key: cord-1056468-uuof00go
authors: Mochizuki, Kota; Inaoka, Daniel Ken; Mazet, Muriel; Shiba, Tomoo; Fukuda, Keisuke; Kurasawa, Hana; Millerioux, Yoann; Boshart, Michael; Balogun, Emmanuel O.; Harada, Shigeharu; Hirayama, Kenji; Bringaud, Frédéric; Kita, Kiyoshi
title: The ASCT/SCS cycle fuels mitochondrial ATP and acetate production in Trypanosoma brucei
date: 2020-08-04
journal: Biochim Biophys Acta Bioenerg
DOI: 10.1016/j.bbabio.2020.148283
sha: c82c6bf55ba7bbc29c79d6f2231ee54cbf6c97dc
doc_id: 1056468
cord_uid: uuof00go

Abstract Acetate:succinate CoA transferase (ASCT) is a mitochondrial enzyme that catalyzes the production of acetate and succinyl-CoA, which is coupled to ATP production with succinyl-CoA synthetase (SCS) in a process called the ASCT/SCS cycle. This cycle has been studied in Trypanosoma brucei (T. brucei), a pathogen of African sleeping sickness, and is involved in (i) ATP and (ii) acetate production and proceeds independent of oxygen and an electrochemical gradient. Interestingly, knockout of ASCT in procyclic form (PCF) of T. brucei cause oligomycin A-hypersensitivity phenotype indicating that ASCT/SCS cycle complements the deficiency of ATP synthase activity. In bloodstream form (BSF) of T. brucei, ATP synthase works in reverse to maintain the electrochemical gradient by hydrolyzing ATP. However, no information has been available on the source of ATP, although ASCT/SCS cycle could be a potential candidate. Regarding mitochondrial acetate production, which is essential for fatty acid biosynthesis and growth of T. brucei, ASCT or acetyl-CoA hydrolase (ACH) are known to be its source. Despite the importance of this cycle, direct evidence of its function is lacking, and there are no comprehensive biochemical or structural biology studies reported so far. Here, we show that in vitro–reconstituted ASCT/SCS cycle is highly specific towards acetyl-CoA and has a higher k cat than that of yeast and bacterial ATP synthases. Our results provide the first biochemical basis for (i) rescue of ATP synthase-deficient phenotype by ASCT/SCS cycle in PCF and (ii) a potential source of ATP for the reverse reaction of ATP synthase in BSF.

Energy metabolism of parasites is quite diverse and essential for their survival in the host [1, 2] . Human African trypanosomiasis (HAT) is a parasitic disease transmitted by the tsetse fly. HAT includes two forms: Trypanosoma brucei gambiense, which causes a slow-progressing form of the disease and is endemic in western and central Africa, and T. b. rhodesiense, which causes a rapidly progressing form endemic in eastern Africa [3] . Although five drugs are available for treating HAT, they are suboptimal in terms of safety and efficacy [4] . T. brucei also infects livestock animals such as cattle and sheep (Nagana), causing considerable economic losses in endemic countries [5, 6] . Although efforts to control African trypanosomiasis have been ongoing for a century, the disease persists, and the outlook for eradication is poor [5, 7, 8] .

During its life cycle, T. brucei undergoes developmental changes including metabolic reprogramming dependent on the host environment (Fig. S1 ). When the bloodstream form (BSF) in mammalian host, which includes long slender (LS) and short stumpy (SS) forms, is ingested by the tsetse fly, the replicative LS form dies and the SS form differentiates into a procyclic form (PCF) in the midgut [9] . The parasite then migrates to the insect salivary glands, where it differentiates into infective metacyclic trypomastigote, which is injected into mammalian hosts during blood meals [9] . In the tsetse fly, the parasite switches from glucose to amino acid metabolism in order to adapt to its new environment. In the PCF in the insect vector, the end products of energy metabolism are, acetate, succinate, alanine, pyruvate and glycine [10] [11] [12] [13] , whereas pyruvate followed by acetate and succinate are the end products in the LS and SS forms in mammalian host [13] [14] [15] . In both of the BSFs, enzymes associated J o u r n a l P r e -p r o o f ACH and SCOT reaction. Abbreviations: AcCoA, acetyl-CoA; Ace, acetate; Suc, succinate;

SucCoA, succinyl-CoA; AcAce, acetoacetate; AcAcCoA, acetoacetyl-CoA.

