Cysteinyl-tRNA synthetase is a direct descendant of the first aminoacyl-tRNA synthetase.

The gene encoding the cysteinyl-tRNA synthetase of E. coli was cloned from an E. coli genomic library made in lambda 2761, a lambda vector which can integrate and which carries a chloramphenicol resistance gene. A thermosensitive cysS mutant of E. coli was lysogenised and chloramphenicol-resistant colonies able to grow at 42 degrees C were selected to isolate phages containing the wild-type cysS gene. The sequence of the gene was determined. It codes for a 461 amino-acid protein and includes the sequences HIGH and KMSK known to be involved in the ATP and tRNA binding respectively of class I synthetases. The cysteinyl enzyme has segments in common with the cytoplasmic leucyl-tRNA synthetase of Neurospora crassa, the tryptophanyl-tRNA synthetase of Bacillus stearothermophilus, and the phenylalanyl-tRNA synthetase of Saccharomyces cerevisiae. Sequence comparisons show that the amino end of the cysteinyl-tRNA synthetase has similarities with prokaryotic elongation factors Tu; this region is close to the equivalent acceptor binding domain of the glutaminyl-tRNA synthetase of E. coli. There is a further similarity with the seryl enzyme (a class II enzyme) which has led us to propose that both classes had a common origin and that this was the ancestor of the cysteinyl-tRNA synthetase.     

Isolation of two cDNAs encoding functional human cytoplasmic cysteinyl-tRNA synthetase

Cysteinyl-tRNA synthetase catalyzes the addition of cysteine to its cognate tRNA. The available eukaryotic sequences for this enzyme contain several insertions that are absent from bacterial sequences. To gain insights into the differences between the bacterial and eukaryotic forms, we previously studied the E. coli cysteinyl-tRNA synthetase. In this study, we sought to clone and express the full-length gene for the human cytoplasmic cysteinyl-tRNA synthetase. Although a gene encoding the human enzyme has been described, the predicted protein sequence, consisting of 638 amino acids, lacks homology with other eukaryotic enzymes in the carboxyl-terminus. This suggested that a further investigation was necessary to obtain the definitive sequence for the human enzyme. Here we report the isolation of a full-length cDNA that encodes a protein of 748 amino acids. The predicted protein sequence shows considerable similarity to other eukaryotic cysteinyl-tRNA synthetases in the carboxyl-terminus. We also found that approximately 20% of the mRNA encoding the cytoplasmic cysteinyl-tRNA synthetase contained an insertion of 8 bases in the 3' coding region of the mRNA. This insertion arises from an alternative splicing between the last two exons of the gene. The alternative splicing alters the reading frame and results in the replacement of the carboxy-terminal 44 amino acids with a novel sequence of 22 amino acids. Expression of the full-length and alternative forms of the enzyme in E. coli generated functional proteins that were active in aminoacylation of human cytoplasmic tRNA(Cys) with cysteine.     

Editing function of Escherichia coli cysteinyl-tRNA synthetase: cyclization of cysteine to cysteine thiolactone.

A cyclic sulfur compound, identified as cysteine thiolactone by several chemical and enzymatic tests, is formed from cysteine during in vitro tRNA(Cys) aminoacylation catalyzed by Escherichia coli cysteinyl-tRNA synthetase. The mechanism of cysteine thiolactone formation involves enzymatic deacylation of Cys-tRNA(Cys) (k = 0.017 s-1) in which nucleophilic sulfur of the side chain of cysteine in Cys-tRNA(Cys) attacks its carboxyl carbon to yield cysteine thiolactone. Nonenzymatic deacylation of Cys-tRNA(Cys) (k = 0.0006 s-1) yields cysteine, as expected. Inhibition of enzymatic deacylation of Cys-tRNA(Cys) by cysteine and Cys-AMP, but not by ATP, indicates that both synthesis of Cys-tRNA(Cys) and cyclization of cysteine to the thiolactone occur in a single active site of the enzyme. The cyclization of cysteine is mechanistically similar to the editing reactions of methionyl-tRNA synthetase. However, in contrast to methionyl-tRNA synthetase which needs the editing function to reject misactivated homocysteine, cysteinyl-tRNA synthetase is highly selective and is not faced with a problem in rejecting noncognate amino acids. Despite this, the present day cysteinyl-tRNA synthetase, like methionyl-tRNA synthetase, still retains an editing activity toward the cognate product, the charged tRNA. This function may be a remnant of a chemistry used by an ancestral cysteinyl-tRNA synthetase.     

