Báo cáo khoa học: Origin and properties of cytoplasmic and mitochondrial isoforms of taurocyamine kinase pptx

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:
(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 ( 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
( [27], sosuisignal

( />submit.html) and signalp ( />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 ( />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.
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