The ASCT/SCS cycle was initially investigated in T. brucei PCF, and is thought to have two functions: i) ATP and ii) acetate production [20, 21] . The role of the ASCT/SCS cycle in ATP production was demonstrated indirectly by the hypersensitivity of TbASCT null mutants (~1000-fold) to oligomycin A (a specific F o /F 1 -ATP synthase inhibitor) [22] , indicating that ATP production by this cycle is essential for ATP synthesis when oxidative phosphorylation (OXPHOS) is impaired. In addition, SLPHOS observed in the presence of pyruvate and succinate was abolished upon RNAi knockdown of SCS β subunit [23] , which further support the role of ATP production by ASCT/SCS cycle in PCF. From previous reports, SCS activity was detected in both BSF and PCF [24] , and shown to be essential by RNAi knockdown [23, 25] . Furthermore, the expression of TbASCT and SCS (α and β subunits) were also confirmed by proteome analyses in both BSF and PCF at levels comparable to other mitochondrial enzymes such as pyruvate dehydrogenase complex [26, 27] .

Regarding acetate production catalyzed by the ASCT/SCS cycle, glucose-and L-threonine-derived acetyl-CoA is converted into acetate by TbASCT. Mitochondrial acetate is also produced by acetyl-CoA hydrolase (TbACH) [10, 22] , which hydrolyzes acetyl-CoA to acetate and CoA (Fig. 1B) . It is important to note that, in contrast to TbASCT, TbACH has no CoA transferase activity, as previously demonstrated [22] . Most of the acetate formed by TbASCT and TbACH is excreted as an end product, but a portion of it is used to supply de novo fatty acid biosynthesis in PCF trypanosomes [28] via a process termed the "acetate shuttle", in which the acetate produced in mitochondria is transported to the cytosol, where it is re-converted to acetyl-CoA by "AMP-forming" acetyl-CoA synthase (TbACS) at the expense of ATP [15, 28] . To date, the acetate shuttle has been demonstrated only in trypanosomes and replaces the ubiquitous citrate shuttle, which does not operate in trypanosomatids [28] . This shuttling is essential for de novo biosynthesis of fatty acids, as J o u r n a l P r e -p r o o f 7

Replacement of the acetyl-CoA hydrolase gene (ACH: Tb927.3.4260) with the phleomycin (BLE) and puromycin (PAC) resistance markers via homologous recombination was performed using DNA fragments containing the resistance marker gene flanked by the ACH UTR sequences, as described previously [22] . Briefly, the pGEMt plasmid was used to clone an HpaI DNA fragment containing the BLE and PAC resistance marker genes preceded by the ACH 5"-UTR fragment (537 or 492 bp) and followed by the ACH 3"-UTR fragment (521 or 485 bp) [22] . The ACH knock-out was generated in the 427 MITat1. 2 13-90 (Hyg-Neo) monomorphic BSF cell line, which constitutively expresses the T7 RNA polymerase gene and the Tet repressor under control of a T7 RNA polymerase promoter for Tet-inducible expression [38] . Transfections were performed as previously reported, using the Nucleofector ® system [39] , and selection of drug-resistant clones, designated Δach (TetR-HYG T7RNAPOL-NEO Δach::BLE/Δach::PURO), was performed in HMI-9 medium containing hygromycin (5 µg/ml), neomycin (2.5 µg/ml), phleomycin (2.5 µg/ml), and puromycin (0.1 µg/ml). After transfection, the cells were re-suspended in 50 ml of conditioned medium and aliquoted into two 25-well titer plates; antibiotics were added the following day.

Inhibition of expression of the ASCT gene (Tb927.11.2690) by RNAi in the 427 MITat1.2 13-90 BSF strain was performed by expression of stem-loop "sense/anti-sense" RNA molecules of the targeted sequences introduced in the pHD1336 expression vector.

J o u r n a l P r e -p r o o f 8 RNA expression in the latter mutant was performed by addition of 10 g/ml tetracycline.

