Evolution of the Cytoplasmic and Mitochondrial Phosphagen Kinases Unique to Annelid Groups

Creatine kinase (CK) is a member of a group of phosphoryl transfer enzymes called phosphagen kinases that play a key role in cellular energy transactions in animals. Three CK isoform gene families are known—cytoplasmic CK (CK), flagellar CK (fCK), and mitochondrial CK (MiCK). Each of the isoforms has a unique gene structure (intron/exon organization). A broad array of other phosphagen kinases is present in animals. Some of these enzymes are found only in annelids and closely related groups including glyocyamine kinase (GK), lombricine kinase (LK), taurocyamine kinase (TK), and a unique arginine kinase (AK) restricted to annelids. Phylogenetic analyses of these annelid phosphagen kinases indicate that they appear to have evolved from a CK-like ancestor. To gain a greater understanding of the relationship of the CK isoforms to the annelid enzymes, we have determined the intron/exon organization of the genes for the following phosphagen kinases: Eisenia LK, Sabellastarte AK, and Arenicola mitochondrial TK (MiTK). Analysis of genomic database for the polychaete Capitella sp. yielded two putative LK genes [cytoplasmic LK and mitochondrial LK (MiLK)]. The intron/exon organization of these genes was compared with available data for cytoplasmic and mitochondrial CKs, and an annelid GK. Surprisingly, these annelid genes, irrespective of whether they are cytoplasmic (LK, AK, and GK) or mitochondrial (MiTK and MiLK), had the same 8-intron/9-exon organization and were strikingly similar to MiCK genes sharing seven of eight splice junctions. These results support the view that the MiCK gene is basal and ancestral to the phosphagen kinases unique to annelids.     

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.     

Origin of the genes for the isoforms of creatine kinase

Creatine kinase (CK) is a member of a family of phosphoryl transfer enzymes called phosphagen (guanidino) kinases which play a central role in cellular energy homeostasis. There are three CK isoform gene groups, each coding for proteins targeted to different intracellular compartments--cytoplasmic (CytCK), mitochondrial (MtCK) and flagellar (FlgCK). The former two CKs are either dimeric or octameric while FlgCKs are contiguous trimers consisting of three fused, complete CK domains. Conventional wisdom supports the view that CKs evolved from a cytoplasmic, monomeric ancestral protein closely related to a phosphagen kinase homologue, arginine kinase (AK). Recently, it has been shown that a demosponge (Phylum Porifera) expresses a true MtCK and two dimeric, protoflagellar CKs (protoflgCK) with great similarity to FlgCKs. To further probe the early evolution of CK, we have obtained additional sequences for Mt- and protoflgCKs from two more demosponges and from three hexactinellid (glass) sponges as well as an MtCK sequence from a basal metazoan cnidarian. Phylogenetic analyses using Maximum Likelihood (ML) of these new CK sequences with other CKs and phosphagen kinases yielded a consensus tree containing an assemblage of MtCKs and a supercluster consisting of protoflg-, Flg- and CytCKs. The MtCKs appear basal in the tree topology consistent with prior results. Within the protoflg-, Flg- and CytCK supercluster, the protoflgCKs appear to be allied to the domains of the FlgCKs, although the support is not robust. PCR amplification of genomic DNA and sequencing of the genes for Mt- and protoflgCK from the demosponge Suberites fuscus showed that the sponge MtCK shares four-five common intron:exon boundaries with invertebrate, protochordate and vertebrate MtCKs supporting a common ancestry and the extreme conservation of intron:exon organization in MtCK genes. The protoflgCK gene organization was highly divergent in relation to other CK genes but shares a common intron:exon boundary with domain 2 of the gene for the FlgCK from the tunicate Ciona intestinalis, providing support for the linkage of the protoflgCKs with the FlgCKs. Our results show that the two, major CK gene lineages are present in arguably the oldest, extant metazoan group, the hexactinellid sponges, indicating that these two genes are ancient and confirming prior work that the MtCK gene is likely basal and ancestral.     

