Origin and properties of cytoplasmic and mitochondrial isoforms of taurocyamine kinase Kouji Uda 1 , Naoto Saishoji 1 , Shuichi Ichinari 1 , W. Ross Ellington 2 and Tomohiko Suzuki 1 1 Laboratory of Biochemistry, Faculty of Science, Kochi University, Japan 2 Institute of Molecular Biophysics and Department of Biological Science, Florida State University, Tallahassee, FL, USA Keywords taurocyamine kinase; creatine kinase; phosphagen kinase; cDNA sequence; mitochondrial Correspondence T. Suzuki, Laboratory of Biochemistry, Faculty of Science, Kochi University, Kochi 780–8520, Japan Fax: +81 88 844 8356 Tel: +81 88 844 8693 E-mail: suzuki@cc.kochi-u.ac.jp (Received 9 March 2005, revised 23 April 2005, accepted 13 May 2005) doi:10.1111/j.1742-4658.2005.04767.x 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 brasil- iensis, 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 anne- lids, 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 taurocyam- ine) 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, construc- ted using CK templates, provided some insight into the structural corre- lation 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 mitochond- rial 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 mitochond- rial TKs, evolved from a CK-like ancestor(s) early in the divergence of the protostome metazoans. Furthermore, our results suggest that the cytoplas- mic and mitochondrial isoforms of TK evolved independently. Abbreviations AK, arginine kinase; CK, creatine kinase; GK, glycocyamine kinase; GS, guanidino specificity; LK, lombricine kinase; MiCK, mitochondrial creatine kinase; MiTK, mitochondrial taurocyamine kinase; TK, taurocyamine kinase. FEBS Journal 272 (2005) 3521–3530 ª 2005 FEBS 3521 Phosphagen kinases are enzymes that catalyze the reversible transfer of the gamma phosphoryl group of ATP to naturally occurring guanidino compounds such as creatine, glycocyamine, taurocyamine, lombricine and arginine, yielding ADP and a phosphorylated guanidine typically referred to as a phosphagen (phos- phocreatine, phosphoglycocyamine, etc.). Members of this enzyme family play a key role in the interconnec- tion between energy production and utilization in ani- mals [1]. In vertebrates, phosphocreatine is the only phosphagen, and the corresponding phosphagen kinase is creatine kinase (CK). In invertebrates, at least six unique phosphagens and corresponding kinases, phosphoglycocyamine (glycocyamine kinase, GK), phosphotaurocyamine (taurocyamine kinase, TK), phospholombricine (lombricine kinase, LK), phospho- opheline (opheline kinase, OK), phosphohypotauro- cyamine (hypotaurocyamine kinase, HTK) and phosphoarginine (arginine kinase, AK), are present in addition to phosphocreatine [2–5]. The former four enzymes, GK, LK, TK and OK, are found only in annelid and annelid-allied worms. Some species of annelids may also contain CK or AK. A broad spectrum of invertebrate AK and verteb- rate, protochordate and invertebrate CK sequences are now available. In terms of phosphagen kinases restric- ted to annelid groups, sequences for polychaete GKs [6–8] and LKs from the earthworm Eisenia [9] and the marine echiuroid worm Urechis [10] have appeared. Comparison of the available CK, AK, GK and LK sequences suggest that they have evolved from a com- mon ancestor [6,9,11], but the evolutionary relation- ships are not fully understood. Phylogenetic analyses have shown that there were two major evolutionary lineages in the phosphagen kinases, CK and AK, which probably diverged from their ancestral gene at the dawn of the radiation of multicellular animals [12]. The available evidence would suggest that GK and LK evolved within the CK lineage after the divergence of the lophotrochozoan and ecdysozoan protostomes [9]. There have been extensive studies on the structure, function and evolution of vertebrate and invertebrate CKs. Recently we showed that three CK isoforms, cytoplasmic (CK), flagellar (fCK) and mitochondrial (MiCK), diverged at an early stage of metazoan evo- lution [13]. MiCK, which is targeted to the inter- membrane compartment of mitochondria and exists primarily as a homo-octamer, plays a key role in intra- cellular energy transport from mitochondria to cyto- plasm [14], and fCK is present in primitive-type spermatozoa in some species of invertebrates as an unusual contiguous trimer [15]. Recently, Sona et al. [16] have shown that sponges, the most primitive of extant multicellular animals, have a true MiCK and what appear to be protoflagellar CKs. TK was first isolated from the body wall muscle of polychaete lugworm Arenicola marina [17,18]. It shows considerable activity for hypotaurocyamine (about 50% of that of the main target substrate, taurocyam- ine), and weak activity for lombricine and glycocyam- ine [19]. TK is a dimeric enzyme like LK and GK [18], and the partial 16 amino acid sequence of an internal peptide was very similar to that of the corresponding peptide of LK [20]. It is interesting to note that anti- sera against TK cross-reacted with LK and OK, but not with CK or AK [21]. The bulk of TK activity in Arenicola marina is cytoplasmic but 6–8% of the activ- ity was associated with the mitochondrial fraction of body wall muscle [19]. Surprisingly, Ellington and Hines [22] could not detect TK activity in the mito- chondria of a congenor Arenicola cristata. The relaxed substrate specificity of TKs for lombri- cine, their dimeric quaternary structure and immuno- logical similarities would suggest that TKs are related to LKs and possibly to the other phosphagen kinases restricted to annelid groups. To probe these relation- ships, the structural correlations of this relaxed sub- strate specificity and the possibility of cytoplasmic and mitochondrial isoforms of TK, we have amplified two cDNAs coding for Arenicola brasiliensis TKs and cloned them into pMAL plasmid. One of the two cDNA-derived amino acid sequences corresponds to a cytoplasmic isoform, and the other appears to be a mitochondrial isoform. Incorporating these new TK sequences in a phylogenetic analysis of phosphagen kinases showed that annelid-specific enzymes, GK, LK and TKs (cytoplasmic and mitochondrial) evolved from a common ancestor, and that they diverged from a primordial gene for CK at an early stage of meta- zoan evolution. Furthermore, the evolution of the cytoplasmic and mitochondrial isoforms of TK may have occurred independently. Our amino acid sequence comparisons with other phosphagen kinases provide insight into the nature of the observed differences in guanidine specificity in these two TKs. Results and Discussion Cytoplasmic and mitochondrial isoforms of TK are present in Arenicola brasiliensis We succeeded in amplifying two complete cDNAs coding for TK from the cDNA pool of the bodywall muscle of Arenicola brasiliensis. One was identified as a cytoplasmic form of TK, and the other with a long N-terminal extension of amino acid sequence was Cytoplasmic and mitochondrial taurocyamine kinases K. Uda et al. 3522 FEBS Journal 272 (2005) 3521–3530 ª 2005 FEBS identified as a mitochondrial isoform (referred to henceforth as MiTK) as described below. The cDNA for the cytoplasmic TK consists of 1811 bp with an ORF of 1098 bp and 5 ¢ and 3¢ untranslated regions of 21 and 692 bp, respectively. The ORF codes for a pro- tein containing 366 amino acid residues (Fig. 1) with a calculated molecular mass of 41 351.39 Da and an esti- mated pI of 7.62. The cDNA sequence of the cytoplas- mic TK from Arenicola brasiliensis has been deposited in DDBJ under the Accession No. AB186411. The cDNA for the MiTK consists of 1680 bp with an ORF of 1239 bp and 5¢ and 3¢ untranslated regions of 68 bp and 373 bp, respectively. The ORF codes for a protein containing 412 amino acid residues (Fig. 1) with a calculated mass of 46 201.38 Da and estimated pI of 8.55. The cDNA sequence of the MiTK has been deposited in DDBJ (AB186412). The deduced amino acid sequence of MiTK appears to have an N-terminal extension of 40 residues com- pared to the cytoplasmic TK (Fig. 1, underlined resi- dues). Analyses of the amino acid sequence of this region revealed that this extension region has a high probability of being a mitochondrial targeting sequence. Alignment of the mitochondrial targeting sequences from two invertebrate MiCKs and four ver- tebrate MiCKs (Fig. 2) indicates that the cleavage site of Ala is conserved also in Arenicola MiTK. The amino acid sequence of Arenicola cytoplasmic TK showed 69% identity with those of annelid LKs, 57–58% with annelid GKs, 49–58% with the three CK isoforms including sequences from annelids, and 25–41% with AKs. The partial amino acid sequence (LGYLGTCPTNIGTGLR) of a tryptic peptide of TK isolated