Abalone myoglobins evolved from indoleamine dioxygenase: The cDNA-derived amino acid sequence of myoglobin fromNordotis madaka

The cDNA for the unusual 41 kD myoglobin of the abaloneNordotis madaka was amplified by polymerase chain reaction (PCR), and the cDNA-derived amino acid sequence of 378 residues was determined. As with the myoglobin of the related abaloneSulculus diversicolor (Suzuki and Takagi,J. Mol. Biol. 228, 698–700, 1992), the sequence ofNordotis myoglobin showed no significant homology with any other globins, but showed high homology (35% identity) with vertebrate indoleamine 2,3-dioxygenase, a tryptophan degrading enzyme containing heme. The amino acid sequence homology betweenNordotis andSulculus myoglobins was 87%. These results support our previous idea that the abalone myoglobins evolved from a gene for indoleamine dioxygenase, but not from a globin gene, and therefore all of the hemoglobins and myoglobins are not homologous. Thus, abalone myoglobins appear to be a typical case of convergent evolution.     

The cDNA-derived amino acid sequence of indoleamine dioxygenase like-myoglobin from the gastropod molluscOmphalius pfeifferi

Myoglobin was isolated from the radular muscle of the archaegastropod molluscOmphalius pfeifferi (Trochidae). The molecular mass was estimated by SDS-PAGE to be about 40 kDa, 2.5 times larger than that of usual myoglobin. The cDNA forOmphalius myoglobin was amplified by polymerase chain reaction, and the cDNA-derived amino acid sequence of 375 residues was determined, of which 73 residues were identified directly by the chemical sequencing of internal peptides. The amino acid sequence ofOmphalius myoglobin showed no significant homology with any other usual 16-kDa globins, but showed 84% and 36% identities with indoleamine dioxygenase-like myoglobins fromBattilus (Turbinidae) andSulculus (Haliotiidae), respectively. It also shows significant homology (26% identity) with human indoleamine 2,3-dioxygenase, a tryptophan-degrading enzyme containing heme. The distribution of indoleamine dioxygenase-like myoglobins suggests that they must have arisen exclusively along the specified lineage including the three families Haliotiidae, Turbinidae, and Trochidae of Archaegastropoda in molluscan evolution.     

Amino Acid Sequence, Spectral, Oxygen-Binding, and Autoxidation Properties of Indoleamine Dioxygenase-Like Myoglobin from the Gastropod Mollusc Turbo cornutus

Myoglobin was isolated from the radular muscle of the archaeogastropod mollusc Turbo cornutus (Turbinidae). This myoglobin is a monomer carrying one protoheme group; the molecular mass was estimated by SDS–PAGE to be about 40 kDa, 2.5 times larger than that of usual myoglobin. The cDNA-derived amino acid sequence of 375 residues was determined, of which 327 residues were identified directly by chemical sequencing of internal peptides. The amino acid sequence of Turbo myoglobin showed no significant homology with any other usual 16-kDa globins, but showed 36% identity with the myoglobin from Sulculus diversicolor (Haliotiidae) and 27% identity with human indoleamine 2,3-dioxygenase, a tryptophan-degrading enzyme containing heme. Thus, the Turbo myoglobin can be counted among the myoglobins which evolved from the same ancestor as that of indoleamine 2,3-dioxygenase. The absorbance ratio of to CT maximum (/CT) of Turbo metmyoglobin was 17.8, indicating that this myoglobin probably possesses a histidine residue near the sixth coordination position of heme iron. The Turbo myoglobin binds oxygen reversibly. Its oxygen equilibrium properties are similar to those of Sulculus myoglobin, giving P ₅₀ = 3.5 mm Hg at pH 7.4 and 20°C. The pH dependence of autoxidation of Turbo oxymyoglobin was quite different from that of mammalian myoglobin, suggesting a unique protein folding around the heme cavity of Turbo myoglobin. A kinetic analysis of autoxidation indicates that the amino acid residue with pK _a = 5.4 is involved in the reaction. The autoxidation reaction was enhanced markedly at pH 7.6, but not at pH 5.5 and 6.3 in the presence of tryptophan. We suggest that a noncatalytic binding site for tryptophan, in which several dissociation groups with pK _a 7.6 are involved, remains in Turbo myoglobin as a relic of molecular evolution.     