Total protein extracts of wild-type or mutant T. brucei BSFs (5×10 6 cells) were size-fractionated by SDS-PAGE (10%) and immunoblotted onto Immobilon-P filters (Millipore) [40] . Immunodetection was performed as described elsewhere [40, 41] using rabbit anti-ASCT (diluted 1:1000) [20] , purified rabbit anti-ACH [22] , and rabbit anti-GPDH (glycerol-3-phosphate dehydrogenase, EC 1.1.1.8; diluted 1:100) [42] as primary antibodies and anti-rabbit IgG conjugated to horseradish peroxidase (BioRad, 1:5,000 dilution) as the secondary antibody. Blots were developed using SuperSignal ® West Pico Chemiluminescent Substrate as described by the manufacturer (Thermo Scientific). Images were acquired and analyzed using a KODAK Image Station 4000 MM.

The TbASCT gene lacking the mitochondrial targeting signal (MTS; Δ1-9 residues) was amplified by PCR from genomic DNA (ILtat 1.4 strain [43] ) and inserted into the pETSUMO vector (Thermo Fisher Scientific) by TA cloning [44] according to the manufacturer"s protocol. The generated plasmid (pETSUMO/TbASCT) was used to transform chemically competent One Shot TM TOP10 (Thermo Fisher Scientific) Escherichia coli. In comparison to the TREU 927 strain, ASCT cloned from ILtat 1.4 had a single substitution of serine to asparagine at position 419, which is located at the protein surface. Next, BL21 star TM (DE3) Pre-equilibrated Ni-NTA agarose (2 ml) was added to the mixture and mixed for 1 hour.

J o u r n a l P r e -p r o o f 50-kDa cutoff; Millipore), glycerol was added to a final concentration of 50% (v/v) and kept at −30°C until use. The purification of TbASCT is summarized in Table S1 .

HsSCOT was purified by a similar protocol as that used for TbASCT purification, with minor modifications. Specifically, after induction, the cells were cultured for 16.5 hours at 37°C, the column was not washed with wash buffer containing ATP, and 50 ml of wash buffer containing 50 mM imidazole was used for the initial wash.

Protein concentration was determined using a Bio-Rad Protein Assay kit with bovine serum albumin as the standard.

The cytosolic, flow-through, elution, and tag-free fractions from each purification step were subjected to discontinuous SDS-PAGE according to Laemmli [45] . The stacking and separating gels were 4% and 12% (w/v) acrylamide, respectively. Samples were mixed 1:3 

Free thiol groups from CoA can reduce nitroblue tetrazolium salt (NBT) in the presence of phenazine methosulfate (PMS) [46, 47] . To assay TbASCT activity (in-gel ASCT-activity J o u r n a l P r e -p r o o f 0.12 mg/ml PMS were added and incubated static in the dark at room temperature.

The in vitro reconstituted ASCT/SCS cycle was assayed by quantification of released CoA (Fig. 1A) using DTNB (Sigma). In this system, DTNB reacts with one molecule of CoA, forming TNB, which can be followed at 412 nm (ε 412 = 14.0 mM −1 cm −1 ) [48] . The rate of CoA production was monitored using a V-660 spectrophotometer (JASCO) equipped with a water bath circulator (Taitec).

The dose-response range of TbASCT was determined at 30°C. ASCT activity at different concentrations of purified TbASCT, ranging from 12.5 to 200 ng/ml, was assayed in 1 ml reaction mixture (100 mM Tris-HCl pH 7.0, 10 mM sodium phosphate, 2 mM MgCl 2 , 1 mM ADP, 0.1 mM DTNB, 0.5 mM acetyl-CoA, 0.44 U/ml SCS) using a black quartz cuvette.

After addition of enzyme, the background was recorded for 5 min, and initial velocity was monitored after addition of succinate to a final concentration of 20 mM.

The optimal temperature for the TbASCT assay was also determined. ASCT activity was assayed in 100 mM Tris-HCl pH 6. 

The substrate specificity of TbASCT was determined in 96-well plates in a reaction mix containing 200 ng/ml TbASCT. CoA donors used in this study included acetyl-CoA,

malonyl-CoA, hexanoyl-CoA, octanoyl-CoA, decanoyl-CoA, and stearoyl-CoA. Two microliters of each CoA donor (10 mM) was transferred to the plate and mixed with 193 μl of reaction mix. After recording the background for 5 min, the reaction was initiated by adding 5 μl of 800 mM succinate. Each condition was assayed in quadruplicate; the ASCT activity in the presence of acetyl-CoA was set to 100%, and relative activity of TbASCT under each condition was then calculated.

