Structure of translation initiation factor 5A from Pyrobaculum aerophilum at 1.75 A resolution

BACKGROUND: Translation initiation factor 5A (IF-5A) is reported to be involved in the first step of peptide bond formation in translation, to be involved in cell-cycle regulation and to be a cofactor for the Rev and Rex transactivator proteins of human immunodeficiency virus-1 and T-cell leukemia virus I, respectively. IF-5A contains an unusual amino acid, hypusine (N-epsilon-(4-aminobutyl-2-hydroxy)lysine), that is required for its function. The first step in the post-translational modification of lysine to hypusine is catalyzed by the enzyme deoxyhypusine synthase, the structure of which has been published recently. RESULTS: IF-5A from the archebacterium Pyrobaculum aerophilum has been heterologously expressed in Escherichia coli with selenomethionine substitution. The crystal structure of IF-5A has been determined by multiwavelength anomalous diffraction and refined to 1.75 A. Unmodified P. aerophilum IF-5A is found to be a beta structure with two domains and three separate hydrophobic cores. CONCLUSIONS: The lysine (Lys42) that is post-translationally modified by deoxyhypusine synthase is found at one end of the IF-5A molecule in an turn between beta strands beta4 and beta5; this lysine residue is freely solvent accessible. The C-terminal domain is found to be homologous to the cold-shock protein CspA of E. coli, which has a well characterized RNA-binding fold, suggesting that IF-5A is involved in RNA binding.     

Barriers to heterologous expression of a selenoprotein gene in bacteria

The specificity parameters counteracting the heterologous expression in Escherichia coli of the Desulfomicrobium baculatum gene (hydV) coding for the large subunit of the periplasmic hydrogenase which is a selenoprotein have been studied. hydV'-'lacZ fusions were constructed, and it was shown that they do not direct the incorporation of selenocysteine in E. coli. Rather, the UGA codon is efficiently suppressed by some other aminoacyl-tRNA in an E. coli strain possessing a ribosomal ambiguity mutation. The suppression is decreased by the strA1 allele, indicating that the hydV selenocysteine UGA codon has the properties of a "normal" and suppressible nonsense codon. The SelB protein from D. baculatum was purified; in gel shift experiments, D. baculatum SelB displayed a lower affinity for the E. coli fdhF selenoprotein mRNA than E. coli SelB did and vice versa. Coexpression of the hydV'-'lacZ fusion and of the selB and tRNA(Sec) genes from D. baculatum, however, did not lead to selenocysteine insertion into the protein, although the formation of the quaternary complex between SelB, selenocysteyl-tRNA(Sec), and the hydV mRNA recognition sequence took place. The results demonstrate (i) that the selenocysteine-specific UGA codon is readily suppressed under conditions where the homologous SelB protein is absent and (ii) that apart from the specificity of the SelB-mRNA interaction, a structural compatibility of the quaternary complex with the ribosome is required.     

Selenocysteine synthesis in mammalia: an identity switch from tRNA(Ser) to tRNA(Sec)

The mechanism of selenocysteine insertion into proteins is distinct from all other amino acids in all lines of descent in that it needs specific protein cofactors and a structurally unique tRNA(Sec). It is first aminoacylated with serine and further recognized among all other serylated serine isoacceptors by a selenocysteine synthase and is converted to selenocysteyl-tRNA(Sec). We present here the complete set of identity elements for selenylation of mammalian seryl-tRNA(Sec) and show that the transplantation of these elements into normal serine tRNA allows its selenylation. Four particular structural motifs differentiate eukaryotic tRNA(Sec) from normal tRNA(Ser): the orientation of the extra arm, the short 4 bp T psi C-stem, the extra long 9 bp acceptor-stem and the elongated 6 bp dihydrouridine-stem. Only the last two are essential and only together sufficient for selenocysteine synthesis, whereby the additional base-pairs of the acceptor-stem may be replaced by non-paired nucleotides. Each exchange of the first three structural motifs mentioned above between tRNA(Ser) and tRNA(Sec) resulted in a significant loss of serylation, indicating that the overall composition of particular structure elements is necessary to maintain normal functions of tRNA(Sec). Since we find that all seryl-tRNAs which are selenylated are also substrates for serine phosphorylation we propose that phosphoseryl-tRNA(Sec) is a storage form of seryl-tRNA(Sec).     