Role of amino-acid residue 95 in substrate specificity of phosphagen kinases

The purpose of this study is to elucidate the mechanisms of guanidine substrate specificity in phosphagen kinases, including creatine kinase (CK), glycocyamine kinase (GK), lombricine kinase (LK), taurocyamine kinase (TK) and arginine kinase (AK). Among these enzymes, LK is unique in that it shows considerable enzyme activity for taurocyamine in addition to its original target substrate, lombricine. We earlier proposed several candidate amino acids associated with guanidine substrate recognition. Here, we focus on amino-acid residue 95, which is strictly conserved in phosphagen kinases: Arg in CK, Ile in GK, Lys in LK and Tyr in AK. This residue is not directly associated with substrate binding in CK and AK crystal structures, but it is located close to the binding site of the guanidine substrate. We replaced amino acid 95 Lys in LK isolated from earthworm Eisenia foetida with two amino acids, Arg or Tyr, expressed the modified enzymes in Escherichia coli as a fusion protein with maltose-binding protein, and determined the kinetic parameters. The K95R mutant enzyme showed a stronger affinity for both lombricine (Km=0.74 mM and kcat/Km=19.34 s(-1) mM(-1)) and taurocyamine (Km=2.67 and kcat/Km=2.81), compared with those of the wild-type enzyme (Km=5.33 and kcat/Km=3.37 for lombricine, and Km=15.31 and kcat/ Km=0.48for taurocyamine). Enzyme activity of the other mutant, K95Y, was dramatically altered. The affinity for taurocyamine (Km=1.93 and kcat/Km=6.41) was enhanced remarkably and that for lombricine (Km=14.2 and kcat/Km=0.72) was largely decreased, indicating that this mutant functions as a taurocyamine kinase. This mutant also had a lower but significant enzyme activity for the substrate arginine (Km=33.28 and kcat/Km=0.01). These results suggest that Eisenia LK is an inherently flexible enzyme and that substrate specificity is strongly controlled by the amino-acid residue at position 95.     

Organization of the gene for an invertebrate mitochondrial creatine kinase: comparisons with genes of higher forms and correlation of exon boundaries with functional domains

Two major gene duplication events are thought to have taken place in the evolution of creatine kinases (CK) in the vertebrates - (1) the formation of distinct mitochondrial (MiCK) and cytoplasmic forms from the primordial gene and (2) subsequent formation of the sarcomeric (sar-) and ubiquitous (ubi-) isoforms of octameric MiCK and muscle (M) and brain (B) isoforms of dimeric, cytoplasmic CK. The genes of these two CK clades reflect a distant divergence as sar- and ubiMiCK genes consistently have nine protein-coding exons while M- and B-CK genes have seven protein-coding exons; these genes share only one common exon. CKs are also widely distributed in the invertebrates and it has recently been shown that MiCKs evolved well before the divergence of the major metazoan groups. In the present communication, we report the structure and topology of the gene for MiCK from the protostome marine worm Chaetopterus variopedatus. The protein-coding region of the gene for this primitive MiCK spans over 10 kb and consists of eight exons, the last five (E4-E8) have identical boundaries to the corresponding exons of sar- and ubiMiCK genes. Exon-3 of the C. variopedatus MiCK gene consists of the corresponding E3 and E4 of the vertebrate MiCKs with no intervening intron. E1 is longer and E2 is shorter in the polychaete MiCK gene than the counterpart sarcomeric and ubiquitous genes. The insertion of the intron in C. variopedatus E3 creating the two exons as well as the rearrangement of the intron between E1 and E2 must have occurred prior to or coincident with the duplication event creating the two vertebrate mitochondrial isoforms. Sarcomeric and ubiMiCKs display substantial differences from their invertebrate MiCK counterparts in properties relating to octamer stability and membrane binding. The evolutionary changes in gene topology may be a component of this functional progression.     