The cDNA-derived amino acid sequence of indoleamine dioxygenase like-myoglobin from the gastropod molluscOmphalius pfeifferi

Myoglobin was isolated from the radular muscle of the archaegastropod molluscOmphalius pfeifferi (Trochidae). The molecular mass was estimated by SDS-PAGE to be about 40 kDa, 2.5 times larger than that of usual myoglobin. The cDNA forOmphalius myoglobin was amplified by polymerase chain reaction, and the cDNA-derived amino acid sequence of 375 residues was determined, of which 73 residues were identified directly by the chemical sequencing of internal peptides. The amino acid sequence ofOmphalius myoglobin showed no significant homology with any other usual 16-kDa globins, but showed 84% and 36% identities with indoleamine dioxygenase-like myoglobins fromBattilus (Turbinidae) andSulculus (Haliotiidae), respectively. It also shows significant homology (26% identity) with human indoleamine 2,3-dioxygenase, a tryptophan-degrading enzyme containing heme. The distribution of indoleamine dioxygenase-like myoglobins suggests that they must have arisen exclusively along the specified lineage including the three families Haliotiidae, Turbinidae, and Trochidae of Archaegastropoda in molluscan evolution.     

Abalone myoglobins evolved from indoleamine dioxygenase: The cDNA-derived amino acid sequence of myoglobin fromNordotis madaka

The cDNA for the unusual 41 kD myoglobin of the abaloneNordotis madaka was amplified by polymerase chain reaction (PCR), and the cDNA-derived amino acid sequence of 378 residues was determined. As with the myoglobin of the related abaloneSulculus diversicolor (Suzuki and Takagi,J. Mol. Biol. 228, 698–700, 1992), the sequence ofNordotis myoglobin showed no significant homology with any other globins, but showed high homology (35% identity) with vertebrate indoleamine 2,3-dioxygenase, a tryptophan degrading enzyme containing heme. The amino acid sequence homology betweenNordotis andSulculus myoglobins was 87%. These results support our previous idea that the abalone myoglobins evolved from a gene for indoleamine dioxygenase, but not from a globin gene, and therefore all of the hemoglobins and myoglobins are not homologous. Thus, abalone myoglobins appear to be a typical case of convergent evolution.     

Amino Acid Sequence, Spectral, Oxygen-Binding, and Autoxidation Properties of Indoleamine Dioxygenase-Like Myoglobin from the Gastropod Mollusc Turbo cornutus

Myoglobin was isolated from the radular muscle of the archaeogastropod mollusc Turbo cornutus (Turbinidae). This myoglobin is a monomer carrying one protoheme group; the molecular mass was estimated by SDS–PAGE to be about 40 kDa, 2.5 times larger than that of usual myoglobin. The cDNA-derived amino acid sequence of 375 residues was determined, of which 327 residues were identified directly by chemical sequencing of internal peptides. The amino acid sequence of Turbo myoglobin showed no significant homology with any other usual 16-kDa globins, but showed 36% identity with the myoglobin from Sulculus diversicolor (Haliotiidae) and 27% identity with human indoleamine 2,3-dioxygenase, a tryptophan-degrading enzyme containing heme. Thus, the Turbo myoglobin can be counted among the myoglobins which evolved from the same ancestor as that of indoleamine 2,3-dioxygenase. The absorbance ratio of γ to CT maximum (γ/CT) of Turbo metmyoglobin was 17.8, indicating that this myoglobin probably possesses a histidine residue near the sixth coordination position of heme iron. The Turbo myoglobin binds oxygen reversibly. Its oxygen equilibrium properties are similar to those of Sulculus myoglobin, giving P ₅₀ = 3.5 mm Hg at pH 7.4 and 20°C. The pH dependence of autoxidation of Turbo oxymyoglobin was quite different from that of mammalian myoglobin, suggesting a unique protein folding around the heme cavity of Turbo myoglobin. A kinetic analysis of autoxidation indicates that the amino acid residue with pK _a = 5.4 is involved in the reaction. The autoxidation reaction was enhanced markedly at pH 7.6, but not at pH 5.5 and 6.3 in the presence of tryptophan. We suggest that a noncatalytic binding site for tryptophan, in which several dissociation groups with pK _a ≥ 7.6 are involved, remains in Turbo myoglobin as a relic of molecular evolution.     

