Activation of Human Mitochondrial Pantothenate Kinase 2 by Palmitoylcarnitine

Home , PANK1, PANK2 (gene), PANK4, Pantothenate kinase

Activation of human mitochondrial pantothenate kinase 2 by palmitoylcarnitine

Roberta Leonardi, Charles O. Rock, Suzanne Jackowski, and Yong-Mei Zhang*

Department of Infectious Diseases, St. Jude Children’s Research Hospital, Memphis, TN 38105

Edited by Gottfried Schatz, University of Basel, Reinach, Switzerland, and approved December 3, 2006 (received for review August 31, 2006) The human isoform 2 of pantothenate kinase (PanK2) is localized ence of iron, leading to free radical generation and cell damage to the mitochondria, and mutations in this protein are associated (10); or (ii) mitochondrial CoA deficiency leading to increased with a progressive neurodegenerative disorder. PanK2 inhibition oxidative stress from free radicals (7). Both of these hypotheses by acetyl-CoA is so stringent (IC50 < 1 ␮M) that it is unclear how the posit that the mutated PanK2 proteins are functionally defective enzyme functions in the presence of intracellular CoA concentra- (13). Although it is clear that a number of PANK2 mutations give tions. Palmitoylcarnitine was discovered to be a potent activator of rise to inactive proteins, recent biochemical analyses show that PanK2 that functions to competitively antagonize acetyl-CoA in- many of the PanK2 missense mutations encode functional hibition. Acetyl-CoA was a competitive inhibitor of purified PanK2 proteins that are regulated by acetyl-CoA (5, 8). Disruption of with respect to ATP. The interaction between PanK2 and acetyl- the Pank2 gene in mice does not produce a neurodegenerative CoA was stable enough that a significant proportion of the purified phenotype (14), although it does result in retinal degeneration, protein was isolated as the PanK2⅐acetyl-CoA complex. The long- often linked with the human disease, and azoospermia, which is chain acylcarnitine activation of PanK2 explains how PanK2 func- associated with reduced PanK activity in Drosophila melano- tions in vivo, by providing a positive regulatory mechanism to gaster (15). Clearly, there is much to be learned regarding the counteract the negative regulation of PanK2 activity by acetyl-CoA. regulatory biochemistry and role of PanK2 in intermediary Our results suggest that PanK2 is located in the mitochondria to metabolism. sense the levels of palmitoylcarnitine and up-regulate CoA biosyn- One of the puzzling features of PanK2 biochemistry is its thesis in response to an increased mitochondrial demand for the extremely high sensitivity to inhibition by acyl-CoA (5, 8). The cofactor to support ␤-oxidation. IC50 for acetyl-CoA is Ͻ1 ␮M at 2.5 mM ATP (8), which is much lower than the cytosolic CoA concentrations estimated at 20–140 carnitine ͉ coenzyme A ͉ ␤-oxidation ͉ pantothenate kinase-associated ␮M, and, if PanK2 resides in the mitochondrial matrix compart- neurodegeneration ment, CoA levels are likely between 2.2 and 5 mM (16, 17). These data make it hard to understand how PanK2 functions in vivo antothenate kinase (PanK) catalyzes the first and rate- because of the high concentration of CoA thioesters. This work Pcontrolling step in the biosynthesis of CoA (for review, see describes the reversal of acetyl-CoA inhibition of PanK2 by ref. 1). In humans, there are three genes that express four palmitoylcarnitine. This new biochemical property of PanK2 isoforms of PanK. PanK1␣ and 1␤ arise from the use of alternate provides an explanation for how PanK2 is activated in vivo and initiation exons of the PANK1 gene (2, 3), and the PANK3 gene suggests that its mitochondrial location is important for its produces a single polypeptide (4). The PANK2 gene differs from function as an acylcarnitine sensor that up-regulates CoA bio- the others in that it encodes a protein that is targeted to the synthesis in response to accelerated demand for mitochondrial mitochondria (5–7). The PanK2 protein translated from the most ␤-oxidation. 