-3-tetrulose 4-phosphate by the dehydrogenase EryB. It is accepted which is then oxidized by the dehydrogenase EryB to L-3-tetrulose- that further steps involve oxidation by the putative dehydrogenase 4-P (10). It is accepted that the next three steps imply the oxida- EryC and subsequent decarboxylation to yield triose-phosphates. Ac- tion of the first carbon of L-3-tetrulose-4-P to an aldehyde by an cordingly, growth on erythritol as the sole C source should require NAD-dependent dehydrogenase, purportedly EryC (12), followed aldolase and fructose-1,6-bisphosphatase to produce essential by dehydrogenation and decarboxylation reactions generating di- hexose-6-monophosphate. However, we observed that a mutant hydroxyacetone-P and carbon dioxide as end products (10). devoid of fructose-1,6-bisphosphatases grew normally on eryth- The use of this pathway to grow on erythritol is not restricted ritol and that EryC, which was assumed to be a dehydrogenase, to brucellae or to their intracellular pathogenic lifestyle but is actually belongs to the xylose isomerase superfamily. Moreover, we shared by other members of the Rhizobiales,suchasOchrobactrum found that TpiA2 and RpiB, distant homologs of triose phosphate anthropi (an environmental bacteria), Rhizobium leguminosarum, isomerase and ribose 5-phosphate isomerase B, were necessary, as and Sinorhizobium meliloti (both plant symbionts) (13, 14). In all previously shown for Rhizobium. By using purified recombinant these bacteria, there is, downstream of the ery operon, a second enzymes, we demonstrated that L-3-tetrulose-4-phosphate was highly conserved operon that contains three ORFs including converted to D-erythrose 4-phosphate through three previously tpiA2 and rpiB, which are homologous to triose phosphate isom- unknown isomerization reactions catalyzed by EryC (tetrulose-4- erase and ribose 5-phosphate isomerase B, respectively (13). In- phosphate racemase), TpiA2 (D-3-tetrulose-4-phosphate isomerase; terestingly, rpiB and tpiA2 mutants of R. leguminosarum (15) are renamed EryH), and RpiB (D-erythrose-4-phosphate isomerase; renamed EryI), a pathway fully consistent with the isotopomer distribu- unable to grow on erythritol as a carbon source. tion of the erythrose-4-phosphate-derived amino acids phenylalanine and tyrosine obtained from bacteria grown on 13C-labeled erythritol. Significance D-Erythrose-4-phosphate is then converted by enzymes of the pentose phosphate pathway to glyceraldehyde 3-phosphate and fructose 6- Erythritol is a preferential substrate for Brucella, a common zoo- phosphate, thus bypassing fructose-1,6-bisphosphatase. This is the notic bacterial pathogen. This four-carbon polyol is found in the first description to our knowledge of a route feeding carbohydrate reproductive organs of several affected species, a feature that metabolism exclusively via D-erythrose 4-phosphate, a pathway that may account for the characteristic viscerotropism of Brucella that may provide clues to the preferential metabolism of erythritol by leads to sterility and abortion. Although described previously as Brucella and its role in pathogenicity. feeding glycolysis via dihydroxyacetone-phosphate, we show here that erythritol is actually converted into D-erythrose-4- Brucella | erythritol | alphaproteobacteria | pentose phosphate cycle | phosphate through a hitherto undescribed set of reactions that