Amino acid sequence of myoglobin from the molluscBursatella leachii

The complete amino acid sequence of myoglobin from the triturative stomach of gastropodic molluscBursatella leachii has been determined. It is composed of 146 amino acid residues, is acetylated at the N-terminus, and contains a single histidine residue at position 95 which corresponds to the heme-binding proximal histidine. The E7 distal histidine, which is conserved widely in myoglobins and hemoglobins, is replaced by valine inBursatella myoglobin. The amino acid sequence ofBursatella myoglobin shows strong homology (73–84%) with those ofAplysia andDolabella myoglobins.     

Amino acid sequence of myoglobin from the chitonLiolophura japonica and a phylogenetic tree for molluscan globins

Myoglobin was isolated from the radular muscle of the chitonLiolophura japonica, a primitive archigastropodic mollusc.Liolophura contains three monomeric myoglobins (I, II, and III), and the complete amino acid sequence of myoglobin I has been determined. It is composed of 145 amino acid residues, and the molecular mass was calculated to be 16,070 D. The E7 distal histidine, which is replaced by valine or glutamine in several molluscan globins, is conserved inLiolophura myoglobin. The autoxidation rate at physiological conditions indicated thatLiolophura oxymyoglobin is fairly stable when compared with other molluscan myoglobins. The amino acid sequence ofLiolophura myoglobin shows low homology (11–21%) with molluscan dimeric myoglobins and hemoglobins, but shows higher homology (26–29%) with monomeric myoglobins from the gastropodic molluscsAplysia, Dolabella, andBursatella. A phylogenetic tree was constructed from 19 molluscan globin sequences. The tree separated them into two distinct clusters, a cluster for muscle myoglobins and a cluster for erythrocyte or gill hemoglobins. The myoglobin cluster is divided further into two subclusters, corresponding to monomeric and dimeric myoglobins, respectively.Liolophura myoglobin was placed on the branch of monomeric myoglobin lineage, showing that it diverged earlier from other monomeric myoglobins. The hemoglobin cluster is also divided into two subclusters. One cluster contains homodimeric, heterodimeric, tetrameric, and didomain chains of erythrocyte hemoglobins of the blood clamsAnadara, Scapharca, andBarbatia. Of special interest is the other subcluster. It consists of three hemoglobin chains derived from the bacterial symbiont-harboring clamsCalyptogena andLucina, in which hemoglobins are supposed to play an important role in maintaining the symbiosis with sulfide bacteria.     

Fluorescence studies on the interaction of myoglobin with mitochondria

To determine the nature and characteristic parameters of the myoglobin-mitochondrion interaction during oxymyoglobin (MbO₂) deoxygenation in the cell, we studied the quenching of the intrinsic mitochondrial flavin and tryptophan fluorescence by different liganded myoglobins in the pH range of 6–8, as well as the quenching of the fluorescence of the membrane probes 1,8-ANS and merocyanine 540 (M 540) embedded into the mitochondrial membrane. Physiologically active MbO₂ and oxidized metmyoglobin (metMb), which are unable to bind oxygen, were used as the quenchers. The absence of quenching of flavin and tryptophan fluorescence implies that myoglobin does not form quenching complexes with either electron transport chain proteins of the inner mitochondrial membrane or with outer membrane proteins. We found, however, that MbO₂ and metMb effectively quench 1,8-ANS and M 540 fluorescence in the pH range of 6–8. Characteristic parameters of 1,8-ANS and M 540 fluorescence quenching by the myoglobins (extent of quenching and quencher binding constant, K _m) are very similar, indicating that both probes are localized in phospholipid sites of the mitochondrial membrane, and myoglobin is complexed with these sites. The dependence of K _m on ionic strength proves the important role of coulombic interactions in the formation of the quenching complex. Since the overall charge of myoglobin is shown not to influence the K _m values, the ionic strength dependence must be due to local electrostatic interactions in which polar groups of some part of the myoglobin molecule participate. The most likely candidates to interact with anionic groups of mitochondrial phospholipids are invariant lysine and arginine residues in the environment of the myoglobin heme cavity, which do not change their ionization state in the pH range investigated.     