5Ј start site is sequentially cleaved at two sites by mitochondrial processing peptidases, generating a long-lived 48-kDa mature Results protein (5). Two shorter PanK2 isoforms have been described, Palmitoylcarnitine Activates PanK2. One of the conundrums of one of which also localizes to mitochondria (7), whereas the research on PanK2 is that it binds acetyl-CoA, and other CoA second does not because of the lack of targeting sequences (6). species, with an apparent Kd that is far below the estimated A common feature of all PanK proteins is that they are feedback intracellular and intramitochondrial CoA concentrations. Ac- inhibited by CoA thioesters (1); however, the isoforms are cordingly, PanK2 expressed and partially purified from 293T distinguished by their unique sensitivities to the CoA thioester cells was inhibited by acetyl-CoA with an apparent IC50 of Ϸ0.3 pool (2–4, 8). A fourth gene, PANK4, lacks the essential catalytic ␮M under the conditions tested (Fig. 1A). These data suggested glutamate residue present in all other enzymes (9) and may not that PanK2 would be inactive in vivo and led us to search for be a functional PanK. ligands that may regulate PanK2. Palmitoylcarnitine was discov- The PanK2 isoform is the focus of much current research ered as an activator of PanK2 activity in 293T cell lysates (Fig. because mutations in the human PANK2 gene give rise to 1B). The enzymatic activity of PanK2 was stimulated to the PanK-associated neurodegeneration (PKAN), an inherited au- highest level by 8 ␮M palmitoylcarnitine. In contrast, carnitine tosomal recessive disease (10). PKAN patients constitute a did not have any effect on the PanK2 activity when included in subset of those diagnosed with neurodegeneration with brain the reactions at the same concentration range (Fig. 1B). We next iron accumulation, formerly known as Hallervorden–Spatz syn- drome (11). PKAN patients have a pathological accumulation of iron in the basal ganglia and a combination of motor symptoms Author contributions: R.L., C.O.R., S.J., and Y.-M.Z. designed research; R.L. and Y.-M.Z. in the forms of dystonia, dysarthria, intellectual impairment, and performed research; R.L., C.O.R., S.J., and Y.-M.Z. analyzed data; and R.L., C.O.R., S.J., and gait disturbance (11). Early-onset patients have a rapidly pro- Y.-M.Z. wrote the paper. gressive disease, and late-onset patients have a slowly progres- The authors declare no conflict of interest. sive, atypical disease, where parkinsonism is common (11, 12). This article is a PNAS direct submission. Two hypotheses have been proposed to explain the clinical Abbreviations: IMS, intermembrane space; OMM, outer mitochondrial membrane; PanK, problems associated with mutations in PANK2:(i) accumulation pantothenate kinase; PKAN, pantothenate kinase-associated neurodegeneration. of cysteine-containing substrates because of inhibition of CoA *To whom correspondence should be addressed. E-mail: [email protected]. synthesis, which may undergo rapid autooxidation in the pres- © 2007 by The National Academy of Sciences of the USA