Methanocaldococcus jannaschii prolyl-tRNA synthetase charges tRNA(Pro) with cysteine

Methanocaldococcus jannaschii prolyl-tRNA synthetase (ProRS) was previously reported to also catalyze the synthesis of cysteinyl-tRNA(Cys) (Cys-tRNA(Cys)) to make up for the absence of the canonical cysteinyl-tRNA synthetase in this organism (Stathopoulos, C., Li, T., Longman, R., Vothknecht, U. C., Becker, H., Ibba, M., and S?ll, D. (2000) Science 287, 479-482; Lipman, R. S., Sowers, K. R., and Hou, Y. M. (2000) Biochemistry 39, 7792-7798). Here we show by acid urea gel electrophoresis that pure heterologously expressed recombinant M. jannaschii ProRS misaminoacylates M. jannaschii tRNA(Pro) with cysteine. The enzyme is unable to aminoacylate purified mature M. jannaschii tRNA(Cys) with cysteine in contrast to facile aminoacylation of the same tRNA with cysteine by Methanococcus maripaludis cysteinyl-tRNA synthetase. Although M. jannaschii ProRS catalyzes the synthesis of Cys-tRNA(Pro) readily, the enzyme is unable to edit this misaminoacylated tRNA. We discuss the implications of these results on the in vivo activity of the M. jannaschii ProRS and on the nature of the enzyme involved in the synthesis of Cys-tRNA(Cys) in M. jannaschii.     

The plant aminoacyl-tRNA synthetases. Purification and characterization of valyl-tRNA, tryptophanyl-tRNA and seryl-tRNA synthetases from yellow-lupin seeds.

Valyl-tRNA, tryptophanyl-tRNA, and seryl-tRNA synthetases from yellow lupin seeds Lupinus luteus were purified to homogeneity by ammonium sulfate fractionation, hydrophobic chromatography on aminohexyl-Sepharose column and affinity chromatography on tRNA-Sepharose column. Valyl-tRNA synthetase consists of one polypeptide chain of molecular weight 125000 as judged by Sephadex G-200 gel filtration and dodecylsulfate-polyacrylamide gel electrophoresis in the presence of reducing agent. Seryl-tRNA synthetase, Mr equals 110000, is composed of two 55000-Mr subunits. Tryptophanyl-tRNA synthetase exhibits molecular weight of 200000 on Sephadex G-200 and 37000 in dodecylsulfate-polyacrylamide gel electrophoresis. This indicates that tryptophanyl-tRNA synthetase consists of several subunits (probably four). Since the seryl-tRNA synthetase exhibits the same mobility on dodecylsulfate-polyacrylamide gels both in the presence and absence of reducing agent it is concluded that there is no covalent bond(s) between the subunits of the enzyme. There is also no covalent bond(s) between the subunits of tryptophanyl-tRNA synthetase. Effect of anti-sulfhydryl reagents, monovalent salts, pH and different buffers on activity of the three synthetases is described. Kinetic constants for the substrates of the synthetases are also given. dATP is a substrate for seryl-tRNA synthetase but not for valyl-tRNA and tryptophanyl-tRNA synthetases.     