Cloning and expression of a lombricine kinase from an echiuroid worm: insights into structural correlates of substrate specificity

Phosphagen kinases constitute a large family of enzymes catalyzing the reversible phosphorylation of guanidino acceptor compounds. These guanidino substrates differ substantially in size and chemical properties. In spite of the appearance of X-ray crystal structures for two members of this family, creatine kinase (CK) and arginine kinase (AK), the structural correlates of substrate specificity remain to be fully elucidated. We have determined the cDNA and deduced amino acid sequences for lombricine (guanidinethylphosphoserine) kinase (LK) from the echiuroid worm Urechis caupo and expressed the cDNA in Escherichia coli. The recombinant protein was purified by affinity chromatography and showed high capacity for phosphorylation of lombricine. Phosphagen kinases consist of a small, N-terminal domain and a much larger domain connected by a linker sequence. A key event in catalysis in CK and AK, and certainly all other phosphagen kinases, is a large conformational change involving involving a rotation of the two domains and the movement of two highly conserved flexible loops (one located in the small domain; the other located in the large domain of these enzymes) which clamp down on the substrates. Multiple sequence alignments of Urechis LK with the only other LK sequence available and CK, AK and glycocyamine kinase sequences, confirm the importance of the small flexible loop located in the N-terminal domain of phosphagen kinases as one component of the structural determinants of guanidine specificity. The role of the other flexible loop in the large domain in terms of substrate specificity remains questionable.     

Variable intron/exon structure in the oligochaete lombricine kinase gene

Lombricine kinase is an annelid enzyme that belongs to the phosphagen kinase family of which creatine kinase and arginine kinase are the typical representatives. The enzymes play important roles in the cellular energy metabolism of animals. Biochemical, physiological and molecular information with respect to lombricine kinase is limited compared to other phosphagen kinases. This study presents data on the cDNA sequences of lombricine kinase from two smaller oligochaetes, Enchytraeus sp. and Stylaria sp. The deduced amino acid sequences are analyzed and compared with other selected phosphagen kinases. The intron/exon structure of the lombricine kinase gene was determined for these two species as well as two additional oligochaetes, Lumbriculus variegatus and Tubifex tubifex, and compared with available data for annelid phosphagen kinases. The data indicate the existence of a variable organization of the proposed 8-intron/9-exon gene structure. The results provide further insights in the evolution and position of these enzymes within the phosphagen kinase family.     

The Early Evolution of the Phosphagen Kinases—Insights from Choanoflagellate and Poriferan Arginine Kinases

Arginine kinase (AK) is a member of a large family of phosphoryl transfer enzymes called phosphagen (guanidino) kinases. AKs are present in certain protozoans, sponges, cnidarians, and both lophotrochozoan and ecdysozoan protostomes. Another phosphagen kinase, creatine kinase (CK), is found in sponges, cnidarians, and both deuterostome and protostome groups but does not appear to be present in protozoans. To probe the early evolution of phosphagen kinases, we have amplified the cDNAs for AKs from three choanoflagellates and from the hexactinellid sponge Aphrocallistes beatrix and the demosponges Suberites fuscus and Microciona prolifera. Phylogenetic analysis using maximum likelihood of these choanoflagellate and sponge AKs with other AK sequences revealed that the AK from the choanoflagellate Monosiga brevicollis clusters with the AK from the glass sponge Aphrocallistes and is part of a larger cluster containing AKs from the demosponges Suberites and Microciona as well as basal and protostome invertebrates. In contrast, AKs from Codonosiga gracilis and Monosiga ovata form a distinct cluster apart from all other AK sequences. tBLASTn searches of the recently released M. brevicollis genome database showed that this species has three unique AK genes—one virtually identical to the M. brevicollis cDNA and the other two showing great similarity to C. gracilis and M. ovata AKs. Three distinct AK genes are likely present in choanoflagellates. Two of these AKs display extensive similarity to both CKs and an AK from sponges. Previous work has shown CK evolved from an AK-like ancestor prior to the divergence of sponges. The present results provide evidence suggesting that the initial gene duplication event(s) leading to the CK lineage may have occurred before the divergence of the choanoflagellate and animal lineages.     

