- Email: [email protected]
Evolution of phosphagen kinase V. cDNA-derived amino acid sequences of two molluscan arginine kinases from the chiton Liolophura japonica and the turbanshell Battilus cornutus
Biochimica et Biophysica Acta 1340 Ž1997. 1–6
Short sequence-paper
Evolution of phosphagen kinase V. cDNA-derived amino acid sequences of two molluscan arginine kinases from the chiton Liolophura japonica and the turbanshell Battilus cornutus Tomohiko Suzuki ) , Tohei Ban, Takahiro Furukohri Department of Biology, Faculty of Science, Kochi UniÕersity, Kochi 780, Japan Received 4 March 1997; revised 8 April 1997; accepted 9 April 1997
Abstract The cDNAs of arginine kinases from the chiton Liolophura japonica ŽPolyplacophora. and the turbanshell Battilus cornutus ŽGastropoda. were amplified by polymerase chain reaction ŽPCR., and the complete nucleotide sequences of 1669 and 1624 bp, respectively, were determined. The open reading frame for Liolophura arginine kinase is 1050 nucleotides in length and encodes a protein with 349 amino acid residues, and that for Battilus is 1077 nucleotides and 358 residues. The validity of the cDNA-derived amino acid sequence was supported by chemical sequencing of internal tryptic peptides. The molecular masses were calculated to be 39 057 and 39 795 Da, respectively. The amino acid sequence of Liolophura arginine kinase showed 65–68% identity with those of Battilus and Nordotis Žabalone. arginine kinases, and the homology between Battilus and Nordotis was 79%. Molluscan arginine kinases also show lower, but significant homology Ž38–43%. with rabbit creatine kinase. The sequences of arginine kinases could be used as a molecular clock to elucidate the phylogeny of Mollusca, one of the most diverse animal phyla. q 1997 Elsevier Science B.V. Keywords: Phosphagen kinase; Arginine kinase; Molecular evolution; Chiton
Phosphagen kinases Žguanidino kinases. are the enzymes that catalyze the reversible transfer of the high energy phosphoryl group of ATP to naturally occurring guanidino compounds such as creatine, glycocyamine, taurocyamine, lombricine and arginine, and play a key role in the interconnection of energy production and utilization in animals w1–3x. The phosphorylated high energy guanidine is referred to as phosphagen. In vertebrates, the only phosphagen is phosphocre-
)
Corresponding author. Fax: q81 888 448356. E-mail: [email protected]
atine, and the corresponding phosphagen kinase is creatine kinase. But in invertebrates, at least six unique phosphagens, phosphoarginine, phosphoglycocyamine, phosphotaurocyamine, phospholombricine, phosphohypotaurocyamine and phosphoopheline, are present in addition to phosphocreatine, and the corresponding kinases for the former four, arginine kinase, glycocyamine kinase, taurocyamine kinase and lombricine kinase, are also identified w1,2x. The molecular mass for the subunit of these enzymes is about 40 kDa, and in some cases, they form a dimer. Arginine kinase is most widely distributed among invertebrates and thought to be closer to an ancestral
0167-4838r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 7 - 4 8 3 8 Ž 9 7 . 0 0 0 6 6 - 6
2
T. Suzuki et al.r Biochimica et Biophysica Acta 1340 (1997) 1–6
phosphagen kinase w1,2,4x. Here we focus on molluscan arginine kinases. They usually exist as a monomer of 40 kDa subunit, but the presence of 80 kDa enzyme is also reported in some species of Eulamellibranchia w1x. In this connection, the arginine kinase with molecular mass of 86 kDa has been isolated from a clam w5x and sequenced by our group. Surprisingly, the 86 kDa arginine kinase was not a dimer of 40 kDa subunit but a two-domain structure resulting from gene duplication ŽKawasaki et al., manuscript in preparation. . The amino acid sequence of molluscan arginine kinase is known only for Nordotis madaka Žarchaegastropodic abalone. w6x. To elucidate the function and evolution of these enzymes, it is inevitably necessary to accumulate amino acid sequences. Thus we decided to determine the sequences of arginine kinase from the chiton Liolophura japonica, since the Polyplacophora is believed to be one of the most primitive molluscan groups, as well as of arginine kinase from another archaegastropod Battilus cornutus Žturbanshell. for comparison. Here we report the cDNA-derived amino acid sequences of arginine kinases from the two