HsSCOT was characterized similarly to TbASCT, as described above. Briefly, the dose-response range of HsSCOT was assayed in 1 ml of reaction mixture containing 100 mM Tris-HCl pH 8.6, 15 mM MgCl 2 , and 1 mM succinyl-CoA in the presence of 58-1860 ng of purified HsSCOT at 37°C. To start the reaction, 7.5 mM acetoacetate was added, and the increase in absorbance at 310 nm, which indicates the formation of acetoacetyl-CoA (ε 310 = 7.8 mM −1 cm −1 ) [49] , was monitored. Next, HsSCOT activity was assayed under different pH and temperature conditions to optimize the assay. The kinetic parameters (K m and V max ) for acetoacetate and succinyl-CoA were determined under optimal conditions (232 ng purified HsSCOT, 100 mM Tris-HCl pH 8.6, 15 mM MgCl 2 and 1 mM succinyl-CoA at 37°C).

TbASCT mutants were obtained by quick change (QIAGEN) according to the supplier"s protocol using pETSUMO/TbASCT as the template. After the desired plasmid was obtained, it was used to transform BL21 star TM (DE3) cells. Each mutant was expressed and purified as described for wild-type TbASCT. Before being flash-cooled in liquid nitrogen, TbASCT crystals were soaked in reservoir solution supplemented with 20% (v/v) glycerol as a cryoprotectant.

X-ray diffraction data were collected using the synchrotron radiation at Spring-8

(beamline BL44XU, Japan). X-ray diffraction data set at 2.01-Å resolution were obtained after processing and scaling using XDS and XSCALE [50] , respectively. The structure of

TbASCT was solved according to the molecular replacement method with the MOLREP [51] program using the structure of HsSCOT (PDB code 3DLX; 52% amino-acid sequence identity with TbASCT) as the initial model and refined using the REFMAC5 [52] and COOT [53] programs. Data collection and refinement statistics are summarized in Supplementary TbACH and TbASCT were expressed in the T. brucei wild type 427 strain, although at a much lower level for TbASCT compared to the PCF ( Fig. 2A and 2B, bottom panels) . This was consistent with the 6-fold higher level of TbASCT expression in PCF trypanosomes compared to LS BSFs quantified in a previous proteome study [54] . Similar result was shown

for TbACH in other studies [26, 27] .

To study the role of TbACH and TbASCT in the BSF, we have prepared mutant cell lines of either ACH knock-out, ASCT knockdown, and its combination. We generated the TbACH 

Recombinant His 6 -SUMO TbASCT was successfully expressed in E. coli BL21 star TM (DE3) cells and purified to homogeneity. Under optimal conditions, bacterial growth reached an OD 600 of 10, from which 48 g of wet cell pellet per 3.6-L culture of TB medium was obtained. Finally, after adding SUMO protease, 26 mg of tag-free TbASCT was purified with a yield of 18% and specific ASCT activity of 119 μmol/min/mg protein, corresponding to a 20-fold increase compared to the cytosolic fraction (Table S1) . Under similar conditions, we purified 0.485 mg of tag-free HsSCOT with a 6% yield and specific SCOT activity of 16.6 μmol/min/mg protein, representing a 325-fold increase compared to the cytosolic fraction (Table S2 ). According to our protocol, both enzymes were purified to homogeneity as judged by SDS-PAGE (Fig. S3 ).

As previously reported assay method of ASCT is not suitable for the detailed kinetic analysis of the enzyme, we established a new system. In the ASCT/SCS cycle, one molecule of acetyl-CoA is consumed by TbASCT, and equimolar amounts of ATP and CoA are produced by SCS (Fig. 1A ) [55] . We developed a new coupled enzyme assay for ASCT based on this cycle and the reaction of 5,5"-Dithiobis(2-nitrobenzoic acid) (DTNB) with the thiol group of CoA to produce one molecule of 2-nitro-5-thiobenzoic acid (TNB), which can be followed colorimetrically.

To validate the in vitro reconstituted ASCT/SCS cycle assay, the dependence on each assay component was investigated (Fig. S4A ). When TbASCT, acetyl-CoA, or succinate was removed, no activity was detected. However, when either SCS, ADP, phosphate or MgCl 2 was removed, a minimal amount of activity remained, ranging from 3.76 to 14.7 μmol/min/mg protein, which was consistent with the non-enzymatic degradation of Table 1 ). At a high concentration of succinate, substrate inhibition was clearly observed, with a K i of 75.7 mM (Fig. 3B) .