Effects of muscle relaxants on catecholamine release from the bovine adrenal medulla

Studies on the structure and substrate specificity of purified rat kidney nuclear (RKN) lysozyme are reported. The carboxyl and amino terminal residues of RKN-lysozyme were found to be leucine and alanine respectively. The amino acid composition indicated similarities and differences as compared with that of hen egg white (HEW) lysozyme. There were alterations in the nine amino acid residues, Lys, His, Arg, Asp, Glu, Pro, 1/2 Cys, Tyr and Trp. The other nine residues were present in identical proportions to those of HEW-lysozyme. The decrease in the arginine and aspartic acid residues was found to be compensated by the increase in the number of lysine, histidine and glutamic acid residues. The overall ratio of the acidic to basic amino acids has thus been conserved in the mammalian enzyme. In addition, RKN-lysozyme contained decreased numbers of Trp, Tyr and 1/2 Cys, and increased numbers of proline residues as found in HEW-lysozyme. RKN-lysozyme did not cross react with heterologous antibodies produced against HEW-lysozyme, and vice versa. RKN-lysozyme showed distinct specificity towards the lysis of M. luteus. Against this substrate, it was three times more efficient than HEW-lysozyme. It also cleaved E. coli B, but its efficiency was half as much as with M. luteus. However, it cleaved P. septica and B. subtilis at a rate similar to HEW-lysozyme under identical conditions.     

Cardiolipin synthase from Escherichia coli

Escherichia coli cardiolipin synthase catalyzes reversible phosphatidyl group transfer from one phosphatidylglycerol molecule to another to form cardiolipin (CL) and glycerol. The enzyme is specified by the cls gene, located at min 28.02 of the E. coli genetic map. Cells with mutations in cls have longer doubling times, tend to lose viability in the stationary phase, are more resistant to 3,4-dihydroxybutyl-1-phosphonate, and have an altered sensitivity to novobiocin. Although cls null mutants appear to lack CL synthase activity, they are still able to form trace quantities of CL. The enzyme appears to be regulated at both the genetic and enzymatic levels. CL synthase's molecular mass is 45-46 kDa, or about 8 kDa less than the polypeptide predicted by the gene sequence, suggesting that posttranslational processing occurs. CL synthase can use various polyols such as mannitol and arabitol to convert CL to the corresponding phosphatidylglycerol analog. When the amino acid sequences of four bacterial CL synthases are compared, three highly conserved regions are apparent. One of these regions contains a conserved pentapeptide sequence, RN(Q)HRK, and another has a conserved HXK sequence. These two sequences may be part of the active site. E. coli CL synthase has been studied by using a mixed micelle assay. The enzyme is inhibited by CL, the product of the reaction, and by phosphatidate. Phosphatidylethanolamine partially offsets inhibition caused by CL but not by phosphatidate. CDP-diacylglycerol does not appear to affect the activity of the purified enzyme but does stimulate the activity associated with crude membrane preparations.     

Localization and in vitro mutagenesis of the active site in the Saccharomyces cerevisiae mRNA capping enzyme