species, and estimate the divergence times of three molluscs from arginine kinase sequences. Total RNA was prepared from the buccal mass of Liolophura japonica and Battilus cornutus, respectively, according to the method of Chomczynski and Sacchi w7x, and polyŽ A.q RNA was purified with a FirstTrack mRNA Isolation Kit ŽInvitrogen. . The single-stranded cDNA was synthesized with avian reverse transcriptase using oligo-dT adaptor, 5X GGATTCGAATTCCCCGGGT17 as a primer. The 3X half of cDNA of arginine kinase was first amplified for 30 cycles, each consisting of 1 min at 948C for denaturation, 1.5 min at 558C for annealing and 2 min at 728C for primer extension by polymerase chain reaction ŽPCR. w8x. Taq DNA polymerase ŽPromega. was used as an enzyme. The primers used are the oligo-dT adaptor and a 512f o ld ‘ u n iv e r s a l’ r e d u n d a n t o lig o m e r ; GTŽ ACGT.TGGŽAG.TŽACGT. AAŽ TC. GAŽAG. GAŽAG.GAŽTC.CA, designed for amplification of phosphagen kinases w6x. This universal oligomer is based on the conserved amino acid sequence of Val-TrpValrIle-Asn-Glu-Glu-Asp-His. The 1000 bp products were subcloned in the SmaI site of pUC18 and
three independent clones were sequenced with a PRISM dye terminator cycle sequencing kit using Model 373-18 DNA sequencer ŽApplied BioSystems.. The 5X half of the cDNA was amplified as follows. The polyŽG. q tail was added to the 3X end of the oligo-dT primed single-stranded cDNA with a terminal deoxynucleotidyl transferase. Then the 5X half of the cDNA was amplified by the same method as described above, using the oligo-dC Ž20 mer. and the non-redundant primer R1 for Liolophura Žsee the sequence in Fig. 1. or R2 for Battilus Ž see the sequence in Fig. 2. . The amplified products were subcloned in the SmaI site of pUC18 and three independent clones were sequenced. Arginine kinases were isolated from foot muscle as described previously w6x. The isolated chains were pyridylethylated and digested with trypsin at an enzymersubstrate ratio of 1:100 in 0.1 M NH 4 HCO 3 ŽpH 8.0. at 378C for 2 h. The digested products were isolated by reverse-phase chromatography. The column ŽCosmosil 5C 18-300, 4.6 = 150 mm. was equilibrated with 0.1% TFA ŽpH 1.9. and eluted with a linear gradient of 0–90% acetonitrile in 0.1% TFA over 90 min at a flow rate of 1 mlrmin. Peptides for sequence analysis were purified further by rechromatography on the same column with a linear gradient of acetonitrile in 10 mM ammonium acetate Ž pH 7.1. w9x. The amino acid sequences of the whole proteins and tryptic peptides were determined by an automated protein sequencer ŽApplied BioSystems 476A.. The complete nucleotide sequence of 1669 bp of cDNA of Liolophura arginine kinase was determined by combination of sequences of 5X and 3X clones ŽFig. 1.. The sequence contains 35 and 584 bp of 5X and 3X untranslated sequences, respectively. A polyadenylation signal, AATAAA Ž boxed in Fig. 1., is present in 27 bp upstream from 3X termini. The open reading frame is 1050 nucleotides long and encodes a protein with 349 amino acid residues. The molecular mass was calculated to be 39 057 Da. The cDNA-derived amino acid sequence was completely consistent with the chemical sequencing Ž39 residues. of tryptic peptides Žunderlined in the amino acid sequence of Fig. 1.. No N-terminal amino acid of the whole protein was detected by protein sequencing, suggesting that the N-terminus is blocked. The complete nucleotide sequence of 1624 bp of
T. Suzuki et al.r Biochimica et Biophysica Acta 1340 (1997) 1–6
cDNA of Battilus arginine kinase was determined by combination of sequences of 5X and 3X clones, has been also determined Ž Fig. 2.. The sequence contains 39 and 508 bp of 5X and 3X untranslated sequences, respectively. A polyadenylation signal, AATAAA Žboxed in Fig. 2., is present in 22 bp upstream from
3
3X termini. The open reading frame is 1077 nucleotides long and encodes a protein with 358 amino acid residues. The molecular mass was calculated to be 39 795 Da. The cDNA-derived amino acid sequence was completely consistent with the chemical sequencing Ž52 residues. of tryptic peptides Žunder-
Fig. 1. Nucleotide and derived amino acid sequences of cDNA of Liolophura arginine kinase. The polyadenylation signal ŽAATAAA. is boxed. Arrows indicate primers used for amplification. Amino acid sequences determined chemically are underlined.