1494–1499 ͉ PNAS ͉ January 30, 2007 ͉ vol. 104 ͉ no. 5 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0607621104 Downloaded by guest on October 1, 2021 Fig. 1. Negative and positive regulators of PanK2 activity. (A) Acetyl-CoA inhibited PanK2 activity in 293T cell lysates with an IC50 value of 0.3 ␮M. (B) Palmitoylcarnitine (F) activated PanK2 in partially puriﬁed 293T cell lysates, whereas carnitine (E) did not affect PanK2 activity. (C) Palmitoylcarnitine (F) reversed the inhibition of PanK2 activity in 293T cell lysates by 0.2 ␮M acetyl-CoA, whereas carnitine (E) did not.

determined whether palmitoylcarnitine was able to reverse the levels higher than those in the absence of acetyl-CoA (Fig. 3A). inhibition of PanK2 by acetyl-CoA (Fig. 1C). Palmitoylcarnitine We tested the effect of palmitic acid and found that it was not was capable of partially reversing the inhibition of PanK2 by 0.2 effective in the reversal of acetyl-CoA inhibition (data not ␮M acetyl-CoA, whereas carnitine was not. Thus, palmitoylcar- shown). A kinetic analysis of the interaction between palmitoyl- nitine was a positive regulator of PanK2 activity. carnitine and acetyl-CoA indicated that palmitoylcarnitine was a competitive antagonist of acetyl-CoA (Fig. 3B). These data Analysis of Acetyl-CoA Regulation of the Purified PanK2. The data in demonstrated that the mechanism of palmitoylcarnitine activa- Fig. 1 support palmitoylcarnitine as a positive regulator of tion was to competitively reverse the inhibition by acetyl-CoA. PanK2; however, interpreting the mechanism of its activation in Paradoxically, palmitoylcarnitine also activated purified PanK2 partially purified lysates was complicated by the lack of control in the absence of added acetyl-CoA (Fig. 4A). Octanoylcarnitine over the concentrations of endogenous regulatory ligands that was not an activator indicating that the activation event is specific might be present. Therefore, the N-terminal His-tagged PanK2 for long-chain species of acylcarnitine (Fig. 4A). Fisher et al. (18) was expressed in Escherichia coli and purified to homogeneity by reported that carnitine was a nonessential activator of partially affinity and size-exclusion chromatography (Fig. 2A). The pure purified PanK activity from rat heart. Carnitine did not activate protein exhibited a specific activity of 0.2421 Ϯ 0.006 pmol/ng purified PanK2; however, we were able to repeat the observa- per min under the standard PanK assay condition. The elution tions of Fisher et al. in cell lysates showing that carnitine position of PanK2 on the gel filtration column was consistent activated PanK2 at higher concentrations (Fig. 4B). Thus, we with previous work in cell lysates that indicated it was a dimer in attributed the activation of PanK2 by carnitine in cell lysates to solution. Acetyl-CoA was a potent inhibitor of purified PanK2 the formation of acylcarnitine from carnitine in the presence of 2ϩ with an IC50 value of Ϸ60 nM (Fig. 2B). We confirmed that the ATP:Mg required for the kinase assay, and we concluded long-chain acyl-CoAs, such as palmitoyl-CoA, also strongly that carnitine was not a regulator of PanK2. inhibited PanK2 activity (5, 8). The kinetic mechanism of acetyl-CoA inhibition was explored, and the graphical analysis of The PanK2⅐Acetyl-CoA Complex. One interpretation of the palmi- the data indicated acetyl-CoA inhibition was competitive with toylcarnitine activation of PanK2 in the absence of added ATP (Fig. 2C). This conclusion was confirmed by using a direct acetyl-CoA in the cell lysates (Fig. 1B) was the contamination of binding assay employing the fluorescent ATP analog, TNP-ATP. the sample with endogenous acetyl-CoA, but a similar activation The binding of TNP-ATP to proteins results in a strong increase of purified PanK2 that had been subjected to two column in fluorescence that is directly proportional to the extent of chromatography steps and dialysis (Fig. 4A) suggested that a binding. The addition of acetyl-CoA to the TNP-ATP binding proportion of PanK2 protein was purified as the PanK2⅐acetyl- assay displaced the ATP analog from the protein (Fig. 2D), CoA complex. Therefore, we analyzed our pure PanK2 prepa- confirming that PanK2⅐acetyl-CoA complex formation compro- rations for bound acetyl-CoA. The UV spectrum of purified mised ATP binding. PanK2 exhibited a shoulder at 260 nm compared with the spectrum that was calculated based on the amino acid compo- Palmitoylcarnitine Regulation of PanK2. Palmitoylcarnitine was able sition (Fig. 5A). Heat denaturation of PanK2 followed by the to reverse the inhibition of purified PanK2 by acetyl-CoA (Fig. removal of the precipitated protein resulted in a supernatant 3A). In this experiment, 60 nM acetyl-CoA was used, which with an absorbance maximum at 260 nm (Fig. 5A), indicating the resulted in Ϸ50% inhibition of PanK2 activity. However, palmi- presence of a bound nucleotide-like molecule. Based on the

toylcarnitine stimulated PanK2 activity to a level that was extinction coefficient of acetyl-CoA and the absorbance at 260 BIOCHEMISTRY Ϸ130% of the control PanK2 activity in the absence of acetyl- nm, we calculated that between 40% and 60% of the protein in CoA. Palmitoylcarnitine also reversed the inhibition of PanK2 by different batches of purified PanK2 contained a bound CoA palmitoyl-CoA (data not shown). Three PKAN-associated point species. Mass spectrometry clearly identified acetyl-CoA as the mutants, which are active and sensitive to acetyl-CoA inhibition nucleotide ligand bound to PanK2 (Fig. 5B). Mass peaks corre- (8), were purified and tested for the regulation by palmitoylcar- sponding to doubly charged (m/z ϭ 403.72) and singly charged nitine. In the presence of 60 nM acetyl-CoA, all three mutants, (m/z ϭ 808.11) acetyl-CoA peaks were detected in the negative- PanK2[R286C], PanK2[N404I], and PanK2[T528M], were inhib- ion scan. The mass spectrum gave no indication for the presence ited by 40–60%. Similar to its effect on the wild-type protein, of nonesterified CoA or other CoA species. The bacterial CoA palmitoylcarnitine stimulated the activities of the mutants to pool consists primarily of acetyl-CoA, but there are also signif-