Synthesis of cysteinyl-tRNA(Cys) by a genome that lacks the normal cysteine-tRNA synthetase

Synthesis of cysteinyl-tRNA(Cys) by cysteine-tRNA synthetase is required for decoding cysteine codons in all known organisms. The genome of the archaeon Methanococcus jannaschii lacks the gene for a normal cysteine-tRNA synthetase. The activity of the enzyme, however, was identified recently, and it allowed the purification of the enzyme and cloning of its gene. Sequence analysis of the gene showed that it encodes proline-tRNA synthetase and, thus, raised the possibility of dual activities in a single aminoacyl-tRNA synthetase. Assays of aminoacyl-adenylate synthesis confirmed the ability of the enzyme to activate proline and cysteine and showed that both activities were independent of tRNA. Assays of tRNA aminoacylation established the specific attachment of proline to tRNA(Pro) and cysteine to tRNA(Cys). However, in contrast to a recent report of comparable activities with cysteine and proline, results here indicate that the adenylate synthesis and aminoacylation activities with cysteine are significantly lower than the respective activity with proline. In addition, there is evidence of overlapping amino acid-binding sites and tRNA-binding sites. These considerations, among others, raised the distinct possibility that the M. jannaschii proline-tRNA synthetase may recruit additional protein or RNA factors to facilitate the synthesis of cysteinyl-tRNA(Cys).     

The homotetrameric phosphoseryl-tRNA synthetase from Methanosarcina mazei exhibits half-of-the-sites activity

Synthesis of cysteinyl-tRNA(Cys) in methanogenic archaea proceeds by a two-step pathway in which tRNA(Cys) is first aminoacylated with phosphoserine by phosphoseryl-tRNA synthetase (SepRS). Characterization of SepRS from the mesophile Methanosarcina mazei by gel filtration and nondenaturing mass spectrometry shows that the native enzyme exists as an alpha4 tetramer when expressed at high levels in Escherichia coli. However, active site titrations monitored by ATP/PP(i) burst kinetics, together with analysis of tRNA binding stoichiometry by fluorescence spectroscopy, show that the tetrameric enzyme binds two tRNAs and that only two of the four chemically equivalent subunits catalyze formation of phosphoseryl adenylate. Therefore, the phenomenon of half-of-the-sites activity, previously described for synthesis of 1 mol of tyrosyl adenylate by the dimeric class I tyrosyl-tRNA synthetase, operates as well in this homotetrameric class II tRNA synthetase. Analysis of cognate and noncognate reactions by ATP/PP(i) and aminoacylation kinetics strongly suggests that SepRS is able to discriminate against the noncognate amino acids glutamate, serine, and phosphothreonine without the need for a separate hydrolytic editing site. tRNA(Cys) binding to SepRS also enhances the capacity of the enzyme to discriminate among amino acids, indicating the existence of functional connectivity between the tRNA and amino acid binding sites of the enzyme.     

Yeast argininosuccinate synthetase. Purification; structural and kinetic properties.

Yeast argininosuccinate synthetase has been purified to homogeneity. The enzyme was found to have a molecular weight of 228,000 as determined by gel sieving. It is composed of identical subunits of Mr 49,000 as shown by gel electrophoresis. The quaternary structure as determined by cross-linking of the subunits with glutaraldehyde, followed by gel electrophoresis with dodecylsulfate, is tetrameric. The saturation functions by citrulline and aspartate are hyperbolic; with MgATP as the variable substrate a sigmoid character, dependent on the concentration of citrulline, aspartate, argininosuccinate and arginine, was observed. The positive cooperativity is reduced by increasing concentrations of citrulline and aspartate; it is increased by argininosuccinate and arginine. Kinetic analysis provided evidence for a random addition of substrates. Initial velocity studies as well as product and dead-end inhibition studies comply with a rapid-equilibrium random model, except for the interconversion of the central quaternary complexes; the different kinetic constants have been established on the basis. Yeast argininosuccinate synthetase has a double metabolic function: anabolic in the biosynthesis of arginine, catabolic as the first enzyme of citrulline utilization as nitrogen source. The kinetic properties of the enzyme point to a physiologically well-adjusted activity for both roles and to an economic and efficient utilization of ATP.     