4
T. Suzuki et al.r Biochimica et Biophysica Acta 1340 (1997) 1–6
lined in the amino acid sequence of Fig. 2.. No N-terminal amino acid of the whole protein was detected by protein sequencing, suggesting that the N-terminus is blocked. The nucleotide and amino acid sequences of Liolophura and Battilus arginine kinases will be submitted to the DDBJ.
The cDNA-derived amino acid sequences of Liolophura and Battilus arginine kinases were aligned with those of Nordotis Žabalone. arginine kinase w6x and rabbit creatine kinase w10x, in Fig. 3, with the algorithm of Feng and Doolittle w11x. In the alignment, there are 202 amino acid residues Žindicated by asterisks. conserved in 3 molluscan enzymes, of
Fig. 2. Nucleotide and derived amino acid sequences of cDNA of Battilus arginine kinase. The polyadenylation signal ŽAATAAA. is boxed. Arrows indicate primers used for amplification. Amino acid sequences determined chemically are underlined.
T. Suzuki et al.r Biochimica et Biophysica Acta 1340 (1997) 1–6
5
Fig. 3. Alignment of amino acid sequences of three molluscan arginine kinases with that of rabbit creatine kinase. This alignment was obtained with the algorithm of Feng and Doolittle w11x. Invariant residues in molluscan enzymes are indicated by asterisks. The residues conserved in all four sequences are marked by q.
which 107 residues are also conserved in rabbit creatine kinase Žindicated by q.. Several functional key residues proposed for vertebrate creatine kinase Žboxed in Fig. 3., Cys-287 located in the center of the active site w3,12x, Trp-213 and 230 involving in the binding site of ATP w13x, and His-96 that is most likely candidate as a general acidrbase catalyst w14,15x, are also conserved in molluscan arginine kinases. A recent crystal structure of chicken cardiac mitochondrial creatine kinase has established the involvement of these residues in its function w16x. The amino acid sequence of Liolophura arginine kinase showed 65–68% identity with those of Battilus and Nordotis Žabalone. arginine kinases, and the homology between Battilus and Nordotis was 79%. Molluscan arginine kinases also show lower, but significant homology Ž38–43%. with rabbit CK. The amino acid sequences of molluscan arginine kinases can be used as a molecular clock to elucidate the phylogeny of Mollusca, one of the most diverse
animal phyla, because the enzymes appear to be derived from a single gene strain and have a moderate evolutionary rate. A UPG tree constructed from
Fig. 4. A UPG-tree for amino acid sequences of molluscan and arthropod Žshrimp. arginine kinases. This tree was obtained with the program in GeneWorks 2.4 using amino acid sequence alignment of arginine kinases from three molluscs and shrimp w6x. MyrBP, million years before present. Standard error is indicated as box at a branching point. The same topology is obtained with the Neighbor-Joining method w18x.
T. Suzuki et al.r Biochimica et Biophysica Acta 1340 (1997) 1–6
6
sequences of molluscan and arthropod arginine kinases allows us to estimate the divergence time of the 3 molluscan species Ž Fig. 4. . If we assume the divergence between Mollusca and Arthropoda occurred about 550 million years before present ŽMyrBP. w17x, the divergence time of the two gastropods Battilus and Nordotis is estimated to be 220 MyrBP, and that of chiton Ž Liolophura. and the other two gastropods is 360 MyrBP. The tree is essentially consistent with the fossil record of molluscan groups w19x.
References w1x D.C. Watts, Advan. Comp. Physiol. Biochem. 3 Ž1968. 1–115. w2x J.F. Morrison, in: P.C. Boyer ŽEd.., The Enzymes, Vol. 8, Academic Press, New York, 1973, pp. 457–486. w3x G.L. Kenyon, G.H. Reed, Adv. Enzymol. 54 Ž1986. 367– 426. w4x S.M. Muhelebach, M. Gross, T. Wirz, T. Wallimann, J.-C. Perriard, M. Wyss, Mol. Cell. Biochem. 133r134 Ž1994. 245–262. w5x T. Soga, Y. Yazawa, Zool. Sci. ŽAbstract. 13 ŽSuppl.. Ž1996. 55. w6x T. Suzuki, T. Furukohri, J. Mol. Biol. 237 Ž1994. 353–357.