Primary structure of otter (Lutra lutra L.) myoglobin. II. Pepsin peptides of trypsin hydrolysate. Reconstruction of the polypeptide chain of the otter myoglobin globin component

13 peptic peptides have been isolated from the insoluble (at pH 5.0) fraction of the tryptic hydrolysate of main chromatographic component of otter myoglobin and their amino acid composition and N-terminal amino acid sequences have been determined. The isolated peptides contain in total 40 amino acid residues. The results obtained, along with those on tryptic peptides and the comparison with homologous portions of myoglobins of the known primary structure, allowed reconstructing the complete amino acid sequence of otter myoglobin.     

Amino acid sequence of myoglobin from the molluscBursatella leachii

The complete amino acid sequence of myoglobin from the triturative stomach of gastropodic molluscBursatella leachii has been determined. It is composed of 146 amino acid residues, is acetylated at the N-terminus, and contains a single histidine residue at position 95 which corresponds to the heme-binding proximal histidine. The E7 distal histidine, which is conserved widely in myoglobins and hemoglobins, is replaced by valine inBursatella myoglobin. The amino acid sequence ofBursatella myoglobin shows strong homology (73–84%) with those ofAplysia andDolabella myoglobins.     

The sequence-immunology correlation revisited: Data for cetacean myoglobins and mammalian lysozymes

Quantitative microcomplement fixation tests employing rabbit antisera were done to compare immunologically 13 cetacean myoglobins and 15 mammalian lysozymes c of known amino acid sequence. In both cases there was a strong correlation between immunological distance (y) and percent sequence difference (x), as had been found for several other globular proteins. For myoglobin the relationship could be described by y = 10.5x and for lysozyme by y = 8.5x. The coefficients in both of these equations are appreciably higher than the values of 5.1–6.9 reported for three other vertebrate globular proteins (bird lysozyme c, mammalian ribonuclease, and mammalian serum albumin), and they imply that rabbit antisera to mammalian myoglobins and lysozymes are more sensitive to evolutionary substitutions. A strong inverse correlation (r = -0.95) was found when the slope of the line relating y to x for these five data sets was plotted against the percent sequence difference between the rabbit's own protein and the proteins immunized with. Specifically, the cetacean myoglobins on average differ in amino acid sequence from rabbit myoglobin by less than 13% and exhibit the steepest slope (10.5), while bird lysozyme sequences differ by nearly 40% from rabbit lysozyme and exhibit the shallowest slope (5.1).     

Evidence of met-form myoglobin from Theliostyla albicilla radular muscle

Gastropod mollusc myoglobins provide interesting clues to the evolution of this family of proteins. In addition to conventional monomeric myoglobins, this group also has dimeric and unusual indoleamine dioxygenase-like myoglobins. We isolated myoglobin from the radular muscle of living gastropod mollusc Theliostyla albicilla. The myoglobin appeared to be present in an oxidized met-form, a physiologically inactive form that is not capable of binding oxygen. Under the same extraction conditions, myoglobins mainly of the physiologically active oxy-form have been isolated from other molluscs. The complete amino acid sequence of 157 residues of Theliostyla myoglobin shows that it has a long N-terminal extension of seven residues and contains three functional key residues: CD1-Phe, E7-His, and F8-His. The metmyoglobin can easily be reduced to a ferrous state with Na(2)S(2)O(4). The autoxidation rate of the oxy-form was comparable to other molluscan myoglobins over a wide pH range, and Theliostyla myoglobin was shown to be stable as an oxygen-binding protein. Thus, the predominantly met-form of myoglobin in Theliostyla can be attributed to the incomplete functioning of the myoglobin reduction system in the radular muscle. Although the function of Theliostyla myoglobin is unclear, it may be a scavenger of H(2)O(2).     

