Crystal structures of human pantothenate kinases. Insights into allosteric regulation and mutations linked to a neurodegeneration disorder

Pantothenate kinase (PanK) catalyzes the first step in CoA biosynthesis and there are three human genes that express four isoforms with highly conserved catalytic core domains. Here we report the homodimeric structures of the catalytic cores of PanK1alpha and PanK3 in complex with acetyl-CoA, a feedback inhibitor. Each monomer adopts a fold of the actin kinase superfamily and the inhibitor-bound structures explain the basis for the allosteric regulation by CoA thioesters. These structures also provide an opportunity to investigate the structural effects of the PanK2 mutations that have been implicated in neurodegeneration. Biochemical and thermodynamic analyses of the PanK3 mutant proteins corresponding to PanK2 mutations show that mutant proteins with compromised activities and/or stabilities correlate with a higher incidence of the early onset of disease.     

Localization and regulation of mouse pantothenate kinase 2

Coenzyme A (CoA) biosynthesis is initiated by pantothenate kinase (PanK) and CoA levels are controlled through differential expression and feedback regulation of PanK isoforms. PanK2 is a mitochondrial protein in humans, but comparative genomics revealed that acquisition of a mitochondrial targeting signal was limited to primates. Human and mouse PanK2 possessed similar biochemical properties, with inhibition by acetyl-CoA and activation by palmitoylcarnitine. Mouse PanK2 localized in the cytosol, and the expression of PanK2 was higher in human brain compared to mouse brain. Differences in expression and subcellular localization should be considered in developing a mouse model for human PanK2 deficiency.     

The properties and regulation of pantothenate kinase from rat heart

Pantothenate kinase (ATP:D-pantothenate 4'-phosphotransferase, EC 2.7.1.33), the first enzyme in the pathway of CoA synthesis, was partially purified from rat heart. A study of the properties of the kinase showed that it possesses a broad pH optimum between 6 and 9, is activated or inhibited nonspecifically by various anions, and has MgATP as the nucleotide substrate. The Km for MgATP is 0.6 mM and that for pantothenate is 18 microM. CoA and acyl esters of CoA are inhibitors of the kinase with the inhibition by acetyl-CoA being only slightly greater than that by free CoA. The inhibition by free CoA is uncompetitive with respect to pantothenate concentration, with a Ki for inhibition of 0.2 microM. L-Carnitine was found to be a nonessential activator of the kinase. This compound had no effect by itself but specifically reversed the inhibition of the kinase by CoA. The Ka for deinhibition by L-carnitine is 0.27 mM. Free carnitine content was measured in perfused hearts and is found to vary in correlation with perfusion conditions that are known to alter rates of intracellular phosphorylation of pantothenate. These properties of pantothenate kinase provide a potential mechanism for the control of CoA synthesis. The enzyme is regulated by feedback inhibition by CoA and its acyl esters and this inhibition is modified by changes in the concentration of free carnitine.     

Purification and various properties of pantothenate kinase from the rat liver

Homogeneous (according to disc gel electrophoresis data) ATP: D-pantothenate-4'-phosphotransferase (pantothenate kinase, EC 2.7.1.33) was obtained from rat liver cytosol of heterogeneous stock rats. The enzyme was purified 199-fold with a 9.3% yield. The enzyme was relatively unstable but retained its activity in the presence of 10% glycerol containing 5.10(-4) M ATP over 10 days at 4 degrees C. The pH optimum was 6.5; the apparent Km values were equal to 1.2 X 10(-5) M and 1.4 X 10(-3) M for pantothenate and ATP, respectively, at the ATP/Mg2+ ratio of 1. Pantetheine produced a competitive inhibition of pantothenate kinase. Pantethine or pantetheine disulfide did not inhibit the enzyme.     