Next, the TbASCT reaction mechanism was examined. The reaction of TbASCT requires two substrates (acetyl-CoA and succinate). When the enzyme activity was determined by varying the concentration of one substrate under a fixed concentration of the second substrate and vice versa, double reciprocal plots of both cases exhibited parallel lines (Fig. 4A, B) ,

indicating that TbASCT follows a ping-pong bi-bi catalytic mechanism (Fig. 4C ) [56] . Values represent the average ± SE (n = 3) calculated using GraphPad Prism 7. N.D. indicates non-detectable level. 

To investigate the oligomeric state of TbASCT in solution, BN-PAGE and in-gel ASCT activity staining were performed (Fig. 4D) . Surprisingly, in-gel ASCT activity staining showed 1,000 times more sensitivity compared with CBB staining (Fig. 4D ) and can be used to detect TbASCT as low as 0.1 ng/lane.

Although acetyl-CoA is known to be a substrate for TbASCT, the specificity of CoA donors has not been demonstrated. Hence, we examined the ability of TbASCT to utilize different CoA donors (Fig. 5A ) and found that in the presence of n-propionyl-CoA, J o u r n a l P r e -p r o o f 19 acetoacetyl-CoA, or malonyl-CoA, the activity remaining relative to acetyl-CoA was 60%, 6%, and 13%, respectively. Other CoA donors tested in this study exhibited negligible CoA-transferase activity (Fig. 5A) . As TbASCT exhibited significantly high CoA-transferase activity with n-propionyl-CoA, we determined the kinetic parameters and found K m , V max , k cat values of 0.018 mM, 49.7 μmol/min/mg, and 42.9 s −1 , respectively. At n-propionyl-CoA concentrations above 0.1 mM, CoA-transferase activity was inhibited, with a K i value of 0.633 mM (Fig. 5B) . 

Because TbASCT shares 52% amino acid sequence identity with HsSCOT, and TbASCT had SCOT activity (Table S5) , we also examined whether HsSCOT exhibits ASCT activity.

Recombinant HsSCOT was purified, and after removal of the His 10 -SUMO tag, a single 52-kDa band corresponding to HsSCOT was detected by SDS-PAGE (Fig. S3B) . HsSCOT activity exhibited a linear dose response over the concentration range of 58 to 1,860 ng/ml in the assay mixture, with an R 2 value of 0.998 (Fig. S5A) ; therefore, subsequent assays were conducted using 232 ng/ml. The optimum temperature and pH for this reaction were determined to be 37°C and 8.6, respectively (Fig. S5B, C) . Under optimal conditions, purified recombinant HsSCOT followed classical Michaelis-Menten kinetics, with K m , V max , and k cat values for acetoacetate and succinyl-CoA of 0.765 mM, 121 μmol/min/mg, and 105 s − 1 and 0.971 mM, 219 μmol/min/mg, and 190 s −1 , respectively (Table S3 ). Acetoacetate exhibited substrate inhibition at high concentrations, with a K i value of 285 mM (Fig. S5D ), which is extremely high and under physiological condition [57] , inhibition by acetoacetate should be insignificant. The ASCT and SCOT activities of TbASCT and HsSCOT were also analyzed and compared, with the activity assayed under optimal conditions. The SCOT activity of WT TbASCT was 0.038 µmol/min/mg, whereas HsSCOT did not exhibit detectable ASCT activity. Thus, TbASCT exhibited 3,000-fold greater ASCT than SCOT activities.

To compare detailed structural difference between ASCT and SCOT, the crystal structure of TbASCT was investigated. The ligand-free TbASCT crystal ( Fig. S6D and S7A The results of this study demonstrate an essential role of mitochondrial acetate production in the T. brucei BSF energy metabolism and provide the first biochemical characterization of an ASCT family enzyme. The gene encoding T. brucei ASCT was identified in 2004 [20] followed by ACH [22] , it has been shown to play an essential role in mitochondrial acetate production. The acetate production catalyzed by ASCT is coupled to the generation of ATP via the ASCT/SCS cycle [22] . Although the RNAi ASCT.i mutant BSF lacks a growth phenotype (Fig. 2) , it has been previously demonstrated in PCF that the knockdown of F 1 β subunit of F o /F 1 -ATP synthase is lethal in Δasct background, indicating that TbASCT and ATP synthase are both responsible for ATP production in mitochondria.

