Archaeal shikimate kinase, a new member of the GHMP-kinase family

Shikimate kinase (EC 2.7.1.71) is a committed enzyme in the seven-step biosynthesis of chorismate, a major precursor of aromatic amino acids and many other aromatic compounds. Genes for all enzymes of the chorismate pathway except shikimate kinase are found in archaeal genomes by sequence homology to their bacterial counterparts. In this study, a conserved archaeal gene (gi1500322 in Methanococcus jannaschii) was identified as the best candidate for the missing shikimate kinase gene by the analysis of chromosomal clustering of chorismate biosynthetic genes. The encoded hypothetical protein, with no sequence similarity to bacterial and eukaryotic shikimate kinases, is distantly related to homoserine kinases (EC 2.7.1.39) of the GHMP-kinase superfamily. The latter functionality in M. jannaschii is assigned to another gene (gi591748), in agreement with sequence similarity and chromosomal clustering analysis. Both archaeal proteins, overexpressed in Escherichia coli and purified to homogeneity, displayed activity of the predicted type, with steady-state kinetic parameters similar to those of the corresponding bacterial kinases: K(m,shikimate) = 414 +/- 33 microM, K(m,ATP) = 48 +/- 4 microM, and k(cat) = 57 +/- 2 s(-1) for the predicted shikimate kinase and K(m,homoserine) = 188 +/- 37 microM, K(m,ATP) = 101 +/- 7 microM, and k(cat) = 28 +/- 1 s(-1) for the homoserine kinase. No overlapping activity could be detected between shikimate kinase and homoserine kinase, both revealing a >1,000-fold preference for their own specific substrates. The case of archaeal shikimate kinase illustrates the efficacy of techniques based on reconstruction of metabolism from genomic data and analysis of gene clustering on chromosomes in finding missing genes.     

Molecular cloning of thehom—thrC—thrB cluster fromBacillus sp. ULM1: Expression of thethrC gene inEscherichia coli and corynebacteria,and evolutionary relationships of the threonine genes

A 6.5 kb DNA fragment containing the gene (thrC) encoding threonine synthase, the last enzyme of the threonine biosynthetic pathway, has been cloned from the DNA ofBacillus sp. ULM1 by complementation ofEscherichia coli andBrevibacterium lactofermentum thrC auxotrophs. Complementation studies showed that thethrB gene (encoding homoserine kinase) is found downstream from thethrC gene, and analysis of nucleotide sequences indicated that thehom gene (encoding homoserine dehydrogenase) is located upstream of thethrC gene. The organization of this cluster of genes is similar to theBacillus subtilis threonine operon (hom—thrC—thrB). An 1.9 kbBclI, fragment from theBacillus sp. ULM1 DNA insert that complementedthrC mutations both inE. coli and in corynebacteria was sequenced, and an ORF encoding a protein of 351 amino acids was found corresponding to a protein of 37462 Da. ThethrC gene showed a low G+C content (39.4%) and the encoded threonine synthase is very similar to theB. subtilis enzyme. Expression of the 1.9 kbBclI DNA fragment inE. coli minicells resulted in the formation of a 37 kDa protein. The upstream region of this gene shows promoter activity inE. coli but not in corynebacteria. A peptide sequence, including a lysine that is known to bind the pyridoxal phosphate cofactor, is conserved in all threonine synthase sequences and also in the threonine and serine dehydratase genes. Amino acid comparison of nine threonine synthases revealed evolutionary relationships between different groups of bacteria. Dedicated to Dr. J. Spížek on the occasion of his 60th birthday     

The Crithidia fasciculata CRK gene encodes a novel cdc2-related protein containing large inserts between highly conserved domains.