from bodywall muscle of Arenicola marina [20] is identical to the corresponding sequence of Arenicola brasiliensis cytoplasmic TK except for one position. It has been shown that the Arenicola marina TK contains a higher number of cysteine residues than other phos- phagen kinases [29]. In agreement with this, the sequence of Arenicola brasiliensis cytoplasmic TK con- tains eight cysteine residues (Fig. 1), the same number of cysteines estimated to be in Arenicola marina TK. Interestingly, the amino acid sequence of Arenicola MiTK showed 63–67% identity with invertebrate MiCKs, 57–62% with cytoplasmic CKs, 56–60% with annelid enzymes (GK, LK and cytoplasmic TK deter- mined in this study), and 24–42% with AKs. Thus the entire sequence of Arenicola MiTK displays a much greater sequence similarity to the MiCKs than to the cytoplasmic TK. The recombinant enzymes were successfully expressed as soluble proteins, purified by affinity chro- matography, and appeared to be nearly homogeneous on SDS ⁄ PAGE. The enzyme activities of Arenicola TKs were measured using the substrates taurocyamine, lombricine, glycocyamine, creatine and arginine (Table 1). Arenicola cytoplasmic TK showed activity for the substrates taurocyamine and lombricine (9% that of taurocyamine) in agreement with the previous report on Arenicola marina TK [19]. On the other hand, mitochondrial TK showed activity for tauro- cyamine, lombricine (30% of that of taurocyamine) and glycocyamine (7% of that of taurocyamine). Recombinant Arenicola MiTK was incapable of phos- phorylating creatine even though this TK has a higher degree of sequence similarity to MiCKs than to LKs and other phosphagen kinases. Clearly, both cytoplas- mic and mitochondrial proteins from Arenicola are true TKs in that taurocyamine is their primary guani- dine substrate. To compare Arenicola cytoplasmic TK and MiTK with each other and typical CKs, homology models for both were constructed using the Swiss-Model auto- mated modeling server [46] with chicken and human MiCKs serving as templates. The predicted structures of TK and MiTK, both in the open (apo-) state, were very similar to each other, except for the length of the GS loop (one of the important determinants of guani- dine substrate recognition) described below (Fig. 3, loop indicated by the arrow). The open catalytic pocket is delineated by the GS loop and the identified residues (discussed below). Arenicola MiTK is very similar to typical mitochondrial creatine kinases Wyss et al. [14] reviewed the functional role of MiCK in intracellular energy transport from the mitochondria to the cytoplasm. The MiCK isoenzymes are specific- ally localized within the intermembrane space of mito- chondria, where creatine is rapidly phosphorylated to phosphocreatine by ATP exiting the adenine nucleotide translocase for export into the cytoplasm. The octa- meric form of MiCK and its targeting to the inter- membrane space evolved before the divergence of the protostomes and deuterstomes [30–32]. These results show that a mitochondrial isoform of TK is present in Arenicola and that this protein displays great similarit- ies to octameric MiCKs. Vertebrate and invertebrate MiCKs typically have higher pI values than their cytoplasmic isoform coun- terparts [14,32], which produces a net positive charge to the enzyme under physiological conditions. Arenicola MiTK also has a higher pI than the cytoplasmic TK. It has been proposed that this would facilitate the binding of MiCK to the negatively charged cardiolipin on the K. Uda et al. Cytoplasmic and mitochondrial taurocyamine kinases FEBS Journal 272 (2005) 3521–3530 ª 2005 FEBS 3523 Fig. 1. Multiple sequence alignment of Arenicola TK and MiTK with other phosphagen kinases, namely AKs from the horsehoe crab Limulus and the silkworm Bombyx,theb subunits of the GK from the polychaete Neanthes and Nereis, the MiCKs from the polychaete Chaetopte- rus and Neanthes, LKs from the oligochaete Eisenia and the echiuroid Urechis, and the cytoplasmic muscle CKs from human and the electric ray Torpedo. The underlined N-terminal sequence in Arenicola MiTK corresponds to a putative mitochondrial targeting sequence. The boxed sequences define the GS region in all phosphagen kinases. Arrow a, Ile69 equivalent residue in the GS region of all CKs; arrow b, position 130 (Arg95 equivalent in CK) containing phosphagen