The yeast mRNA capping enzyme is composed of 52 (alpha) and 80 kDa (beta) polypeptides, which are responsible for its mRNA guanylyltransferase and RNA 5'-triphosphatase activities, respectively. We isolated the gene encoding the alpha subunit (CEG1) and showed that CEG1 is essential for yeast cell growth [Shibagaki et al., (1992) J. Biol. Chem. 267, 9521-9528]. In this study, CEG1 was expressed in Escherichia coli and the alpha subunit protein was purified to near homogeneity. A [32P]GMP-bound tryptic peptide derived from the recombinant enzyme-[32P]GMP covalent reaction intermediate was converted to a [32P]phosphoryl-peptide through periodate oxidation followed by beta-elimination. Hydrolysis of the [32P]phosphoryl-peptide with alkali resulted in [32P]N epsilon-phospholysine as the only phosphoamino acid, indicating that GMP in the enzyme-GMP complex is bound to a lysine residue via a phosphoamide linkage. Microsequencing of the [32P]GMP-peptide showed that the GMP binding site was located in the region between amino acids 60 and 75, which contained an internal trypsin-resistant lysine at position 70. CEG1 was subjected to site-directed mutagenesis and the mutant proteins were expressed in E. coli. Substitution of His or Ile for Lys70 entirely abolished the enzyme-GMP formation activity, and this mutation was lethal to yeast in vivo, supporting the notion that the active site in the alpha subunit is located at Lys70. Replacement of Lys70 with Arg reduced the ability to form the enzyme-GMP complex; however, yeast cells bearing this allele were not viable. A series of mutations, including 8 amino acid replacements and 3 insertions, near the active site (Lys70-Thr-Asp-Gly motif) were also introduced and the mutant polypeptides were examined for catalytic activity in vitro as well as yeast cell viability in vivo. There was a good correlation between the in vitro and in vivo functions of the mutant proteins, except when Asp72 was replaced with Glu, which allowed formation of the enzyme-GMP complex but failed to support cell growth. The results with Lys70 to Arg and Asp72 to Glu substitutions indicated that guanylyltransfer to RNA and/or additional roles besides cap formation per se are impaired in these mutant proteins.     

Imaging of en bloc renal transplants: normal and abnormal postoperative findings

Escherichia coli possesses a hexameric citrate synthase that exhibits allosteric kinetics and regulatory sensitivity, and for which the gene (gltA) has previously been cloned and sequenced. A citrate-synthase-deficient strain of E. coli (K114) has been mutated to generate a revertant (K114r4) that produces a dimeric citrate synthase with altered kinetic and regulatory properties. On cloning and sequencing the gltA gene from both K114 and K114r4, a single mutation was found that caused the replacement of Asp362 with Asn. Asp362 has been previously shown to be a catalytically essential residue in E. coli citrate synthase, and we demonstrate that the hexameric enzyme produced on expression of the gltA gene from K114 and K114r4 is inactive. The dimeric citrate synthase from K114r4 has been purified and shown to be immunologically distinct from the wild-type hexameric enzyme. Determination of its N-terminal amino acid sequence demonstrates that the mutant citrate synthase is encoded by a gene distinct from the E. coli gltA gene. The N-terminal sequence is compared with those of other eukaryotic, eubacterial and archaebacterial citrate synthases.     

Thermostable farnesyl diphosphate synthase of Bacillus stearothermophilus: molecular cloning, sequence determination, overproduction, and purification

The structural gene for thermostable farnesyl diphosphate synthase from Bacillus stearothermophilus was cloned, sequenced, and overexpressed in Escherichia coli cells. A 1,260-nucleotide sequence of the cloned fragment was determined. This sequence specifies an open reading frame of 891 nucleotides for farnesyl diphosphate synthase. The deduced amino acid sequence shows a 42% similarity with that of E. coli FPP synthase [Fujisaki et al. (1990) J. Biochem. 108, 995-1000]. Comparison with prenyltransferases from a wide range of organisms, from bacteria to human, revealed the presence of seven highly conserved regions. In contrast to thermolabile prenyltransferases, which have four to six cysteine residues, the thermostable farnesyl diphosphate synthase carries only two cysteine residues. This enzyme is also unique in that some of the amino acids that are fully conserved in equivalents from other sources are replaced by functionally different amino acids. Construction of an overproducing strain provided a sufficient supply of this enzyme and it was purified to homogeneity. The purified recombinant enzyme is immunochemically identical with the native B. stearothermophilus enzyme, and it is not inactivated even after treatment at 65 degrees C for 70 min.     