tetrose metabolism involves three isomerases and that allows hexose-mono- BIOCHEMISTRY phosphate synthesis and growth by feeding the pentose phos- rucellosis is a widespread bacterial zoonosis caused by the phate shunt. Elucidation of this unique carbohydrate pathway, Bfacultative intracellular pathogens Brucella spp. Domestic which also applies to the Rhizobiales plant endosymbionts, opens ruminants and swine, which altogether represent more than 4 the way for further research on the metabolic adaptation of an billion animals worldwide, are highly susceptible, and in this important facultative intracellular pathogen to target organs. livestock, the brucellae characteristically target the genital organs Author contributions: T.B., F.C., E.V.S., and J.-J.L. designed research; T.B., F.C., A.Z.-R., T.G., J.B., and cause abortion and infertility (1). Close to parturition, these and C.W. performed research; T.B., F.C., A.Z.-R., I.M., T.G., J.B., C.W., E.V.S., and J.-J.L. analyzed 14 bacteria reach exceedingly high numbers (up to 10 bacteria per data; and T.B., F.C., A.Z.-R., I.M., T.G., J.B., C.W., E.V.S., and J.-J.L. wrote the paper. conceptus) (2), thus making brucellosis abortion a highly conta- The authors declare no conflict of interest. gious condition. This article is a PNAS Direct Submission. It is accepted that this tropism and extensive multiplication relate 1T.B. and F.C. contributed equally to this work. to the high erythritol concentration in the genital organs of those 2E.V.S. and J.-J.L. contributed equally to this work. animals (3, 4). In vitro, this four-carbon polyol is a preferential car- 3To whom correspondence may be addressed. Email: [email protected] bon source of Brucella, promotes growth, and up-regulates the ex- or [email protected]. pression of major virulence factors and key enzymes of central This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. metabolism (5–9). In vivo, it has been observed that parenteral 1073/pnas.1414622111/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1414622111 PNAS | December 16, 2014 | vol. 111 | no. 50 | 17815–17820 Downloaded by guest on September 25, 2021 Fig. 1. Erythritol catabolism by Brucella.(A) The two first reactions of the “accepted” and the “revised” pathways are similar (highlighted in gray and further characterized in Figs. S1 and S2 and Table S1). Although it was previously admitted that L-3-tetrulose-4-P is afterward oxidized to dihydroxyacetone-phos-

phate and CO2, we now show that this intermediate is converted to D-erythrose-4-P via three isomerization reactions catalyzed by EryC, TpiA2, and RpiB. The relevant enzyme-encoding genes are organized as illustrated in two operons also comprising two regulators (EryD and DeoR). (B) D-erythrose-4-P is further processed by enzymes of the pentose phosphate pathway to generate fructose-6-P and glyceraldehyde-3-P, two intermediates of glycolysis/gluconeogenesis (C). The proteins whose genes are induced by erythritol (9) are circled in red. The new proposed gene names are indicated in parentheses below the old designations. Fba, fructose bisphosphate aldolase; Fbp, fructose bisphosphatase; G6P, glucose-6-P; Gapdh, glyceraldehyde-3-P dehydrogenase; Pgk, phosphoglycerate kinase; Pgm, phosphoglycerate mutase; R5P, ribose-5-P; Rpe, ribulose-5-P epimerase; Rpi, ribose-5-P isomerase; Ru5P, ribulose-5-P; S7P, sedo- heptulose-7-P; Tal, transaldolase; Tkt, transketolase; Tpi, triose-P isomerase; X5P, xylulose-5-P.

Because the involvement of two putative isomerases is not sole carbon source requires isomerization of dihydroxyacetone-P considered in the accepted pathway, where a dehydrogenase and to glyceraldehyde-3-P, triose-P condensation by aldolase, and a decarboxylase are predicted to catalyze the last two steps, we dephosphorylation of fructose-1,6-P2 by fructose-1,6-bisphos- reassessed the erythritol pathway. A combination of genetic, phatase (FBPase). Brucella spp. carry only two FBPase genes [fbp biochemical, and isotopic evidence brought us to the conclusion and glpX (16)], and surprisingly, a double FBPase mutant (Bru- Δ Δ that erythritol metabolism involves three as-yet undescribed isom- cella abortus 2308 fbp glpx) showed no growth defect when provided with erythritol as the only carbon source (Fig. S3A), erization reactions catalyzed by EryC, TpiA2, and RpiB, which indicating that this polyol is converted not into trioses-P but into together lead to the formation of D-erythrose-4-P, rather than a product that can generate fructose-6-P without involvement of dihydroxyacetone-P. Erythrose-4-P can then be converted into fructose-1,6-P2. Because D-erythrose-4-P can be converted to fruc- glyceraldehyde-3-P and fructose-6-P through reactions catalyzed tose-6-P and glyceraldehyde-3-P via the pentose phosphate pathway by enzymes of the pentose phosphate pathway (Fig. 1B). (Fig. 1B), we hypothesized that L-3-tetrulose-4-P is converted to D-erythrose-4-P by a series of isomerization reactions (Fig. 1A). Results Fructose-1,6-Bisphosphatases Are Dispensable for Growth on Erythritol. Three Putative Isomerases/Epimerases Are Required Instead of Because hexose-monophosphates are essential biomass precursors, Dehydrogenases. Our hypothesis was supported by sequence the accepted pathway predicts that growth on erythritol as the comparisons showing that EryC was not a dehydrogenase