Selenium Metabolism in Neptunia amplexicaulis

ATP sulfurylase (EC 2.7.7.4), cysteinyl-tRNA synthetase (EC 6.1.1.16), and methionyl-tRNA synthetase (EC 6.1.1.10) from Neptunia amplexicaulis have been purified approximately 162-, 140- and 185-fold, respectively. Purified ATP sulfurylase in the presence of purified inorganic pyrophosphatase catalyzed the incorporation of sulfate into adenosine 5′-phosphosulfate; evidence of an analogous reaction with selenate is presented. Crude extracts catalyzed both the sulfate- and the adenosine 5′-phosphosulfate-dependent NADH oxidation in the adenosine 5′-phosphosulfate kinase assay of Burnell and Whatley (1977 Biochim Biophys Acta 481: 266-278), but an analogous reaction with selenate could not be detected. Both purified cysteinyl-tRNA synthetase and methionyl-tRNA synthetase used selenium-containing analogs as substrates in both the ATP-pyrophosphate exchange and the aminoacylation assays.     

Reduction of DL-selenocystine and isolation of L-seleoncysteine

Cystine, selenocytsine, and several analogs were reduced by dithiothreitol (DTT), beta-mercaptoethanol (ME) and sodium borohydride (NaBH4). DTT was the most effective; DTT to cystine ratios from 10 to 80 were equally effective. With selenocysteine, however, absorption was considerably reduced at all ratios. Selenocysteine was identified as the reduction product by reaction with Gaitonde's reagent, comparison of absorption spectra, paper chromatograhy, utilization by cysteinyl-tRNA synthetase fro Paracoccus denitrificans and Vigna radiata, changes in solubility after DTT treatment, and comparison of infrared spectra. During the ATP-PPi exchange assay, DTT and ME convert cysteine and selenocysteine derivatives to cysteine and selenocysteine which serve as substrates for cysteinyl-tRNA synthetase.     

Cysteinyl-tRNA synthetase from Escherichia coli does not need an editing mechanism to reject serine and alanine. High binding energy of small groups in specific molecular interactions.

The cysteinyl-tRNA synthetase from Escherichia coli only very slowly activates serine, alanine, and alpha-aminobutyrate, the possible competitors of cysteine. The upper limits on the values of kcat/KM for the amino acid dependent ATP/pyrophosphate exchange reactions, relative to that of cysteine, are less than 10(-8), 2 x 10(-7), and 3 x 10(-6), respectively. It is calculated from these data and the concentrations of the amino acids in vivo that the error rates for the misincorporation of serine and alanine for cysteine are less than 10(-9) and 5 x 10(-8), respectively. There is no need for an error correcting mechanism and no evidence has been found to implicate one: there is no detectable ATP/pyrophosp hatase activity of the enzyme in the presence of tRNACys and alanine; Ala-tRNACys has been synthesized by the reductive desulfurization of Cys-tRNACys and has been found to be relatively resistant to the enzyme-catalyzed deacylation. Part of the high selectivity of the enzyme for the -SH group of cysteine (approximately 5 kcal/mol) appears to be caused by dispersion forces: simple calculations suggest that the dispersion energy between sulfur and a methylene group is about 2.5 times greater than that between two methylene groups. This high "hydrophobicity" of sulfur is consistent with the relative binding energies of substrates of the methionyl-tRNA synthetase. The rest of the high binding energy of the-SH group may come from hydrogen bonding.     

Cysteinyl-tRNA Synthetase from Phaseolus aureus: Purification and Properties

l-Cysteinyl-tRNA synthetase (EC 6.1.1.16) from Phaseolus aureus was purified approximately 300-fold and was free of contaminating aminoacyl-tRNA synthetases. Optimum assay conditions were determined and substrate specificity and inhibitor properties were investigated using the ATP-PPi exchange reaction. The Km values for l-cysteine, ATP, and PPi were 6.20 x 10(-5)m, 1.15 x 10(-3)m, and 1 x 10(-3)m, respectively. Both l-selenocysteine (Km = 5 x 10(-5)m) and alpha-l-aminobutyric acid (Km = 1 x 10(-2)m) acted as alternative substrates of the purified cysteinyl-tRNA synthetase. The enzyme was sensitive to sulfhydryl group reagents; it was inhibited by sulfide, 0-acetylserine, and reduced glutathione.     

