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The characterization of a unique Trypanosoma brucei β-hydroxybutyrate dehydrogenase
Molecular & Biochemical Parasitology 179 (2011) 100–106
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Molecular & Biochemical Parasitology
The characterization of a unique Trypanosoma brucei -hydroxybutyrate dehydrogenase Tina D. Shah, Meghan C. Hickey, Kathryn E. Capasso, Jennifer B. Palenchar ∗ Department of Chemistry, Villanova University, 800 E. Lancaster Ave., Villanova, PA 19085, United States
a r t i c l e
i n f o
Article history: Received 2 May 2011 Received in revised form 13 June 2011 Accepted 2 July 2011 Available online 7 July 2011 Keywords: Trypanosoma brucei Hydroxybutyrate dehydrogenase Cofactor preference Short dehydrogenase/reductase superfamily Energy metabolism
a b s t r a c t A putative -hydroxybutyrate dehydrogenase (HBDH) ortholog was identified in Trypanosoma brucei, the unicellular eukaryotic parasite responsible for causing African Sleeping Sickness. The trypanosome enzyme has greater sequence similarity to bacterial sources of soluble HBDH than to membranebound Type I HBDH found in higher eukaryotes. The HBDH gene was cloned from T. brucei genomic DNA and active, recombinant His-tagged enzyme (His10 -TbHBDH) was purified to approximate homogeneity from E. coli. HBDH catalyzes the reversible NADH-dependent conversion of acetoacetate to d-3-hydroxybutyrate. In the direction of d-3-hydroxybutyrate formation, His10 -TbHBDH has a kcat value of 0.19 s−1 and a KM value of 0.69 mM for acetoacetate. In the direction of acetoacetate formation, His10 TbHBDH has a kcat value of 11.2 s−1 and a KM value of 0.65 mM for d-3-hydroxybutyrate. Cofactor preference was examined and His10 -TbHBDH utilizes both NAD(H) and NADP(H) almost equivalently, distinguishing the parasite enzyme from other characterized HBDHs. Furthermore, His10 -TbHBDH binds NAD(P)+ in a cooperative fashion, another unique characteristic of trypanosome HBDH. The apparent native molecular weight of recombinant His10 -TbHBDH is 112 kDa, corresponding to tetramer, as determined through size exclusion chromatography. RNA interference studies in procyclic trypanosomes were carried out to evaluate the importance of TbHBDH in vivo. Upon knockdown of TbHBDH, a small reduction in parasite growth was observed suggesting HBDH has an important physiological role in T. brucei. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Beta hydroxybutyrate dehydrogenase (HBDH, E.C. 1.1.1.30) catalyzes the NAD(H)-dependent interconversion of acetoacetate to d-3-hydroxybutyrate. HBDH is involved in the synthesis of ketone bodies, namely acetoacetate, d-3-hydroxybutyrate, and acetone, from acetyl-CoA. Ketone body production occurs when carbohydrates are scarce and acetyl-CoA is present in excess, such as during periods of starvation. When acetyl-CoA is in excess it does not enter the TCA cycle; rather, it is metabolized to yield ketone bodies. d-3-Hydroxybutyrate synthesized by HBDH in the liver is exported to extrahepatic tissues, where HBDH participates in the pathway that converts d-3-hydroxybutyrate back to acetyl-CoA, by catalyzing the conversion of d-3-hydroxybutyrate to acetoacetate. Acetyl-CoA is subsequently utilized in the TCA cycle in extrahepatic tissues, ultimately providing fuel for the organism.
Abbreviations: HBDH, beta-hydroxybutyrate dehydrogenase; Tb, Trypanosoma brucei; RNAi, RNA interference. ∗ Corresponding author. Tel.: +1 610 519 4868; fax: +1 610 519 7167. E-mail address: [email protected] (J.B. Palenchar). 0166-6851/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.molbiopara.2011.07.001
There are fundamental differences between eukaryotic and bacterial HBDHs. Eukaryotic HBDH (Type 1) is bound to the inner mitochondrial membrane and requires lipid for activity [1,2]. More recently, a cytosolic eukaryotic Type 2 HBDH (human DHRS6) has been discovered, although its physiological function is yet to be determined [3]. Bacterial HBDHs, by contrast, are not membrane bound and do not require lipid for activation [4]. In bacteria, HBDH is part of the poly-hydroxybutyrate (PHB) cycle. Many bacteria possess a complement of enzymes to polymerize d-3-hydroxybutyrate to form PHB and subsequently breakdown the PHB polymer according to the nutritional state of the bacteria [5]. Poly-hydroxybutyrate serves as an energy store, protects bacteria from stresses [6], and functions as an electron and carbon sink [7]. Trypanosoma brucei is the eukaryotic, unicellular protozoan that causes African Sleeping Sickness. The parasite’s lifecycle is split between its tsetse fly vector and its vertebrate host. There are unique features to the parasite’s energy metabolism in these two different environments. In the vertebrate host, the bloodstream form of the parasite relies solely upon glycolysis for energy generation; pyruvate is the excreted end product [8,9]. The tsetse fly midgut procyclic form of the parasite employs a more complex energy metabolism, wherein both glucose and amino acids are degraded. In procyclic parasites, acetate and succinate are the major
T.D. Shah et al. / Molecular & Biochemical Parasitology 179 (2011) 100–106
end products excreted [10,11]. Acetyl-CoA is not degraded to CO2 through the TCA cycle; rather, acetyl-CoA is metabolized through a two-enzyme cycle comprised of succinate:acetate CoA-transferase (ASCT) and succinyl CoA synthetase, giving rise to the production of ATP and succinate [12,13]. Another unique aspect of trypanosome metabolism is the ability of procyclic parasites to utilize acetate for the production of cytosolic acetyl-CoA by the enzyme acetyl-CoA synthetase to meet the demands of de novo fatty acid synthesis [14]. We have identified a putative HBDH ortholog in Trypanosoma species that resembles the bacterial sources of the enzyme, but there are no apparent orthologs of the enzymes involved in the bacterial PHB cycle present in trypanosomes. T. brucei has been reported to excrete low levels of d-3-hydroxybutyrate [12,15], indicating a functional HBDH. Curiously, the related trypanosomatid Leishmania species do not possess an identifiable HBDH ortholog, based on database mining. Moreover, the vast majority of singlecelled eukaryotes lack a HBDH ortholog. Among protists, only T. brucei, Trypanosoma cruzi, Tetrahymena thermophila, and Dictyostelium discoideum have an apparent HBDH ortholog, while among fungi, only Aspergillus fumigates contains an identifiable HBDH ortholog [16]. Thus, the function of HBDH in trypanosomatid metabolism is unclear. To characterize the trypanosome HBDH, a recombinant form of the T. brucei HBDH protein was expressed, isolated, and characterized kinetically to assess the activity of the putative trypanosome HBDH. An unusual cofactor preference was observed and cooperative binding of the oxidized form of the cofactor was experimentally determined, both traits unique to the parasite enzyme. The importance of TbHBDH in vivo was assessed through RNA interference studies in procyclic parasites. Parasites in which TbHBDH is depleted exhibit slowed growth, indicating an important role for this enzyme in vivo. 2. Materials and methods 2.1. Cloning, expression and purification of T. brucei ˇHBDH The full length TbHBDH gene (GeneDB ID: Tb927.10.11930) was amplified from T. brucei 427 wild-type genomic DNA and cloned into pET16b (Novagen). The construct, pMH1, encodes for an amino-terminal 10 histidine-tagged TbHBDH. The sequences of all constructs were verified by DNA sequencing (Genewiz, Inc.). Soluble His10 -TbHBDH was overexpressed in Rosetta 2 cells (Novagen) grown at 27 ◦ C, induced with 50 M IPTG for 12 h, and purified to approximate homogeneity using Ni affinity chromatography. Purified His10 -TbHBDH was stored stably without loss of activity at −20 ◦ C in 50 mM sodium phosphate, 120 mM sodium chloride, and 250 mM imidazole, pH 8.5 containing 50% glycerol (storage buffer). Recombinant His10 -TbHBDH was used to generate custom polyclonal antibodies in rabbits (Lampire Biologicals, Pipersville, PA). 2.2. Substrate determination, kinetic analysis and inhibition studies