17816 | www.pnas.org/cgi/doi/10.1073/pnas.1414622111 Barbier et al. Downloaded by guest on September 25, 2021 homolog but, rather, a member of the metal-dependent xylose The experiments performed on the products resulting from isomerase-like superfamily (17). Two other hypothetical iso- incubations of either D-erythrose-4-P or L-3-tetrulose-4-P with merases, TpiA2 and RpiB, which are homologous to triose-P the three isomerases alone or in combinations (Fig. 3) led to the isomerase and ribose-P isomerase, respectively, are required following conclusions: First, EryC acts on L-3-tetrulose-4-P for growth of R. leguminosarum on erythritol (15). Similarly, without moving the position of the oxo function (Fig. 3 B, C, E, B. abortus mutants (ΔtpiA2 and ΔrpiB) in the orthologous genes, and F), confirming therefore that it is a 3-tetrulose-4-P racemase which are downstream of the ery operon, did not grow on (Fig. 1A). Second, TpiA2 moves reversibly the oxo group be- erythritol (Fig. S3 B and C). These results, together with previous tween the second and the third carbons (Fig. 3 B and C), thus evidence on the indispensability of EryC (12), indicate that three interconverting a 3-tetrulose-4-P and an erythrulose-4-P. It is putative isomerases are in fact involved in erythritol metabolism. specific for D-3-tetrulose-4-P because it does not act directly on the product of EryB, but well after its racemization with EryC. EryC, TpiA2, and RpiB Convert L-3-Tetrulose-4-P to D-Erythrose-4-P. To As illustrated in Fig. 1A, it acts in the reverse direction on the confirm the role of EryC, TpiA2, and RpiB, L-3-tetrulose-4-P isomer of erythrulose-4-P made from erythrose-4-P by RpiB (i.e., was produced from L-erythritol-4-P with EryB, purified, and the D-isomer; Fig. 3 E and F). Not unexpectedly, the stereo- tested for conversion to D-erythrose-4-P by the purified proteins. chemistry of the TpiA2 reaction is similar to that of the ho- Using an assay in which D-erythrose-4-P formation was deter- mologous enzyme triose-phosphate isomerase (TPI in Fig. S6). mined spectrophotometrically, with purified Escherichia coli As shown in Fig. S7, the replacement of the highly conserved erythrose-4-P dehydrogenase (18) as the coupling enzyme, we Asn10 in triose phosphate isomerase by a serine in TpiA2 pre- found that D-erythrose-4-P was formed from the product of EryB sumably allows the accommodation of a larger substrate; that is, when EryC, TpiA2, and RpiB were present together in the assay a tetrose-P instead of a triose-P. Finally, RpiB reversibly moves mixture (Fig. 2A). No reaction was observed if any of these three the oxo group between C1 and C2 (Fig. 3 D and E), thus cata- proteins or the coupling enzyme was omitted or if EDTA was lyzing the isomerization of D-erythrose-4-P with D-erythrulose-4-P present, in all likelihood because of the metal-dependency of (Fig. 1A). EryC (Fig. S4). These findings confirmed that all three isomer- Isotopic Labeling of Phenylalanine and Tyrosine Supports the Revised ases are required and D-erythrose-4-P is the end-product of Pathway. Because phenylalanine and tyrosine are formed from erythritol catabolism. one molecule of D-erythrose-4-P and two molecules of phosphoenolpyruvate (Fig. 4A), analysis of the isotopomers of these Identification of the Sequence for the Three Isomerization Reactions. 12 We took advantage of the metal-dependency of EryC to check amino acids in cells grown with a mixture of 80% [ C] and 20% [U-13C] (uniformly labeled) erythritol should allow us to dis- whether this protein acted directly on L-3-tetrulose-4-P. This “ ” “ ” compound was preincubated with EryC, the reaction was stop- criminate the accepted and the revised pathways. In the former, the M+4 isotopomer of D-erythrose-4-P directly results ped with EDTA, and the formation of D-erythrose-4-P was from [U-13C] erythritol metabolism and amounts therefore measured in the presence of TpiA2 and RpiB (Fig. 2B, second to ∼ 20% of total D-erythrose-4-P. In the latter, D-erythrose-4-P