Biochemical and motile properties of Myo1b splice isoforms

Myo1b is a widely expressed myosin-I isoform that concentrates on endosomal and ruffling membranes and is thought to play roles in membrane trafficking and dynamics. Myo1b is alternatively spliced within the regulatory domain of the molecule, yielding isoforms with six (myo1b(a)), five (myo1b(b)), or four (myo1b(c)) non-identical IQ motifs. The calmodulin binding properties of the myo1b IQ motifs have not been investigated, and the mechanical and cell biological consequences of alternative splicing are not known. Therefore, we expressed the alternatively spliced myo1b isoforms truncated after the final IQ motif and included a sequence at their C termini that is a substrate for bacterial biotin ligase. Site-specific biotinylation allows us to specifically attach the myosin to motility surfaces via a biotin-streptavidin linkage. We measured the ATPase and motile properties of the recombinant myo1b splice isoforms, and we correlated these properties with calmodulin binding. We confirmed that calcium-dependent changes in the ATPase activity are due to calcium binding to the calmodulin closest to the motor. We found that calmodulin binds tightly to some of the IQ motifs (Kd < 0.2 microM) and very weakly to the others (Kd > 5 microM), suggesting that a subset of the IQ motifs are not calmodulin bound under physiological conditions. Finally, we found the in vitro motility rate to be dependent on the myo1b isoform and the calmodulin concentration and that the myo1b regulatory domain acts as a rigid lever arm upon calmodulin binding to the high affinity and low affinity IQ motifs.     

Origin and properties of cytoplasmic and mitochondrial isoforms of taurocyamine kinase

Taurocyamine kinase (TK) is a member of the highly conserved family of phosphagen kinases that includes creatine kinase (CK) and arginine kinase. TK is found only in certain marine annelids. In this study we used PCR to amplify two cDNAs coding for TKs from the polychaete Arenicola brasiliensis, cloned these cDNAs into the pMAL plasmid and expressed the TKs as fusion proteins with the maltose-binding protein. These are the first TK cDNA and deduced amino acid sequences to be reported. One of the two cDNA-derived amino acid sequences of TKs shows a high amino acid identity to lombricine kinase, another phosphagen kinase unique to annelids, and appears to be a cytoplasmic isoform. The other sequence appears to be a mitochondrial isoform; it has a long N-terminal extension that was judged to be a mitochondrial targeting peptide by several on-line programs and shows a higher similarity in amino acid sequence to mitochondrial creatine kinases from both vertebrates and invertebrates. The recombinant cytoplasmic TK showed activity for the substrates taurocyamine and lombricine (9% of that of taurocyamine). However, the mitochondrial TK showed activity for taurocyamine, lombricine (30% of that of taurocyamine) and glycocyamine (7% of that of taurocyamine). Neither TK catalyzed the phosphorylation of creatine. Comparison of the deduced amino acid sequences of mitochondrial CK and TK indicated that several key residues required for CK activity are lacking in the mitochondrial TK sequence. Homology models for both cytoplasmic and mitochondrial TK, constructed using CK templates, provided some insight into the structural correlation of differences in substrate specificity between the two TKs. A phylogenetic analysis using amino acid sequences from a broad spectrum of phosphagen kinases showed that annelid-specific phosphagen kinases (lombricine kinase, glycocyamine kinase and cytoplasmic and mitochondrial TKs) are grouped in one cluster, and form a sister-group with CK sequences from vertebrate and invertebrate groups. It appears that the annelid-specific phosphagen kinases, including cytoplasmic and mitochondrial TKs, evolved from a CK-like ancestor(s) early in the divergence of the protostome metazoans. Furthermore, our results suggest that the cytoplasmic and mitochondrial isoforms of TK evolved independently.     

Spinach pyruvate kinase isoforms : partial purification and regulatory properties

Pyruvate kinase from spinach (Spinacea oleracea L.) leaves consists of two isoforms, separable by blue agarose chromatography. Both isoforms share similar pH profiles and substrate and alternate nucleotide K_m values. In addition, both isoforms are inhibited by oxalate and ATP and activated by AMP. The isoforms differ in their response to three key metabolites; citrate, aspartate, and glutamate. The first isoform is similar to previously reported plant pyruvate kinases in its sensitivity to citrate inhibition. The K_i for this inhibition is 1.2 millimolar citrate. The second isoform is not affected by citrate but is regulated by aspartate and glutamate. Aspartate is an activator with a K_a of 0.05 millimolar, and glutamate is an inhibitor with a K_i of 0.68 millimolar. A pyruvate kinase with these properties has not been previously reported. Based on these considerations, we suggest that the activity of the first isoform is regulated by respiratory metabolism. The second isoform, in contrast, may be regulated by the demand for carbon skeletons for use in ammonia assimilation.     