w7x P. Chomczynski, N. Sacchi, Anal. Biochem. 162 Ž1987. 156–159. w8x R.K. Saiki, D. Gelfand, S. Stoffel, S. Scharf, R. Higuchi, G. Horn, K. Mullis, H. Erlich, Science 239 Ž1988. 487–491. w9x T. Suzuki, T. Takagi, T. Gotoh, J. Biol. Chem. 265 Ž1990. 12168–12177. w10x S. Putney, W. Herlihy, N. Royal, P. Pang, H.V. Aposhian, L. Pickering, R. Belagaje, K. Biemann, D. Page, S. Kuby, P. Schimmel, J. Biol. Chem. 259 Ž1984. 14317–14320. w11x D.A. Feng, R.F. Doolittle, J. Mol. Evol. 25 Ž1987. 351–360. w12x R. Furter, E.M. Furter-Graves, T. Wallimann, Biochemistry 32 Ž1993. 7022–7029. w13x M. Vasak, K. Nagayama, K. Wuthrich, M.L. Mertens, J.H.R. Kagi, Biochemistry 18 Ž1979. 5050–5055. w14x R.P. Rosevear, P. Desmeules, G.L. Kenyon, A.S. Mildvan, Biochemistry 20 Ž1981. 6155–6164. w15x L.H. Chen, C.L. Borders, J.R. Vasquez, G.L. Kenyon, Biochemistry 35 Ž1996. 7895–7902. w16x K. Fritz-Wolf, T. Schnyder, T. Wallimann, W. Kabsch, Nature 381 Ž1996. 341–345. w17x M. Goodman, J. Pedwaydon, J. Czelusniak, T. Suzuki, T. Gotoh, L. Moens, F. Shishikura, D. Walz, S. Vinogradov, J. Mol. Evol. 27 Ž1988. 236–249. w18x J. Felsenstein, PHYLIP ŽPhylogeny Inference Package. version 3.5c. Distributed by the author. Department of Genetics, University of Washington, Seattle, USA, 1993. w19x L.A. Cox, in: R.C. Moore ŽEd.., Treatise on invertebrate paleontology, part I, University of Kansas Press, Lawrence, 1960, pp. 85–169.
Short sequence-paper
Evolution of phosphagen kinase V. cDNA-derived amino acid sequences of two molluscan arginine kinases from the chiton Liolophura japonica and the turbanshell Battilus cornutus Tomohiko Suzuki ) , Tohei Ban, Takahiro Furukohri Department of Biology, Faculty of Science, Kochi UniÕersity, Kochi 780, Japan Received 4 March 1997; revised 8 April 1997; accepted 9 April 1997
Abstract The cDNAs of arginine kinases from the chiton Liolophura japonica ŽPolyplacophora. and the turbanshell Battilus cornutus ŽGastropoda. were amplified by polymerase chain reaction ŽPCR., and the complete nucleotide sequences of 1669 and 1624 bp, respectively, were determined. The open reading frame for Liolophura arginine kinase is 1050 nucleotides in length and encodes a protein with 349 amino acid residues, and that for Battilus is 1077 nucleotides and 358 residues. The validity of the cDNA-derived amino acid sequence was supported by chemical sequencing of internal tryptic peptides. The molecular masses were calculated to be 39 057 and 39 795 Da, respectively. The amino acid sequence of Liolophura arginine kinase showed 65–68% identity with those of Battilus and Nordotis Žabalone. arginine kinases, and the homology between Battilus and Nordotis was 79%. Molluscan arginine kinases also show lower, but significant homology Ž38–43%. with rabbit creatine kinase. The sequences of arginine kinases could be used as a molecular clock to elucidate the phylogeny of Mollusca, one of the most diverse animal phyla. q 1997 Elsevier Science B.V. Keywords: Phosphagen kinase; Arginine kinase; Molecular evolution; Chiton
Phosphagen kinases Žguanidino kinases. are the enzymes that catalyze the reversible transfer of the high energy phosphoryl group of ATP to naturally occurring guanidino compounds such as creatine, glycocyamine, taurocyamine, lombricine and arginine, and play a key role in the interconnection of energy production and utilization in animals w1–3x. The phosphorylated high energy guanidine is referred to as phosphagen. In vertebrates, the only phosphagen is phosphocre-
)
Corresponding author. Fax: q81 888 448356. E-mail: [email protected]