Leonardi et al. PNAS ͉ January 30, 2007 ͉ vol. 104 ͉ no. 5 ͉ 1495 Downloaded by guest on October 1, 2021 Fig. 2. Acetyl-CoA is a competitive inhibitor of PanK2 with respect to ATP. (A) His-tagged PanK2 was overexpressed in E. coli and purified to homogeneity by Ni-affinity and size exclusion chromatography. (Inset) An SDS gel showing the purity of the PanK2 preparation. (B) Acetyl-CoA inhibited purified PanK2 with an IC50 of 60 nM. (C) Lineweaver–Burk plots of PanK2 in the absence (■) or presence of 30 nM (Œ)or60nM() acetyl-CoA. The lines intercept on the y axis, indicating that acetyl-CoA was a competitive inhibitor of PanK2 with respect to ATP. (D) Acetyl-CoA displaced the ATP analog TNP-ATP from the PanK2 active site. Fluorescent emission spectra of 5 ␮M TNP-ATP only (dashed line), 5 ␮M TNP-ATP plus 1 ␮M PanK2 (dotted line), and 5 ␮M TNP-ATP plus 1 ␮M PanK2 and 0.4 ␮M acetyl-CoA (solid line). The fluorescence of the environmentally sensitive ATP analog increased on binding to PanK2, but the addition of acetyl-CoA displaced TNP-ATP from PanK2.

icant concentrations of nonesterified CoA, succinyl-CoA, and to the IMS because: (i) like known IMS proteins such as malonyl-CoA (19). Thus, we concluded that PanK2 selectively cytochrome b2, cytochrome c1, and cytochrome peroxidase, copurified with acetyl-CoA. These data show that a proportion PanK2 possesses two cleavable mitochondrial targeting signals of the total PanK2 protein survived as the PanK2⅐acetyl-CoA (20, 21), predicting an IMS localization; and (ii) the IMS complex during the multistep purification protocol underscoring localization enables ready transport of pantothenate into IMS the tight binding of acetyl-CoA to PanK2. The presence of the for PanK2 activity and product release across the OMM to the PanK2⅐acetyl-CoA complex explained palmitoylcarnitine activa- cytosol where the downstream reactions in the CoA biosynthetic tion of PanK2 in the absence of added acetyl-CoA (Fig. 4A). pathway are located. In animal tissues, the cytosolic concentrations of total CoA range from 20 to 140 ␮M (16, 17, 22). Discussion Approximately 30% (6–40 ␮M) of the total CoA pool are The positive and negative regulation of PanK2 by intermediates acyl-CoAs including both short- and long-chain species. Carni- in mitochondrial metabolism provides a mechanistic basis for tine is more abundant than CoA. The concentration of total understanding the biological function of the enzyme (Fig. 6). carnitine is 2 mM in liver and 4 mM in heart (23). Long-chain The mature form of human PanK2 resides in the mitochondria acylcarnitine species constitute 10% of the carnitine pool, where it phosphorylates pantothenate to 4Ј-phosphopantothe- leading to a concentration ranging from 200 to 400 ␮M. Thus, the nate. Because the enzymes catalyzing the subsequent reactions IMS compartment contains more activators of PanK2 than in the CoA biosynthetic pathway are cytosolic, the product of inhibitors. The concentrations of the effectors used in our assays PanK2 exits the mitochondria and is converted to CoASH. To are achievable in vivo. We also tested and confirmed the ability put PanK2 in the proper physiological context with its activator of palmitoylcarnitine to reverse the acetyl-CoA inhibition at a and inhibitors, one has to understand where PanK2 localizes higher acetyl-CoA concentration (1 or 10 ␮M). within the mitochondria: the intermembrane space (IMS) or the The carnitine shuttle system transports acyl groups into the matrix. Because of the presence of an abundant outer mitochon- mitochondrial matrix and is composed of three proteins: the drial membrane (OMM) protein named porin, the OMM is OMM carnitine palmitoyltransferase I that releases acylcarni- highly permeable to small molecules with molecular masses Ͻ5 tine into the IMS, the inner membrane acylcarnitine:carnitine kDa. Thus, the cytosolic concentrations of the CoA and carnitine translocase, and the inner membrane carnitine palmitoyltrans- species equilibrate with the IMS. Experimental data are not ferase II that transfers the acyl group from carnitine to CoASH available to pinpoint in which mitochondrial compartment in the matrix (24). Thus, matrix CoASH is required to initiate PanK2 is located. However, we propose that PanK2 is localized mitochondrial fatty acid ␤-oxidation, and a deficiency in CoASH

