The characterization of novel monomeric creatine kinases in the early branching Alveolata species, Perkinsus marinus: Implications for phosphagen kinase evolution.

The genome of the unicellular molluscan parasite Perkinsus marinus contains at least five genes coding for putative creatine kinases (CK), a phosphoryl transfer enzyme which plays a key role in cellular energy transactions. Expression and kinetic analyses of three of the P. marinus CKs revealed them to be true CKs with catalytic properties in the range of typical metazoan CKs. A sequence comparison of the P. marinus CKs with a range of CK dimers and other dimeric phosphoryl transfer enzymes in this family (phosphagen kinases) showed that the P. marinus CKs lacked some of the critical residues involved in dimer stabilization, a trait all previously characterized CKs share. Size exclusion chromatography of all three expressed P. marinus CK constructs indicated they are monomeric, consistent with the observed lack of some critical dimer stabilizing residues. Phylogenetic analyses of the P. marinus CKs and putative dinoflagellate CKs with a broad range of monomeric and dimeric phosphagen kinases revealed that the Perkinsus CKs form a distinct, well-supported clade with dinoflagellate CKs which also lack the dimer stabilizing residues. Analysis of the genomic data for P. marinus showed the presence of putative genes for the two enzymes associated with creatine biosynthesis. CK in higher organisms plays a critical role in energy buffering in cell types displaying high and variable rates of ATP turnover. The presence of multiple CKs and the creatine biosynthetic pathway in P. marinus indicates that this unicellular parasite has the full complement of molecular machinery for CK-mediated energy buffering.

A diverse array of creatine kinase and arginine kinase isoform genes is present in the starlet sea anemone Nematostella vectensis, a cnidarian model system for studying developmental evolution.

Phosphagen (guanidino) kinases (PK) constitute a family of homologous phosphotransferases catalyzing the reversible transfer of the high-energy phosphoryl group of ATP to naturally occurring guanidine compounds. Prior work has shown that PKs can be phylogenetically separated into two distinct groups- an arginine kinase (AK) subfamily and a creatine kinase (CK) subfamily. The latter includes three CK isoforms- cytoplasmic CK (CyCK), mitochondrial CK (MiCK) and three-domain flagellar CK (fCK). In the present study we identified six unique PK genes from the draft genome sequence of the starlet sea anemone Nematostella vectensis, a well-known model organism for understanding metazoan developmental evolution. Using reverse transcription polymerase chain reaction (RTPCR) methods, full length cDNAs were amplified for all of these PKs. These cDNAs were cloned and expressed in Escherichia coli as 6x His-tagged fusion proteins. The six PKs were identified as the three typical CK isoforms (CyCK, MiCK and fCK), two unusual AKs (a two-domain AK (2DAK) and a three-domain AK (3DAK)) and a PK which phosphorylated arginine. The latter enzyme had a very low AK activity (its apparent V(max) value being less than 0.2% that of 3DAK), lacks several key residues necessary for AK enzyme activity, and was tentatively designated as AK1. As far as we know, this constitutes the first report of an AK with the three fused AK domains. The Bayesian tree suggested that the third domain of 3DAK likely evolved from the gene for domain 2 of typical two-domain AK found widely in cnidarians. Construction of phylogenetic trees and comparison of exon-intron organizations of their respective genes indicated that the N. vectensis three-domain fCK and 3DAK evolved independently, and both enzymes are likely to be targeted to cell membranes since they have a myristoylation signal at their respective N-termini. These results complement prior work on other basal invertebrates showing that multiple CK and AK isoform genes were present at the dawn of the radiation of metazoans. The presence of isoform diversity in an organism lacking in structural complexity reflects an early imperative for targeting of PKs to particular cellular contexts such as muscle fibers, neurons, ciliated/flagellated epithelia and spermatozoa.

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.

The structure of lombricine kinase: implications for phosphagen kinase conformational changes.

Lombricine kinase is a member of the phosphagen kinase family and a homolog of creatine and arginine kinases, enzymes responsible for buffering cellular ATP levels. Structures of lombricine kinase from the marine worm Urechis caupo were determined by x-ray crystallography. One form was crystallized as a nucleotide complex, and the other was substrate-free. The two structures are similar to each other and more similar to the substrate-free forms of homologs than to the substrate-bound forms of the other phosphagen kinases. Active site specificity loop 309-317, which is disordered in substrate-free structures of homologs and is known from the NMR of arginine kinase to be inherently dynamic, is resolved in both lombricine kinase structures, providing an improved basis for understanding the loop dynamics. Phosphagen kinases undergo a segmented closing on substrate binding, but the lombricine kinase ADP complex is in the open form more typical of substrate-free homologs. Through a comparison with prior complexes of intermediate structure, a correlation was revealed between the overall enzyme conformation and the substrate interactions of His(178). Comparative modeling provides a rationale for the more relaxed specificity of these kinases, of which the natural substrates are among the largest of the phosphagen substrates.