atine, and the corresponding phosphagen kinase is creatine kinase. But in invertebrates, at least six unique phosphagens, phosphoarginine, phosphoglycocyamine, phosphotaurocyamine, phospholombricine, phosphohypotaurocyamine and phosphoopheline, are present in addition to phosphocreatine, and the corresponding kinases for the former four, arginine kinase, glycocyamine kinase, taurocyamine kinase and lombricine kinase, are also identified w1,2x. The molecular mass for the subunit of these enzymes is about 40 kDa, and in some cases, they form a dimer. Arginine kinase is most widely distributed among invertebrates and thought to be closer to an ancestral
0167-4838r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 7 - 4 8 3 8 Ž 9 7 . 0 0 0 6 6 - 6
2
T. Suzuki et al.r Biochimica et Biophysica Acta 1340 (1997) 1–6
phosphagen kinase w1,2,4x. Here we focus on molluscan arginine kinases. They usually exist as a monomer of 40 kDa subunit, but the presence of 80 kDa enzyme is also reported in some species of Eulamellibranchia w1x. In this connection, the arginine kinase with molecular mass of 86 kDa has been isolated from a clam w5x and sequenced by our group. Surprisingly, the 86 kDa arginine kinase was not a dimer of 40 kDa subunit but a two-domain structure resulting from gene duplication ŽKawasaki et al., manuscript in preparation. . The amino acid sequence of molluscan arginine kinase is known only for Nordotis madaka Žarchaegastropodic abalone. w6x. To elucidate the function and evolution of these enzymes, it is inevitably necessary to accumulate amino acid sequences. Thus we decided to determine the sequences of arginine kinase from the chiton Liolophura japonica, since the Polyplacophora is believed to be one of the most primitive molluscan groups, as well as of arginine kinase from another archaegastropod Battilus cornutus Žturbanshell. for comparison. Here we report the cDNA-derived amino acid sequences of arginine kinases from the two species, and estimate the divergence times of three molluscs from arginine kinase sequences. Total RNA was prepared from the buccal mass of Liolophura japonica and Battilus cornutus, respectively, according to the method of Chomczynski and Sacchi w7x, and polyŽ A.q RNA was purified with a FirstTrack mRNA Isolation Kit ŽInvitrogen. . The single-stranded cDNA was synthesized with avian reverse transcriptase using oligo-dT adaptor, 5X GGATTCGAATTCCCCGGGT17 as a primer. The 3X half of cDNA of arginine kinase was first amplified for 30 cycles, each consisting of 1 min at 948C for denaturation, 1.5 min at 558C for annealing and 2 min at 728C for primer extension by polymerase chain reaction ŽPCR. w8x. Taq DNA polymerase ŽPromega. was used as an enzyme. The primers used are the oligo-dT adaptor and a 512f o ld ‘ u n iv e r s a l’ r e d u n d a n t o lig o m e r ; GTŽ ACGT.TGGŽAG.TŽACGT. AAŽ TC. GAŽAG. GAŽAG.GAŽTC.CA, designed for amplification of phosphagen kinases w6x. This universal oligomer is based on the conserved amino acid sequence of Val-TrpValrIle-Asn-Glu-Glu-Asp-His. The 1000 bp products were subcloned in the SmaI site of pUC18 and
three independent clones were sequenced with a PRISM dye terminator cycle sequencing kit using Model 373-18 DNA sequencer ŽApplied BioSystems.. The 5X half of the cDNA was amplified as follows. The polyŽG. q tail was added to the 3X end of the oligo-dT primed single-stranded cDNA with a terminal deoxynucleotidyl transferase. Then the 5X half of the cDNA was amplified by the same method as described above, using the oligo-dC Ž20 mer. and the non-redundant primer R1 for Liolophura Žsee the sequence in Fig. 1. or R2 for Battilus Ž see the sequence in Fig. 2. . The amplified products were subcloned in the SmaI site of pUC18 and three independent clones were sequenced. Arginine kinases were isolated from foot muscle as described previously w6x. The isolated chains were pyridylethylated and digested with