kinase specific residues; arrow c, Trp304 present in all MiCKs; arrow d, Val325 equival- ent present in all CKs. The basic residues in the C-terminus of Arenicola MiTK that could potentially mediate membrane interaction are shown in bold. Cytoplasmic and mitochondrial taurocyamine kinases K. Uda et al. 3524 FEBS Journal 272 (2005) 3521–3530 ª 2005 FEBS outer portion of the inner mitochondrial membrane [14]. The amino acid residues responsible for membrane binding have been identified as six or seven basic amino acid residues in the C-terminal region of the protein [33–35]. In the Arenicola MiTK sequence, five lysine residues (Lys401, Lys405, Lys407, Lys408, Lys418) and two arginines (Arg395, Arg402) are conserved in the C-terminal region (Fig. 1, bold). An internal lysine resi- due in MiCKs (Lys110 in chicken sarcomeric MiCK) has also been implicated in membrane interaction [35]; this residue is absolutely conserved in all MiCKs [12]. This Lys110 equivalent residue is also present in Areni- cola MiTK (Fig. 1, position 149), but not in cytoplas- mic TK, LK, GK and CK. These results suggest that Arenicola MiTK will also interact electrostatically with the inner mitochondrial membrane. A tryptophan residue (Trp264 in chicken sarcomeric MiCK) plays a key role in octamer stability of MiCKs as demonstrated by site-directed mutagenesis studies and by several X-ray crystal structures (reviewed in Fig. 3. Prediction of three-dimensional structures of Arenicola TK and MiTK by Swiss-Model [46]. Four key amino acid residues, a–d in Fig. 1, are shown. The GS loop is indicated by the arrow and the N-terminus of each protein is denoted by ‘N’. Fig. 2. Alignment of mitochondrial targeting sequences of vertebrate and invertebrate MiCKs and Arenicola MiTK. Sequences correspond to ubiquitous (uMiCK) and sarcomeric (sMiCK) MiCKs from man and chicken and MiCKs from the polychaetes Neanthes and Chaetopterus. The boxed region is the cleavage site. Table 1. Enzyme activity of recombinant Arenicola TK and MiTK for various guanidino compounds. Percentages are relative to the activity for the taurocyamine. v values were obtained in the presence of 4.75 m M guanidino compounds. NA, No activity. Arenicola TK Arenicola MiTK v(lmolÆmin )1 Æmg protein )1 )(%) v(lmolÆmin )1 Æmg protein )1 )(%) Taurocyamine 28.71 ± 1.06 100 17.82 ± 1.24 100 Lombricine 2.541 ± 0.297 8.9 5.16 ± 0.380 29 Glycocyamine NA – 1.32 ± 0.079 7.4 Arginine NA – NA – Creatine NA – NA – K. Uda et al. Cytoplasmic and mitochondrial taurocyamine kinases FEBS Journal 272 (2005) 3521–3530 ª 2005 FEBS 3525 [35]). This residue is absolutely conserved in all proto- stome and deuterostome MiCKs but is not present in cytoplasmic and flagellar CKs as well as in other phosphagen kinases such as AK, LK and GK [12,13,32]. In the MiCK from the sponge Tethya aurantia, however, this residue is replaced by a tyro- sine, and the MiCK forms dimers, not octamers [16]. The Arenicola MiTK has this conserved Trp residue (Figs 1 and 3, arrow c) suggesting that this protein has the potential to form octamers. The equivalent posi- tion in Arenicola cytoplasmic TK is an Arg residue (Figs 1 and 3). It seems highly likely that this MiTK exists in the octameric state in vivo where it can effect- ively interact with membranes in the intermembrane space. Expression and characterization of the oligo- meric state of the mature Arenicola MiTK should be most revealing. Evolution of cytoplasmic and mitochondrial taurocyamine kinases To evaluate the evolutionary relationships of cytoplas- mic and mitochondrial TKs with other phosphagen kinases, a phylogenetic tree was constructed from the amino acid sequences of cytoplasmic TK, MiTK, LK, GK and CK isoforms by the neighbor-joining method (Fig. 4). The neighbor-joining tree separates the sequences into two major groups: a group for CK iso- forms (presented schematically) and a group for annelid-specific enzymes, GK, LK and TK. Arenicola cytoplasmic TK is grouped with the other annelid- specific enzymes, especially adjacent to LKs, in accord with their immunological cross-reactivity and enzy- matic nature. Arenicola MiTK is clustered just outside the annelid cytoplasmic enzymes. Clearly, the annelid- specific phosphagen kinases (including cytoplasmic and mitochondrial TKs) and the cytoplasmic, mitochond- rial and flagellar CKs evolved from a common