Since in BSF, the ATP synthase functions to maintain the electrochemical gradient by pumping protons using ATP (reverse reaction) [16, 17] , the TbASCT/SCS cycle can be the main or the only source of mitochondrial ATP.

Nevertheless, the growth inhibition observed in the Δach/ RNAi ASCT.i double mutant generated in this study demonstrated that mitochondrial acetate production is indeed essential not only in PCF [22] , but also in BSF. It is noteworthy that the Δach/ RNAi ASCT cell line obtained in this study exhibited an important leakage (i.e., reduced TbASCT expression even in the absence of tetracycline) (Day 0 post-induction in Fig. 2D ). TbASCT was rapidly re-expressed (Day 7 in Fig. 2D ) with restoration of normal growth at 7 days post-induction.

Re-expression of RNAi-targeted genes upon induction is commonly observed in the BSF as well as PCF when the targeted gene is essential for their growth [59, 60] . The quite early reversion of growth, correlated with TbASCT re-expression supports an essential role for ASCT-ACH genes in the BSF. Thus, collectively, these data demonstrate that although

TbASCT and TbACH catalyze distinct reactions, they share a common role in terms of acetate production to feed the de novo fatty acid biosynthesis [15, 28] , act redundantly, and are indispensable for growth of BSF trypanosomes as previously observed for the PCF [22] .

Biochemical and structural biology studies of ASCT have thus far been hampered due to a difficulty to express and purify the recombinant enzyme because of its low expression level and solubility and high degree of instability [20, 33, 34] . Expression of recombinant TbASCT fused with a His 6 -SUMO tag at the N-terminus in this study is therefore critical for increasing the expression level as well as solubility of the enzyme [61] [62] [63] [64] , and the addition of glycerol J o u r n a l P r e -p r o o f Saccharomyces cerevisiae (120 s −1 ) [70] , clearly demonstrating that the ASCT/SCS cycle produces ATP with high efficiency. The F o /F 1 -ATP synthase is a multi-subunit mega-complex enzyme, with the simplest form of E. coli composed of 8 subunits, α 3 β 3 γδϵ (F o ) and ab 2 c 10 (F 1 ) [71] . Compared to the reported ATP synthases, the ASCT/SCS cycle is encoded by a total of three genes (for ASCT α 4 and SCS αβ subunits) and does not interact each other. The most prominent advantage of the ASCT/SCS cycle is the ability to synthesize ATP and acetate independently of the presence of oxygen, an electrochemical gradient, and OXPHOS, hence, this cycle fuels and boosts ATP and acetate production in the parasite"s mitochondria.

Such an observation is consistent with the fact that the ASCT/SCS cycle is conserved in the hydrogenosomes and mitochondria of many anaerobic protists and helminths [21] , in which J o u r n a l P r e -p r o o f

HsSCOT does not have ASCT activity, in accordance with the lack of experimental evidence of β-oxidation in T. brucei [72] . Moreover, we investigated the reaction mechanism of TbASCT and found that TbASCT employs a ping-pong bi-bi mechanism of catalysis, similar to other family IA CoA transferases, including SCOTs [21] . We also analyzed the substrate specificity in terms of CoA donor and found that TbASCT can utilize n-propionyl-CoA with an affinity higher than that for acetyl-CoA, with similar catalytic efficiency (k cat /K m ): 2,375 and 2,267 s −1 mM −1 , respectively. Interestingly, F. hepatica excretes acetate and propionate as end products of glucose catabolism [73] , and it was demonstrated that F. hepatica ASCT utilizes both propionyl-CoA and acetyl-CoA as CoA donors [33] . It is important to note that T.

brucei does not accumulate propionate [10] [11] [12] [13] [14] ; consistent with these reports, n-propionyl-CoA was not detected in a previous metabolome analysis [74] . Taken together,

our results indicate that acetyl-CoA is the physiological and the only CoA donor for TbASCT in vivo.