Evolution of the diverse array of phosphagen systems present in annelids.

Annelids as a group express a variety of phosphagen kinases including creatine kinase (CK), glyocyamine kinase (GK), lombricine kinase (LK), taurocyamine kinase (TK) and a unique arginine kinase (AK) restricted to annelids. In prior work, we have determined and compared the intron/exon organization of the annelid genes for cytoplasmic GK, LK, AK, and mitochondrial TK and LK (MiTK and MiLK, respectively), and found that these annelid genes, irrespective of cytoplasmic or mitochondrial, have the same 8-intron/9-exon organization strikingly similar to mitochondrial CK (MiCK) genes. These results support the view that the MiCK gene is basal and ancestral to the phosphagen kinases unique to annelids. To gain a greater understanding of the evolutionary processes leading to the diversity of annelid phosphagen kinases, we determined for the first time the intron/exon organization of a cytoplasmic CK gene from a polychaete as well as that of another polychaete MiCK gene. These gene structures, coupled with a phylogenetic analyses of annelid enzymes and assessment of the fidelity of substrate specificity of some these phosphagen kinases, provide insight into the pattern of radiation of the annelid enzymes. Annelid phosphagen kinases appeared to have diverged in the following order (earliest first): (1) cytoplasmic AK, LK and TK, (2) GK, and (3) mitochondrial MiLK and MiTK. Interestingly, phylogenetic analyses showed that the above phosphagen kinases appear to be basal to all CK isoforms (mitochondrial, cytoplasmic and flagellar CKs). This somewhat paradoxical placement of CKs most likely reflects a higher rate of evolution and radiation of the annelid-specific LK, TK and GK genes than the CK isoform genes.

Two-domain arginine kinase from the deep-sea clam Calyptogena kaikoi--evidence of two active domains.

The cDNA and deduced amino acid sequences for arginine kinase (AK) from the deep-sea clam Calyptogena kaikoi have been determined revealing an unusual two-domain (2D) structure with molecular mass of 80 kDa, twice that of normal AK. The amino acid sequences of both domains contain most of the residues thought to be required for substrate binding found in the horseshoe crab Limulus polyphemus AK, a well studied system for which several X-ray crystal structures exist. However, two highly conserved residues, D62 and R193, that form a salt bridge thereby stabilizing the substrate-bound structure have been replaced by G and N in domain 1, and G and P in domain 2, respectively. The present effort probes whether both domains of Calyptogena AK are catalytically competent. Recombinant constructs of the wild-type enzyme of both single domains, and of selected mutants of the Calyptogena AK have been expressed as fusion proteins with the maltose-binding protein. The wild-type two-domain enzyme (2D[WT]) had high AK activity (k(cat)=23 s(- 1), average value of the two domains), and the single domain 2 (D2[WT]) showed 1.5-times higher activity (k(cat)=38 s(- 1)) than the wild-type 2D[WT]. Interestingly, the single domain 1 (D1[WT]) showed only a very low activity (k(cat) approximately 0.016 s(- 1)). Introduction of a Y68A mutation in both domains virtually abolished catalytic activity. On the other hand, significant residual activity was observed (k(cat)=2.8 s(- 1)), when the Y68A mutation was introduced only into domain 2 of the two-domain enzyme. A similar mutation in domain 1 of the two-domain enzyme reduced activity to a much lower extent (k(cat)=11.1 s(- 1)). Although the domains of this "contiguous" dimeric AK each have catalytic capabilities, the presence of domain 2 strongly influences the stability and activity of domain 1.

Contributions to catalysis and potential interactions of the three catalytic domains in a contiguous trimeric creatine kinase.

Three separate creatine kinase (CK) isoform families exist in animals. Two of these (cytoplasmic and mitochondrial) are obligate oligomers. A third, flagellar, is monomeric but contains the residues for three complete CK domains. It is not known whether the active sites in each of the contiguous flagellar domains are catalytically competent, and, if so, whether they are capable of acting independently. Here we have utilized site-directed mutagenesis to selectively disable individual active sites and all possible combinations thereof. Kinetic studies showed that these mutations had minimal impact on substrate binding and synergism. Interestingly, the active sites were not catalytically equivalent, and were in fact interdependent, a phenomenon that has previously been reported only in the oligomeric CK isoforms.