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formation, the reaction contained 70 g of His10 -TbHBDH and varying amounts of either acetoacetate or NAD(P)H in 50 mM citric acid, pH 6.0. d-3-Hydroxybutyrate formation was monitored spectrophotometrically at 370 nm due to high absorbance limitations at 340 nm. We determined and used a molar extinction coefficient of 2567 M−1 cm−1 for NAD(P)H at 370 nm. Kinetic constants were determined by using either a Lineweaver–Burk plot or by fitting the Hill equation to the data (SigmaPlot v. 11, Jandel Corporation). All kinetic constants were determined in triplicate. All other potential substrate molecules were examined for NAD+ -dependent dehydrogenase activity. l-3-Hydroxybutyrate, lactate, malonate, and cacodylate were tested as inhibitors. Enzyme activity was monitored in the presence of 70 g of His10 -TbHBDH, 4 mM NAD+ , 0.01–3.0 mM d-3-hydroxybutyrate and one of the following inihibtors: 10 mM l-hydroxybutyrate, 15 mM lactate, 1 mM cacodylate, or 15 mM malonate. Results were analyzed using Lineweaver–Burk plots. 2.3. Molecular mass determination The oligomeric state of His10 -TbHBDH was determined through size exclusion chromatography using Ultrogel AcA-34 resin equilibrated with storage buffer. Blue dextran, bovine intestinal alkaline phosphatase, gamma-globin, bovine albumin, and egg grade III albumin (all from Sigma), were applied to the column at 3 mg/mL and their elution monitored by absorbances at either 600 nm (blue dextran) or 280 nm. His10 -TbHBDH was applied to the column at 1.0 mg/mL in storage buffer. His10 -TbHBDH elution was monitored by -hydroxybutyrate dehdyrogenase activity. 2.4. RNA interference studies To target TbHBDH by RNAi, a 400 bp region from nucleotide 173 to 572 of the open reading frame of Tb927.10.11930 was amplified from T. brucei genomic DNA and inserted into p2T7177 [17] to yield pMH2. The RNAi construct for TbASCT (GeneDB ID: Tb11.02.0290) targeted the same region described in Ref. [12], which was cloned into p2T7-177 and named pMH3. Both TbHBDH and TbASCT were targeted simultaneously by inserting the TbHBDH target region into pMH3 at the HindIII site to yield pMH4. All primers can be found in supplemental Table 1. These constructs were then transfected into T. brucei procyclic cell line 29-13 [18] according the protocol of McCulloch et al. [19]. Clonal cell lines were generated through limiting dilution. RNA interference was induced through the addition of 2 g/mL tetracycline to the media daily. RNAi-induced and non-induced cells were grown in parallel and in replicate. Cell density was determined every 24 h and 8 × 106 cells were removed from the RNAi-induced and RNAi-non induced cell cultures daily for Western blot analysis. TbHBDH was detected using custom TbHBDH rabbit polyclonal antibodies (serum used at a dilution of 1:500) and goat anti-rabbit-IgG conjugated with alkaline phosphatase used at a 1:20,000 dilution. 3. Results and discussion 3.1. Identification of a Trypanosome ˇHBDH
All substrates were of reagent grade. pH rate profiles were carried out to determine the optimal pH for maximal activity in both directions. For the enzyme activity assay in the direction of acetoacetate formation, the 1 mL reaction contained 70 g of His10 TbHBDH, and varying amounts of either d-3-hydroxybutyrate or NAD(P)+ in 50 mM sodium phosphate, pH 8.5. Enzyme activity was monitored spectrophotometrically at 340 nm (Beckman D 640 Spectrophotometer), using a molar extinction coefficient of 6220 M−1 cm−1 for NAD(P)H. In the direction of hydroxybutyrate
BLAST searches of the T. brucei database (www.genedb.org) revealed one potential HBDH ortholog, Tb927.10.11930, annotated as an NAD or NADP dependent oxidoreductase, putative, short chain dehydrogenase. In an attempt to identify as many HBDH orthologs as possible, BLAST searches of the T. brucei genome with several bacterial, Type I and Type II HBDHs still yielded only Tb927.10.11930 as a significant match. This is in contrast to multicellular eukaryotes, which contain both Type I
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Fig. 1. TbHBDH resembles bacterial sources of HBDH. (A) Multiple sequence alignment of bacterial and trypanosome HBDHs. Shown in the sequence alignment are HBDH from Trypansoma brucei (GenBank ID: XP 823399), T. cruzi (GenBank ID: XP 811840), Trypanosoma vivax (GeneDB ID: TvY486 1011560), Pseudomonas fragi (GenBank ID: BAD86668.1), Azospirillum brasilense (GenBank ID: AF355575.1), Ralstonia picketti (GenBank ID: BAE72685), and A. vinelandii (YP 002798947.1). A black background indicates complete conservation of an amino acid, a gray background with white lettering indicates the amino acid is conserved in 80% of the sequences shown, and a gray background with black lettering indicates the amino acid is conserved in 60% of the sequences shown. Residues of the catalytic tetrad are indicated with an *, the conserved TGXXXGXG motif found in the SDR family is indicated with a black line above the alignment. Residues involved in NAD+ binding are indicated with a closed circle, residues involved in substrate binding are indicated with an open circle, and the hinge residue involved in conformational change is indicated by an open square. (B) Phylogentic tree of bacterial, trypanosomal, and eukaryotic type 1 and 2 HBDHs. In addition to the bacterial and trypanosome enzymes from (A) are Dania rerio type 1 (GenBank ID: NP 001082978.1) and type 2 (GenBank ID: NP 001017809.1), Rattus norvegicus type 1 (GenBank ID: NP 446447.2) and 2 (GenBank ID: NP 001099943.1), and Homo sapiens type 1 (GenBank ID: Q02338.3) and 2 (GenBank ID: NP 064524).
and Type II HBDH. Bacteria possess only one HBDH. Only the Trypanosoma spp. of the parasite have the ortholog; curiously, Leishmania spp. do not. By sequence analysis, the eukaryotic trypanosome protein more closely resembles the bacterial sources of HBDH (Fig. 1A and B). The most closely related annotated HBDH to T. brucei HBDH is Azotobacter vinelandii HBDH (GenBank ID: YP 002798947.1). The two enzymes are 55% identical and 19% strongly similar [20] in their sequences. Notably, T. bru-
cei hydroxymethyl glutaryl (HMG)-CoA synthetase (GenBank ID: XP 847441.1) and HMG-CoA lyase (GenBank ID: XP 844420.1), which, along with HBDH, comprise the pathway required for ketone body synthesis, also appear bacterial in origin by BLAST and phylogenetic analysis. Multiple sequence alignments and phylogenetic trees comparing bacterial, trypanosome, and eukaryotic sources of HMG-CoA synthetase and lyase are provided in Supplemental Figs. 1–4.
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a -hydroxybutyrate dehydrogenase. As a control, an unrelated His-tagged trypanosome protein was expressed, purified, and assayed in an identical fashion to that of His10 -TbHBDH. No HBDH activity was detected using this control protein, indicating the observed enzyme activity is not due to trace amounts of contaminating bacterial HBDH.
Fig. 2. Purification of His10 -TbHBDH. Purification of recombinant His10 -TbHBDH from E. coli using nickel affinity chromatography. Electrophoresis in 12% polyacrylamide containing 0.1% sodium dodecyl sulfate. Protein samples were: (S) protein standards, (1) pellet post-sonication, (2) clarified supernatant post-sonication, (3) column flow-through, (4) column wash 20 mM imidazole, (5) column wash 50 mM imidazole, (6) 250 mM imidazole elution, and (7) 250 mM imidazole elution. The position of His10 -TbHBDH is indicated with an asterisk. The theoretical molecular weight of recombinant His10 -TbHBDH is 30.3 kDa.