The catalytic glutamate residue located within the SENG motif was identified in

SsSCOT by site-directed mutagenesis [75] and analysis of the crystal structure in complex with CoA (PDB: 3OXO) [58] . Although TbASCT exhibited extremely low SCOT activity (0.038 µmol/min/mg), the enzyme has 52% identity with SsSCOT and the SENG motif is conserved. In contrast to homodimeric mammalian SCOTs, the TbASCT forms a tight homotetramer as revealed by the crystal structure obtained in this study (Fig. S8 ) and by BN-PAGE followed by CBB and activity staining (Fig. 4D) . The RMS deviation of monomers within TbASCT (Chain A) and SsSCOT (Chain E, PDB: 3OXO) was 1.50 Å between the two structures for 454 C atoms. In the crystal structure of SsSCOT, CoA was found covalently bound to E344, which corresponds to E319 of TbASCT. In addition, the carbonyl group of M423 located within the X-CoA tunnel from SsSCOT, which is not conserved in TbASCT and is replaced by P398, is found making hydrogen bond with the amine group of β-mercaptoethylamine of bound CoA. As the ASCT activity of the E319A and P398A mutants were completely abolished (Table. S4 and S5), we confirmed that E319

is the catalytic glutamate residue and P398 is located in the predicted X-CoA tunnel from

TbASCT.

In the structure of SsSCOT bound to CoA, a second tunnel, adjacent to CoA binding site, is found with potential to bind the CoA acceptor (Fig. S6 ). In the second tunnel, several residues conserved in mammalian SCOTs (L327, L350, Y353), which are not conserved in trypanosomatid ASCTs (T302, M325, F328) are identified (Fig. S7) . However, TbASCT mutants targeting T302, M325 and F328 showed no drastic changes in ASCT or SCOT activities (Table S4 and S5) and thus, we conclude that the second tunnel is not involved in J o u r n a l P r e -p r o o f 25 catalysis. As a crystal structure of ligand free TbASCT was obtained (PDB: 6LP1), study on the binding site of the CoA acceptor by solving the crystal structure of TbASCT in complex with acetate or succinate is on going.

In conclusion, we demonstrated that mitochondrial acetate production is essential for growth of the BSF of trypanosomes. Previously, we have reported the remarkable effect of combination of ascofuranone and glycerol targeting TAO and glycerol kinase, respectively, for trypanocidal activity against BSF [76] . Considering the growth phenotype of Δach/ RNAi ASCT.i double mutant described in this study, a dual inhibitor targeting TbASCT and TbACH can be a suitable drug candidate to combat African trypanosomes due to their common function in terms of acetate production, although they catalyze distinct reaction.

Furthermore, the absence of the ACH gene in the T. cruzi (American trypanosome) genome suggests that ASCT is the sole source of mitochondrial acetate and may be a potential drug target against Chagas disease. Because of the high degree of amino acid identity, the kinetic characterization of TbASCT reported here might facilitate the development of high-throughput screening systems and development of useful tools for structure-based design of ASCT-specific inhibitors targeting African and American trypanosomiasis.

The Transparency document associated with this article can be found, in online version.

The Table S4 and S5 include mutant analyses data. The ligand-free structure of TbASCT with PDB accession code 6LP1 is described in METHODS. Source data for Fig. S6 and 7 can be found in Table S6 . 

The synchrotron beamline BL44XU at SPring-8 was used under the Cooperative Research Program of the Institute for Protein Research, Osaka University. Funding source This work was supported in part by Infectious Disease Control from the Science and Technology Research Partnership for Sustainable Development

); a Grant-in-aid for the Bilateral Joint Research Project (no. 16035611-000722 to K.K.); a Grant-in-aid for Scientific Research on Priority Areas 18073004 (to K.K.); a Creative Scientific

Japan Society for the Promotion of Science; a Grant-in-aid from the Program for the Promotion of Basic and Applied Research for Innovations in Bio-Oriented Industry (BRAIN) (no. 26020A to

Grants-in-aid for Scientific Research (B) 16K19114 to D.K.I and (C) 23570131 and 26234567 to T.S.; and The Leading Initiative for Excellent Young Researchers (LEADER) 16811362 to D.K.I. and (B) 19H03436 to

) from the Agency for Medical Research and Development (AMED); a Grant-in-aid for research on emerging and re-emerging infectious diseases from the Japanese Ministry of Health and Welfare (to K.K.); and the Strategic Japanese-French Cooperative Program 11102218 (to K.K.). This work was also supported by the Centre National de la Recherche Scientifique (CNRS), the Université de Bordeaux, the Agence Nationale de la Recherche (ANR) through grants ACETOTRYP of the ANR-BLANC-2010 call and GLYCONOV of the