Cysteinyl-tRNA synthetase: determination of the last E. coli aminoacyl-tRNA synthetase primary structure.

The gene coding for E. coli cysteinyl-tRNA synthetase (cysS) was isolated by complementation of a strain deficient in cysteinyl-tRNA synthetase activity at high temperature (43 degrees C). Sequencing of a 2.1 kbp DNA fragment revealed an open reading frame of 1383 bp coding for a protein of 461 amino acid residues with a Mr of 52,280, a value in close agreement with that observed for the purified protein, which behaves as a monomer. The sequence of CysRS bears the canonical His-Ile- Gly -His (HIGH) and Lys-Met-Ser-Lys-Ser (KMSKS) motifs characteristic of the group of enzymes containing a Rossmann fold; furthermore, it shows striking homologies with MetRS (an homodimer of 677 residues) and to a lesser extent with Ile-, Leu-, and ValRS (monomers of 939, 860, and 951 residues respectively). With its monomeric state and smaller size, CysRS is probably more closely related to the primordial aminoacyl-tRNA synthetase from which all have diverged.     

Purification and properties of acetyl coenzyme A synthetase from bakers' yeast.

Acetyl-CoA synthetase, utilized in a coupled reaction system, has been shown to be applicable to the spectrophotometric determination of propionic and methylmalonic acids in biological fluids. The isolation of acetyl-CoA synthetase from yeast is simpler than the purification from mammalian sources. This study also presents some properties of the yeast enzyme and compares it to the more extensively studied enzyme isolated from ammmalian tissue. Isolation and purification yielded a preparation with a specific activity of 44 units/mg at 25 degrees. The purified acetyl-CoA synthetase was apparently homogeneous by sodium dodecyl sulfate-poly-acrylamide gel electrophoresis with an estimated subunit molecular weight of 78,000. Polyacrylamide gel electrophoresis in the presence of ATP revealed a single protein band which contained all of the enzyme activity. Analytical ultra-centrifuge studies indicated the presence of a single protein with a molecular wright of 151,000 and sedimentation velocity analysis revealed a single peak with a sedimentation coefficient of 8.65 So20,w. Similar to the enzyme from mammalian sources, yeast acetyl-CoA synthetase has a high degree of substrate specificity and is active only on acetate and propionate. In addition, the reaction mechanism, as demonstrated by initial velocity patterns obtained from substrate pairs, appeared to be identical to the enzyme from bovine heart. However, the apparent Michaelis constants for the substrates were significantly different from the mammalian enzyme. The yeast-derived enzyme also differed from the mammalian in terms of molecular weight, amino acid composition, pH optimum, effect of monovalent cations, and stability characteristics. Thus, yeast acetyl-CoA synthetase is more easily purified than the mammalian enzyme and provides an excellent preparation for the assay of propionic and methylmalonic acids.     

Studies on prephenoloxidase-activating enzyme from cuticle of the silkworm Bombyx mori. II. Purification and characterization of the enzyme

Prephenoloxidase-activating enzyme has been purified approximately 4800-fold from cuticular extract of the silkworm, and the preparation seems to be homogeneous as judged by disc- and dodecylsulfate-polyacrylamide gel electrophoresis. By means of gel filtration through Sephadex G-100, it has been supposed that the enzyme exists as mono- and dimeric forms at slightly acidic pH, while a monomeric form is predominant under slightly alkaline condition. The molecular weight of the monomer was estimated to be 33,000–35,000 by dodecylsulfate-polyacrylamide gel electrophoresis and gel filtration.It has been demonstrated that ester substrates for trypsin, benzoyl-l-arginine ethyl ester and tosyl-l-arginine methyl ester, can be hydrolyzed by the purified enzyme. Several lines of evidence indicating that a single protein is involved in both activation and esterolytic reactions have been presented. Some enzymatic properties of the purified preparation as esterase have also been described.In connection to esterase activity of the purified enzyme, a mechanism of prephenoloxidase activation in the silkworm system has briefly been discussed.     