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.

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 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.

Early evolution of the creatine kinase gene family and the capacity for creatine biosynthesis and membrane transport.

The creatine kinase (CK)/phosphocreatine (PCr) energy buffering system is widespread in animal groups. Recent genomic sequencing and experimental results support the view that the capacity for creatine biosynthesis and membrane transport may have evolved quite early, perhaps coincident with CK. Conventional wisdom would suggest that CK evolved from an ancestral protein most similar to the CK homologue, arginine kinase. This early CK gene subsequently diverged into the cytoplasmic, mitochondrial and flagellar CK gene families. It is now clear that both the mitochondrial and cytoplasmic-flagellar genes were present prior to the divergence of sponges from the multi-cellular animal (metazoan) lineage, possibly as long as a billion years ago. Sponges constitute the most ancient, extant metazoan group. It is likely that the primary function of the CK-PCr system in these primitive animals was to mitigate reaction-diffusion constraints in highly polarized cells such as spermatozoa and choanocytes, the water current generating cells in sponges.

Evolution of the arginine kinase gene family.

Arginine kinase (AK), catalyzing the reversible transfer of phosphate from MgATP to arginine yielding phosphoarginine and MgADP, is widely distributed throughout the invertebrates and is also present in certain protozoa. Typically, these proteins are found as monomers targeted to the cytoplasm, but true dimeric and contiguous dimeric AKs as well as mitochondrial AK activities have been observed. In the present study, we have obtained the sequences of the genes for AKs from two distantly related molluscs-the cephalopod Nautilus pompilius and the bivalve Crassostrea gigas. These new data were combined with available gene structure data (exon/intron organization) extracted from EST and genome sequencing project databases. These data, comprised of 23 sequences and gene structures from Protozoa, Cnidaria, Platyhelminthes, Mollusca, Arthropoda and Nematoda, provide great insight into the evolution and divergence of the AK family. Sequence and phylogenetic analyses clearly show that the AKs are homologous having arisen from some common ancestor. However, AK gene organization is highly divergent and variable. Molluscan AK genes typically have a highly conserved six-exon/five-intron organization, a structure that is very similar to that of the platyhelminth Schistosoma mansoni Arthropod and nematode AK genes have fewer introns, while the cnidarian and protozoan genes each display unique exon/intron organization when compared to the other AK genes. The non-conservative nature of the AK genes is in sharp contrast to the relatively high degree of conservation of intron positions seen in a homologous enzyme creatine kinase (CK). The present results also show that gene duplication and subsequent fusion events forming unusual two-domain AKs occurred independently at least four times as these contiguous dimers are present in Protozoa, Cnidaria, Platyhelminthes and Mollusca. Detailed analyses of the amino acid sequences indicate that two AKs (one each from Drosophila and Caenorhabditis) have what appear to be N-terminal mitochondrial targeting sequences, providing the first evidence for true mitochondrial AK genes. The AK gene family is ancient and the lineage has undergone considerable divergence as well as multiple duplication and fusion events.

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.

Over-expression, purification and characterization of the oligomerization dynamics of an invertebrate mitochondrial creatine kinase.

Mitochondrial creatine kinase (MtCK) plays a central role in energy homeostasis within cells that display high and variable rates of ATP turnover. Vertebrate MtCKs exist primarily as octamers but readily dissociate into constituent dimers under a variety of circumstances. MtCK is an ancient protein that is also found in invertebrates including sponges, the most primitive of all multi-cellular animals. We have cloned, expressed, and purified one of these invertebrate MtCKs from a marine polychaete worm, Chaetopterus variopedatus (CVMtCK). Size exclusion chromatography and dynamic light scattering (DLS) were used to characterize oligomeric state in comparison with that of octameric chicken sarcomeric isoform (SarMtCK). At protein concentrations >1 mg/ml, CVMtCK was predominantly octameric (>90%). When diluted to 0.1 mg/ml, CVMtCK dissociated into dimers much more rapidly than SarMtCK when observed under identical conditions. The rate of dissociation for both MtCKs increased as temperature rose from 2 to 28 degrees C, and in CVMtCK, fell at higher incubation temperatures. The fraction of octameric CVMtCK at equilibrium increased with temperature and then fell. Temperature transition studies showed that octamers and dimers were rapidly interconvertible on a similar time scale. Importantly, when CVMtCK was converted to the transition state analog complex (TSAC), both size exclusion chromatography and DLS showed that there was minimal dissociation of octamers into dimers while SarMtCK octamers were highly unstable as the TSAC. These results clearly show distinct differences in octamer stability between CVMtCK and SarMtCK, which could impact function under physiological circumstances. Furthermore, the large yield of recombinant protein and high stability of CVMtCK in the TSAC suggest that this protein might be a good target for crystallization efforts.