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Comparative proteomics of two life cycle stages of stable isotope-labeled Trypanosoma brucei reveals novel components of the parasite's host adaptation machinery

Quantitative Proteomics Uncovers Novel Factors Involved in Developmental Differentiation of Trypanosoma brucei

Acetate produced in the mitochondrion is the essential precursor for lipid biosynthesis in procyclic trypanosomes

Biochemistry and evolution of anaerobic energy metabolism in eukaryotes

An anthelmintic compound, nafuredin, shows selective inhibition of complex I in helminth mitochondria

Anaerobic NADH-fumarate reductase system is predominant in the respiratory chain of Echinococcus multilocularis, providing a novel target for the chemotherapy of alveolar echinococcosis

Structural Insights into the Molecular Design of Flutolanil Derivatives Targeted for Fumarate Respiration of Parasite Mitochondria

Acetate:succinate

CoA-transferase in the anaerobic mitochondria of Fasciola hepatica

Acetate:succinate CoA-transferase in the hydrogenosomes of Trichomonas vaginalis: identification and characterization

Disorders of ketone production and utilization

Pathways and control of ketone body metabolism: on the fringe of lipid biochemistry

New vectors for co-expression of proteins: structure of Bacillus subtilis ScoAB obtained by high-throughput protocols

A tightly regulated inducible expression system for conditional gene knock-outs and dominant-negative genetics in Trypanosoma brucei

Highly efficient stable transformation of bloodstream forms of Trypanosoma brucei

Antibodies: A Laboratory Manual

Molecular cloning: a laboratory manual

Affinity chromatography using trypanocidal arsenical drugs identifies a specific interaction between glycerol-3-phosphate dehydrogenase from Trypanosoma brucei and Cymelarsan

Analysis of antigenic types appearing in first relapse populations of clones of Trypanosoma brucei

Cleavage of structural proteins during the assembly of the head of bacteriophage T4

Handbook of Detection of Enzymes on Electrophoretic Gels

Method for the Accurate Measurement of Freezing

Mechanism-based fragmentation of coenzyme A transferase. Comparison of alpha 2-macroglobulin and coenzyme A transferase thiol ester reactions

Integration, scaling, space-group assignment and post-refinement

MOLREP: an Automated Program for Molecular Replacement

Refinement of macromolecular structures by the maximum-likelihood method

Coot: model-building tools for molecular graphics

Proteome remodelling during development from blood to insect-form Trypanosoma brucei quantified by SILAC and mass spectrometry

Histidine-phosphorylation of succinyl CoA synthetase from Trypanosoma brucei

Enzyme kinetics

Adaptation of myocardial substrate metabolism to a ketogenic nutrient environment

Catalytic role of the conformational change in succinyl-CoA:3-oxoacid CoA transferase on binding CoA

Mitochondrial heat shock protein machinery hsp70/hsp40 is indispensable for proper mitochondrial DNA maintenance and replication

The role of the zinc finger protein ZC3H32 in bloodstream-form Trypanosoma brucei

SUMO fusions and SUMO-specific protease for efficient expression and purification of proteins

Expression and purification of SARS coronavirus proteins using SUMO-fusions

Comparison of SUMO fusion technology with traditional gene fusion systems: enhanced expression and solubility with SUMO

Expression, purification, and crystallization of type 1 isocitrate dehydrogenase from Trypanosoma brucei brucei

A mitochondrial NADH-dependent fumarate reductase involved in the production of succinate excreted by procyclic Trypanosoma brucei

Characterization of the dihydroorotate dehydrogenase as a soluble fumarate reductase in Trypanosoma cruzi

Characterization of Trypanosoma brucei dihydroorotate dehydrogenase as a possible drug target; structural, kinetic and RNAi studies

Efficient ATP synthesis by thermophilic Bacillus FoF1-ATP synthase

ATP synthesis catalyzed by the ATP synthase of Escherichia coli reconstituted into liposomes

Proton transport coupled ATP synthesis by the purified yeast H+ -ATP synthase in proteoliposomes

Rotation and structure of FoF1-ATP synthase

Triacylglycerol Storage in Lipid Droplets in Procyclic Trypanosoma brucei

Studies on the carbohydrate metabolism of the liver fluke Fasciola hepatica

We thank all of the staff members of beamline BL44XU (SPring-8) and BL-17A 

The authors declare no conflict of interests.