1496 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0607621104 Leonardi et al. Downloaded by guest on October 1, 2021 Fig. 3. Effect of palmitoylcarnitine on the inhibition of PanK2 by acetyl-CoA. Fig. 4. Activation of PanK2 was specific for palmitoylcarnitine. (A) Palmi- (A) Palmitoylcarnitine reversed the inhibition of purified wild-type (ᮀ)or toylcarnitine (■) activated PanK2 by using the purified PanK2 preparation, mutant ([R286C], ■; [N404I], F; [T528M], E) PanK2 by 60 nM acetyl-CoA. (B) whereas octanoylcarnitine (ᮀ) did not. (B) Carnitine activated PanK2 from the PanK2 activity was assayed in the presence of different concentrations of 293T cell lysates (■). In contrast, carnitine did not activate purified PanK2 (ᮀ). acetyl-CoA in the absence (F) or presence of 2 ␮M(E)or4␮M(■) palmitoylcarnitine. Dixon plots of the reciprocal of velocity versus the acetyl-CoA concentration showed that the lines intercepted on the y axis, indicating that model for the regulation of PanK2 by palmitoylcarnitine suggests palmitoylcarnitine activation was competitive with respect to acetyl-CoA. that the absence of this enzyme activity in PKAN patients may lead to a defect in the regulation of mitochondrial matrix CoASH and the impairment of fatty acid ␤-oxidation. The presence of would lead to the accumulation of long-chain acylcarnitine. The other PanK isoforms ensures that PKAN patients are not potent inhibition of PanK2 by acyl-CoAs maintains the enzyme severely deficient in total CoA; thus, the mitochondrial dysfunc- in a normally off position by preventing the binding of tion in this disease would be milder than in severe mitochondrial ATP:Mg2ϩ; however, the presence of long-chain acylcarnitine ␤-oxidation disorders, perhaps accounting for the variability of antagonizes the inhibitory action of acyl-CoA and activates symptoms among PKAN patients (12). The severity of symptoms PanK2. This regulatory effect on the rate-controlling enzyme in in ␤-oxidation disorders is highly dependent on diet, and the CoA biosynthesis boosts the cytosolic concentration of CoASH, seriousness of the clinical symptoms of the human ␤-oxidation which is actively transported into the mitochondrial matrix by the disorders can be ameliorated, in some cases, by a high- Leu5p protein, an inner membrane carrier (25). The mitochon- carbohydrate, low-fat diet supplemented with carnitine (32). drial localization of PanK2 places it in an ideal subcellular This diet effect suggests that a similar strategy should be location to sense the levels of long-chain acylcarnitine and the considered in the management of PKAN disease; however, the ␤ status of mitochondrial -oxidation. failure of PanK2 knockout mice to accurately recapitulate the In addition to its essential role in transport described in Fig. properties of the human disease (14) makes the experimental 6, carnitine is also involved in modulating the acyl-CoA:CoASH verification of this idea problematical in an animal model. ratio to release CoASH to support ␤-oxidation, pyruvate dehydrogenase and ␣-ketoglutarate dehydrogenase activities, and the Materials and Methods BIOCHEMISTRY tricarboxylic acid cycle (26, 27). The cellular carnitine concen- Transfection of HEK 293T Cells and Preparation of Cell Lysates. The tration can approach 3 mM, and Ϸ90% of the total carnitine is coding sequence for the mature form of human PanK2 protein located in the cytosol (28). Like CoA, carnitine exists as free (from residue 141 of the full-length protein) was cloned into carnitine and short- and long-chain carnitine esters, and the ratio pcDNA3.1-HisA (Invitrogen, Carlsbad, CA) to yield pKM56 (8), of free to esterified carnitine varies depending on the availability which was used to transfect HEK 293T cells. The cells were of oxidative substrates (26, 29). In human disorders that disrupt cultured in DMEM and 10% FCS (Atlanta Biologicals, Law- mitochondrial ␤-oxidation, acylcarnitines accumulate and are renceville, GA) after transfection with FuGENE 6 according to secreted in the urine (30, 31), consistent with the role of carnitine the manufacturer’s recommendation (Roche, Basel, Switzer- in releasing CoASH to support intermediary metabolism. Our land). Cells were collected and lysed 48 h after transfection, and