Amino acid sequence of myoglobin from the chitonLiolophura japonica and a phylogenetic tree for molluscan globins

Myoglobin was isolated from the radular muscle of the chitonLiolophura japonica, a primitive archigastropodic mollusc.Liolophura contains three monomeric myoglobins (I, II, and III), and the complete amino acid sequence of myoglobin I has been determined. It is composed of 145 amino acid residues, and the molecular mass was calculated to be 16,070 D. The E7 distal histidine, which is replaced by valine or glutamine in several molluscan globins, is conserved inLiolophura myoglobin. The autoxidation rate at physiological conditions indicated thatLiolophura oxymyoglobin is fairly stable when compared with other molluscan myoglobins. The amino acid sequence ofLiolophura myoglobin shows low homology (11–21%) with molluscan dimeric myoglobins and hemoglobins, but shows higher homology (26–29%) with monomeric myoglobins from the gastropodic molluscsAplysia, Dolabella, andBursatella. A phylogenetic tree was constructed from 19 molluscan globin sequences. The tree separated them into two distinct clusters, a cluster for muscle myoglobins and a cluster for erythrocyte or gill hemoglobins. The myoglobin cluster is divided further into two subclusters, corresponding to monomeric and dimeric myoglobins, respectively.Liolophura myoglobin was placed on the branch of monomeric myoglobin lineage, showing that it diverged earlier from other monomeric myoglobins. The hemoglobin cluster is also divided into two subclusters. One cluster contains homodimeric, heterodimeric, tetrameric, and didomain chains of erythrocyte hemoglobins of the blood clamsAnadara, Scapharca, andBarbatia. Of special interest is the other subcluster. It consists of three hemoglobin chains derived from the bacterial symbiont-harboring clamsCalyptogena andLucina, in which hemoglobins are supposed to play an important role in maintaining the symbiosis with sulfide bacteria.     

Molecular Cloning and Characterization of the Myostatin Gene in Croceine Croaker, Pseudosciaena crocea

Myostatin (MSTN) is a negative regulator of skeletal muscle mass and has a potential application in aquaculture. We reported the characterization of the myostatin gene and its expression in the croceine croaker, Pseudosciaena crocea. The myostatin gene had three exons encoding 376 amino acids. The cDNA was 1,906 bp long with a 5′-UTR and 3′-UTR of 108 bp and 667 bp, respectively. A microsatellite sequence, CA₃₀ and CA₂₆ separated by TA, existed in the 3′-UTR. Intron I and II were 343 bp and 758 bp in length, respectively. The deduced amino acid sequence was highly conserved, and had more than 90% identical to shi drum, gilthead seabream, striped sea-bass, white perch, and white bass proteins. The myostatin of croceine croaker had a putative amino terminal signal sequence (residues 1–22), a transforming growth factor-beta (TGF-β) propeptide domain (residues 41–256), a RXXR proteolytic processing site (RARR, residues 264–267, matching the RXXR consensus site), and a TGF-β domain (residues 282–376). There were 13 conserved cysteine residues in croceine croaker myostatin, nine of which are common to all TGF-β superfamily members. The most conserved region of vertebrate myostatins is the TGF-β domain, which was the mature bioactive domain of the myostatin protein. The myostatin gene was expressed not only in the skeletal muscle, but also in the other tissues.     

Crystal structure of myoglobin form a synthetic gene

Crystal have been grown of myoglobin produced in Escherichia coli from a synthetic gene, and the structure has been solved to 1.9 Å resolution. The space group of the crystals is P6, which is different from previously solved myoglobin crystal forms. The synthetic myoglobin is essentially identical to myoglobin isolated from sperm whale tissue, except for the retention of the initiator methionine at the N-terminus and the substitution of asparagine for aspartic acid at position 122. Superposition of the coordinates of native and synthetic sperm whale myoglobins reveals only minor changes in the positions of main chain atoms and roeientation of some surface side chains. Crystals of variant of the “synthetic” myoglobin have also been grown for structural analysis of the role of key amino acid residues in ligand and specificity.     

Isolation and cDNA-derived amino acid sequences of hemoglobin and myoglobin from the deep-sea clam Calyptogena kaikoi