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.

The active site cysteine of arginine kinase: structural and functional analysis of partially active mutants.

Arginine kinase buffers cellular ATP levels by catalyzing reversible phosphoryl transfer between ATP and arginine. A conserved cysteine has long been thought important in catalysis. Here, cysteine 271 of horseshoe crab arginine kinase has been mutated to serine, alanine, asparagine, or aspartate. Catalytic turnover rates were 0.02-1.0% of wild type, but the activity of uncharged mutations could be partially rescued with chloride. Steady-state binding constants were slightly increased, more so for phospho-L-arginine than ADP. Substrate binding synergy observed in many phosphagen kinases was reduced or eliminated in mutant enzymes. The crystallographic structure of the alanine mutant at 2.3 A resolution, determined as a transition state analogue complex with arginine, nitrate, and MgADP, was nearly identical to wild type. Enzyme-substrate interactions are maintained as in wild type, and substrates remain at least roughly aligned for in-line phosphoryl transfer. Homology models with serine, asparagine, or aspartate replacing the active site cysteine similarly show only minor structural changes. Most striking, however, is the presence in the C271A mutant crystallographic structure of a chloride ion within 3.5 A of the nonreactive N(eta) substrate nitrogen, approximating the position of the sulfur in the wild-type's cysteine. Together, the results contradict prevailing speculation that the cysteine mediates a substrate-induced conformational change, confirm that it is the thiolate form that is relevant to catalysis, and suggest that one of its roles is to help to enhance the catalytic rate through electrostatic stabilization of the transition state.

Alternative splicing produces transcripts coding for alpha and beta chains of a hetero-dimeric phosphagen kinase.

Glycocyamine kinase (GK) catalyzes the reversible phosphorylation of glycocyamine (guanidinoacetate), a reaction central to cellular energy homeostasis in certain animals. GK is a member of the phosphagen kinase enzyme family and appears to have evolved from creatine kinase (CK) early in the evolution of multi-cellular animals. Prior work has shown that GK from the polychaete Neanthes (Nereis) diversicolor exits as a hetero-dimer in vivo and that the two polypeptide chains (termed alpha and beta) are coded for by unique transcripts. In the present study, we demonstrate that the GK from a congener Nereis virens is also hetero-dimeric and is coded for by alpha and beta transcripts, which are virtually identical to the corresponding forms in N. diversicolor. The GK gene from N. diversicolor was amplified by PCR. Sequencing of the PCR products showed that the alpha and beta chains are the result of alternative splicing of the GK primary mRNA transcript. These results also strongly suggest that this gene underwent an early tandem exon duplication event. Full-length cDNAs for N. virens GKalpha and GKbeta were individually ligated into expression vectors and the resulting constructs used to transform Escherichia coli expression hosts. Regardless of expression conditions, minimal GK activity was observed in both GKalpha and GKbeta constructs. Inclusion bodies for both were harvested, unfolded in urea and alpha chains, beta chains and mixtures of alpha and beta chains were refolded by sequential dialysis. Only modest amounts of GK activity were observed when alpha and beta were refolded individually. In contrast, when refolded the alpha and beta mixture yielded highly active hetero-dimers, as validated by size exclusion chromatography, electrophoresis and mass spectrometry, with a specific activity comparable to that of natural GK. The above evidence suggests that there is a preference for hetero-dimer formation in the GKs from these two polychaetes. The evolution of the alternate splicing and an additional exon in these GKs, producing alpha and beta transcripts, can be viewed as a possible compensation for a mutation(s) in the original gene, which most likely coded for a homo-dimeric protein.

Cloning and expression of mitochondrial and protoflagellar creatine kinases from a marine sponge: implications for the origin of intracellular energy transport systems.