trypsin at an enzymersubstrate ratio of 1:100 in 0.1 M NH 4 HCO 3 ŽpH 8.0. at 378C for 2 h. The digested products were isolated by reverse-phase chromatography. The column ŽCosmosil 5C 18-300, 4.6 = 150 mm. was equilibrated with 0.1% TFA ŽpH 1.9. and eluted with a linear gradient of 0–90% acetonitrile in 0.1% TFA over 90 min at a flow rate of 1 mlrmin. Peptides for sequence analysis were purified further by rechromatography on the same column with a linear gradient of acetonitrile in 10 mM ammonium acetate Ž pH 7.1. w9x. The amino acid sequences of the whole proteins and tryptic peptides were determined by an automated protein sequencer ŽApplied BioSystems 476A.. The complete nucleotide sequence of 1669 bp of cDNA of Liolophura arginine kinase was determined by combination of sequences of 5X and 3X clones ŽFig. 1.. The sequence contains 35 and 584 bp of 5X and 3X untranslated sequences, respectively. A polyadenylation signal, AATAAA Ž boxed in Fig. 1., is present in 27 bp upstream from 3X termini. The open reading frame is 1050 nucleotides long and encodes a protein with 349 amino acid residues. The molecular mass was calculated to be 39 057 Da. The cDNA-derived amino acid sequence was completely consistent with the chemical sequencing Ž39 residues. of tryptic peptides Žunderlined in the amino acid sequence of Fig. 1.. No N-terminal amino acid of the whole protein was detected by protein sequencing, suggesting that the N-terminus is blocked. The complete nucleotide sequence of 1624 bp of
T. Suzuki et al.r Biochimica et Biophysica Acta 1340 (1997) 1–6
cDNA of Battilus arginine kinase was determined by combination of sequences of 5X and 3X clones, has been also determined Ž Fig. 2.. The sequence contains 39 and 508 bp of 5X and 3X untranslated sequences, respectively. A polyadenylation signal, AATAAA Žboxed in Fig. 2., is present in 22 bp upstream from
3
3X termini. The open reading frame is 1077 nucleotides long and encodes a protein with 358 amino acid residues. The molecular mass was calculated to be 39 795 Da. The cDNA-derived amino acid sequence was completely consistent with the chemical sequencing Ž52 residues. of tryptic peptides Žunder-
Fig. 1. Nucleotide and derived amino acid sequences of cDNA of Liolophura arginine kinase. The polyadenylation signal ŽAATAAA. is boxed. Arrows indicate primers used for amplification. Amino acid sequences determined chemically are underlined.
4
T. Suzuki et al.r Biochimica et Biophysica Acta 1340 (1997) 1–6
lined in the amino acid sequence of Fig. 2.. No N-terminal amino acid of the whole protein was detected by protein sequencing, suggesting that the N-terminus is blocked. The nucleotide and amino acid sequences of Liolophura and Battilus arginine kinases will be submitted to the DDBJ.
The cDNA-derived amino acid sequences of Liolophura and Battilus arginine kinases were aligned with those of Nordotis Žabalone. arginine kinase w6x and rabbit creatine kinase w10x, in Fig. 3, with the algorithm of Feng and Doolittle w11x. In the alignment, there are 202 amino acid residues Žindicated by asterisks. conserved in 3 molluscan enzymes, of
Fig. 2. Nucleotide and derived amino acid sequences of cDNA of Battilus arginine kinase. The polyadenylation signal ŽAATAAA. is boxed. Arrows indicate primers used for amplification. Amino acid sequences determined chemically are underlined.
T. Suzuki et al.r Biochimica et Biophysica Acta 1340 (1997) 1–6
5
Fig. 3. Alignment of amino acid sequences of three molluscan arginine kinases with that of rabbit creatine kinase. This alignment was obtained with the algorithm of Feng and Doolittle w11x. Invariant residues in molluscan enzymes are indicated by asterisks. The residues conserved in all four sequences are marked by q.