column). We found that under these conditions, about 50% of would result from recombination of [U-13C] triose-phosphate the L-3-tetrulose-4-P had been converted by EryC during the with a 13C-labeled carbon from another intermediate of the preincubation step to a substrate for TpiA2 and RpiB, suggesting pentose phosphate pathway, thus amounting to only about 4% of that EryC catalyzed the racemization of L-3-tetrulose-4-P to its total D-erythrose-4-P (Table S2). The observed distribution of D-isomer. Experiments in which L-3-tetrulose-4-P was preincubated the M+1toM+8 isotopomers in carbons C2–C9 of phenylalanine with EryC and either TpiA2 or RpiB indicated that TpiA2, but and tyrosine were in much better agreement with the prediction not RpiB, increased the amount of tetrose-4-phosphate that be- of the revised than of the accepted pathway (Fig. 4 B and C), came available for conversion to erythrose-4-P and erythronate- particularly if we took into account the possibility that part of 4-P in the subsequent assay (Fig. 2B, columns 3 and 4; see SI 13 13 13 D-[U- C]erythrose-4-P may be converted to [2,3,4- C] and [1- Results for a more detailed interpretation of this experiment). C] erythrose-4-P through exchange reactions catalyzed by Accordingly, the last specific step of the pathway should be the transaldolase and transketolase. isomerization catalyzed by RpiB. This was confirmed by showing that incubation of D-erythrose 4-phosphate with RpiB (although Discussion not with EryC or TpiA2) caused about a 90% decrease in its This work leads to a substantial revision of the erythritol path- concentration (Fig. 2C), a result consistent with the fact that the way, most particularly of the steps beyond L-3-tetrulose-4-P. equilibrium of the D-erythrulose-4-P/erythrose-4-P isomerization is These steps involve three enzymes that have not been described much in favor of D-erythrulose-4-P (19). for any other species. Because the role of TpiA2 and RpiB in the BIOCHEMISTRY metabolism of erythritol is now established, we propose to Identification of the Intermediates with Tritiated Borohydride and rename their genes eryH and eryI [the designations eryE, eryF, Periodate Oxidation. To analyze further the reactions catalyzed and eryG have already been proposed for the erythritol transport by the three isomerases, we examined the nature of the succes- system of B. abortus (21)]. The gene encoding the putative reg- sively formed tetrose-4-Ps by taking advantage of the fact that ulatory protein DeoR, which precedes tpiA2 on the chromosome, reduction of erythrose-4-P, erythrulose-4-P, and 3-tetrulose-4-P has been designated eryR by Geddes and colleagues (13). Ex- with tritiated borohydride leads to specific incorporation of tri- pression of these genes, including eryA,B,C,D, is induced in the tium on their C1, C2, and C3, respectively. Oxidation with presence of erythritol (9, 12). This regulation is partly based on periodate breaks down polyols according to well-known rules the release by erythritol of the EryD-dependent repression of (20) predicting that the first carbon of tetritol-4-P will be con- eryA,B,C,D expression (12), whereas the role of EryR remains verted to formaldehyde, the second one to formate, and the third unknown. By analogy with other members of the DeoR family, and fourth ones to phosphoglycolaldehyde (see the expected EryR binds in all likelihood a phosphate ester, presumably an patterns in Fig. S5). These breakdown products, which keep the intermediate of the revised erythritol pathway (15). It would thus original hydrogen–carbon linkages, can be easily separated by participate, together with EryD, in the regulation of the ex- anion-exchange chromatography. The nature of the radiolabeled pression of enzymes involved in erythritol catabolism. breakdown products thus informs directly on the position of the Of note, adjacent homologs of tpiA2 (with the typical Asn oxo group in the intermediates. to Ser substitution, Fig. S7) and rpiB are also found always