Prediction of three different isoforms of the human UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase

The bifunctional enzyme UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) is the key enzyme of the biosynthesis of sialic acids, terminal components of glycoconjugates associated with a variety of cellular processes. Two novel isoforms of human GNE, namely GNE2 and GNE3, which possess extended and deleted N-termini, respectively, were characterized. GNE2 was also found in other species like apes, rodents, chicken or fish, whereas GNE3 seems to be restricted to primates. Both, GNE2 and GNE3, displayed tissue specific expression patterns, therefore may contribute to the complex regulation of sialic acid metabolism.     

Structure-activity relationships and enzyme inhibition of pantothenamide-type pantothenate kinase inhibitors

A set of novel pantothenamide-type analogues of the known Staphylococcus aureus pantothenate kinase (SaPanK) inhibitors, N-pentyl, and N-heptylpantothenamide, was synthesized in three series. The first series of analogues (1-3) were designed as molecular probes of the PanK binding site to elucidate important structure-activity relationships (SAR). The second series of analogues (4-16) were designed using structural information obtained from the Escherichia coli PanK (EcPanK) structure by targeting the pantothenate binding site and the adjacent phenylalanine-lined lipophilic pocket. Insight into the antimicrobial effect of N-pentylpantothenamide (N5-Pan) through its conversion to the antimetabolite ethyldethia-CoA and further incorporation into an inactive acyl carrier protein analogue drove the development of the third series of analogues (17-25) to enhance this effect using substrate-like substitutions. Each of the analogues was screened for enzyme inhibition activity against a panel of pantothenate kinases consisting of EcPanK, Aspergillus nidulans (AnPanK), SaPanK, and the murine isoform (MmPanK1alpha). Series 1 demonstrated only modest inhibitory activity, but did reveal some important SAR findings including stereospecific binding. Series 2 demonstrated a much higher inhibition rate for the entire series and significant inhibition was seen with analogues containing alkyl substituents. Series 3 demonstrated the most preferential inhibition profile, with the highest inhibitory activity against the SaPanK and MmPanK1alpha. The MmPanK1alpha protein was inhibited by a broad spectrum of the compounds, whereas the E. coli enzyme showed greater selectivity. The overall activity data from these analogues suggest a complex and non-enzyme specific SAR for pantothenamide substrate/inhibitors of the different PanK enzymes.     

Regulation of pantothenate kinase by coenzyme A and its thioesters

Pantothenate kinase catalyzes the rate-controlling step in the coenzyme A (CoA) biosynthetic pathway, and its activity is modulated by the size of the CoA pool. The effect of nonesterified CoA (CoASH) and CoA thioesters on the activity of pantothenate kinase was examined to determine which component of the CoA pool is the most effective regulator of the enzyme from Escherichia coli. CoASH was five times more potent than acetyl-CoA or other CoA thioesters as an inhibitor of pantothenate kinase activity in vitro. Inhibition by CoA thioesters was not due to their hydrolysis to CoASH. CoASH inhibition was competitive with respect to ATP, thus providing a mechanism to coordinate CoA production with the energy state of the cell. There were considerable differences in the size and composition of the CoA pool in cells grown on different carbon sources, and a carbon source shift experiment was used to test the inhibitory effect of the different CoA species in vivo. A shift from glucose to acetate as the carbon source resulted in an increase in the CoASH:acetyl-CoA ratio from 0.7 to 4.3. The alteration in the CoA pool composition was associated with the selective inhibition of pantothenate phosphorylation, consistent with CoASH being a more potent regulator of pantothenate kinase activity in vivo. These results demonstrate that CoA biosynthesis is regulated through feedback inhibition of pantothenate kinase primarily by the concentration of CoASH and secondarily by the size of the CoA thioester pool.     