A gene (CRK) encoding a cdc2-related protein has been identified in the trypanosomatid Crithidia fasciculata. CRK has a high degree of sequence identity with the human cdc2 gene and contains the sixteen amino acid PSTAIR motif, characteristic of p34cdc2 protein-serine/threonine kinases, with four amino acid substitutions in the motif. In addition, two inserts of more than sixty amino acids have been found between conserved domains of this putative protein-serine/threonine kinase. CRK is a single copy gene and is expressed on a 3.8 kb mRNA. Anti-CRK antibodies detect a 53kDa protein in extracts of C.fasciculata in agreement with the size predicted from the nucleotide sequence of the cloned gene. These antibodies also recognize proteins of 48 and 60 kDa in extracts of the trypanosomatid Leishmania tarentolae. Antibodies against the human PSTAIR peptide detect the p34cdc2 protein in human nuclear extracts but fail to detect a 34 kDa protein in C.fasciculata extracts. These results suggest that novel higher molecular weight forms of the cdc2 protein family may be involved in cell cycle control in trypanosomes.     

Saccharomyces cerevisiae homoserine kinase is homologous to prokaryotic homoserine kinases

The Saccharomyces cerevisiae gene (THR1) encoding homoserine kinase (HK; EC 2.7.1.39) was cloned by complementation in yeast. Disruption of the THR1 gene results in threonine auxotrophy in yeast. Comparison of the amino acid sequences of yeast and bacterial HKs reveals substantial similarity.     

Identification of the gene encoding homoserine kinase from Arabidopsis thaliana and characterization of the recombinant enzyme derived from the gene

Homoserine kinase (EC 2.7.1.39) catalyzes the formation of O-phospho-l-homoserine, a branch point intermediate in the pathways for Met and Thr in plants. A genomic open reading frame located on the top arm of chromosome II and a corresponding cDNA have been identified from Arabidopsis thaliana that encode homoserine kinase. The HSK gene is composed of an 1113-bp continuous open reading frame that could produce a 38-kDa protein. The gene product has homology with homoserine kinase from bacteria and fungi. It contains a conserved motif, known as GHMP, found in a group of ATP-dependent metabolite kinases and thought to comprise the ATP binding site. The amino-terminal 50 amino acids of the HSK protein show features of a transit peptide for localization to plastids. Genomic blot analysis revealed that there is a single locus in A. thaliana to which the HSK cDNA hybridizes. The HSK protein expressed as a His-tagged construct in Escherichia coli shows a specific activity in an l-homoserine-dependent ADP synthesis assay of 3.09 +/- 0.25 micromol min(-1) mg(-1) protein at pH 8.5 and 37 degrees C. The apparent K(m) values are 0.40 mM for l-homoserine and 0.32 mM for Mg-ATP. Other hydroxylated compounds are not used as substrates. The enzyme requires 40 mM K(+) and 3 mM Mg(2+) for activity. It has an unusually high temperature optimum, yet it is very unstable, losing more than 80% of its activity after a single cycle of freeze-thawing. The HSK enzyme shows no significant regulation by amino acids in vitro.     

Nucleotide sequence of a novel kanamycin resistance gene, aphA-7, from Campylobacter jejuni and comparison to other kanamycin phosphotransferase genes

A novel kanamycin phosphotransferase gene, aphA-7, was cloned from a 14-kb plasmid obtained from a strain of Campylobacter jejuni and the nucleotide sequence of the gene was determined. The presumed open reading frame of the aphA-7 structural gene was 753 bp in length and encoded a protein of 251 amino acids with a calculated weight of 29,691 Da. A 29-kDa protein was demonstrated in Escherichia coli maxicells containing the cloned aphA-7 gene. A ribosomal binding site corresponding to 5 of 8 bases of the 3' end of the E. coli 16S rRNA was 8 bp upstream of the start codon. Sequences corresponding to the -35 and -10 regions of the consensus promoter sequences of E. coli were upstream of the presumed initiation codon of the gene. The DNA sequence was most closely related to the aphA-3 gene from Streptococcus faecalis, showing 55.4% sequence similarity. There was 45.6% identity at the amino acid level between the aphA-3 and the aphA-7 proteins. Of the three conserved regions noted previously in phosphotransferase genes, the aphA-7 amino acid sequence was identical to the six conserved amino acids in motif 3, but differed in one of the five conserved amino acids in motif 1 (if gaps are permitted) and 3 of the 10 conserved residues in motif 2. The 32.8% G + C ratio in the open reading frame of the aphA-7 kanamycin resistance gene, which is similar to that of the C. jejuni chromosome, suggests that the aphA-7 may be indigenous to Campylobacters.     