Heparin-binding properties of selenium-containing thioredoxin reductase from HeLa cells and human lung adenocarcinoma cells

Mammalian selenocysteine-containing thioredoxin reductase (TR) isolated from HeLa cells and from human lung adenocarcinoma cells was separated into two major enzyme species by heparin-agarose affinity chromatography. The low-affinity enzyme forms that were not retained on heparin agarose showed strong crossreactivity in immunoblot assays with anti-rat liver TR polyclonal antibodies, whereas the high-affinity enzyme forms that were retained by the heparin column were not detected. Both low and high heparin-affinity enzyme forms contained FAD, were indistinguishable on SDS/PAGE analysis, and exhibited similar catalytic activities in the NADPH-dependent DTNB [5,5'-dithiobis(2-nitrobenzoate)] assay. The C-terminal amino acid sequences of 75Se-labeled tryptic peptides from lung adenocarcinoma low- and high heparin-affinity enzyme forms were identical to the predicted C-terminal sequence of human placental TR. These two determined peptide sequences were -Ser-Gly-Ala-Ser-Ile-Leu-Gln-Ala-Gly-Cys-Secys-(Gly). Occurrence of the Se-carboxymethyl derivative of radioactive selenocysteine in the position corresponding to TGA in the gene confirmed that UGA is translated as selenocysteine. The presence of cysteine followed by a reactive selenocysteine residue in this C-terminal region of the protein may explain some of the unusual properties of the mammalian TRs.     

Rat and calf thioredoxin reductase are homologous to glutathione reductase with a carboxyl-terminal elongation containing a conserved catalytically active penultimate selenocysteine residue

We have determined the sequence of 23 peptides from bovine thioredoxin reductase covering 364 amino acid residues. The result was used to identify a rat cDNA clone (2.19 kilobase pairs), which contained an open reading frame of 1496 base pairs encoding a protein with 498 residues. The bovine and rat thioredoxin reductase sequences revealed a close homology to glutathione reductase including the conserved active site sequence (Cys-Val-Asn-Val-Gly-Cys). This also confirmed the identity of a previously published putative human thioredoxin reductase cDNA clone. Moreover, one peptide of the bovine enzyme contained a selenocysteine residue in the motif Gly-Cys-SeCys-Gly (where SeCys represents selenocysteine). This motif was conserved at the carboxyl terminus of the rat and human enzymes, provided that TGA in the sequence GGC TGC TGA GGT TAA, being identical in both cDNA clones, is translated as selenocysteine and that TAA confers termination of translation. The 3'-untranslated region of both cDNA clones contained a selenocysteine insertion sequence that may form potential stem loop structures typical of eukaryotic selenocysteine insertion sequence elements required for the decoding of UGA as selenocysteine. Carboxypeptidase Y treatment of bovine thioredoxin reductase after reduction by NADPH released selenocysteine from the enzyme with a concomitant loss of enzyme activity measured as reduction of thioredoxin or 5,5'-dithiobis(2-nitrobenzoic acid). This showed that the carboxyl-terminal motif was essential for the catalytic activity of the enzyme.     

Mutational analysis of yeast mRNA capping enzyme

RNA guanylyltransferase (capping enzyme) catalyzes the transfer of GMP from GTP to the 5'-diphosphate end of mRNA. The capping reaction proceeds via an enzyme-guanylate intermediate in which GMP is linked covalently to a lysine residue of the enzyme. In the capping enzyme of Saccharomyces cerevisiae, GMP is attached to a 52-kDa polypeptide, identified as the product of the essential CEG1 gene. The amino acid sequence of the CEG1 protein includes a motif, Lys70-Thr-Asp-Gly, that is conserved at the active site of vaccinia virus RNA guanylyltransferase and which is similar to the KXDG sequence found at the active sites of RNA and DNA ligases. To evaluate the role of this motif in the function of the yeast enzyme, we have expressed the CEG1 protein in active form in Escherichia coli. Replacement of Lys70 or Gly73 with alanine abrogated enzyme-guanylate formation in vitro; in contrast, alanine substitutions at Thr71 or Asp72 merely reduced activity relative to wild-type enzyme. The K70A and G73A mutations were lethal to yeast, whereas yeast carrying the T71A and D72A alleles of CEG1 were viable. These results implicate Lys70 as the active site of yeast guanylyltransferase and provide evidence that cap formation per se is an essential function in eukaryotic cells.     