Based on the bacterial nature of the T. brucei enzyme, we compared T. brucei HBDH with known bacterial HBDHs to determine whether key residues for enzyme function are conserved in the trypanosome protein (Fig. 1A). Beta-hydroxybutyrate dehydrogenase is a member of the short-chain dehydrogenases/reductases (SDR) superfamily, a broad group of enzymes that catalyze NAD(P)(H)dependent oxidoreductions [21]. In this superfamily, NAD(P)(H) binding occurs in the N-terminal region of the protein and is associated with the GXXXGXG motif in the ‘classical’ subfamily [21]. This motif is present in the parasite sources of the enzyme (Fig. 1A). Short-chain dehydrogenases/reductases superfamily members also contain a well-conserved catalytic tetrad comprised of residues Asn114, Ser142, Tyr155, and Lys159, which is conserved in trypanosome HBDH [22]. (All residue numbering corresponds to the Pseudomonas fragi HBDH enzyme.) Several crystal structures of HBDH, in the absence and presence of substrate analog and cofactor, have been obtained from bacterial enzyme sources [23–26]. These structures provide a wealth of information related to substrate binding, cofactor binding, and conformational change in the enzyme. The amino acids involved in binding hydroxybutyrate [26] and the cofactor NAD+ [23] are conserved in most cases or contain a conservative substitution in the parasite enzyme (Fig. 1A). The only notable substitution of an amino acid involved in cofactor binding is found at position 36, where phenylalanine is present in bacterial sources of the enzyme. Positively charged lysine or arginine occupies the equivalent position in the trypanosome protein. Finally, an amino acid key to the conformational change from open to closed form of the enzyme, Thr190, is conserved in the trypanosome enzymes [26]. The conservation of important catalytic, binding, and structural residues in the trypanosome protein, coupled with knowledge that the putative T. brucei HBDH is expressed in the procyclic form of the parasite based on a previous large scale protein expression study [27], prompted us to further study this protein. The putative T. brucei HBDH was cloned from T. brucei genomic DNA, expressed as soluble, recombinant His10 -TbHBDH in bacteria, and purified to approximate homogeneity using nickel affinity chromatography (Fig. 2). 3.2. Kinetic analysis of the putative T. brucei ˇHBDH Beta-hydroxybutyrate dehydrogenase catalyzes the reversible, NAD(H)-dependent conversion of acetoacetate to d-3hydroxybutyrate. The recombinant His10 -TbHBDH protein exhibits activity with both substrates confirming its identity as
3.2.1. Kinetic characterization in the direction of 3-d-hydroxybutyrate formation In the direction of d-3-hydroxybutyrate formation, the pH for maximum activity was determined to be 6.0. In this direction, recombinant His10 -TbHBDH was found to have a specific activity value of 0.38 mol/min/mg and a KM value of 0.69 mM for acetoacetate (Table 1). The turnover number in this direction is 0.19 s−1 . From the limited kinetic data that is published for activity in this direction, the His10 -TbHBDH KM value for acetoacetate is similar to the values for both bacterial and eukaryotic sources of the enzyme; for example, P. fragi, 0.37 mM [26] and R. norveticus heart, 0.31 mM [28] However, the turnover number for the T. brucei enzyme is lower; for example, the kcat value reported for the P. fragi enzyme is 120 s−1 . Cofactor preference was also examined. During d-3hydroxybutyrate formation, NADH is oxidized. Unexpectedly, His10 -TbHBDH binds NADH and NADPH almost equivalently, with KM values of 0.05 mM for each. Furthermore, enzyme activity is highly similar between the two cofactors, yielding specific activity values of 0.19 and 0.36 mol/min/mg for NADH and NADPH, respectively. His10 -TbHBDH binds NADH with similar affinity as other HBDHs; for comparison, the P. fragi HBDH KM value for NADH is 0.01 mM [26] and R. norveticus heart HBDH KM value is 0.029 mM [28]. Importantly, to the best of our knowledge, this is the first demonstration of a HBDH that can utilize NADPH. 3.2.2. Kinetic characterization in the direction of acetoacetate formation. In the direction of acetoacetate formation, the reaction was determined to occur optimally at pH 8.5. Recombinant His10 TbHBDH has a specific activity value of 22.25 mol/min/mg, a KM value of 0.65 mM for d-3-hydroxybutyrate, and a kcat value of 11.23 s−1 (Table 1). The trypanosome enzyme binds d-3hydroxybutyrate comparably to other sources of the enzyme, but the activity is lower than most values reported. For example, the P. fragi enzyme has a KM value of 0.8 mM for d-3-hydroxybutyrate but a kcat value of 370 s−1 [26]. However, His10 -TbHBDH activity is still 350-fold higher than the cytosolic human Type II HBDH [3]. Cofactor preference was also examined in the direction of acetoacetate formation and the enzyme was able to utilize both NAD+ and NADP+ . Surprisingly, the trypanosome enzyme does not obey Michaelis–Menten kinetics when d-3-hydroxybutyrate is present at saturating levels and either NAD+ or NADP+ concentrations are varied. A sigmoidal plot of activity vs. cofactor concentration was observed, suggesting cooperativity. The data were fit to the Hill equation using Sigmaplot and the Hill coefficients were determined to be 2.13 ± 0.10 and 2.01 ± 0.11 for NAD+ and NADP+ , respectively (Fig. 3). As a control, the data from the determination of the kinetic parameters for both acetoacetate and d-3-hydroxybutyrate, which exhibited Michaelis–Menten kinetics, was fit to the Hill equation and yielded Hill coefficients of approximately 1, as expected for non-cooperativity. Based on our survey of the literature, the trypanosome enzyme is the first-reported HBDH that exhibits cooperativity. Furthermore, the ability of TbHBDH to use NADP+ is of interest. The only other HBDH reported to be able to utilize NADP+ is the P. fragi enzyme [23]. Other characterized HBDHs are not reported to utilize NADP(H). For P. fragi HBDH, the KM value for NADP+ is 57fold higher than the value for NAD+ . The crystal structure of P. fragi
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Table 1 Substrate specificity and summary of His10 -TbHBDH kinetics in the directions of acetoacetate and d-3-hydroxybutyrate formation. Substrate or cofactor
KM (mM)
Specific activity (mol/min × mg)
kcat (s−1 )
kcat /KM (s−1 mM−1 )
d-3-Hydroxybutyrate l-3-Hydroxybutyrate dl-Lactate Malonate d or l-serine d or l-threonine NAD+ b NADP+ b
0.65 ± 0.34
22.25 ± 2.25 n.d.a n.d. n.d. n.d. n.d.
11.23
19.68
Acetoacetate NADH NADPH
0.69 ± 0.16 0.05 ± 0.01 0.05 ± 0.02
0.38 ± 0.06 0.19 ± 0.01 0.35 ± 0.1
0.19 0.10 0.18
0.28 1.81 3.52
a b
No detectable activity indicated by n.d. Non-Michaelis–Menten kinetics observed.
HBDH with NAD+ bound indicates that the hydroxyl groups of the NAD+ ribose ring form hydrogen bonds with the side chain hydroxyl of Thr13 and the Phe36 main chain nitrogen. In the trypanosome enzyme, Phe36 is replaced with either an Arg or a Lys, while in other HBDHs representing Type I, II, or bacterial enzymes, a hydrophobic residue is present at the corresponding position. Phenylalanine 36 is found in a region of the enzyme sequence that corresponds to the second beta sheet in the structure. The coenzyme preference of SDR family members has been extensively evaluated [29]. In this work, many NADP(H) preferring enzymes contain a basic residue following this particular beta sheet. Given that the trypanosome enzymes contain either a Lys or Arg at this position, we hypothesize the ability of the trypanosome enzyme to utilize NADP(H) results from having a basic amino acid in the region following the second beta sheet. Likely, the negatively charged phosphate moiety of NADPH can interact with the positively charged side chains of either Lys or Arg. When saturating amounts of either cofactor were present and the concentration of d-3-hydroxybutyrate was varied, the enzyme exhibited Michaelis–Menten kinetics; thus, the cooperative behavior is limited to cofactor binding. Indeed, HBDH enzymes are known to bind cofactor and substrate sequentially, with cofactor binding preceding substrate binding [30]. We speculate that
TbHBDH cooperatively binds cofactor and is subsequently poised to bind substrate. Indeed, a recent structure of the P. fragi enzyme crystallized in the presence of NAD+ only suggests that cofactor binding readies the enzyme to adopt the closed conformation associated with catalysis [26] Members of the SDR superfamily catalyze a broad range of reactions. Furthermore, the activity of HBDHs is known to extend beyond the substrates d-3-hydroxybutyrate and acetoacetate; for example, some HBDHs are able to utilize threonine as substrate, albeit with greatly reduced activity [23]. Thus, the trypanosome enzyme was tested for activity with l-3-hydroxybutyrate, dllactate, malonate, d and l-serine, and d and l-threonine in the presence of saturating amounts of NAD+ . His10 -TbHBDH yielded no detectable activity with these potential substrate molecules. Finally, we examined l-3-hydroxybutyrate, dl-lactate, and malonate as potential inhibitors of His10 -TbHBDH. All three molecules inhibited the enzyme in a competitive fashion and yielded Ki values of 2.6, 10.8, and 1.8 mM, respectively. These values correspond well to the values determined for the P. fragi enzyme for these inhibitors: 1.6 mM, 0.9 mM, 3.8 mM, respectively, further supporting the similarity of this eukaryotic enzyme to bacterial HBDH [23]. Thus, the kinetic characterization of trypanosomal HBDH reveals that the enzyme resembles characterized HBDHs with respect to substrate affinity and inhibition, but is strikingly different in its cofactor preference and cooperative binding of the oxidized form of NAD(P)+ . 3.3. Oligomeric state of His10 -TbˇHBDH
Fig. 3. TbHBDH binds oxidized cofactor in a cooperative fashion. A Hill plot of NAD+ and NADP+ binding. Data were fit to the Hill equation using Sigma Plot. Closed black circles represent NAD+ data and open squares represent NADP+ data.
The cooperative behavior of His10 -TbHBDH prompted us to examine the oligomeric state of the enzyme. To date, all characterized HBDHs are tetramers. The subunit molecular weight of His10 -TbHBDH is 30.3 kDa. Gel filtration using ACA34 resin, which has a fractionation range of 20–250 kDa, was carried out at 4 ◦ C. To ensure the active oligomeric state of enzyme was detected, elution of His10 -TbHBDH from the column was monitored by enzyme activity using saturating amounts of d-3-hydroxybutyrate and NAD+ . The retention volume corresponding to the peak His10 TbHBDH activity was used to determine the apparent molecular weight of the active enzyme (Fig. 4). To determine the molecular weight, (Ve /V0 ) vs. log molecular weight was plotted for the protein standards (inset, Fig. 4). His10 -TbHBDH was found to have an apparent molecular weight of 112 kDa for the active enzyme. This weight is intermediate to that expected for either the trimeric (90 kDa) or tetrameric (120 kDa) forms of the enzyme. We con-
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Fig. 4. His10 -TbHBDH is a multimeric enzyme. The elution profile of recombinant His10 -TbHBDH activity from an Ultrogel AcA-34 column. The oligomeric state of His10 TbHBDH was determined using the following protein standards: bovine intestinal alkaline phosphatase (160 kDa), bovine blood gamma-globin (140 kDa), bovine albumin (132 and 66 kDa), and egg grade III albumin (45 kDa). The maximum Abs280 of the elution of the standards are indicated above with arrows and weights. The column void volume (V0 ) was determined with blue dextran. The total column volume (Vt ) was 36 mL. The inset shows the calibration curve used to determine the apparent molecular weight of His10 -TbHBDH, which had a Ve value of 18 mL, corresponding to an apparent molecular weight of 112 kDa.