Cloning of deoxynucleoside monophosphate kinase genes and biosynthesis of deoxynucleoside diphosphates

The genes encoding four deoxynucleoside monophosphate kinase (dNMP kinase) enzymes, including ADK1 for deoxyadenylate monophosphate kinase (AK), GUK1 for deoxyguanylate monophosphate kinase (GK), URA6 for deoxycytidylate monophosphate kinase (CK), and CDC8 for deoxythymidylate monophosphate kinase (TK), were isolated from the genome of Saccharomyces cerevisiae ATCC 2610 strain and cloned into E. coli strain BL21(DE3). Four recombinant plasmids, pET17b-JB1 containing ADK1, pET17b-JB2 containing GUK1, pET17b-JB3 containing URA6, and pET17b-JB4 containing CDC8, were constructed and transformed into E. coli strain for over-expression of AK, GK, CK, and TK. The amino acid sequences of these enzymes were analyzed and a putative conserved peptide sequence for the ATP active site was proposed. The four deoxynucleoside diphosphates (dNDP) including deoxyadenosine diphosphate (dADP), deoxyguanosine diphosphate (dGDP), deoxycytidine diphosphate (dCDP), and deoxythymidine diphosphate (dTDP), were synthesized from the corresponding deoxynucleoside monophosphates (dNMP) using the purified AK, GK, CK, and TK, respectively. The effects of pH and magnesium ion concentration on the dNDP biosynthesis were found to be important. A kinetic model for the synthetic reactions of dNDP was developed based on the Bi-Bi random rapid equilibrium mechanism. The kinetic parameters including the maximum reaction velocity and Michaelis-Menten constants were experimentally determined. The study on dNDP biosynthesis reported in this article are important to the proposed bioprocess for production of deoxynucleoside triphosphates (dNTP) that are used as precursors for in vitro DNA synthesis. There is a significant advantage of using enzymatic biosyntheses of dNDP as compared to the chemical method that has been in commercial use.     

The role of Arg-96 in Danio rerio creatine kinase in substrate recognition and active center configuration

In creatine kinases (CKs), the amino acid residue-96 is a strictly conserved arginine. This residue is not directly associated with substrate binding, but it is located close to the binding site of the substrate creatine. On the other hand, the residue-96 is known to be involved in expression in the substrate specificity of various other phosphagen (guanidino) kinases, since each enzyme has a specific residue at this position: arginine kinase (Tyr), glycocyamine kinase (Ile), taurocyamine kinase (His) and lombricine kinase (Lys). To gain a greater understanding of the role of residue-96 in CKs, we replaced this residue in zebra fish Danio rerio cytoplasmic CK with other 19 amino acids, and expressed these constructs in Escherichia coli. All the twenty recombinant enzymes, including the wild-type, were obtained as soluble form, and their activities were determined in the forward direction. Compared with the activity of wild-type, the R96K mutant showed significant activity (8.3% to the wild-type), but 10 mutants (R96Y, A, S, E, H, T, F, C, V and N) showed a weak activity (0.056–1.0%). In the remaining mutants (R96Q, G, M, P, L, W, D and I), the activity was less than 0.05%. Our mutagenesis studies indicated that Arg-96 in Danio CK can be substituted for partially by Lys, but other replacements caused remarkable loss of activity. From careful inspection of the crystal structures (transition state analog complex (TSAC) and open state) of Torpedo cytoplasmic CK, we found that the side chain of R96 forms hydrogen bonds with A339 and D340 only in the TSAC structure. Based on the assumption that CKs consist of four dynamic domains (domains 1–3, and fixed domain), the above hydrogen bonds act to link putative domains 1 and 3 in TSAC structure. We suggest that residue-96 in CK and equivalent residues in other phosphagen kinases, which are structurally similar, have dual roles: (1) one involves in distinguishing guanidino substrates, and (2) the other plays a key role in organizing the hydrogen-bond network around residue-96 which offers an appropriate active center for the high catalytic turnover. The mode of development of the network appears to be unique each phosphagen kinase, reflecting evolution of each enzyme.     