Characterization of chitin synthase 2 of Saccharomyces cerevisiae. Implication of two highly conserved domains as possible catalytic sites

Chitin synthase 2 of Saccharomyces cerevisiae was characterized by means of site-directed mutagenesis and subsequent expression of the mutant enzymes in yeast cells. Chitin synthase 2 shares a region whose sequence is highly conserved in all chitin synthases. Substitutions of conserved amino acids in this region with alanine (alanine scanning) identified two domains in which any conserved amino acid could not be replaced by alanine to retain enzyme activity. These two domains contained unique sequences, Glu561-Asp562-Arg563 and Gln601-Arg602-Arg603-Arg604-Trp605, that were conserved in all types of chitin synthases. Glu561 or arginine at 563, 602, and 603 could be substituted by glutamic acid and lysine, respectively, without significant loss of enzyme activity. However, even conservative substitutions of Asp562 with glutamic acid, Gln601 with asparagine, Arg604 with lysine, or Trp605 with tyrosine drastically decreased the activity, but did not affect apparent Km values for the substrate significantly. In addition to these amino acids, Asp441 was also found in all chitin synthase. The mutant harboring a glutamic acid substitution for Asp441 severely lost activity, but it showed a similar apparent Km value for the substrate. Amounts of the mutant enzymes in total membranes were more or less the same as found in the wild type. Furthermore, Asp441, Asp562, Gln601, Arg604, and Trp605 are completely conserved in other proteins possessing N-acetylglucosaminyltransferase activity such as NodC proteins of Rhizobium bacterias. These results suggest that Asp441, Asp562, Gln601, Arg604, and Trp605 are located in the active pocket and that they function as the catalytic residues of the enzyme.     

Cloning, sequencing, and heterologous expression of rat methionine synthase cDNA

Methionine synthase catalyzes cobalamin-dependent methyl transfer reaction from 5-methyltetrahydrofolate to homocysteine, forming methionine. Rat methonine synthase cDNA was cloned and analyzed by RT-PCR, 3'- and 5'-RACE techniques. The cDNA consists of a 0.3-kb upstream untranslated region, a 3.8-kb coding region, and a 0.4-kb downstream untranslated region. The open reading frame encoded a polypeptide of 1,253 amino acid residues with a calculated molecular weight of 139,162. This molecular weight was in good agreement with the observed one (143,000) of the purified rat liver enzyme. The deduced amino acid sequence was 53, 92, and 64% identical with those of the Escherichia coli, human, and presumptive Caenorhabditis elegans enzymes, respectively. All the fingerprint sequences, forming parts of the cobalamin- and S-adenosylmethionine-binding sites, were completely conserved in the rat methionine synthase. A high-level expression of catalytically active enzyme in insect cells was done by infection with a baculovirus containing the rat methionine synthase cDNA.     

Characterization of a second lysine decarboxylase isolated from Escherichia coli

We report here on the existence of a new gene for lysine decarboxylase in Escherichia coli K-12. The hybridization experiments with a cadA probe at low stringency showed that the homologous region of cadA was located in lambda Kohara phage clone 6F5 at 4.7 min on the E. coli chromosome. We cloned the 5.0-kb HindIII fragment of this phage clone and sequenced the homologous region of cadA. This region contained a 2,139-nucleotide open reading frame encoding a 713-amino-acid protein with a calculated molecular weight of 80,589. Overexpression of the protein and determination of its N-terminal amino acid sequence defined the translational start site of this gene. The deduced amino acid sequence showed 69.4% identity to that of lysine decarboxylase encoded by cadA at 93.7 min on the E. coli chromosome. In addition, the level of lysine decarboxylase activity increased in strains carrying multiple copies of the gene. Therefore, the gene encoding this lysine decarboxylase was designated Idc. Analysis of the lysine decarboxylase activity of strains containing cadA, ldc, or cadA ldc mutations indicated that ldc was weakly expressed under various conditions but is a functional gene in E. coli.     