clude that His10 -TbHBDH is a tetramer and the slightly lower than expected mass can be attributed to protein shape. 3.4. TbˇHBDH RNA interference (RNAi) studies We next used RNA interference to begin to examine the role of TbHBDH in vivo in procyclic parasites. Trypanofan (http://trypanofan.path.cam.ac.uk/trypanofan/main/) was used to identify a unique region in the Tb927.10.11930 gene to target by RNAi. A small but reproducible difference in the growth was noted between the TbHBDH RNAi-induced and non-induced cells at approximately day 3 (Supplemental Fig. 5). TbHBDH depletion was confirmed through Western blot analysis. There were no gross morphological changes in the TbHBDH RNAi-induced cells and these cells continued to replicate, but at a reproducibly slower rate than the non-induced cells. Growth studies of the TbHBDH RNAi cell line were also carried out in minimal media ME-83 [31] (Supplemental Fig. 5C) and a similar growth phenotype was observed. A recent high-throughput RNA interference screen in T. brucei identified genes with essential functions [32]. This work revealed that TbHBDH is essential for T. brucei differentiation from the bloodstream form of the parasite to the procyclic form. Furthermore, the reduction of TbHBDH results in the loss of viability of blood stream form parasites 6 days post-induction. Taken together, these data reveal an important physiological role for TbHBDH throughout the parasite lifecycle: TbHBDH is important for growth in procyclic parasites, is essential in bloodstream form parasites, and is essential during differentiation. Earlier work in which acetyl:succinate CoA-transferase (ASCT) was either knocked out or depleted from the T. brucei AnTat1.1 cell line resulted in increased levels of excreted hydroxybutyrate [12]. Accordingly, we examined the interactions between TbASCT and TbHBDH. We initially constructed an RNAi cell line that targeted TbASCT in a manner identical to that described in Ref. [12], but began with the procyclic T. brucei 29-13 cell line. A reduced growth phenotype was observed upon induction of RNAi using tetracycline
(Supplemental Fig. 5D). Next, we constructed a cell line in T. brucei 29-13 that targeted both TbASCT and TbHBDH (Supplemental Fig. 5E). In this dual-knockdown cell line, a more pronounced growth phenotype was observed upon RNAi induction and the combination becomes lethal for the cells by day 8 of RNAi induction, demonstrating a synthetic lethal interaction for the two genes. 4. Conclusions Through this work, T. brucei HBDH is shown to have unique kinetic properties among HBDHs studied to date. Distinctly, the parasite enzyme is able to utilize both NAD(H) and NADP(H); furthermore, TbHBDH binds the oxidized form of the cofactor in a cooperative fashion. The reaction catalyzed by HBDH is readily reversible; thus, the cooperative binding of only NAD(P)+ may impart directionality to the reaction in vivo. We are currently working to elucidate the role of TbHBDH in vivo, an enzyme that is either important or essential in both stages of the parasite lifecycle. It is possible that TbHBDH plays a role in maintaining cellular redox balances. The unusual ability of TbHBDH to utilize NADP(H) may also allow the enzyme to contribute to the parasite’s stress response. Trypanothione protects the parasites against oxidative stress and is maintained in the reduced state by the NADPH-dependent trypanothione-disulfide reductase [33]. When physiological conditions dictate, TbHBDH conceivably could play a role in replenishing the required form of the cofactor for this essential system. Indeed, Ralstonia picketti HBDH has been shown to play a role in neutralizing redox stresses [34]. Role of funding sources This work was funded by the Research Corporation for Science Advancement (Cottrell College Science Award to J.B.P.), the Villanova Center for Undergraduate Research and Fellowships (to M.C.H.), and the Villanova University Presidential Graduate Fellowship for Underrepresented Students (to T.D.S.). None of these
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organizations had involvement in study design, data collection, data interpretation, or in preparing this manuscript. Acknowledgments We thank the Research Corporation for Science Advancement (Cottrell College Science Award to J.B.P.), the Villanova Center for Undergraduate Research and Fellowships (to M.C.H.), and the Villanova University Presidential Graduate Fellowship for Underrepresented Students (to T.D.S.) for funding. We also thank Dr. Barry Selinsky for critical reading of the manuscript. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.molbiopara.2011.07.001. References [1] McIntyre JO, Bock HG, Fleischer S. The orientation of d-beta-hydroxybutyrate dehydrogenase in the mitochondrial inner membrane. Biochim Biophys Acta 1978;513:255–67. [2] Gazzotti P, Bock H, Fleischer S. Interaction of d-beta-hydroxybutyrate apodehydrogenase with phospholipids. J Biol Chem 1975;250:5782–90. [3] Guo K, Lukacik P, Papagrigoriou E, Meier M, Lee WH, Adamski J, et al. Characterization of human DHRS6, an orphan short chain dehydrogenase/reductase enzyme: a novel, cytosolic type 2 R-beta-hydroxybutyrate dehydrogenase. J Biol Chem 2006;281:10291–7. [4] Kruger K, Lang G, Weidner T, Engel AM. Cloning and functional expression of the d-beta-hydroxybutyrate dehydrogenase gene of Rhodobacter sp. DSMZ 12077. Appl Microbiol Biotechnol 1999;52:666–9. [5] Anderson AJ, Dawes EA. Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates. Microbiol Rev 1990;54:450–72. [6] Kadouri D, Jurkevitch E, Okon Y. Involvement of the reserve material polybeta-hydroxybutyrate in Azospirillum brasilense stress endurance and root colonization. Appl Environ Microbiol 2003;69:3244–50. [7] Senior PJ, Dawes EA. The regulation of poly-beta-hydroxybutyrate metabolism in Azotobacter beijerinckii. Biochem J 1973;134:225–38. [8] Hellemond JJ, Bakker BM, Tielens AG. Energy metabolism and its compartmentation in Trypanosoma brucei. Adv Microb Physiol 2005;50:199–226. [9] Hannaert V, Bringaud F, Opperdoes FR, Michels PA. Evolution of energy metabolism and its compartmentation in Kinetoplastida. Kinetoplastid Biol Dis 2003;2:11. [10] van Weelden SW, Fast B, Vogt A, van der Meer P, Saas J, van Hellemond JJ, et al. Procyclic Trypanosoma brucei do not use Krebs cycle activity for energy generation. J Biol Chem 2003;278:12854–63. [11] van Grinsven KW, Van Den Abbeele J, Van den Bossche P, van Hellemond JJ, Tielens AG. Adaptations in the glucose metabolism of procyclic Trypanosoma brucei isolates from tsetse flies and during differentiation of bloodstream forms. Eukaryot Cell 2009;8:1307–11. [12] Riviere L, van Weelden SW, Glass P, Vegh P, Coustou V, Biran M, et al. Acetyl:succinate CoA-transferase in procyclic Trypanosoma brucei, gene identification and role in carbohydrate metabolism. J Biol Chem 2004;279: 45337–46. [13] Van Hellemond JJ, Opperdoes FR, Tielens AG. Trypanosomatidae produce acetate via a mitochondrial acetate:succinate CoA transferase. Proc Natl Acad Sci USA 1998;95:3036–41.