The heterodont clam Calyptogena kaikoi, living in the cold-seep area at a depth of 3761 m of the Nankai Trough, Japan, has abundant hemoglobins and myoglobins in erythrocytes and adductor muscle, respectively. Two types of hemoglobins (Hb I and Hb II) were isolated, and the complete amino acid sequences of Hb I (145 residues) and Hb II (137 residues) were obtained with combination of cDNA and protein sequencing. The amino acid sequences of C. kaikoi Hbs I and II differed from homologous chains of the congeneric clam Calyptogena soyoae in eight and five positions, respectively. The distal (E7) His, one of the functionally important residues in hemoglobin and myoglobin, was replaced by Gln in hemoglobins of C. kaikoi. A phylogenetic analysis of clam hemoglobins indicates that the evolutionary rate of Calyptogena hemoglobins is rather faster than those of other clams, suggesting that the mutation rate might be accelerated in the deep-sea animals around the areas of cold seeps or hydrothermal vents. On the other hand, it was found unexpectedly that two myoglobins Mbs I and II, isolated from the red adductor muscle, are identical in amino acid sequence Hbs I and II, respectively. Thus it was assumed that genes for Hbs I and II are also expressed in the muscle of C. kaikoi in substitution for myoglobin gene. This suggests that the major physiological role of globins in C. kaikoi is storage of oxygen under the low oxygen conditions, rather than circulating of oxygen.     

Amino acid sequence of myoglobin from the mollusc Dolabella auricularia

The complete amino acid sequence of the myoglobin from Dolabella auricularia, a common gastropodic mollusc on the Japanese coast, has been determined. The myoglobin is composed of 146 amino acid residues, is acetylated at the NH2 terminus, and contains a single histidine residue at position 95 which most likely corresponds to the heme-binding proximal histidine. The sequence of Dolabella myoglobin shows strong homology (72-77%) with those of Aplysia myoglobins. The autoxidation rate of Dolabella oxymyoglobin (MbO2) was examined in 0.1 M buffer at 25 degrees C over pH range 4.8-12. Dolabella MbO2 was extremely unstable between pH 7 and 11, and the pH dependence of the stability was quite different from that of sperm whale MbO2. This property may be partly due to the absence of a distal (E7) histidine in Dolabella myoglobin.     

Bacterial expression and characterization of molluscan IDO-like myoglobin

The indoleamine 2,3-dioxygenase (IDO)-like myoglobin (Mb) is a unique type of Mb isolated from the buccal mass of several archgastropod species. Here, we expressed Sulculus diversicolor IDO-like Mb as a GST-fusion protein in bacteria. The visible spectrum of GST-fusion IDO-like Mb shows characteristic α- and β-peaks, indicating that it binds oxygen. To identify residues important in heme and oxygen binding, we constructed site-directed mutants. We initially replaced each of the 7 histidines of S. diversicolor IDO-like Mb with alanine. The spectra of three mutants (H74A, H288A, and H332A) revealed a remarkable loss of absorbance around 414 nm, indicating that they cannot bind heme. His⁷⁴, His²⁸⁸, and His³³² were also replaced by arginine or tyrosine. Neither H332R nor H332Y contains heme, suggesting that His³³² is the proximal ligand of IDO-like Mb. In contrast, both H74R and H288Y mutants were isolated in the heme-binding oxy-form. The autoxidation rates of these two mutants showed that they can bind oxygen as stably as wild-type. His⁷⁴ and His²⁸⁸ might be partially associated with heme-binding, but do not act as the distal ligand. The S. diversicolor IDO-like Mb seems to stably bind oxygen in a different manner from normal myoglobins.     

Purification, characterization, and molecular cloning of organic-solvent-tolerant cholesterol esterase from cyclohexane-tolerant Burkholderia cepacia strain ST-200

Extracellular cholesterol esterase of Burkholderia cepacia strain ST-200 was purified from the culture supernatant. Its molecular mass was 37 kDa. The enzyme was stable at pH 5.5–12 and active at pH 5.5–6, showing optimal activity at pH 7.0 at 45°C. Relative to the commercially available cholesterol esterases, the purified enzyme was highly stable in the presence of various water-miscible organic solvents. The enzyme preferentially hydrolyzed long-chain fatty acid esters of cholesterol, except for that of cholesteryl palmitate. The enzyme exhibited lipolytic activity toward various p-nitrophenyl esters. The hydrolysis rate of p-nitrophenyl caprylate was enhanced 3.5- to 7.2-fold in the presence of 5–20% (vol/vol) water-miscible organic solvents relative to that in the absence of organic solvents. The structural gene encoding the cholesterol esterase was cloned and sequenced. The primary translation product was predicted to be 365 amino acid residues. The mature product is composed of 325 amino acid residues. The amino acid sequence of the product showed the highest similarity to the lipase LipA (87%) from B. cepacia DSM3959.