Structural and functional implications of the amino acid sequences of dimeric, cytoplasmic and octameric mitochondrial creatine kinases from a protostome invertebrate.

The cDNA and deduced amino-acid sequences for dimeric and octameric isoforms of creatine kinase (CK) from a protostome, the polychaete Chaetopterus variopedatus, were elucidated and then analysed in the context of available vertebrate CK sequences and the recently determined crystal structure of chicken sarcomeric mitochondrial CK (MiCK). As protostomes last shared a common ancestor with vertebrates roughly 700 million years ago, observed conserved residues may serve to confirm or reject contemporary hypotheses about the roles of particular amino acids in functional/structural processes such as dimer/octamer formation and membrane binding. The isolated cDNA from the dimeric CK consisted of 1463 nucleotides with an open reading frame of 1116 nucleotides encoding a 372-amino-acid protein having a calculated molecular mass of 41.85 kDa. The percentage identity of C. variopedatus dimeric CK to vertebrate CK is as high as 69%. The octameric MiCK cDNA is composed of 1703 nucleotides with an open reading frame of 1227 nucleotides. The first 102 nucleotides of the open reading frame encode a 34-amino-acid leader peptide whereas the mature protein is composed of 375 amino acids with a calculated molecular mass of 42.17 kDa. The percentage identity of C. variopedatus MiCK to vertebrate CK is as high as 71%. This similarity is also evident in residues purported to be important in the structure and function of dimeric and octameric CK: (a) presence of seven basic amino acids in the C-terminal end thought to be important in binding of MiCK to membranes; (b) presence of a lysine residue (Lys110 in chicken MiCK) also thought to be involved in membrane binding; and (c) presence of a conserved tryptophan thought to be important in dimer stabilization which is present in all dimeric and octameric guanidino kinases. However, C. variopedatus MiCK lacks the N-terminal heptapeptide present in chicken MiCK, which is thought to mediate octamer stabilization. In contrast with vertebrate MiCK, polychaete octamers are very stable indicating that dimer binding into octamers may be mediated by additional and/or other residues. Phylogenetic analyses showed that both octamer and dimer evolved very early in the CK lineage, well before the divergence of deuterostomes and protostomes. These results indicate that the octamer is a primitive feature of CK rather than being a derived and advanced character.     

Comparison of kinetic constants of creatine kinase isoforms

The purpose of this study was to elucidate the functional differences between the CK isoforms by cloning the cDNAs of 12 CK isoforms: the M and B cytoplasmic forms and uMiCK from mouse, the M1, M2 and B cytoplasmic forms from Danio rerio, M1 and M2 cytoplasmic forms from the lower vertebrate Lampetra japonica, a cytoplasmic CK and a MiCK from the marine worm Neanthes diversicolor, and a cytoplasmic CK and a MiCK from the soft coral Dendronephthya gigantea. These were expressed in Escherichia coli as a fusion protein with maltose-binding protein, and kinetic constants (K(m), K(d) and k(cat)) of all the recombinant enzymes, except for the unstable Dendronephthya cytoplasmic CK, were determined for the forward reaction. The kinetic constants of the M- and B-forms of the mouse and Danio cytoplasmic CKs differed significantly, with the K(m) for creatine (K(m)Cr) of M-CK being three- to nine-fold higher than that of B-CK, possibly reflecting differences in the concentration of creatine in muscle and brain cells. The mouse uMiCK had the lowest K(m)Cr value among the CK isoforms. In addition, it also exhibited a strong synergism for substrate binding (K(d)/K(m)=11.8). These results indicate that uMiCK has unique characteristics compared with other CK isoforms. Two subisoforms of M-CK were found in the lower vertebrate L. japonica, and the kinetic constants of recombinant M1- and M2-CKs differed significantly. The M1- and M2-CKs were expressed in skeletal muscle with a ratio of 7:3, while M1-CK was the predominant subisoform in the testis. The kinetic constants of cytoplasmic CK from the marine worm Neanthes were significantly different from those of Neanthes MiCK, possibly indicating that functional differences among CK isoforms occurred at least before the divergence of annelids from other protostome invertebrates.     