[14] Riviere L, Moreau P, Allmann S, Hahn M, Biran M, Plazolles N, et al. Acetate produced in the mitochondrion is the essential precursor for lipid biosynthesis in procyclic trypanosomes. Proc Natl Acad Sci USA 2009;106:12694–9. [15] Coustou V, Biran M, Breton M, Guegan F, Riviere L, Plazolles N, et al. Glucoseinduced remodeling of intermediary and energy metabolism in procyclic Trypanosoma brucei. J Biol Chem 2008;283:16342–54. [16] Kanehisa M, Goto S. KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res 2000;28:27–30. [17] Wickstead B, Ersfeld K, Gull K. Targeting of a tetracycline-inducible expression system to the transcriptionally silent minichromosomes of Trypanosoma brucei. Mol Biochem Parasitol 2002;125:211–6. [18] Wirtz E, Hoek M, Cross GA. Regulated processive transcription of chromatin by T7 RNA polymerase in Trypanosoma brucei. Nucleic Acids Res 1998;26:4626–34. [19] McCulloch R, Vassella E, Burton P, Boshart M, Barry JD. Transformation of monomorphic and pleomorphic Trypanosoma brucei. Methods Mol Biol 2004;262:53–86. [20] Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 1994;22:4673–80. [21] Kavanagh KL, Jornvall H, Persson B, Oppermann U. Medium- and short-chain dehydrogenase/reductase gene and protein families: the SDR superfamily: functional and structural diversity within a family of metabolic and regulatory enzymes. Cell Mol Life Sci 2008;65:3895–906. [22] Filling C, Berndt KD, Benach J, Knapp S, Prozorovski T, Nordling E, et al. Critical residues for structure and catalysis in short-chain dehydrogenases/reductases. J Biol Chem 2002;277:25677–84. [23] Ito K, Nakajima Y, Ichihara E, Ogawa K, Katayama N, Nakashima K, et al. d-3Hydroxybutyrate dehydrogenase from Pseudomonas fragi: molecular cloning of the enzyme gene and crystal structure of the enzyme. J Mol Biol 2006;355:722–33. [24] Hoque MM, Shimizu S, Juan EC, Sato Y, Hossain MT, Yamamoto T, et al. Structure of d-3-hydroxybutyrate dehydrogenase prepared in the presence of the substrate d-3-hydroxybutyrate and NAD+ . Acta Crystallogr Sect F: Struct Biol Cryst Commun 2009;65:331–5. [25] Paithankar KS, Feller C, Kuettner EB, Keim A, Grunow M, Strater N. Cosubstrateinduced dynamics of d-3-hydroxybutyrate dehydrogenase from Pseudomonas putida. FEBS J 2007;274:5767–79. [26] Nakashima K, Ito K, Nakajima Y, Yamazawa R, Miyakawa S, Yoshimoto T. Closed complex of the d-3-hydroxybutyrate dehydrogenase induced by an enantiomeric competitive inhibitor. J Biochem 2009;145:467–79. [27] Jones A, Faldas A, Foucher A, Hunt E, Tait A, Wastling JM, et al. Visualisation and analysis of proteomic data from the procyclic form of Trypanosoma brucei. Proteomics 2006;6:259–67. [28] Tucker GA, Dawson AP. The kinetics of rat liver and heart mitochondrial betahydroxybutyrate dehydrogenase. Biochem J 1979;179:579–81. [29] Persson B, Kallberg Y, Oppermann U, Jornvall H. Coenzyme-based functional assignments of short-chain dehydrogenases/reductases (SDRs). Chem Biol Interact 2003;143–144:271–8. [30] Kluger R, Nakaoka K, Wing-Cheong T. Substrate analog studies of the specificity and catalytic mechanism of d-3-hydroxybutyrate dehydrogenase. Journal of the American Chemical Society 1978;100:7388–92. [31] Seebeck T, Kurath U. Two simple media for biochemical experimentation with cultured procyclic Trypanosoma brucei. Acta Trop 1985;42:127–36. [32] Alsford S, Turner D, Obado S, Sanchez-Flores A, Glover L, Berriman M, et al. High throughput phenotyping using parallel sequencing of RNA interference targets in the African trypanosome. Genome Res 2011. [33] Krauth-Siegel RL, Comini MA. Redox control in trypanosomatids, parasitic protozoa with trypanothione-based thiol metabolism. Biochim Biophys Acta 2008;1780:1236–48. [34] Takanashi M, Shiraki M, Saito T. Characterization of a novel 3-hydroxybutyrate dehydrogenase from Ralstonia pickettii T1. Antonie Van Leeuwenhoek 2009;95:249–62.
Contents lists available at ScienceDirect
Molecular & Biochemical Parasitology
The characterization of a unique Trypanosoma brucei -hydroxybutyrate dehydrogenase Tina D. Shah, Meghan C. Hickey, Kathryn E. Capasso, Jennifer B. Palenchar ∗ Department of Chemistry, Villanova University, 800 E. Lancaster Ave., Villanova, PA 19085, United States
a r t i c l e
i n f o
Article history: Received 2 May 2011 Received in revised form 13 June 2011 Accepted 2 July 2011 Available online 7 July 2011 Keywords: Trypanosoma brucei Hydroxybutyrate dehydrogenase Cofactor preference Short dehydrogenase/reductase superfamily Energy metabolism
a b s t r a c t A putative -hydroxybutyrate dehydrogenase (HBDH) ortholog was identified in Trypanosoma brucei, the unicellular eukaryotic parasite responsible for causing African Sleeping Sickness. The trypanosome enzyme has greater sequence similarity to bacterial sources of soluble HBDH than to membranebound Type I HBDH found in higher eukaryotes. The HBDH gene was cloned from T. brucei genomic DNA and active, recombinant His-tagged enzyme (His10 -TbHBDH) was purified to approximate homogeneity from E. coli. HBDH catalyzes the reversible NADH-dependent conversion of acetoacetate to d-3-hydroxybutyrate. In the direction of d-3-hydroxybutyrate formation, His10 -TbHBDH has a kcat value of 0.19 s−1 and a KM value of 0.69 mM for acetoacetate. In the direction of acetoacetate formation, His10 TbHBDH has a kcat value of 11.2 s−1 and a KM value of 0.65 mM for d-3-hydroxybutyrate. Cofactor preference was examined and His10 -TbHBDH utilizes both NAD(H) and NADP(H) almost equivalently, distinguishing the parasite enzyme from other characterized HBDHs. Furthermore, His10 -TbHBDH binds NAD(P)+ in a cooperative fashion, another unique characteristic of trypanosome HBDH. The apparent native molecular weight of recombinant His10 -TbHBDH is 112 kDa, corresponding to tetramer, as determined through size exclusion chromatography. RNA interference studies in procyclic trypanosomes were carried out to evaluate the importance of TbHBDH in vivo. Upon knockdown of TbHBDH, a small reduction in parasite growth was observed suggesting HBDH has an important physiological role in T. brucei. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Beta hydroxybutyrate dehydrogenase (HBDH, E.C. 1.1.1.30) catalyzes the NAD(H)-dependent interconversion of acetoacetate to d-3-hydroxybutyrate. HBDH is involved in the synthesis of ketone bodies, namely acetoacetate, d-3-hydroxybutyrate, and acetone, from acetyl-CoA. Ketone body production occurs when carbohydrates are scarce and acetyl-CoA is present in excess, such as during periods of starvation. When acetyl-CoA is in excess it does not enter the TCA cycle; rather, it is metabolized to yield ketone bodies. d-3-Hydroxybutyrate synthesized by HBDH in the liver is exported to extrahepatic tissues, where HBDH participates in the pathway that converts d-3-hydroxybutyrate back to acetyl-CoA, by catalyzing the conversion of d-3-hydroxybutyrate to acetoacetate. Acetyl-CoA is subsequently utilized in the TCA cycle in extrahepatic tissues, ultimately providing fuel for the organism.
Abbreviations: HBDH, beta-hydroxybutyrate dehydrogenase; Tb, Trypanosoma brucei; RNAi, RNA interference. ∗ Corresponding author. Tel.: +1 610 519 4868; fax: +1 610 519 7167. E-mail address: [email protected] (J.B. Palenchar). 0166-6851/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.molbiopara.2011.07.001
There are fundamental differences between eukaryotic and bacterial HBDHs. Eukaryotic HBDH (Type 1) is bound to the inner mitochondrial membrane and requires lipid for activity [1,2]. More recently, a cytosolic eukaryotic Type 2 HBDH (human DHRS6) has been discovered, although its physiological function is yet to be determined [3]. Bacterial HBDHs, by contrast, are not membrane bound and do not require lipid for activation [4]. In bacteria, HBDH is part of the poly-hydroxybutyrate (PHB) cycle. Many bacteria possess a complement of enzymes to polymerize d-3-hydroxybutyrate to form PHB and subsequently breakdown the PHB polymer according to the nutritional state of the bacteria [5]. Poly-hydroxybutyrate serves as an energy store, protects bacteria from stresses [6], and functions as an electron and carbon sink [7]. Trypanosoma brucei is the eukaryotic, unicellular protozoan that causes African Sleeping Sickness. The parasite’s lifecycle is split between its tsetse fly vector and its vertebrate host. There are unique features to the parasite’s energy metabolism in these two different environments. In the vertebrate host, the bloodstream form of the parasite relies solely upon glycolysis for energy generation; pyruvate is the excreted end product [8,9]. The tsetse fly midgut procyclic form of the parasite employs a more complex energy metabolism, wherein both glucose and amino acids are degraded. In procyclic parasites, acetate and succinate are the major