Effect of gene amplification on threonine production by yeast

In this work, we have studied the effect of amplifying different alleles involved in the threonine biosynthesis on the amino acid production by Saccharomyces cerevisiae. The genes used were wild-type HOM3, HOM2, HOM6, THR1, and THR4, and two mutant alleles of HOM3 (namely HOM3-R2 and HOM3-R6), that code for feedback-insensitive aspartate kinases. The results show that only the amplification of the HOM3 alleles leads to threonine and, in some instances, to homoserine overproduction. In terms of the regulation of the pathway, the data indicate that the main control is exerted by inhibition of the aspartate kinase and that, probably, a second and less important regulation takes place at the level of the homoserine kinase, the THR1 gene product. However, amplification of THR1 in two related Hom3-R2 strains does not increase the amount of threonine but, in one of them, it does induce accumulation of more homoserine. This result probably reflects differences between these strains in some undetermined genetic factor/s related with threonine metabolism. In general, the data indicate that the common laboratory yeast strains are genetically rather heterogeneous and, thus, extrapolation of conclusions must be done carefully. (c) 1996 John Wiley & Sons, Inc.     

Pkg2, a Novel Transmembrane Protein Ser/Thr Kinase of Streptomyces granaticolor

A 4.2-kb SphI-BamHI fragment of chromosomal DNA from Streptomyces granaticolor was cloned and shown to encode a protein with significant sequence similarity to the eukaryotic protein serine/threonine kinases. It consists of 701 amino acids and in the N-terminal part contains all conserved catalytic domains of protein kinases. The C-terminal domain of Pkg2 contains seven tandem repeats of 11 or 12 amino acids with similarity to the tryptophan-docking motif known to stabilize a symmetrical three-dimensional structure called a propeller structure. The pkg2 gene was overexpressed in Escherichia coli, and the gene product (Pkg2) has been found to be autophosphorylated at serine and threonine residues. The N- and C-terminal parts of Pkg2 are separated with a hydrophobic stretch of 21 amino acids which translocated a PhoA fusion protein into the periplasm. Thus, Pkg2 is the first transmembrane protein serine/threonine kinase described for streptomycetes. Replacement of the pkg2 gene by the spectinomycin resistance gene resulted in changes in the morphology of aerial hyphae.     

Changes in ascorbic acid content and ascorbate peroxidase activity during the development of Acetabularia mediterranea

We cloned ras-related sequences from goldfish genomic libraries constructed as recombinants using the lambda phage. Restriction enzyme mapping of the clones obtained revealed three kinds of ras-related sequences among approximately 350,000 genomic clones. One of these clones was partially sequenced. Comparison with the nucleotide sequences of mammalian ras genes showed that the determined sequences covered the predicted amino acid coding regions and parts of the intervening regions. The predicted amino acid sequences of the cloned ras-related goldfish gene suggested that the coding region is localized separately in DNA, and that its exon-intron boundaries are exactly the same as those of corresponding mammalian genes. The nucleotide and amino acid sequences of the goldfish ras-related gene may have extensive homologies to mammalian p 21 protein. Among the three mammalian ras proteins, the predicted amino acid sequence of the sequenced ras-related goldfish clone is most closely homologous (96%) to the Kirsten ras protein. Differences in the predicted amino acid sequence were greatest in the sequence predicted from the fourth exon; fewer differences were found in the sequence from the third exon, and only slight or no differences were found in the sequence predicted for the first and second exons. The 12th and 61st amino acids from the N-terminal of the protein, which are thought to be critical positions for GTP binding and catalysis, are both conserved in the goldfish protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cloning and sequencing of a copper-containing,topa quinone-containing monoamine oxidase from human placenta