Evolution and Divergence of the Genes for Cytoplasmic,Mitochondrial, and Flagellar Creatine Kinases

Creatine kinase (CK) plays a central role in energy homeostasis in cells that display high and variable rates of energy turnover. A number of CK genes exist, each being targeted to particular intracellular compartments. In the vertebrates, two genes code for proteins which form homo- and heterodimers targeted to the cytoplasm, while two additional genes code for primarily octameric proteins targeted to the mitochondrial intermembrane space. Yet another gene is present in certain groups which codes for three fused, complete CK domains and is typically targeted to the flagellar membrane of primitive-type spermatozoa. CK is widely distributed in protochordates and both protostome and deuterostome invertebrate groups. The evolutionary relationships of these CK genes have not been fully elucidated. The present communication reports new cDNA-derived deduced amino acid sequences for four cytoplasmic and three mitochondrial CKs and one flagellar CK from lophotrochozoan, protostome invertebrates as well as a new cytoplasmic CK sequence from a protochordate tunicate. These new sequences, coupled with available sequences in the databases and sequences extracted from genome sequencing projects, provide revealing insights into the evolution and divergence of CK genes. Phylogenetic analyses showed that single cytoplasmic, mitochondrial, and flagellar CK genes were present prior to the divergence of the protostomes and deuterostomes. The flagellar CK gene may have evolved within the cytoplasmic gene clade, although the evidence is somewhat equivocal. The two cytoplasmic genes in the vertebrates, and most likely the two mitochondrial genes, evolved after the divergence of the craniates from the protochordates. Comparison of the structure of the genes for selected cytoplasmic, mitochondrial, and flagellar CKs revealed two identical intron boundaries, further reinforcing the notion of a common evolutionary origin, but also showed patterns of changes in structure consistent with each gene type. These studies show that the cytoplasmic, mitochondrial, and flagellar CK genes are rather ancient and that there has been a systematic pattern of duplication and divergence consistent with changing nature of energy demands and physicochemical environment in the cells where they are expressed.[Reviewing Editor: Martin Kreitman]     

Gene duplication events producing muscle (M) and brain (B) isoforms of cytoplasmic creatine kinase: cDNA and deduced amino acid sequences from two lower chordates.