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end products excreted [10,11]. Acetyl-CoA is not degraded to CO2 through the TCA cycle; rather, acetyl-CoA is metabolized through a two-enzyme cycle comprised of succinate:acetate CoA-transferase (ASCT) and succinyl CoA synthetase, giving rise to the production of ATP and succinate [12,13]. Another unique aspect of trypanosome metabolism is the ability of procyclic parasites to utilize acetate for the production of cytosolic acetyl-CoA by the enzyme acetyl-CoA synthetase to meet the demands of de novo fatty acid synthesis [14]. We have identified a putative HBDH ortholog in Trypanosoma species that resembles the bacterial sources of the enzyme, but there are no apparent orthologs of the enzymes involved in the bacterial PHB cycle present in trypanosomes. T. brucei has been reported to excrete low levels of d-3-hydroxybutyrate [12,15], indicating a functional HBDH. Curiously, the related trypanosomatid Leishmania species do not possess an identifiable HBDH ortholog, based on database mining. Moreover, the vast majority of singlecelled eukaryotes lack a HBDH ortholog. Among protists, only T. brucei, Trypanosoma cruzi, Tetrahymena thermophila, and Dictyostelium discoideum have an apparent HBDH ortholog, while among fungi, only Aspergillus fumigates contains an identifiable HBDH ortholog [16]. Thus, the function of HBDH in trypanosomatid metabolism is unclear. To characterize the trypanosome HBDH, a recombinant form of the T. brucei HBDH protein was expressed, isolated, and characterized kinetically to assess the activity of the putative trypanosome HBDH. An unusual cofactor preference was observed and cooperative binding of the oxidized form of the cofactor was experimentally determined, both traits unique to the parasite enzyme. The importance of TbHBDH in vivo was assessed through RNA interference studies in procyclic parasites. Parasites in which TbHBDH is depleted exhibit slowed growth, indicating an important role for this enzyme in vivo. 2. Materials and methods 2.1. Cloning, expression and purification of T. brucei ˇHBDH The full length TbHBDH gene (GeneDB ID: Tb927.10.11930) was amplified from T. brucei 427 wild-type genomic DNA and cloned into pET16b (Novagen). The construct, pMH1, encodes for an amino-terminal 10 histidine-tagged TbHBDH. The sequences of all constructs were verified by DNA sequencing (Genewiz, Inc.). Soluble His10 -TbHBDH was overexpressed in Rosetta 2 cells (Novagen) grown at 27 ◦ C, induced with 50 M IPTG for 12 h, and purified to approximate homogeneity using Ni affinity chromatography. Purified His10 -TbHBDH was stored stably without loss of activity at −20 ◦ C in 50 mM sodium phosphate, 120 mM sodium chloride, and 250 mM imidazole, pH 8.5 containing 50% glycerol (storage buffer). Recombinant His10 -TbHBDH was used to generate custom polyclonal antibodies in rabbits (Lampire Biologicals, Pipersville, PA). 2.2. Substrate determination, kinetic analysis and inhibition studies
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formation, the reaction contained 70 g of His10 -TbHBDH and varying amounts of either acetoacetate or NAD(P)H in 50 mM citric acid, pH 6.0. d-3-Hydroxybutyrate formation was monitored spectrophotometrically at 370 nm due to high absorbance limitations at 340 nm. We determined and used a molar extinction coefficient of 2567 M−1 cm−1 for NAD(P)H at 370 nm. Kinetic constants were determined by using either a Lineweaver–Burk plot or by fitting the Hill equation to the data (SigmaPlot v. 11, Jandel Corporation). All kinetic constants were determined in triplicate. All other potential substrate molecules were examined for NAD+ -dependent dehydrogenase activity. l-3-Hydroxybutyrate, lactate, malonate, and cacodylate were tested as inhibitors. Enzyme activity was monitored in the presence of 70 g of His10 -TbHBDH, 4 mM NAD+ , 0.01–3.0 mM d-3-hydroxybutyrate and one of the following inihibtors: 10 mM l-hydroxybutyrate, 15 mM lactate, 1 mM cacodylate, or 15 mM malonate. Results were analyzed using Lineweaver–Burk plots. 2.3. Molecular mass determination The oligomeric state of His10 -TbHBDH was determined through size exclusion chromatography using Ultrogel AcA-34 resin equilibrated with storage buffer. Blue dextran, bovine intestinal alkaline phosphatase, gamma-globin, bovine albumin, and egg grade III albumin (all from Sigma), were applied to the column at 3 mg/mL and their elution monitored by absorbances at either 600 nm (blue dextran) or 280 nm. His10 -TbHBDH was applied to the column at 1.0 mg/mL in storage buffer. His10 -TbHBDH elution was monitored by -hydroxybutyrate dehdyrogenase activity. 2.4. RNA interference studies To target TbHBDH by RNAi, a 400 bp region from nucleotide 173 to 572 of the open reading frame of Tb927.10.11930 was amplified from T. brucei genomic DNA and inserted into p2T7177 [17] to yield pMH2. The RNAi construct for TbASCT (GeneDB ID: Tb11.02.0290) targeted the same region described in Ref. [12], which was cloned into p2T7-177 and named pMH3. Both TbHBDH and TbASCT were targeted simultaneously by inserting the TbHBDH target region into pMH3 at the HindIII site to yield pMH4. All primers can be found in supplemental Table 1. These constructs were then transfected into T. brucei procyclic cell line 29-13 [18] according the protocol of McCulloch et al. [19]. Clonal cell lines were generated through limiting dilution. RNA interference was induced through the addition of 2 g/mL tetracycline to the media daily. RNAi-induced and non-induced cells were grown in parallel and in replicate. Cell density was determined every 24 h and 8 × 106 cells were removed from the RNAi-induced and RNAi-non induced cell cultures daily for Western blot analysis. TbHBDH was detected using custom TbHBDH rabbit polyclonal antibodies (serum used at a dilution of 1:500) and goat anti-rabbit-IgG conjugated with alkaline phosphatase used at a 1:20,000 dilution. 3. Results and discussion 3.1. Identification of a Trypanosome ˇHBDH
All substrates were of reagent grade. pH rate profiles were carried out to determine the optimal pH for maximal activity in both directions. For the enzyme activity assay in the direction of acetoacetate formation, the 1 mL reaction contained 70 g of His10 TbHBDH, and varying amounts of either d-3-hydroxybutyrate or NAD(P)+ in 50 mM sodium phosphate, pH 8.5. Enzyme activity was monitored spectrophotometrically at 340 nm (Beckman D 640 Spectrophotometer), using a molar extinction coefficient of 6220 M−1 cm−1 for NAD(P)H. In the direction of hydroxybutyrate
BLAST searches of the T. brucei database (www.genedb.org) revealed one potential HBDH ortholog, Tb927.10.11930, annotated as an NAD or NADP dependent oxidoreductase, putative, short chain dehydrogenase. In an attempt to identify as many HBDH orthologs as possible, BLAST searches of the T. brucei genome with several bacterial, Type I and Type II HBDHs still yielded only Tb927.10.11930 as a significant match. This is in contrast to multicellular eukaryotes, which contain both Type I
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Fig. 1. TbHBDH resembles bacterial sources of HBDH. (A) Multiple sequence alignment of bacterial and trypanosome HBDHs. Shown in the sequence alignment are HBDH from Trypansoma brucei (GenBank ID: XP 823399), T. cruzi (GenBank ID: XP 811840), Trypanosoma vivax (GeneDB ID: TvY486 1011560), Pseudomonas fragi (GenBank ID: BAD86668.1), Azospirillum brasilense (GenBank ID: AF355575.1), Ralstonia picketti (GenBank ID: BAE72685), and A. vinelandii (YP 002798947.1). A black background indicates complete conservation of an amino acid, a gray background with white lettering indicates the amino acid is conserved in 80% of the sequences shown, and a gray background with black lettering indicates the amino acid is conserved in 60% of the sequences shown. Residues of the catalytic tetrad are indicated with an *, the conserved TGXXXGXG motif found in the SDR family is indicated with a black line above the alignment. Residues involved in NAD+ binding are indicated with a closed circle, residues involved in substrate binding are indicated with an open circle, and the hinge residue involved in conformational change is indicated by an open square. (B) Phylogentic tree of bacterial, trypanosomal, and eukaryotic type 1 and 2 HBDHs. In addition to the bacterial and trypanosome enzymes from (A) are Dania rerio type 1 (GenBank ID: NP 001082978.1) and type 2 (GenBank ID: NP 001017809.1), Rattus norvegicus type 1 (GenBank ID: NP 446447.2) and 2 (GenBank ID: NP 001099943.1), and Homo sapiens type 1 (GenBank ID: Q02338.3) and 2 (GenBank ID: NP 064524).
and Type II HBDH. Bacteria possess only one HBDH. Only the Trypanosoma spp. of the parasite have the ortholog; curiously, Leishmania spp. do not. By sequence analysis, the eukaryotic trypanosome protein more closely resembles the bacterial sources of HBDH (Fig. 1A and B). The most closely related annotated HBDH to T. brucei HBDH is Azotobacter vinelandii HBDH (GenBank ID: YP 002798947.1). The two enzymes are 55% identical and 19% strongly similar [20] in their sequences. Notably, T. bru-
cei hydroxymethyl glutaryl (HMG)-CoA synthetase (GenBank ID: XP 847441.1) and HMG-CoA lyase (GenBank ID: XP 844420.1), which, along with HBDH, comprise the pathway required for ketone body synthesis, also appear bacterial in origin by BLAST and phylogenetic analysis. Multiple sequence alignments and phylogenetic trees comparing bacterial, trypanosome, and eukaryotic sources of HMG-CoA synthetase and lyase are provided in Supplemental Figs. 1–4.
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a -hydroxybutyrate dehydrogenase. As a control, an unrelated His-tagged trypanosome protein was expressed, purified, and assayed in an identical fashion to that of His10 -TbHBDH. No HBDH activity was detected using this control protein, indicating the observed enzyme activity is not due to trace amounts of contaminating bacterial HBDH.
Fig. 2. Purification of His10 -TbHBDH. Purification of recombinant His10 -TbHBDH from E. coli using nickel affinity chromatography. Electrophoresis in 12% polyacrylamide containing 0.1% sodium dodecyl sulfate. Protein samples were: (S) protein standards, (1) pellet post-sonication, (2) clarified supernatant post-sonication, (3) column flow-through, (4) column wash 20 mM imidazole, (5) column wash 50 mM imidazole, (6) 250 mM imidazole elution, and (7) 250 mM imidazole elution. The position of His10 -TbHBDH is indicated with an asterisk. The theoretical molecular weight of recombinant His10 -TbHBDH is 30.3 kDa.