A 4040-bp cDNA was cloned from a human placenta library by screening with a polymerase chain reaction-amplified fragment. The fragment was generated from the library using primers corresponding to conserved sequences encompassing the topa quinone (TPQ) cofactor sites of the copper-containing proteins, bovine serum amine oxidase (BSAO) and human kidney diamine oxidase (DAO). The cloned cDNA contains a coding sequence from positions 161 to 2449. Between bases 2901 and 2974, in a very long 1591-bp 3′-untranslated region, there is a G/A-rich region in the minus strand, which contains a (AGG)₅ tandem repeat. The human placenta cDNA sequence and its translated amino acid sequence are 84% and 81% identical to the corresponding BSAO sequences, while the identities for the placenta sequences and those for human kidney DAO are 60% and 41%, respectively. The TPQ consensus nucleotide and protein sequences are identical for the placenta enzyme and BSAO, but the corresponding sequences for human kidney DAO are nonidentical. Three His residues that have been identified as Cu(II) ligands in other amine oxidases are conserved in the human placenta amine oxidase protein sequence. It was concluded that the placenta cDNA open-reading frame codes for a copper-containing, TPQ-containing monoamine oxidase. A putative 19-amino acid signal peptide was identified for human placenta amine oxidase. The resulting mature protein would be composed of 744 amino acids, and would have a M_r of 82 525. Comparison of the human placenta amine oxidase with DNA sequences found in GenBank suggests that the gene for this enzyme is located in the q21 region of human chromosome 17, near the BRCA1 gene.     

Regulation of aspartate-derived amino acid biosynthesis in the yeastSaccharomyces cerevisiae

The activity of three enzymes, aspartokinase, homoserine dehydrogenase, and homoserine kinase, has been studied in the industrial strainSaccharomyces cerevisiae IFI256 and in the mutants derived from it that are able to overproduce methionine and/or threonine. Most of the mutants showed alteration of the kinetic properties of the enzymes aspartokinase, which was less inhibited by threonine and increased its affinity for aspartate, and homoserine dehydrogenase and homoserine kinase, which both lost affinity for homoserine. Furthermore, they showed in vitro specific activities for aspartokinase and homoserine kinase that were higher than those of the wild type, resulting in accumulation of aspartate, homoserine, threonine, and/or methionine/S-adenosyl-methionine (Ado-Met). Together with an increase in the specific activity of both aspartokinase and homoserine kinase, there was a considerable and parallel increase in methionine and threonine concentration in the mutants. Those which produced the maximal concentration of these amino acids underwent minimal aspartokinase inhibition by threonine. This supports previous data that identify aspartokinase as the main agent in the regulation of the biosynthetic pathway of these amino acids. The homoserine kinase in the mutants showed inhibition by methionine together with a lack or a reduction of the inhibition by threonine that the wild type undergoes, which finding suggests an important role for this enzyme in methionine and threonine regulation. Finally, homoserine dehydrogenase displayed very similar specific activity in the mutants and the wild type in spite of the changes observed in amino acid concentrations; this points to a minor role for this enzyme in amino acid regulation.     

Vesicular stomatitis virus NS proteins: structural similarity without extensive sequence homology.

The complete nucleotide sequence of the NS mRNA of vesicular stomatitis virus (New Jersey serotype) was established from two cDNA clones spanning the entire coding region of the mRNA. The gene is 856 nucleotides long and can code for a polypeptide of 274 amino acids. Comparison with the nucleotide sequence of the NS gene of the Indiana serotype revealed only 41% sequence homology. The deduced amino acid sequences of the NS proteins were only 32% homologous, with no identical stretches of more than five amino acids. However, at the C-terminal domain there was a conserved region of 21 amino acids with greater than 90% homology. Surprisingly, relative hydropathicity plots also demonstrated the presence of a large number of hydrophilic amino acids sequestered similarly over the N-terminal half of the protein. In addition, the total number of serine and threonine residues, presumptive phosphorylation sites, was similar and included seven serine and three threonine residues located at identical positions. It appears that during divergent evolution of these two vesicular stomatitis virus serotypes from a common ancestor, considerable mutation occurred in the main body of the gene but the overall structure of the protein was retained. The function of the NS protein in relation to the evolution of the two viruses is discussed.     