Biochemical and molecular characterization of diseases linked to motor proteins

Recent studies have revealed that kinesin, dynein and myosin each form large superfamilies and participate in many different intracellular transport systems. Importantly, these motor proteins play significant roles in the pathogenesis of a variety of diseases. Studies using knockout mice for kinesin KIF1B have led to the identification of the cause of a human hereditary neuropathy, Charcot-Marie-Tooth disease type 2A. The function of members of the dynein superfamily whose existence has previously only been confirmed through genome databases, has been revealed by studies of immotile cilia syndrome. Unconventional myosins have been shown to function in the inner-ear cells by examination of hereditary human hearing impairment and studies using mouse models. In addition, some diseases are caused by mutations, not in the motor itself, but in the proteins associated with the motor proteins. Here, we discuss the relationship of these motor proteins and how they contribute to disease in molecular terms.     

Marked differences between two isoforms of human pyruvate dehydrogenase kinase

Pyruvate dehydrogenase kinase (PDK) isoforms 2 and 3 were produced via co-expression with the chaperonins GroEL and GroES and purified with high specific activities in affinity tag-free forms. By using human components, we have evaluated how binding to the lipoyl domains of the dihydrolipoyl acetyltransferase (E2) produces the predominant changes in the rates of phosphorylation of the pyruvate dehydrogenase (E1) component by PDK2 and PDK3. E2 assembles as a 60-mer via its C-terminal domain and has mobile connections to an E1-binding domain and then two lipoyl domains, L2 and L1 at the N terminus. PDK3 was activated 17-fold by E2; the majority of this activation was facilitated by the free L2 domain (half-maximal activation at 3.3 microm L2). The direct activation of PDK3 by the L2 domain resulted in a 12.8-fold increase in k(cat) along with about a 2-fold decrease in the K(m) of PDK3 for E1. PDK3 was poorly inhibited by pyruvate or dichloroacetate (DCA). PDK3 activity was stimulated upon reductive acetylation of L1 and L2 when full activation of PDK3 by E2 was avoided (e.g. using free lipoyl domains or ADP-inhibited E2-activated PDK3). In marked contrast, PDK2 was not responsive to free lipoyl domains, but the E2-60-mer enhanced PDK2 activity by 10-fold. E2 activation of PDK2 resulted in a greatly enhanced sensitivity to inhibition by pyruvate or DCA; pyruvate was effective at significantly lower levels than DCA. E2-activated PDK2 activity was stimulated >/=3-fold by reductive acetylation of E2; stimulated PDK2 retained high sensitivity to inhibition by ADP and DCA. Thus, PDK3 is directly activated by the L2 domain, and fully activated PDK3 is relatively insensitive to feed-forward (pyruvate) and feed-back (acetylating) effectors. PDK2 was activated only by assembled E2, and this activated state beget high responsiveness to those effectors.     

Characterization of protein kinase C and its isoforms in human T lymphocytes

Protein kinase C (PKC) regulates numerous T cell functions and is present in abundance in normal human T cells and certain T cell lines. Although crude Triton X-100 soluble material obtained from T cell pellets contains minimal PKC activity, DEAE chromatography revealed that 12 to 37% of cellular PKC was membrane associated, probably due to removal of an inhibitor through column chromatography. As in other tissues, PKC from lymphoid tissue was phospholipid and Ca2+ dependent and diolein reduced the Ca2+ requirements for enzyme activity. Hydroxylapatite chromatography revealed that T cells possess two major peaks of PKC activity. Although, the enzyme in these peaks had similar m.w. and identical iso-electric mobility, the proteins differed with respect to their autophosphorylation sites and immunoreactivity toward an isoform specific antibody. Furthermore, differences in their activities in the presence of phospholipid, diolein, and limiting amounts of Ca2+ imply that these isoforms may be differentially activated. We discuss optimal conditions for activation of PKC and its isoforms for study of T lymphocyte cellular function.     