Leonardi et al. PNAS ͉ January 30, 2007 ͉ vol. 104 ͉ no. 5 ͉ 1497 Downloaded by guest on October 1, 2021 Fig. 6. A model illustrating the positive and negative regulation of PanK2 activity. Human PanK2 is proposed to localize in the IMS of mitochondria, where it phosphorylates pantothenate (Pan) to form 4Ј-phosphopantothe- nate (P-Pan). P-Pan translocates to the cytosol and is converted to CoA by the CoA biosynthetic pathway. Fatty acid ␤-oxidation in the matrix requires the carnitine shuttle system, which converts acyl-CoA to acylcarnitine in the IMS by the action of carnitine palmitoyltransferase I (CPTI); acylcarnitine is shuttled into the matrix by the carnitine:acylcarnitine translocase (CT) and converted back to acyl-CoA by carnitine palmitoyltransferase II (CPTII). Acyl-CoAs, including acetyl-CoA (Ac-CoA), potently inhibit PanK2 activity by competing with the ATP substrate. Palmitoylcarnitine reverses the acetyl-CoA inhibition on PanK2 by competing with acetyl-CoA for the enzyme. The existence of both negative and positive regulation of PanK2 activity provides a mechanism to ensure ample supply of CoA for ␤-oxidation by controlling the activity of the mitochondria-localized PanK2, a rate-limiting step in the CoA biosynthetic pathway. OMM, outer mitochondrial membrane; IMM, inner mitochondrial membrane. Fig. 5. Identiﬁcation of the PanK2-bound inhibitor by UV-visible spectros- copy and electrospray mass spectrometry (ES-MS). (A) UV-visible spectra of PanK2 (0.9 mg/ml, solid thin line) and the bound inhibitor (solid thick line) macia Biotech, Piscataway, NJ) equilibrated in buffer (20 mM released after heat denaturation of the protein sample and removal of the Tris⅐HCl, pH 7.5) containing 300 mM NaCl. Three PKAN precipitate by centrifugation. The peak at 260 nm was characteristic of a ␮ disease-associated point mutations, [R286C], [N404I], and nucleotide-like molecule, and the spectrum of a 25 M acetyl-CoA standard [T528M] (numbering was based on the full-length protein), were solution (dashed line) is shown. (B) ES-MS spectrum of the supernatant from a heat-denatured sample of PanK2. The spectrum revealed the presence of introduced into pKM44 by using the Quikchange mutagenesis kit peaks at m/z 403.72 and 808.11 corresponding to the acetyl-CoA [M-2H]2Ϫ and (Stratagene) according to the manufacturer’s protocol. The [M-H]Ϫ ions, respectively. His-tagged recombinant mutant PanK2 proteins were expressed and purified as the wild-type enzyme.