Based on the bacterial nature of the T. brucei enzyme, we compared T. brucei HBDH with known bacterial HBDHs to determine whether key residues for enzyme function are conserved in the trypanosome protein (Fig. 1A). Beta-hydroxybutyrate dehydrogenase is a member of the short-chain dehydrogenases/reductases (SDR) superfamily, a broad group of enzymes that catalyze NAD(P)(H)dependent oxidoreductions [21]. In this superfamily, NAD(P)(H) binding occurs in the N-terminal region of the protein and is associated with the GXXXGXG motif in the ‘classical’ subfamily [21]. This motif is present in the parasite sources of the enzyme (Fig. 1A). Short-chain dehydrogenases/reductases superfamily members also contain a well-conserved catalytic tetrad comprised of residues Asn114, Ser142, Tyr155, and Lys159, which is conserved in trypanosome HBDH [22]. (All residue numbering corresponds to the Pseudomonas fragi HBDH enzyme.) Several crystal structures of HBDH, in the absence and presence of substrate analog and cofactor, have been obtained from bacterial enzyme sources [23–26]. These structures provide a wealth of information related to substrate binding, cofactor binding, and conformational change in the enzyme. The amino acids involved in binding hydroxybutyrate [26] and the cofactor NAD+ [23] are conserved in most cases or contain a conservative substitution in the parasite enzyme (Fig. 1A). The only notable substitution of an amino acid involved in cofactor binding is found at position 36, where phenylalanine is present in bacterial sources of the enzyme. Positively charged lysine or arginine occupies the equivalent position in the trypanosome protein. Finally, an amino acid key to the conformational change from open to closed form of the enzyme, Thr190, is conserved in the trypanosome enzymes [26]. The conservation of important catalytic, binding, and structural residues in the trypanosome protein, coupled with knowledge that the putative T. brucei HBDH is expressed in the procyclic form of the parasite based on a previous large scale protein expression study [27], prompted us to further study this protein. The putative T. brucei HBDH was cloned from T. brucei genomic DNA, expressed as soluble, recombinant His10 -TbHBDH in bacteria, and purified to approximate homogeneity using nickel affinity chromatography (Fig. 2). 3.2. Kinetic analysis of the putative T. brucei ˇHBDH Beta-hydroxybutyrate dehydrogenase catalyzes the reversible, NAD(H)-dependent conversion of acetoacetate to d-3hydroxybutyrate. The recombinant His10 -TbHBDH protein exhibits activity with both substrates confirming its identity as
3.2.1. Kinetic characterization in the direction of 3-d-hydroxybutyrate formation In the direction of d-3-hydroxybutyrate formation, the pH for maximum activity was determined to be 6.0. In this direction, recombinant His10 -TbHBDH was found to have a specific activity value of 0.38 mol/min/mg and a KM value of 0.69 mM for acetoacetate (Table 1). The turnover number in this direction is 0.19 s−1 . From the limited kinetic data that is published for activity in this direction, the His10 -TbHBDH KM value for acetoacetate is similar to the values for both bacterial and eukaryotic sources of the enzyme; for example, P. fragi, 0.37 mM [26] and R. norveticus heart, 0.31 mM [28] However, the turnover number for the T. brucei enzyme is lower; for example, the kcat value reported for the P. fragi enzyme is 120 s−1 . Cofactor preference was also examined. During d-3hydroxybutyrate formation, NADH is oxidized. Unexpectedly, His10 -TbHBDH binds NADH and NADPH almost equivalently, with KM values of 0.05 mM for each. Furthermore, enzyme activity is highly similar between the two cofactors, yielding specific activity values of 0.19 and 0.36 mol/min/mg for NADH and NADPH, respectively. His10 -TbHBDH binds NADH with similar affinity as other HBDHs; for comparison, the P. fragi HBDH KM value for NADH is 0.01 mM [26] and R. norveticus heart HBDH KM value is 0.029 mM [28]. Importantly, to the best of our knowledge, this is the first demonstration of a HBDH that can utilize NADPH. 3.2.2. Kinetic characterization in the direction of acetoacetate formation. In the direction of acetoacetate formation, the reaction was determined to occur optimally at pH 8.5. Recombinant His10 TbHBDH has a specific activity value of 22.25 mol/min/mg, a KM value of 0.65 mM for d-3-hydroxybutyrate, and a kcat value of 11.23 s−1 (Table 1). The trypanosome enzyme binds d-3hydroxybutyrate comparably to other sources of the enzyme, but the activity is lower than most values reported. For example, the P. fragi enzyme has a KM value of 0.8 mM for d-3-hydroxybutyrate but a kcat value of 370 s−1 [26]. However, His10 -TbHBDH activity is still 350-fold higher than the cytosolic human Type II HBDH [3]. Cofactor preference was also examined in the direction of acetoacetate formation and the enzyme was able to utilize both NAD+ and NADP+ . Surprisingly, the trypanosome enzyme does not obey Michaelis–Menten kinetics when d-3-hydroxybutyrate is present at saturating levels and either NAD+ or NADP+ concentrations are varied. A sigmoidal plot of activity vs. cofactor concentration was observed, suggesting cooperativity. The data were fit to the Hill equation using Sigmaplot and the Hill coefficients were determined to be 2.13 ± 0.10 and 2.01 ± 0.11 for NAD+ and NADP+ , respectively (Fig. 3). As a control, the data from the determination of the kinetic parameters for both acetoacetate and d-3-hydroxybutyrate, which exhibited Michaelis–Menten kinetics, was fit to the Hill equation and yielded Hill coefficients of approximately 1, as expected for non-cooperativity. Based on our survey of the literature, the trypanosome enzyme is the first-reported HBDH that exhibits cooperativity. Furthermore, the ability of TbHBDH to use NADP+ is of interest. The only other HBDH reported to be able to utilize NADP+ is the P. fragi enzyme [23]. Other characterized HBDHs are not reported to utilize NADP(H). For P. fragi HBDH, the KM value for NADP+ is 57fold higher than the value for NAD+ . The crystal structure of P. fragi
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Table 1 Substrate specificity and summary of His10 -TbHBDH kinetics in the directions of acetoacetate and d-3-hydroxybutyrate formation. Substrate or cofactor
KM (mM)
Specific activity (mol/min × mg)
kcat (s−1 )
kcat /KM (s−1 mM−1 )
d-3-Hydroxybutyrate l-3-Hydroxybutyrate dl-Lactate Malonate d or l-serine d or l-threonine NAD+ b NADP+ b
0.65 ± 0.34
22.25 ± 2.25 n.d.a n.d. n.d. n.d. n.d.
11.23
19.68
Acetoacetate NADH NADPH
0.69 ± 0.16 0.05 ± 0.01 0.05 ± 0.02
0.38 ± 0.06 0.19 ± 0.01 0.35 ± 0.1
0.19 0.10 0.18
0.28 1.81 3.52
a b
No detectable activity indicated by n.d. Non-Michaelis–Menten kinetics observed.
HBDH with NAD+ bound indicates that the hydroxyl groups of the NAD+ ribose ring form hydrogen bonds with the side chain hydroxyl of Thr13 and the Phe36 main chain nitrogen. In the trypanosome enzyme, Phe36 is replaced with either an Arg or a Lys, while in other HBDHs representing Type I, II, or bacterial enzymes, a hydrophobic residue is present at the corresponding position. Phenylalanine 36 is found in a region of the enzyme sequence that corresponds to the second beta sheet in the structure. The coenzyme preference of SDR family members has been extensively evaluated [29]. In this work, many NADP(H) preferring enzymes contain a basic residue following this particular beta sheet. Given that the trypanosome enzymes contain either a Lys or Arg at this position, we hypothesize the ability of the trypanosome enzyme to utilize NADP(H) results from having a basic amino acid in the region following the second beta sheet. Likely, the negatively charged phosphate moiety of NADPH can interact with the positively charged side chains of either Lys or Arg. When saturating amounts of either cofactor were present and the concentration of d-3-hydroxybutyrate was varied, the enzyme exhibited Michaelis–Menten kinetics; thus, the cooperative behavior is limited to cofactor binding. Indeed, HBDH enzymes are known to bind cofactor and substrate sequentially, with cofactor binding preceding substrate binding [30]. We speculate that
TbHBDH cooperatively binds cofactor and is subsequently poised to bind substrate. Indeed, a recent structure of the P. fragi enzyme crystallized in the presence of NAD+ only suggests that cofactor binding readies the enzyme to adopt the closed conformation associated with catalysis [26] Members of the SDR superfamily catalyze a broad range of reactions. Furthermore, the activity of HBDHs is known to extend beyond the substrates d-3-hydroxybutyrate and acetoacetate; for example, some HBDHs are able to utilize threonine as substrate, albeit with greatly reduced activity [23]. Thus, the trypanosome enzyme was tested for activity with l-3-hydroxybutyrate, dllactate, malonate, d and l-serine, and d and l-threonine in the presence of saturating amounts of NAD+ . His10 -TbHBDH yielded no detectable activity with these potential substrate molecules. Finally, we examined l-3-hydroxybutyrate, dl-lactate, and malonate as potential inhibitors of His10 -TbHBDH. All three molecules inhibited the enzyme in a competitive fashion and yielded Ki values of 2.6, 10.8, and 1.8 mM, respectively. These values correspond well to the values determined for the P. fragi enzyme for these inhibitors: 1.6 mM, 0.9 mM, 3.8 mM, respectively, further supporting the similarity of this eukaryotic enzyme to bacterial HBDH [23]. Thus, the kinetic characterization of trypanosomal HBDH reveals that the enzyme resembles characterized HBDHs with respect to substrate affinity and inhibition, but is strikingly different in its cofactor preference and cooperative binding of the oxidized form of NAD(P)+ . 3.3. Oligomeric state of His10 -TbˇHBDH
Fig. 3. TbHBDH binds oxidized cofactor in a cooperative fashion. A Hill plot of NAD+ and NADP+ binding. Data were fit to the Hill equation using Sigma Plot. Closed black circles represent NAD+ data and open squares represent NADP+ data.