ances- tor. The oldest extant metazoans (sponges) have both mitochondrial and protoflagellar CK genes; the proto- flagellar CKs are probably ancestral not only to the fCKs but also to the cytoplasmic CKs [16]. Given this fact, we suggest that both cytoplasmic and mitochond- rial TKs evolved from CK ancestors. In fact an attractive, albeit speculative, scenario based on the sequence, catalytic and phylogenetic results, is that cytoplasmic TK and mitochondrial TK evolved inde- pendently; this latter event potentially took place much later in time in the course of annelid evolution, as there is no evidence for mitochondrial LK activities [22]. We have shown previously that echinoderm AKs, and probably all deuterostome AKs, evolved secondar- ily from a CK ancestor [36]. Structural basis for the catalytic properties of Arenicola cytoplasmic TK and MiTK A previous amino acid sequence alignment of phos- phagen kinases indicated that the guanidino specificity (GS) region, having significant amino acid deletions, is Fig. 4. Neighbor-joining tree for the amino acid sequences of phosphagen kinases. The tree was constructed using the program available on the home page of DDBJ (http://www.ddbj.nig.ac.jp/). Bootstrap values are shown at the branch points. The cluster of the CK portion is shown schematically by the representatives of three isoforms; cytoplasmic, mitochondrial and flagellar (a total of 71 CK sequences, available on the database of DDBJ, were used for tree construction). Limulus AK was used as the outgroup. The position of the Arenicola MiTK sequence appears to be unstable. If the number of CK sequences is reduced during phylogenetic tree construction, the Arenicola MiTK sequence is, in some cases, included in the CK cluster. Cytoplasmic and mitochondrial taurocyamine kinases K. Uda et al. 3526 FEBS Journal 272 (2005) 3521–3530 ª 2005 FEBS a possible candidate for the guanidine-recognition site [9] (Fig. 1, boxed region). There is a proportional rela- tionship between the size of the deletion in the GS region and the mass of the phosphagen substrate. LK and AK each have five amino acid deletions in this region and use relatively large guanidine substrates. CK has one such amino acid deletion while GK, which uses the smallest substrate glycocyamine, has no dele- tions (Fig. 1). The GS region encompasses part of the flexible loop in the N-terminal domain of the crystal structures of Limulus AK and Torpedo CK [37,38]. Our previous studies, using Nautilus AK, Stichopus AK, Danio CK [39–41] and Eisenia LK [42], showed that amino acid mutations introduced in the GS region greatly reduced their enzymatic activity. Interestingly, replacement by site-directed mutagenesis of the entire GS loop of Limulus AK with the equivalent and longer loop of CK resulted in a construct displaying reduced but appreciable AK activity [43]. The extended loop of CK in this AK construct did not preclude arginine ⁄ phosphoarginine binding. Arenicola cytoplasmic TK has a five amino acid deletion in the GS region as in LK and AK, in agree- ment with the proposed relationship between the size of guanidine substrate and the number of amino acids deleted [9]. In addition, the amino acid sequence of GS region of cytoplasmic TK was very similar to that of Eisenia LK (Fig. 1). This feature is consistent with the following enzymatic properties: LK shows considerable activity for taurocyamine (about one-third that of the main target substrate, lombricine) [42], while TK shows considerable activity for lombricine (Table 1). Phylogenetic analysis also suggests that TK and LK have evolved from a common ancestor (Fig. 4). Areni- cola MiTK unexpectedly had only one amino acid deletion in the GS region, unlike cytoplasmic TK but similar to MiCKs. This does not fit with the proposed relationship between the size of guanidine substrate and the number of deletions in the GS region. The dif- ference in the length of the GS region is easily seen in the homology models (Fig. 3, arrows). However, if we consider that Arenicola MiTK shows broader substrate specificity than cytoplasmic TK (Table 1), the five- residue deletion in the GS region is preferable to the original target activity for taurocyamine. It has recently been shown in rabbit muscle CK that two key residues form a ‘specificity pocket’ [44]. Ile69, which is in the so-called GS loop, and Val325 