which 107 residues are also conserved in rabbit creatine kinase Žindicated by q.. Several functional key residues proposed for vertebrate creatine kinase Žboxed in Fig. 3., Cys-287 located in the center of the active site w3,12x, Trp-213 and 230 involving in the binding site of ATP w13x, and His-96 that is most likely candidate as a general acidrbase catalyst w14,15x, are also conserved in molluscan arginine kinases. A recent crystal structure of chicken cardiac mitochondrial creatine kinase has established the involvement of these residues in its function w16x. The amino acid sequence of Liolophura arginine kinase showed 65–68% identity with those of Battilus and Nordotis Žabalone. arginine kinases, and the homology between Battilus and Nordotis was 79%. Molluscan arginine kinases also show lower, but significant homology Ž38–43%. with rabbit CK. The amino acid sequences of molluscan arginine kinases can be used as a molecular clock to elucidate the phylogeny of Mollusca, one of the most diverse
animal phyla, because the enzymes appear to be derived from a single gene strain and have a moderate evolutionary rate. A UPG tree constructed from
Fig. 4. A UPG-tree for amino acid sequences of molluscan and arthropod Žshrimp. arginine kinases. This tree was obtained with the program in GeneWorks 2.4 using amino acid sequence alignment of arginine kinases from three molluscs and shrimp w6x. MyrBP, million years before present. Standard error is indicated as box at a branching point. The same topology is obtained with the Neighbor-Joining method w18x.
T. Suzuki et al.r Biochimica et Biophysica Acta 1340 (1997) 1–6
6
sequences of molluscan and arthropod arginine kinases allows us to estimate the divergence time of the 3 molluscan species Ž Fig. 4. . If we assume the divergence between Mollusca and Arthropoda occurred about 550 million years before present ŽMyrBP. w17x, the divergence time of the two gastropods Battilus and Nordotis is estimated to be 220 MyrBP, and that of chiton Ž Liolophura. and the other two gastropods is 360 MyrBP. The tree is essentially consistent with the fossil record of molluscan groups w19x.
References w1x D.C. Watts, Advan. Comp. Physiol. Biochem. 3 Ž1968. 1–115. w2x J.F. Morrison, in: P.C. Boyer ŽEd.., The Enzymes, Vol. 8, Academic Press, New York, 1973, pp. 457–486. w3x G.L. Kenyon, G.H. Reed, Adv. Enzymol. 54 Ž1986. 367– 426. w4x S.M. Muhelebach, M. Gross, T. Wirz, T. Wallimann, J.-C. Perriard, M. Wyss, Mol. Cell. Biochem. 133r134 Ž1994. 245–262. w5x T. Soga, Y. Yazawa, Zool. Sci. ŽAbstract. 13 ŽSuppl.. Ž1996. 55. w6x T. Suzuki, T. Furukohri, J. Mol. Biol. 237 Ž1994. 353–357.
w7x P. Chomczynski, N. Sacchi, Anal. Biochem. 162 Ž1987. 156–159. w8x R.K. Saiki, D. Gelfand, S. Stoffel, S. Scharf, R. Higuchi, G. Horn, K. Mullis, H. Erlich, Science 239 Ž1988. 487–491. w9x T. Suzuki, T. Takagi, T. Gotoh, J. Biol. Chem. 265 Ž1990. 12168–12177. w10x S. Putney, W. Herlihy, N. Royal, P. Pang, H.V. Aposhian, L. Pickering, R. Belagaje, K. Biemann, D. Page, S. Kuby, P. Schimmel, J. Biol. Chem. 259 Ž1984. 14317–14320. w11x D.A. Feng, R.F. Doolittle, J. Mol. Evol. 25 Ž1987. 351–360. w12x R. Furter, E.M. Furter-Graves, T. Wallimann, Biochemistry 32 Ž1993. 7022–7029. w13x M. Vasak, K. Nagayama, K. Wuthrich, M.L. Mertens, J.H.R. Kagi, Biochemistry 18 Ž1979. 5050–5055. w14x R.P. Rosevear, P. Desmeules, G.L. Kenyon, A.S. Mildvan, Biochemistry 20 Ž1981. 6155–6164. w15x L.H. Chen, C.L. Borders, J.R. Vasquez, G.L. Kenyon, Biochemistry 35 Ž1996. 7895–7902. w16x K. Fritz-Wolf, T. Schnyder, T. Wallimann, W. Kabsch, Nature 381 Ž1996. 341–345. w17x M. Goodman, J. Pedwaydon, J. Czelusniak, T. Suzuki, T. Gotoh, L. Moens, F. Shishikura, D. Walz, S. Vinogradov, J. Mol. Evol. 27 Ž1988. 236–249. w18x J. Felsenstein, PHYLIP ŽPhylogeny Inference Package. version 3.5c. Distributed by the author. Department of Genetics, University of Washington, Seattle, USA, 1993. w19x L.A. Cox, in: R.C. Moore ŽEd.., Treatise on invertebrate paleontology, part I, University of Kansas Press, Lawrence, 1960, pp. 85–169.