The cooperative behavior of His10 -TbHBDH prompted us to examine the oligomeric state of the enzyme. To date, all characterized HBDHs are tetramers. The subunit molecular weight of His10 -TbHBDH is 30.3 kDa. Gel filtration using ACA34 resin, which has a fractionation range of 20–250 kDa, was carried out at 4 ◦ C. To ensure the active oligomeric state of enzyme was detected, elution of His10 -TbHBDH from the column was monitored by enzyme activity using saturating amounts of d-3-hydroxybutyrate and NAD+ . The retention volume corresponding to the peak His10 TbHBDH activity was used to determine the apparent molecular weight of the active enzyme (Fig. 4). To determine the molecular weight, (Ve /V0 ) vs. log molecular weight was plotted for the protein standards (inset, Fig. 4). His10 -TbHBDH was found to have an apparent molecular weight of 112 kDa for the active enzyme. This weight is intermediate to that expected for either the trimeric (90 kDa) or tetrameric (120 kDa) forms of the enzyme. We con-
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Fig. 4. His10 -TbHBDH is a multimeric enzyme. The elution profile of recombinant His10 -TbHBDH activity from an Ultrogel AcA-34 column. The oligomeric state of His10 TbHBDH was determined using the following protein standards: bovine intestinal alkaline phosphatase (160 kDa), bovine blood gamma-globin (140 kDa), bovine albumin (132 and 66 kDa), and egg grade III albumin (45 kDa). The maximum Abs280 of the elution of the standards are indicated above with arrows and weights. The column void volume (V0 ) was determined with blue dextran. The total column volume (Vt ) was 36 mL. The inset shows the calibration curve used to determine the apparent molecular weight of His10 -TbHBDH, which had a Ve value of 18 mL, corresponding to an apparent molecular weight of 112 kDa.
clude that His10 -TbHBDH is a tetramer and the slightly lower than expected mass can be attributed to protein shape. 3.4. TbˇHBDH RNA interference (RNAi) studies We next used RNA interference to begin to examine the role of TbHBDH in vivo in procyclic parasites. Trypanofan (http://trypanofan.path.cam.ac.uk/trypanofan/main/) was used to identify a unique region in the Tb927.10.11930 gene to target by RNAi. A small but reproducible difference in the growth was noted between the TbHBDH RNAi-induced and non-induced cells at approximately day 3 (Supplemental Fig. 5). TbHBDH depletion was confirmed through Western blot analysis. There were no gross morphological changes in the TbHBDH RNAi-induced cells and these cells continued to replicate, but at a reproducibly slower rate than the non-induced cells. Growth studies of the TbHBDH RNAi cell line were also carried out in minimal media ME-83 [31] (Supplemental Fig. 5C) and a similar growth phenotype was observed. A recent high-throughput RNA interference screen in T. brucei identified genes with essential functions [32]. This work revealed that TbHBDH is essential for T. brucei differentiation from the bloodstream form of the parasite to the procyclic form. Furthermore, the reduction of TbHBDH results in the loss of viability of blood stream form parasites 6 days post-induction. Taken together, these data reveal an important physiological role for TbHBDH throughout the parasite lifecycle: TbHBDH is important for growth in procyclic parasites, is essential in bloodstream form parasites, and is essential during differentiation. Earlier work in which acetyl:succinate CoA-transferase (ASCT) was either knocked out or depleted from the T. brucei AnTat1.1 cell line resulted in increased levels of excreted hydroxybutyrate [12]. Accordingly, we examined the interactions between TbASCT and TbHBDH. We initially constructed an RNAi cell line that targeted TbASCT in a manner identical to that described in Ref. [12], but began with the procyclic T. brucei 29-13 cell line. A reduced growth phenotype was observed upon induction of RNAi using tetracycline
(Supplemental Fig. 5D). Next, we constructed a cell line in T. brucei 29-13 that targeted both TbASCT and TbHBDH (Supplemental Fig. 5E). In this dual-knockdown cell line, a more pronounced growth phenotype was observed upon RNAi induction and the combination becomes lethal for the cells by day 8 of RNAi induction, demonstrating a synthetic lethal interaction for the two genes. 4. Conclusions Through this work, T. brucei HBDH is shown to have unique kinetic properties among HBDHs studied to date. Distinctly, the parasite enzyme is able to utilize both NAD(H) and NADP(H); furthermore, TbHBDH binds the oxidized form of the cofactor in a cooperative fashion. The reaction catalyzed by HBDH is readily reversible; thus, the cooperative binding of only NAD(P)+ may impart directionality to the reaction in vivo. We are currently working to elucidate the role of TbHBDH in vivo, an enzyme that is either important or essential in both stages of the parasite lifecycle. It is possible that TbHBDH plays a role in maintaining cellular redox balances. The unusual ability of TbHBDH to utilize NADP(H) may also allow the enzyme to contribute to the parasite’s stress response. Trypanothione protects the parasites against oxidative stress and is maintained in the reduced state by the NADPH-dependent trypanothione-disulfide reductase [33]. When physiological conditions dictate, TbHBDH conceivably could play a role in replenishing the required form of the cofactor for this essential system. Indeed, Ralstonia picketti HBDH has been shown to play a role in neutralizing redox stresses [34]. Role of funding sources This work was funded by the Research Corporation for Science Advancement (Cottrell College Science Award to J.B.P.), the Villanova Center for Undergraduate Research and Fellowships (to M.C.H.), and the Villanova University Presidential Graduate Fellowship for Underrepresented Students (to T.D.S.). None of these
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organizations had involvement in study design, data collection, data interpretation, or in preparing this manuscript. Acknowledgments We thank the Research Corporation for Science Advancement (Cottrell College Science Award to J.B.P.), the Villanova Center for Undergraduate Research and Fellowships (to M.C.H.), and the Villanova University Presidential Graduate Fellowship for Underrepresented Students (to T.D.S.) for funding. We also thank Dr. Barry Selinsky for critical reading of the manuscript. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.molbiopara.2011.07.001. References [1] McIntyre JO, Bock HG, Fleischer S. The orientation of d-beta-hydroxybutyrate dehydrogenase in the mitochondrial inner membrane. Biochim Biophys Acta 1978;513:255–67. [2] Gazzotti P, Bock H, Fleischer S. Interaction of d-beta-hydroxybutyrate apodehydrogenase with phospholipids. J Biol Chem 1975;250:5782–90. [3] Guo K, Lukacik P, Papagrigoriou E, Meier M, Lee WH, Adamski J, et al. Characterization of human DHRS6, an orphan short chain dehydrogenase/reductase enzyme: a novel, cytosolic type 2 R-beta-hydroxybutyrate dehydrogenase. J Biol Chem 2006;281:10291–7. [4] Kruger K, Lang G, Weidner T, Engel AM. Cloning and functional expression of the d-beta-hydroxybutyrate dehydrogenase gene of Rhodobacter sp. DSMZ 12077. Appl Microbiol Biotechnol 1999;52:666–9. [5] Anderson AJ, Dawes EA. Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates. Microbiol Rev 1990;54:450–72. [6] Kadouri D, Jurkevitch E, Okon Y. Involvement of the reserve material polybeta-hydroxybutyrate in Azospirillum brasilense stress endurance and root colonization. Appl Environ Microbiol 2003;69:3244–50. [7] Senior PJ, Dawes EA. The regulation of poly-beta-hydroxybutyrate metabolism in Azotobacter beijerinckii. Biochem J 1973;134:225–38. [8] Hellemond JJ, Bakker BM, Tielens AG. Energy metabolism and its compartmentation in Trypanosoma brucei. Adv Microb Physiol 2005;50:199–226. [9] Hannaert V, Bringaud F, Opperdoes FR, Michels PA. Evolution of energy metabolism and its compartmentation in Kinetoplastida. Kinetoplastid Biol Dis 2003;2:11. [10] van Weelden SW, Fast B, Vogt A, van der Meer P, Saas J, van Hellemond JJ, et al. Procyclic Trypanosoma brucei do not use Krebs cycle activity for energy generation. J Biol Chem 2003;278:12854–63. [11] van Grinsven KW, Van Den Abbeele J, Van den Bossche P, van Hellemond JJ, Tielens AG. Adaptations in the glucose metabolism of procyclic Trypanosoma brucei isolates from tsetse flies and during differentiation of bloodstream forms. Eukaryot Cell 2009;8:1307–11. [12] Riviere L, van Weelden SW, Glass P, Vegh P, Coustou V, Biran M, et al. Acetyl:succinate CoA-transferase in procyclic Trypanosoma brucei, gene identification and role in carbohydrate metabolism. J Biol Chem 2004;279: 45337–46. [13] Van Hellemond JJ, Opperdoes FR, Tielens AG. Trypanosomatidae produce acetate via a mitochondrial acetate:succinate CoA transferase. Proc Natl Acad Sci USA 1998;95:3036–41.
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