Creatine kinase (CK) plays a central role in energy transactions in cells displaying high and variable rates of ATP turnover. Cytoplasmic and mitochondrial CK genes code for isoforms which are targeted to distinct intracellular compartments often in close physical proximity to sites of ATP hydrolysis or synthesis. In certain lower groups a third CK gene is present which codes for a flagellar CK isoform consisting of three complete, fused CK domains. Recent work has shown that cytoplasmic, mitochondrial, and flagellar CKs are present in protochordates and in deuterostome and protostome invertebrates. We report here that the marine sponge Tethya aurantia, a representative of the oldest of all multi-cellular animal groups, expresses three unique CK transcripts. One of these CK transcripts codes for protein that has a mitochondrial targeting sequence and in a phylogenetic analysis is positioned at the base of the cluster containing mitochondrial CK sequences from invertebrates, protochordates, and vertebrates; it is clearly a mitochondrial CK. When expressed in Escherichia coli the mitochondrial form from T. aurantia was found to be dimeric unlike all other mitochondrial CKs which are typically octameric. The other two T. aurantia transcripts code for proteins that appear to be more closely related to flagellar CKs. These protoflagellar CKs were found to be dimers when expressed in Escherichia coli. Sponges last shared a common ancestor with higher animals as long as one billion years ago. The antiquity of intracellular localization, as evidenced by the presence of a true mitochondrial CK and protoflagellar CKs in the sponge T. aurantia, indicates that physical constraints on cellular energy transport were key, early driving forces in the evolution of this key enzyme system.

Elements of the major myofibrillar binding peptide motif are present in the earliest of true muscle type creatine kinases.

Most vertebrates possess two genes for cytoplasmic creatine kinase (CK) coding for muscle (M-CK) and brain (B-CK) isoforms which assemble into homo-dimeric (MM, BB) and hetero-dimeric (MB) active enzymes. In mammals and birds, a significant fraction of MM-CK is bound to the myofibrillar M-line where it is thought to facilitate energy buffering and transport. Myofibrillar binding is mediated by major and minor lysine charge clamp motifs (K104/K115 [major] and K8/K24 [minor] in chicken M-CK) located in the N-terminal region [J. Cell Biol. 149 (2000) 1225]. We have obtained the cDNA and deduced amino acid sequences for cytoplasmic CKs from two hagfish, Myxine glutinosa and Eptatretus stoutii, non-vertebrate craniates, and the sequences for two cytoplasmic CKs from the lamprey Lampetra japonica, a jawless true vertebrate. All four cDNAs code for CKs consisting of approximately 380 residues. Phylogenetic analyses showed that the hagfish and lamprey CKs are coded for by genes which are clearly muscle type (M) creatine kinases. Two of these four M-CKs have the K104/K115-equivalent residues of the major myofibrillar binding region while the other two have the K115 equivalent but lack the corresponding K104 residue. All four M-CKs lack the K8/K24 equivalent elements of the minor myofibrillar binding region. Comparison of these sequences to corresponding sequences of cytoplasmic CKs from two protochordates (tunicate, amphioxus) and M- and B-CKs from true fish and above reveal a pattern of acquisition (and loss) of key lysine residues consistent with the physiological context in which these enzymes operate.

The role of phosphagen specificity loops in arginine kinase.

Phosphagen kinases catalyze the reversible transfer of a phosphate between ATP and guanidino substrates, a reaction that is central to cellular energy homeostasis. Members of this conserved family include creatine and arginine kinases and have similar reaction mechanisms, but they have distinct specificities for different guanidino substrates. There has not been a full structural rationalization of specificity, but two loops have been implicated repeatedly. A small domain loop is of length that complements the size of the guanidino substrate, and is located where it could mediate a lock-and-key mechanism. The second loop contacts the substrate with a valine in the methyl-substituted guanidinium of creatine, and with a glutamate in the unsubstituted arginine substrate, leading to the proposal of a discriminating hydrophobic/hydrophilic minipocket. In the present work, chimeric mutants were constructed with creatine kinase loop elements inserted into arginine kinase. Contrary to the prior rationalizations of specificity, most had measurable arginine kinase activity but no creatine kinase activity or enhanced phosphocreatine binding. Guided by structure, additional mutations were introduced in each loop, recovering arginine kinase activities as high as 15% and 64% of wild type, respectively, even though little activity would be expected in the constructs if the implicated sites had dominant roles in specificity. An atomic structure of the mismatched complex of arginine kinase with creatine and ADP indicates that specificity can also be mediated by an active site that allows substrate prealignment that is optimal for reactivity only with cognate substrates and not with close homologs that bind but do not react.