Creatine kinase (CK) is coded for by at least four loci in higher vertebrates--two cytoplasmic isoforms, muscle (M) and brain (B), and two mitochondrial isoforms, sarcomeric and ubiquitous. M is expressed primarily in skeletal muscle, while B is expressed in a variety of cells, including cardiac and smooth muscle fibers, neurons, transport epithelia, and photoreceptors. M and B subunits form very stable homodimers (MM [M-CK], BB [B-CK]) and heterodimers (MB). M-CK is capable of binding to the M line of the myofibril, thereby creating an energy transfer microcompartment; BB and MB CKs are not. M- and B-like CKs are present in all vertebrates yet examined, including fish. Cytoplasmic, dimeric CKs are widely distributed in the invertebrates. The only available amino acid sequence for an invertebrate dimeric CK, that of the protostome polychaete Chaetopterus variopedatus, is just as similar to the vertebrate M isoform as to the B isoform. Echinoderms lack dimeric, cytoplasmic CKs, which appear to be replaced by a dimeric arginine kinase which evolved secondarily from CK. Thus, it is likely that the gene duplication event producing the M and B isoforms occurred after the divergence of the chordates from echinoderms. To narrow down the timing of this duplication event, we obtained the cDNA and deduced amino acid sequences of dimeric CKs from the tunicate Ciona intestinalis (subphylum Urochordata) and the lancelet Branchiostoma floridae (subphylum Cephalochordata). Our results show that these CKs are strikingly similar to both invertebrate and vertebrate CKs. However, phylogenetic analyses by neighbor-joining and parsimony show that these two enzymes appeared to have diverged before the point of divergence of the M and B isoforms. Thus, the gene duplication event for formation of the muscle and brain isoforms of CK most likely occurred during the radiation of the fish, a time noted for gene duplication events at a variety of other loci.     

Hypotaurocyamine kinase evolved from a gene for arginine kinase

Hypotaurocyamine kinase (HTK) is a member of the highly conserved family of phosphagen kinases that includes creatine kinase (CK) and arginine kinase (AK). HTK is found only in sipunculid worms, and it shows activities for both the substrates hypotaurocyamine and taurocyamine. Determining how HTK evolved in sipunculids is particularly insightful because all sipunculid-allied animals have AK and only some sipunculids have HTK. We determined the cDNA sequence of HTK from the sipunculid worm Siphonosoma cumanense for the first time, cloned it in pMAL plasmid and expressed it in E. coli as a fusion protein with maltose-binding protein. The cDNAderived amino acid sequence of Siphonosoma HTK showed high amino acid identity with molluscan AKs. Nevertheless, the recombinant enzyme of Siphonosoma HTK showed no activity for the substrate arginine, but showed activity for taurocyamine. Comparison of the amino acid sequences of HTK and AK indicated that the amino acid residues necessary for the binding of the substrate arginine in AK have been completely lost in Siphonosoma HTK sequence. The phylogenetic analysis indicated that the HTK amino acid sequence was placed just outside the molluscan AK cluster, which formed a sister group with the arthropod and nematode AKs. These results suggest that Siphonosoma HTK evolved from a gene for molluscan AK. Moreover, to confirm this assertion, we determined by PCR that the gene for Siphonosoma HTK has a 5-exon/4-intron structure, which is homologous with that of the molluscan AK genes. Further, the positions of splice junctions were conserved exactly between the two genes. Thus, we conclude that Siphonosoma HTK has evolved from a primordial gene for molluscan AK.     

Arginine kinase isoforms in the closest protozoan relative of metazoans

The genome of the choanoflagellate Monosiga brevicollis contains at least three genes for the phosphoryl transfer enzyme, arginine kinase (AK; EC 2.7.3.3). Bioinformatic analyses of the deduced amino acid sequences of the proteins coded for by two of these genes showed that one of these AKs is cytoplasmic (denoted AK1) while the other appears to have an N-terminal mitochondrial targeting peptide (denoted AK2). Cloning and expression of the cDNA for AK1 yielded considerable soluble AK activity. Three AK2 constructs were expressed - one corresponding to the full length protein and two corresponding to truncated versions in which the signal peptide had been deleted. Expression of the former construct yielded minimal soluble activity. In contrast, significant AK activity was found in both truncated constructs confirming the importance of removal of the targeting peptide for proper folding and catalytic activity. Both AK1 and AK2 are functional oligomers unlike typical AKs which are monomeric. A phylogenetic analysis showed that these choanoflagellate AKs group more closely with a supercluster consisting of cytoplasmic and mitochondrial CKs and invertebrate AKs that evolved secondarily from a CK-like ancestor. Reaction-diffusion constraints in choanoflagellates are likely mitigated by the presence of AK isoforms which facilitate energy transport in these highly polarized cells.