Barbier et al. PNAS | December 16, 2014 | vol. 111 | no. 50 | 17817 Downloaded by guest on September 25, 2021 downstream of a putative dihydroxyacetone kinase in bacterial genomes devoid of eryAanderyB(Fig. S8). We propose that they are involved in the metabolism of L-erythrulose (i.e., D-3-tetrulose), which would be phosphorylated by the “dihydroxyacetone kinase” to D-3-tetrulose-4-P and isomerized to D-erythrose-4-P by the TpiA2 and RpiB homologs. The revised erythritol pathway is remarkable not only because it involves three enzymes that have never been previously iden- tified but also because it provides the first example, to our knowledge, of a substrate that feeds carbohydrate metabolism exclusively via D-erythrose-4-P. We propose that the latter is converted to intermediates of glycolysis and gluconeogenesis by reactions involving the pentose phosphate pathway enzymes (Fig. 1B). The balance of this set of reactions is the net formation of two molecules of glyceraldehyde-3-P and one molecule of fructose-6-P from three molecules of D-erythrose-4-P. A flow of metabolites coming from erythritol and reaching first the erythrose-4-P pool may be advantageous to Brucella. Indeed, this tetrose-P feeds, via chorismate, the synthesis of aromatic amino acids and of the siderophore 2,3-dihydroxybenzoic acid (22), the production of which is, in fact, increased by erythritol under iron- limiting conditions (23). These characteristics of the revised pathway may explain why the brucellae reach exceedingly high numbers in genital tissues. The highly abortifacient effect of some currently used brucellosis vaccines, most notably Rev1 (Revertant 1), may be related with their ability to multiply in genital organs and to con- sume erythritol (24). Accordingly, B. abortus S19, a vaccine that is not able to catabolize erythritol, is associated with a low risk for abortion (25). The risk for abortion is a serious drawback in mass vaccination, which is the only control strategy applicable in large areas of the world (26). A full understanding of erythritol metabolism may provide clues for developing safer vaccines. Further- more, as none of the five enzymes involved in erythritol metabolism is shared with the host, it opens perspectives for the development of new therapeutic agents aimed at inhibiting this pathway. Materials and Methods Construction of Deletion Strains and Growth of B. abortus. Clean deletion mutants of B. abortus 2308 for fbp, glpx, tpiA2, and rpiB were made by allelic replacement and complemented as described in the SI Materials and Methods. Brucella was grown at 37 °C on rich medium 2YT or on a chemically defined medium (modified Plommet medium), as detailed in SI Mate- rials and Methods. The growth was monitored by following the OD (600 nm) during 48–72 h in an automated plate reader (Bioscreen C, Lab Systems) with continuous shaking.