Initial position of aminoacylation of individual Escherichia coli, yeast, and calf liver transfer RNAs.

Transfer RNAs from Escherichia coli, yeast (Sacharomyces cerevisiae), and calf liver were subjected to controlled hydrolysis with venom exonuclease to remove 3'-terminal nucleotides, and then reconstructed successively with cytosine triphosphate (CTP) and 2'- or 3'-deoxyadenosine 5'-triphosphate in the presence of yeast CTP(ATP):tRNA nucleotidyltransferase. The modified tRNAs were purified by chromatography on DBAE-cellulose or acetylated DBAE-cellulose and then utilized in tRNA aminoacylation experiments in the presence of the homologous aminoacyl-tRNA synthetase activities. The E. coli, yeast, and calf liver aminoacyl-tRNA synthetases specific for alanine, glycine, histidine, lysine, serine, and threonine, as well as the E. coli and yeast prolyl-tRNA synthetases and the yeast glutaminyl-tRNA synthetase utilized only those homologous modified tRNAs terminating in 2'-deoxyadenosine (i.e., having an available 3'-OH group). This is interpreted as evidence that these aminoacyl-tRNA synthetases normally aminoacylate their unmodified cognate tRNAs on the 3'-OH group. The aminoacyl-tRNA synthetases from all three sources specific argining, isoleucine, leucine, phenylalanine, and valine, as well as the E. coli and yeast enzymes specific for methionine and the E. coli glutamyl-tRNA synthetase, used as substrates exclusively those tRNAs terminating in 3'-deoxyadenosine. Certain aminoacyl-tRNA synthetases, including the E. coli, yeast, and calf liver asparagine and tyrosine activating enzymes, the E. coli and yeast cysteinyl-tRNA synthetases, and the aspartyl-tRNA synthetase from yeast, utilized both isomeric tRNAs as substrates, although generally not at the same rate. While the calf liver aspartyl- and cysteinyl-tRNA synthetases utilized only the corresponding modified tRNA species terminating in 2'-deoxyadenosine, the use of a more concentrated enzyme preparation might well result in aminoacylation of the isomeric species. The one tRNA for which positional specificity does seem to have changed during evolution is tryptophan, whose E. coli aminoacyl-tRNA synthetase utilized predominantly the cognate tRNA terminating in 3'-deoxyadenosine, while the corresponding yeast and calf liver enzymes were found to utilize predominantly the isomeric tRNAs terminating in 2'-deoxyadenosine. The data presented indicate that while there is considerable diversity in the initial position of aminoacylation of individual tRNA isoacceptors derived from a single source, positional specificity has generally been conserved during the evolution from a prokaryotic to mammalian organism.     

Redundant synthesis of cysteinyl-tRNACys in Methanosarcina mazei

A subset of methanogenic archaea synthesize the cysteinyl-tRNA(Cys) (Cys-tRNA(Cys)) needed for protein synthesis using both a canonical cysteinyl-tRNA synthetase (CysRS) as well as a set of two enzymes that operate via a separate indirect pathway. In the indirect route, phosphoseryl-tRNA(Cys) (Sep-tRNA(Cys)) is first synthesized by phosphoseryl-tRNA synthetase (SepRS), and this misacylated intermediate is then converted to Cys-tRNA(Cys) by Sep-tRNA:Cys-tRNA synthase (SepCysS) via a pyridoxal phosphate-dependent mechanism. Here, we explore the function of all three enzymes in the mesophilic methanogen Methanosarcina mazei. The genome of M. mazei also features three distinct tRNA(Cys) isoacceptors, further indicating the unusual and complex nature of Cys-tRNA(Cys) synthesis in this organism. Comparative aminoacylation kinetics by M. mazei CysRS and SepRS reveals that each enzyme prefers a distinct tRNA(Cys) isoacceptor or pair of isoacceptors. Recognition determinants distinguishing the tRNAs are shown to reside in the globular core of the molecule. Both enzymes also require the S-adenosylmethione-dependent formation of (m1)G37 in the anticodon loop for efficient aminoacylation. We further report a new, highly sensitive assay to measure the activity of SepCysS under anaerobic conditions. With this approach, we demonstrate that SepCysS functions as a multiple-turnover catalyst with kinetic behavior similar to bacterial selenocysteine synthase and the archaeal/eukaryotic SepSecS enzyme. Together, these data suggest that both metabolic routes and all three tRNA(Cys) species in M. mazei play important roles in the cellular physiology of the organism.     