stabilize the methyl group of creatine ⁄ phosphocreatine. The equivalent Ile69 residue is lacking in AKs, GKs and LKs, while the Val325 equivalent is an absolutely con- served Glu residue in these latter three phosphagen kinases [44]. All noncreatine phosphagens and guanidine substrates lack the characteristic methyl group of creatine but instead have a proton in this position. These results show that in both Arenicola TKs the Ile69 equivalent residue is not present but for different reasons; firstly the cytoplasmic TK has the characteristic GS loop deletions, including the Ile69 equivalent, and secondly the MiTK has the four CK- like GS loop insertions but has a threonine residue (Fig. 3, Thr103) instead of the Ile69 equivalent (Fig. 1, arrow a in the boxed GS region). Note that both cyto- plasmic TK and MiTK have the characteristic Glu residue near the C-terminal region (Figs 1 and 3, arrow d). Because AK, LK, GK and TK lack the equivalent CK Ile69 and Val325 residues that form a specificity pocket for creatine in CKs, what are the structural correlates of guanidine specificity for the other phos- phagen kinases? How can one explain the somewhat broader specificity for TKs (and LKs) and the differ- ences in capacity for utilization of lombricine and glyco- cyamine by cytoplasmic TK and MiTK? The amino acid residue at position 130 (equivalent to position 95 in rabbit muscle CK) in the alignment of Fig. 1 (arrow b) is strictly conserved in each of phosphagen kinases, namely Arg in CK, Ile in GK, Tyr in AK and Lys in LK. While this residue is not directly involved in sub- strate binding in CK and AK crystal structures, it is located close to the guanidine substrate binding site. The replacement of this residue dramatically reduced the activity of rabbit muscle CK [45]. Moreover, replacement of Lys by Tyr in Eisenia LK altered the major target substrate of the enzyme from lombricine to taurocyamine [42]. Arenicola cytoplasmic TK has a histidine at position 130 (Fig. 3) that is a unique resi- due compared to the other phosphagen kinases; this residue may tune the active site to enhance substrate specificity for taurocyamine and minimize the activity with other guanidine substrates. Arenicola MiTK, like LK, has a Lys (Fig. 3), in accordance with the higher activity of MiTK for lombricine than that of cytoplas- mic TK (Table 1). Catalysis in AK and CK involves the very precise positioning of the substrates in the active site through a variety of intermolecular contacts [37,38]. A similar array of such contacts, likely to be present, remains to be elucidated in TKs and, in fact, the other annelid-specific phosphagen kinases. General conclusions Both cytoplasmic and mitochondrial TKs appear to have evolved independently in the annelid lineage. Arenicola MiTK evolved from a mitochondrial CK, and still potentially retains many of the features of K. Uda et al. Cytoplasmic and mitochondrial taurocyamine kinases FEBS Journal 272 (2005) 3521–3530 ª 2005 FEBS 3527 MiCKs including octameric quaternary structure and capacity for binding to the inner mitochondrial mem- brane. Both cytoplasmic TK and MiTK utilize tauro- cyamine as their primary substrate but MiTK is less specific and utilizes lombricine and glycocyamine to some extent. This relaxed specificity can be partially explained by differences in key amino acid residues in these two TKs. Experimental procedures cDNA amplification and sequence determination of cytoplasmic TK and mitochondrial TK (MiTK) from A. brasiliensis A specimen of A. brasiliensis was collected on the sea shore at Tokushima, Japan. Total RNA was isolated from the body wall muscle by the acid guanidinium thiocyanate ⁄ phenol ⁄ chloroform extraction method [23]. mRNA was purified from total RNA using a poly(A) + isolation kit (Nippon Gene, Tokyo, Japan). The single stranded cDNA was synthesized with Ready-To-Go You-Prime First-Strand Beads (Amersham Pharmacia Biotech, Piscataway, NJ, USA) with a lock-docking oligo(dT) primer [24]. The 3 ¢ half of the TK cDNA was amplified using the lock-docking oligo(dT) primer and a 256-fold ‘universal’ phosphagen kinase primer (5¢-GTNTGGGTNAAYGAR GARGAYCA-3¢) designed from the highly conserved sequences of phosphagen kinases [6]. Ex Taq DNA poly- merase (Takara, Kyoto, Japan) was used as the amplifying enzyme. PCR amplification was performed for 30 