Protein Purification. Bifidus crudilactis phosphoketolase gene and Brucella eryA, eryB, eryC, tpiA2, and rpiB were inserted into pET15b vectors, which allow expression of N-terminal His-tagged proteins in E. coli Bl21pLysS. Proteins were purified by chromatography on His-trap columns to at least 80% homogeneity. A membrane pellet derived from E. coli expressing EryB was used as source of this enzyme, as it could not be purified. Details are provided in SI Materials and Methods.

Fig. 2. Conversion of L-3-tetrulose-4-P to D-erythrose-4-P: requirement of Synthesis of L-Erythritol-4-P, L-3-Tetrulose-4-P, and D-Erythrose-4-P. L-Erythritol- EryC, TpiA2, and RpiB (A), and determination of the sequence of their 4-phosphate was prepared enzymatically (11), using EryA, and purified. L-3- L reactions (B and C). (A) -3-tetrulose-4-P was incubated with EryC, TpiA2, and tetrulose-4-P was prepared by oxidation of L-erythritol-4-P, using EryB and D RpiB, alone or in combinations, and the formation of -erythrose-4-P was p-benzoquinone as an electron acceptor. D-Erythrose-4-P was produced en- monitored spectrophotometrically in the presence of NAD and erythrose-4-P zymatically with fructose-6-P phosphoketolase from B. crudilactis.SeeSI dehydrogenase (E4PDH). (B) L-3-tetrulose-4-P was preincubated with EryC, Materials and Methods for details. alone or in combination with TpiA2 or RpiB or both. The preincubation was stopped with EDTA, and then TpiA2 or RpiB were added to the incubation Enzymatic Assays. The erythritol kinase activity of EryA was assayed at 30 °C mixtures from which they had been omitted, and the formation of spectrophotometrically by following ADP formation, coupled with the oxi- D-erythrose-4-P was monitored with E4PDH. The five values shown are signifi- dation of NADH. The A was measured either on a spectrophotometer cantly different from each other (P < 0.05 by Student t test), with the exception 340nm or on a plate reader. The L-erythritol-4-P dehydrogenase activity of EryB was of the comparison of “EryC” with “EryC+RpiB,” where there is no difference. (C) D-erythrose-4-P was incubated with EryC, TpiA2, or RpiB. These enzymes were removed from the incubation medium before spectrophotometric mea- surement of the remaining D-erythrose-4-P in the presence of E4PDH. After- reaction (e.g., dephosphorylation by a hypothetical contaminating phos- ward, the enzyme used during the preincubation was added back, as indicated, phatase). In all panels, error bars represent the SD for three distinct to ensure that the metabolite had not been depleted because of an unrelated experiments. These error bars are too small to be visible in some cases.

17818 | www.pnas.org/cgi/doi/10.1073/pnas.1414622111 Barbier et al. Downloaded by guest on September 25, 2021 3 Fig. 3. Analysis by [ H]borohydride labeling/periodate oxidation of the products made from L-3-tetrulose-4-P (A–C) and D-erythrose-4-P (D–F) by EryC, TpiA2, 3 and RpiB. Erythrose-4-P and L-3-tetrulose-4-P were incubated alone, with EryC, TpiA2, and Rpib or in combination, as indicated. [ H]borohydride was added to the reaction mixture and the incubation pursued for 2 h. The samples were acidified with perchloric acid and neutralized. The tetritol-phosphates were purified by anion exchange chromatography and submitted to periodate oxidation. The products were then analyzed by anion-exchange chromatography to separate [3H]-formaldehyde (A and D), [3H]-formate (B and E), and [3H]-phosphoglycolaldehyde (C and F). Tritiated formaldehyde, formate, and phosphoglycolaldehyde are produced in this manner from D-erythrose-4-P, D-erythrulose-4-P, and D-orL-3-tetrulose-4-P, respectively, because of the fixation of tritium on the carbons boxed in the right panels. Mean of two experiments, with the two extremities of the error bars corresponding to the individual values.

assayed at 30 °C by following A600nm in a mixture containing 2,6-dichloro- erythrose-4-P on incubation with the indicated enzymes. Briefly, L-3-tetrulose- phenolindophenol (DCPIP) as an artificial electron acceptor. 4-P (0.1 mM) was incubated (30 min at 30 °C) in a mixture (100 μL) containing The activity of EryC, TpiA2, and RpiB were measured indirectly by measuring 25 mM Tris at pH 8.0 and 1 μg EryC, TpiA2, or RpiB (alone or in combination). the formation of D-erythrose-4-P from L-3-tetrulose-4-P or the depletion of D- EDTA was added to a 2-mM final concentration, and TpiA2 or RpiB were BIOCHEMISTRY