Western blotting was used to confirm the expression of the PanK Activity Assay. Briefly, the standard PanK assays (3, 33) mature form of human PanK2 in the cell lysate as described (8). contained 45 ␮M D-[1-14C]pantothenate (specific activity, 55 mCi/mmol; Amersham Biosciences), 250 ␮M ATP (pH 7.0), 10 Purification of His-Tagged PanK2. The PANK2 gene fragments mM MgCl2, 0.1 M Tris⅐HCl (pH 7.5), and the indicated amount encoding the mature form (amino acids 141–570) was subcloned of protein from the cell extracts containing PanK2 or the purified into expression plasmid pET-28a (Novagen, Darmstadt, Ger- PanK2 proteins in a total volume of 40 ␮l. The mixture was many) between the NheI and BamHI restriction sites. The incubated at 37°C for 10 min. The radioactive product was resultant plasmid pKM44 was transformed into the E. coli quantitated by scintillation counting as described (8). The effect BL21(DE3) (Stratagene, La Jolla, CA) strain to express the of different CoA or carnitine species on the enzymatic activity mature form of PanK2 with an N-terminal His-tag. Transformed was determined by including the indicated concentrations of cells were grown at 37°C in terrific broth in the presence of 30 CoA or carnitine species in the assay mix before the addition of ␮g/ml kanamycin to an A600 of 2 and induced with 1 mM IPTG PanK2. To determine the inhibitory mechanism of acetyl-CoA at 18°C for 20 h. The cells were harvested by centrifugation, on the mature form of PanK2, the Km of PanK2 for ATP in the resuspended in MCAC-20 (20 mM Tris⅐HCl, pH 8/500 mM presence or absence of acetyl-CoA was determined in standard NaCl/20 mM imidazole/10% glycerol) buffer containing 1 mM reaction mixtures containing 180 ␮M D-[1-14C]pantothenate and PMSF, and lysed with a Microfluidizer high-pressure fluids increasing concentrations of both ATP and the CoA thioester, as processor. The cell-free extract was precipitated with 50% indicated. saturated ammonium sulfate, and the protein pellet was resuspended in MCAC-20 buffer containing 50 mM KSCN before UV-Visible Spectra of PanK2 and Mass Spectrometry Analysis. UV- being loaded onto a 5-ml HiTrap Chelating HP column. The visible spectra of PanK2 [0.9 mg/ml in 20 mM Tris⅐HCl (pH 7.4), his-tagged PanK2 protein was eluted by using a linear gradient 300 mM NaCl, 1 mM EDTA, 1 mM DTT] were recorded on an of imidazole from 60 to 300 mM. The fractions containing Agilent 8453 spectrophotometer before and after removal of the PanK2 protein were pooled, concentrated, and further purified enzyme by heat denaturation and centrifugation. The extinction to homogeneity by a Superdex 200 column (Amersham Phar- coefficient at 260 nm for acetyl-CoA was determined to be

1498 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0607621104 Leonardi et al. Downloaded by guest on October 1, 2021 11,500 MϪ1⅐cmϪ1 from the absorbance of standard solutions and half maximum. Instrument control and data acquisition were used to estimate the amount bound to PanK2. performed with the Finnigan Xcalibur (version 1.4 SR1) soft- The supernatant (100 ␮l) from a heat-denatured sample of ware (Thermo Electron Corporation). PanK2 [1.15 mg/ml in 50 mM ammonium acetate (pH 7.0)] was desalted on a C-8 MicroTip Column (Harvard Apparatus) for TNP-ATP Fluorescence Spectra. The ATP analog TNP-ATP (Mo- mass spectrometry (MS) analysis. The column was washed with lecular Probes, Eugene, OR) was excited at 410 nm, and the water, and the protein-bound small molecules were eluted with fluorescence emission spectra were recorded at 25°C on a ␮ methanol (100 l), dried under N2 flow, and resuspended in FluoroLog spectrofluorimeter (Horiba Jobin Yvon, Edison, NJ) methanol:water (1:1 vol/vol, 10 ␮l). MS analysis was performed equipped with a circulating water bath. Binding of TNP-ATP to by using a Finnigan TSQ Quantum (Thermo Electron Corpo- PanK2 and subsequent displacement by acetyl-CoA were de- ration, San Jose, CA) triple quadrupole mass spectrometer tected by the change in fluorescence of a 5 ␮M TNP-ATP equipped with the Nanospray Ion Source. Samples were intro- solution in 100 mM Tris⅐HCl (pH 7.5), 10 mM MgCl2,5% duced via static nanoelectrospray by using EconTips (New glycerol, after the addition of 1 ␮M protein and 400 nM Ojective, Woburn, MA). The instrument was operated in the acetyl-CoA. negative ion mode by using single MS (Q1) scanning. Ion source parameters were spray voltage 1,600 V, capillary temperature Ϫ We thank Ruobing Zhou and Karen Miller for their expert technical 270°C, and capillary offset 35 V, and tube lens offset was set assistance and Phil Poston (Hartwell Center of St. Jude Children’s by infusion of the polytyrosine tuning and calibration solution Research Hospital) for the mass spectrometry experiments. This work (Thermo Electron Corporation) in electrospray mode. MS ac- was supported by National Institutes of Health Grants GM 62896 (to quisition parameters for Q1 scanning were as follows: scan range S.J.), Cancer Center (CORE) Support Grant CA 21765, and the 250–1,100 m/z; scan time, 0.85 s; peak width Q1, 0.7 full width, American Lebanese Syrian Associated Charities.