Subunit II (b') and not subunit I (b) of photosynthetic ATP synthases is equivalent to subunit b of the ATP synthases from nonphotosynthetic eubacteria. Evidence for a new assignment of b-type F0 subunits

Subunit I of chloroplast ATP synthase is reviewed until now to be equivalent to subunit b of Escherichia coli ATP synthase, whereas subunit II is suggested to be an additional subunit in photosynthetic ATP synthases lacking a counterpart in E. coli. After publication of some sequences of subunits II a revision of this assignment is necessary. Based on the analysis of 51 amino acid sequences of b-type subunits concerning similarities in primary structure, isoelectric point and a discovered discontinuous structural feature, our data provide evidence that chloroplast subunit II (subunit b' of photosynthetic eubacteria) and not chloroplast subunit I (subunit b of photosynthetic eubacteria) is the equivalent of subunit b of nonphotosynthetic eubacteria, and therefore does have a counterpart in e.g. E. coli. In consequence, structural features essential for function should be looked for on subunit II (b').     

Modulation of the catalytic activity of brain succinic semialdehyde reductase by reaction with pyridoxal 5'-phosphate

An NADPH-dependent succinic semialdehyde reductase from bovine brain was inactivated by pyridoxal 5'-phosphate. Spectral evidence is presented to indicate that the inactivation proceeds through formation of a Schiff's base with amino groups of the enzyme. After sodium borohydride reduction of the inactivated enzyme, it was observed that 1 mol phosphopyridoxyl residue was incorporated/mol enzyme monomer. The coenzyme, NADPH, protected the enzyme against inactivation by pyridoxal 5'-phosphate. After tryptic digestion of the enzyme modified with pyridoxal 5'-phosphate in the presence and absence of NADPH followed by [1H]NaBH4 reduction, a radioactive peptide absorbing at 310 nm was isolated by reverse-phase HPLC. The amino acid sequence of the peptide identified a portion of the pyridoxal-5'-phosphate-binding site as the region containing the sequence I-L-E-N-I-Q-V-F-X-K, where X indicates that the phenylthiohydantoin amino acid could not be assigned. The missing residue, however, can be designated as a phosphopyridoxyl lysine as interpreted from the result of amino acid composition of the peptide. It is suggested that the catalytic function of succinic semialdehyde reductase is modulated by binding of pyridoxal 5'-phosphate to a specific lysyl residue at or near the coenzyme-binding site of the protein.     

Deletion of the carboxyl-terminal region of 1-aminocyclopropane-1-carboxylic acid synthase, a key protein in the biosynthesis of ethylene, results in catalytically hyperactive, monomeric enzyme

1-Aminocyclopropane-1-carboxylic acid (ACC) synthase is a key enzyme regulating biosynthesis of the plant hormone ethylene. The expression of an enzymatically active, wound-inducible tomato (Lycopersicon esculentum L. cv Pik-Red) ACC synthase (485 amino acids long) in a heterologous Escherichia coli system allowed us to study the importance of hypervariable COOH terminus in enzymatic activity and protein conformation. We constructed several deletion mutants of the gene, expressed these in E. coli, purified the protein products to apparent homogeneity, and analyzed both conformation and enzyme kinetic parameters of the wild-type and truncated ACC syntheses. Deletion of the COOH terminus through Arg429 results in complete inactivation of the enzyme. Deletion of 46-52 amino acids from the COOH terminus results in an enzyme that has nine times higher affinity for the substrate S-adenosylmethionine than the wild-type enzyme. The highly efficient, truncated ACC synthase was found to be a monomer of 52 +/- 1.8 kDa as determined by gel filtration, whereas the wild-type ACC synthase, analyzed under similar conditions, is a dimer. These results demonstrate that the non-conserved COOH terminus of ACC synthase affects its enzymatic function as well as dimerization.     

Cloning and sequencing of the gene for the Thiobacillus ferrooxidans ATCC33020 glutamate synthase (GOGAT) small subunit and complementation of an Escherichia coli gltD mutant

A recombinant plasmid which contains the gltD gene coding for the glutamate synthase (GOGAT) small subunit was isolated from a Thiobacillus ferrooxidans ATCC33020 gene bank by complementation of an Escherichia coli gltD mutant. The sequence of gltD was determined. The deduced amino acid sequence shows strong similarity to the two other prokaryote gltD sequences available, namely those of E. coli and A. brasilense (53% and 45% identity, respectively). A cosmid containing the gltBD region was isolated from a T. ferrooxidans cosmid gene bank, but was unable to complement an E. coli gltB mutant.