Fig. 4. (A) Origin of the different carbons in phenylalanine and tyrosine and their expected (B) and observed (C) mass isotopomer distribution formed in wild-type (WT) and FBPase-deficient (mut) cells grown on 20% [U-13C] erythritol. The analyzed [M-159] fragment ions (SI Materials and Methods) comprise

carbons C2–C9 of phenylalanine (Phe, m/z 234) or tyrosine (Tyr, m/z 364). These carbons are derived from one molecule of erythrose-4-P and from C2–C3 of two molecules of phosphoenolpyruvate. The expected distributions of isotopomers were calculated as described in SI Materials and Methods for the “accepted” pathway and the “revised” pathway, either assuming (revised*) or not (revised) 25% exchange reactions between labeled and unlabeled D-erythrose-4-P.

Barbier et al. PNAS | December 16, 2014 | vol. 111 | no. 50 | 17819 Downloaded by guest on September 25, 2021 added to the incubation mixtures in which they were omitted and D-erythrose- Isotopic Labeling Experiments and GC/MS Analysis. B. abortus 2308 was grown 4-P was quantified enzymatically. D-erythrose-4-P (0.1 mM) was incubated in chemically defined medium with erythritol as sole carbon source com- (30 min at 30 °C) in a mixture (800 μL) containing 25 mM Tris at pH 8.0 and 5 μg posed of 80% [U-12C] and 20% [U-13C]. During exponential growth, cells EryC, TpiA2, or RpiB (alone or in combination). Enzymes were removed by were harvested, washed, and hydrolyzed with 6-M HCl at 100 °C for 24 h. mixing with 50 μL Ni-NTA resin for 30 min at room temperature, followed by Samples were then lyophilized, amino acid derivatization was carried out, centrifugation, and D-erythrose-4-P was quantified enzymatically (SI Materials and the tert-butyl-dimethylsilyl (tBDMS) derivatives were then analyzed by and Methods). GC-MS (further details in SI Materials and Methods).

3 Characterization of Tetrose-Phosphates by [ H]borohydride Reduction and ACKNOWLEDGMENTS. The E.V.S. laboratory is supported by a Welbio grant Periodate Oxidation. L-3-tetrulose-4-P or D-erythrose-4-P (0.25 mM) was in- of the Walloon Region and by a grant from the Fonds de la Recherche cubated at 30 °C in a mixture (100 μL) containing 25 mM Hepes at pH 7.4, Scientifique Médicale. J.-J.L.’s team is supported by an FNRS (Fonds National

1 mM MgCl2, and 2 μg of the indicated enzyme or enzymes (see Results); de la Recherche Scientifique) grant (Fonds de la Recherche Fondamentale after 30 min, 1 mCi tritiated sodium borohydride (final concentration, Collective Grant N° 2452110) and by the Interuniversity Attraction Poles Pro- 2.5 mM) was added and reduction was allowed to proceed on ice for 2 h. gramme initiated by the Belgian Science Policy Office. T.B. has a PhD grant as “Aspirant FNRS.” I.M.’s laboratory is supported by grants from the Ministerio Perchloric acid was then added to a final concentration of 10% (wt/vol) to de Economía y Competitividad of Spain (AGL2011-30453-C04-00) and the destroy residual borohydride and denatured enzymes. After 2 h at room Fundación para la Investigación Médica Aplicada. A.Z.-R. has a postdoctoral temperature, samples were neutralized and the tetritol-phosphates were grant from FIMA. The research team of C.W. acknowledges financial support purified and oxidized with periodate, and their breakdown product was from the German Research Foundation through Schwerpunktprogramm analyzed, as described in SI Materials and Methods. 1316 “Host-Pathogen Interaction.”

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