1. Leonardi R, Zhang YM, Rock CO, Jackowski S (2005) Prog Lipid Res 16. Idell-Wenger J, Grotyohann L, Neely JR (1978) J Biol Chem 253:4310–4318. 44:125–153. 17. Williamson J, Corkey B (1979) Methods Enzymol 55:200–222. 2. Rock CO, Calder RB, Karim MA, Jackowski S (2000) J Biol Chem 275:1377– 18. Fisher MN, Robishaw JD, Neely JR (1985) J Biol Chem 260:15745–15751. 1383. 19. Vallari DS, Jackowski S, Rock CO (1987) J Biol Chem 262:2468–2471. 3. Rock CO, Park HW, Jackowski S (2003) J Bacteriol 185:3410–3415. 20. Gasser SM, Ohashi A, Daum G, Bohni PC, Gibson J, Reid GA, Yonetani T, 4. Zhang YM, Rock CO, Jackowski S (2005) J Biol Chem 280:32594–32601. Schatz G (1982) Proc Natl Acad Sci USA 79:267–271. 5. Kotzbauer PT, Truax AC, Trojanowski JQ, Lee VMY (2005) J Neurosci 21. Van Loon AP, Brandli AW, Pesold-Hurt B, Blank D, Schatz G (1987) EMBO 25:689–698. J 6:2433–2439. 6. Ho¨rtnagel K, Prokisch H, Meitinger T (2003) Hum Mol Genet 12:321–327. 22. Horie S, Isobe M, Suga T (1986) J Biochem 99:1345–1352. 7. Johnson MA, Kuo YM, Westaway SK, Parker SM, Ching KH, Gitschier J, 23. Ramsay RR (1994) Essays Biochem 28:47–61. Hayflick SJ (2004) Ann NY Acad Sci 1012:282–298. 24. Kerner J, Hoppel C (2000) Biochim Biophys Acta 1486:1–17. 8. Zhang YM, Rock CO, Jackowski S (2006) J Biol Chem 281:107–114. 25. Prohl C, Pelzer W, Diekert K, Kmita H, Bedekovics T, Kispal G, Lill R (2001) 9. Hong BS, Yun MK, Zhang YM, Chohnan S, Rock CO, White SW, Jackowski Mol Cell Biol 21:1089–1097. S, Park HW, Leonardi R (2006) Structure (London) 14:1251–1261. 26. Ramsay RR, Zammit VA (2004) Mol Aspects Med 25:475–493. 10. Zhou B, Westaway SK, Levinson B, Johnson MA, Gitschier J, Hayflick SJ 27. Bieber LL (1988) Annu Rev Biochem 57:261–283. (2001) Nat Genet 28:345–349. 28. Hutter JF, Alves C, Soboll S (1990) Biochim Biophys Acta 1016:244–252. 11. Thomas M, Hayflick SJ, Jankovic J (2004) Mov Disord 19:36–42. 29. Ramsay RR, Arduini A (1993) Arch Biochem Biophys 302:307–314. 12. Hayflick SJ, Westaway SK, Levinson B, Zhou B, Johnson MA, Ching KH, 30. Sim KG, Carpenter K, Hammond J, Christodoulou J, Wilcken B (2002) Gitschier J (2003) N Engl J Med 348:33–40. Metabolism 51:366–371. 13. Hayflick SJ (2003) J Neurol Sci 207:106–107. 31. Wanders RJ, Vreken P, den Boer ME, Wijburg FA, van Gennip AH, Ijlst L 14. Kuo YM, Duncan JL, Westaway SK, Yang H, Nune G, Xu EY, Hayflick SJ, (1999) J Inherit Metab Dis 22:442–487. Gitschier J (2005) Hum Mol Genet 14:49–57. 32. Vockley J, Whiteman DA (2002) Neuromuscul Disord 12:235–246. 15. Afshar K, Gonczy P, DiNardo S, Wasserman SA (2001) Genetics 157:1267–1276. 33. Rock CO, Karim MA, Zhang YM, Jackowski S (2002) Gene 291:35–43. BIOCHEMISTRY

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