High glucose upregulates pantothenate kinase 4 (PanK4) and thus affects M2-type pyruvate kinase (Pkm2)

A new Rattus norvegicus PanK gene was isolated by mRNA differential display from high concentration glucose-stimulated rat, which encodes a human PanK4-like protein with KOG2201 and KOG4584 domain. Proteins that interact with rat PanK4 were identified by the application of the yeast two-hybrid system. One of the components, Pkm2, was found to be associated with rat PanK4 and its two domains under both in vitro and in vivo conditions. Immunofluorescence staining and confocal scanning experiments showed that PanK4 could transiently co-express with Pkm2 in the cytoplasm of HeLa cell and HEK293T cell. These findings suggest that PanK4 interacts with Pkm2 and thereby may modulate the glucose metabolism through regulating the activity of Pkm2.     

Characterization of isoforms of human mitochondrial creatine kinase by isoelectric focusing

High enzyme activity of mitochondrial creatine kinase (creatine-N-phosphotransferase, mCK, EC 2.7.3.2) was detected in serum from a patient with advanced carcinoma of the rectum and its isoforms were characterized by means of isoelectric focusing (IEF). Three forms of mCK, membrane-bound (pI 6.9–7.0), octameric (pI 7.0–7.9) and dimeric (pI 6.7, 6.8, 6.9 and 7.0), were detected in the fresh serum. These three forms of mCK were converted to five dimeric isoforms, and these were characterized as one reduced form (pI 7.0) and four oxidized (pI 6.6, 6.7, 6.8 and 6.9) forms upon treatment with urea, hydrogen peroxide or 2-mercaptoethanol (2-ME). The C-terminal of the mCKs was concluded to be a lysine residue because the mCKs treated with carboxypeptidase B migrated to positions closer to the anode than did those not treated with carboxypeptidase B. Therefore, four bands were concluded to represent one reduced-delysined isoform (pI 6.4) and three oxidized-delysined isoforms (pI 6.1, 6.2 and 6.3). The broad octameric mCK band disappeared and a narrow band focused at pI 6.8–6.9 appeared upon probable delysination of the mCKs. Thus, the number of lysine residues at the C-terminal of the octamer was concluded to be variable due to variable catalysis by carboxypeptidase N in the plasma. mCKs seemed to be inactivated during conversion from a membrane-bound form to dimeric oxidized-delysined forms via the octameric, dimeric reduced and oxidized forms.     

Differential regulatory mechanism of Ca2+/calmodulin-dependent protein kinase kinase isoforms.

We have previously demonstrated that the alpha isoform of Ca(2+)/calmodulin-dependent protein kinase kinase (CaM-KKalpha) is strictly regulated by an autoinhibitory mechanism and activated by the binding of Ca(2+)/CaM [Tokumitsu, H., Muramatsu, M., Ikura, M., and Kobayashi, R. (2000) J. Biol. Chem. 275, 20090-20095]. In this study, we find that rat brain extract contains Ca(2+)/CaM-independent CaM-KK activity. This result is consistent with an enhanced Ca(2+)/CaM-independent activity (60-70% of total activity) observed with the recombinant CaM-KKbeta isoform. By using various truncation mutants of CaM-KKbeta, we have identified a region of 23 amino acids (residues 129-151) located at the N-terminus of the catalytic domain as an important regulatory element of the autonomous activity. A CaM-KKbeta deletion mutant of this domain shows a significant increase of Ca(2+)/CaM dependency for the CaM-KK activity as well as for the autophosphorylation activity. The activities of CaM-KKalpha and CaM-KKbeta chimera, in which autoinhibitory sequences were replaced by each other, were completely dependent on Ca(2+)/CaM, suggesting that the autoinhibitory regions of CaM-KKalpha and CaM-KKbeta are functional. These results establish for the first time that residues 129-151 of CaM-KKbeta participate in the release of the autoinhibitory domain from its catalytic core, resulting in generation of autonomous activity.