A novel transmembrane serine/threonine protein kinase gene from a rifamycin SV-producing amycolatopsis mediterranei U32.

Genomic DNA sequencing in the vicinity of methylmalonyl-CoA mutase gene (mutAB) from a rifamycin SV-producing Amycolatopsis mediterranei U32 allowed us to clone, sequence, and identify a gene encoding a novel serine/threonine protein kinase (amk). The sequence contains a complete ORF of 1821 base pairs encoding a predicted protein of 606 amino acids in length. The N-terminal domain of the protein shows significant homology to the catalytic domain of other protein kinases from both prokaryotic and eukaryotic sources. It also contains all the structural features that are highly conserved in active protein kinases, including the Gly-X-Gly-X-X-Gly motif of ATP-binding and the essential amino acids known to be important for the recognition of the correct hydroxyamino acid in serine/threonine protein kinase. This protein kinase gene was expressed in Escherichia coli and was shown to have the ability of autophosphorylation. The autophosphorylated site was found to be the threonine at position 164 by labeled phosphoamino acid analysis and site-directed mutagenesis. The C-terminal half of protein kinase was found to contain strong transmembrane structures by PhoA fusion protein analysis, suggesting that Amk protein kinase is a transmembrane protein. A Southern hybridization experiment showed that this type of protein kinase is distributed ubiquitously and might play significant physiological roles in the various species of streptomycetes. However, overexpression of amk gene in Streptomyces cinnamonensis showed no effect on methylmalonyl-CoA mutase activity, monensin production and the hyphae morphology. Although its biological role is still unknown, Amk protein kinase is the first transmembrane serine/threonine protein kinase described for genus Amycolatopsis.     

Isolation and characterization of a <Emphasis Type="Italic">Paramecium</Emphasis> cDNA clone encoding a putative serine/threonine protein kinase

Mitogen-activated protein (MAP) kinases, a closely related family of protein kinases, are involved in cell cycle regulation and differentiation in yeast and human cells. They have not been documented in ciliates. We used PCR to amplify DNA sequences of a ciliated protozoan—Paramecium caudatum—using primers corresponding to amino acid sequences that are common to MAP kinases. We isolated and sequenced one putative MAP kinase-like serine/threonine kinase cDNA from P. caudatum. This cDNA, called pcstk1 (Paramecium caudatum Serine/Threonine Kinase 1) shared approximately 35% amino acid identity with MAP kinases from yeast. MAP kinases are activated by phosphorylation of specific threonine and tyrosine residues. These two amino acid residues are conserved in the PCSTK1 sequence at positions Thr 159 and Tyr 161. The PSTAIRE motif, which is characteristic of the CDK2 gene family, cannot be found in ORF of PCSTK1. The highest homology score was to human STK9, which contains MAP type kinase domains. Comparisons of expression level have shown that pcstk1 is expressed equally in cells at different stages (sexual and asexual). We discussed the possibility, as in other organisms, that a family of MAP kinase genes exists in P. caudatum.     

The mouse muscle creatine kinase cDNA and deduced amino acid sequences: Comparison to evolutionarily related enzymes

Summary The nucleotide sequence of cloned DNA corresponding to full-length mouse muscle creatine kinase mRNA has been determined. This 1415 base pair DNA sequence and the deduced 381 amino acid sequence of the protein have been compared to creatine kinase sequences from other vertebrate species and to invertebrate guanidino kinase sequences. These comparisons show that the vertebrate muscle creatine kinases constitute a remarkably conserved protein family with a unit evolutionary period of 30. The creatine kinases also retain marked sequence similarity with the more distantly related invertebrate guanidino kinases. A portion of the sequence, presumably part of the ATP binding site, shows similarity to other nucleotide binding proteins with diverse functions. Comparisons of the untranslated regions of the creatine kinase cDNA sequences show that the 5 untranslated regions are more highly conserved than are the 3 untranslated regions; this may point to some regulatory function in the 5 region.