Acyl-coenzyme-A synthetase I from Candida lipolytica. Purification, properties and immunochemical studies.

Acyl-coenzyme-A synthetase I from Candida lipolytica has been purified to homogeneity as evidenced by polyacrylamide gel electrophoresis in the presence and absence of dodecylsulfate as well as by Ouchterlony double-diffusion analysis. The purification procedure involves resolution of cellular particles with Triton X-100 and chromatography on phosphocellulose, Blue-Sepharose and Sephadex G-100. The purified enzyme exhibits a specific activity of 20--24 U/mg protein at 25 degree C, which is about 100-fold higher than those of long-chain acyl-CoA synthetases hitherto reported. The molecular weight of the enzyme has been estimated by polyacrylamide gel electrophoresis in the presence of dodecylsulfate to be approximately 84 000. The enzyme is specific for fatty acids with 14--18 carbon atoms regardless of the degree of unsaturation. Studies with the use of specific antibody to acyl-CoA synthetase I have indicated that this enzyme is immunochemically distinguishable from acyl-CoA synthetase II.     

Molecular characterization of a thermostable cyanophycin synthetase from the thermophilic cyanobacterium Synechococcus sp. strain MA19 and in vitro synthesis of cyanophycin and related polyamides.

The thermophilic cyanobacterium Synechococcus sp. strain MA19 contained the structural genes for cyanophycin synthetase (cphA) and cyanophycinase (cphB), which were identified, cloned, and sequenced in this study. The translation products of cphA and cphB exhibited high levels of similarity to corresponding proteins of other cyanobacteria, such as Anabaena variabilis and Synechocystis sp. Recombinant cells of Escherichia coli harboring cphA colinear with lacPO accumulated cyanophycin that accounted for up to 25% (wt/wt) of the dry cell matter in the presence of isopropyl-beta-D-thiogalactopyranoside (IPTG). The cyanophycin synthetase was enriched 123-fold to electrophoretic homogeneity from the soluble fraction of the recombinant cells by anion-exchange chromatography, affinity chromatography, and gel filtration chromatography. The purified cyanophycin synthetase maintained the parental thermophilic character and was active even after prolonged incubation at 50 degrees C; in the presence of ectoine the enzyme retained 90% of its activity even after 2 h of incubation. The in vitro activity of the enzyme depended on ATP, primers, and both substrates, L-arginine and L-aspartic acid. In addition to native cyanophycin, the purified enzyme accepted a modified cyanophycin containing less arginine, alpha-arginyl aspartic acid dipeptide, and poly-alpha,beta-DL-aspartic acid as primers and also incorporated beta-hydroxyaspartic acid instead of L-aspartic acid or L-canavanine instead of L-arginine at a significant rate. The lack of specificity of this thermostable enzyme with respect to primers and substrates, the thermal stability of the enzyme, and the finding that the enzyme is suitable for in vitro production of cyanophycin make it an interesting candidate for biotechnological processes.     

Cysteinyl-tRNA formation and prolyl-tRNA synthetase

Aminoacyl-tRNA (AA-tRNA) formation is a key step in protein biosynthesis. This reaction is catalyzed with remarkable accuracy by the AA-tRNA synthetases, a family of 20 evolutionarily conserved enzymes. The lack of cysteinyl-tRNA (Cys-tRNA) synthetase in some archaea gave rise to the discovery of the archaeal prolyl-tRNA (Pro-tRNA) synthetase, an enzyme capable of synthesizing Pro-tRNA and Cys-tRNA. Here we review our current knowledge of this fascinating process.