cycles, each consisting of 30 s at 94 °C for denaturation, 30 s at 60 °C for annealing and 2 min at 72 °C for primer exten- sion. The amplified products were purified by agarose gel electrophoresis and subcloned into the pGEM-T Easy Vec- tor (Promega, Madison, WI, USA). Nucleotide sequences were determined with an ABI PRISM 3100-Avant DNA sequencer using a BigDye Terminators v3.1 Cycle Sequen- cing Kit (Applied Biosystems, Foster City, CA, USA). A poly(G) + tail was added to the 3¢ end of the Arenicola cDNA pool with terminal deoxynucleotidyl transferase (Promega). The 5¢ half of the TK cDNA was then amplified using the oligo(dC) primer (5¢-GAATTC 18 -3¢) and a specific primer (5¢-GGCCCTTGGCCTTCATCAGG-3¢ for cyto- plasmic TK, or 5¢-CTCGAAGACCTGCTTCATGTTTC-3¢ for MiTK) designed from the sequence of the 3¢ region. The amplified products were purified, subcloned and sequenced as described above. Cloning and expression of Arenicola TKs The open reading frames (ORFs) of Arenicola TKs were amplified and cloned into the EcoRI ⁄ HindIII site of pMAL-c2 (New England Biolabs, Beverly, MA, USA). In the case of MiTK, the mitochondrial targeting sequence was removed. The maltose binding protein (MBP)-TK fusion protein was expressed in Escherichia coli TB1 cells by induction with 1 mm isopropyl thio-b-d-galactoside at 25 °C for 24 h. The cells were resuspended in 5· Tris ⁄ EDTA buffer, sonicated, and the soluble protein was extracted. Recombinant TK was purified by affinity chro- matography using amylose resin (New England Biolabs). The purity of the recombinant enzyme was verified by SDS ⁄ PAGE. The enzymes were placed on ice until use, and enzymatic activity was determined within 12 h. Analyses of N-terminal amino acid sequences of Arenicola TK and MiTK Analyses were done using several on-line tools, the targetp (http://www.cbs.dtu.dk/services/TargetP/) [27], sosuisignal (http://sosui.proteome.bio.tuat.ac.jp/sosuisignal/sosuisignal_ submit.html) and signalp (http://www.cbs.dtu.dk/services/ SignalP/) [28]. Modeling of three-dimensional structures Predictions of the three-dimensional structures of Arenicola TK and MiTK were made by using the Swiss-Model auto- mated modeling server (http://www.expasy.org/swissmod/ SWISS-MODEL.html; the First Approach Method set at default parameters) [46]. Swiss-Pdb viewer version 3.7 was used to generate a three-dimensional image. Under these conditions, models for Arenicola TK and MiTK were con- structed, based on the structures of chicken and human MiCKs. Alignment o f amino ac id sequences o f phosphagen kinases and construction of phylogenetic tree Multiple sequence alignment of Arenicola TK and MiTK and other phosphagen kinases was done with the clustalw program available on DDBJ homepage (http://www. ddbj.nig.ac.jp/Welcome-j.html). The phylogenetic tree was constructed with the neighbor-joining method available on the DDBJ homepage. Amino acid sequences were taken from DDBJ and GenBank. Limulus AK was used as the outgroup. Enzyme assays Enzyme activity was measured using the NADH-linked spectrophotometric assay at 25 °C [25] and determined for the forward reaction (phosphagen synthesis). Details are as described previously [26]. Protein concentration was estima- ted from the absorbance at 280 nm (0.77 at 280 nm in a 1 cm cuvette corresponds to 1 mg proteinÆmL )1 ). Cytoplasmic and mitochondrial taurocyamine kinases K. Uda et al. 3528 FEBS Journal 272 (2005) 3521–3530 ª 2005 FEBS Acknowledgements This work was supported by a grant from the presi- dent of Kochi University to TS, a grant (17570062) from the Grants-In-Aid for Scientific Research of Japan to TS, and a grant from the U.S. National Sci- ence Foundation (IBN-0130024) to WRE. References 1 Ellington WR (2001) Evolution and physiological roles of phosphagen systems. Annu Rev Physiol 63, 289–325. 2 van Thoai N (1968) Homologous phosphagen phospho- kinases. Homologous Enzymes and Biochemical Evolution (van Thoai N & Roche J, eds), pp. 199–229. Gordon and Breach, NY. 3 Watts DC (1968) The origin and evolution of phospha- gen phosphotransferases. Homologous Enzymes and Bio- chemical Evolution (van Thoai N & Roche J, eds), pp. 279–296. Gordon and Breach, NY. 4 Morrison JF (1973) Arginine kinase and other inverte- brate guanidino kinases. The Enzymes (Boyer PC, ed), pp. 457–486. 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