Crystal structure of threonine synthase from Arabidopsis thaliana

Threonine synthase (TS) is a PLP-dependent enzyme that catalyzes the last reaction in the synthesis of threonine from aspartate. In plants, the methionine pathway shares the same substrate, O-phospho-L-homoserine (OPH), and TS is activated by S-adenosyl-methionine (SAM), a downstream product of methionine synthesis. This positive allosteric effect triggered by the product of another pathway is specific to plants. The crystal structure of Arabidopsis thaliana apo threonine synthase was solved at 2.25 A resolution from triclinic crystals using MAD data from the selenomethionated protein. The structure reveals a four-domain dimer with a two-stranded beta-sheet arm protruding from one monomer onto the other. This domain swap could form a lever through which the allosteric effect is transmitted. The N-terminal domain (domain 1) has a unique fold and is partially disordered, whereas the structural core (domains 2 and 3) shares the functional domain of PLP enzymes of the same family. It also has similarities with SAM-dependent methyltransferases. Structure comparisons allowed us to propose potential sites for pyridoxal-phosphate and SAM binding on TS; they are close to regions that are disordered in the absence of these molecules.     

Structure and function of threonine synthase from yeast.

Threonine synthase catalyzes the final step of threonine biosynthesis, the pyridoxal 5'-phosphate (PLP)-dependent conversion of O-phosphohomoserine into threonine and inorganic phosphate. Threonine is an essential nutrient for mammals, and its biosynthetic machinery is restricted to bacteria, plants, and fungi; therefore, threonine synthase represents an interesting pharmaceutical target. The crystal structure of threonine synthase from Saccharomyces cerevisiae has been solved at 2.7 A resolution using multiwavelength anomalous diffraction. The structure reveals a monomer as active unit, which is subdivided into three distinct domains: a small N-terminal domain, a PLP-binding domain that covalently anchors the cofactor and a so-called large domain, which contains the main of the protein body. All three domains show the typical open alpha/beta architecture. The cofactor is bound at the interface of all three domains, buried deeply within a wide canyon that penetrates the whole molecule. Based on structural alignments with related enzymes, an enzyme-substrate complex was modeled into the active site of yeast threonine synthase, which revealed essentials for substrate binding and catalysis. Furthermore, the comparison with related enzymes of the beta-family of PLP-dependent enzymes indicated structural determinants of the oligomeric state and thus rationalized for the first time how a PLP enzyme acts in monomeric form.     

Osmotin is a homolog of mammalian adiponectin and controls apoptosis in yeast through a homolog of mammalian adiponectin receptor

The antifungal activity of the PR-5 family of plant defense proteins has been suspected to involve specific plasma membrane component(s) of the fungal target. Osmotin is a tobacco PR-5 family protein that induces apoptosis in the yeast Saccharomyces cerevisiae. We show here that the protein encoded by ORE20/PHO36 (YOL002c), a seven transmembrane domain receptor-like polypeptide that regulates lipid and phosphate metabolism, is an osmotin binding plasma membrane protein that is required for full sensitivity to osmotin. PHO36 functions upstream of RAS2 in the osmotin-induced apoptotic pathway. The mammalian homolog of PHO36 is a receptor for the hormone adiponectin and regulates cellular lipid and sugar metabolism. Osmotin and adiponectin, the corresponding "receptor" binding proteins, do not share sequence similarity. However, the beta barrel domain of both proteins can be overlapped, and osmotin, like adiponectin, activates AMP kinase in C2C12 myocytes via adiponectin receptors.     

Drosophila acid DNase is a homolog of mammalian DNase II

Mammalian DNase II enzymes and the Caenorhabditis elegans homolog NUC-1 have recently been shown to be critically important during engulfment-mediated clearance of DNA. In this report, we describe the cloning and characterization of the gene encoding Drosophila DNase II. Database queries using the C. elegans NUC-1 protein sequence identified a highly homologous open reading frame in Drosophila (CG7780) that could encode a similar enzyme. Analysis of crude protein extracts revealed that wild-type Drosophila contain a potent acid endonuclease activity with cleavage preferences similar to DNase II/NUC1, while the same activity was markedly reduced in an acid DNase hypomorphic mutant line. Furthermore, the pattern of cleavage products generated from an end-labeled substrate by hypomorphic-line extracts was significantly altered in comparison to the pattern generated by wild-type extracts. Sequence analysis of CG7780 DNA and mRNA revealed that the hypomorphic line contains a missense mutation within the coding region of this gene. Additionally, Northern analysis demonstrated that CG7780 expression is normal in the mutant line, which in combination with the lowered/altered enzymatic activity and sequencing data suggested a defect in the CG7780 protein. To conclusively determine if CG7780 encoded the Drosophila equivalent of DNase II/NUC-1, transgenic lines expressing wild-type CG7780 in the mutant background were generated and subsequently shown to complement the mutant phenotype. Our results, therefore, provide compelling evidence that the predicted gene CG7780 encodes Drosophila DNase II (dDNase II), an enzyme related in sequence and activity to mammalian DNase II. Interestingly, overexpression of CG7780 both ubiquitously and in specific tissues failed to elicit any discernable phenotype.     

Evolution of biosynthetic pathways: a common ancestor for threonine synthase, threonine dehydratase and D-serine dehydratase.

The Bacillus subtilis genes encoding threonine synthase (thrC) and homoserine kinase (thrB) have been cloned via complementation of Escherichia coli thr mutants. Determination of their nucleotide sequences indicates that the thrC stop codon overlaps the thrB start codon; this genetic organization suggests that the two genes belong to the same operon, as in E. coli. However, the gene order is thrC-thrB in B. subtilis whereas it is thrB-thrC in the thr operon of E. coli. This inversion of the thrC and thrB genes between E. coli and B. subtilis is indicative of a possible independent construction of the thr operon in these two organisms. In other respects, comparison of the predicted amino acid sequences of the B. subtilis and E. coli threonine synthases with that of Saccharomyces cerevisiae threonine dehydratase and that of E. coli D-serine dehydratase revealed extensive homologies between these pyridoxal phosphate-dependent enzymes. This sequence homology, which correlates with similarities in the catalytic mechanisms of these enzymes, indicates that these proteins, catalyzing different reactions in different metabolic pathways, may have evolved from a common ancestor.     

A platypus' eye view of the mammalian genome

The genome of monotremes, like the animals themselves, is unique and strange. The importance of monotremes to genomics depends on their position as the earliest offshoot of the mammalian lineage. Although there has been controversy in the literature over the phylogenetic position of monotremes, this traditional interpretation is now confirmed by recent sequence comparisons. Characterizing the monotreme genome will therefore be important for studying the evolution and organization of the mammalian genome, and the proposal to sequence the platypus genome has been received enthusiastically by the genomics community. Recent investigations of X-chromosome inactivation, genomic imprinting and sex chromosome evolution provide good examples of the power of the monotreme genome to inform us about mammalian genome organization and evolution.     

Atm-dependent interactions of a mammalian Chk1 homolog with meiotic chromosomes

Background: Checkpoint pathways prevent cell-cycle progression in the event of DNA lesions. Checkpoints are well defined in mitosis, where lesions can be the result of extrinsic damage, and they are critical in meiosis, where DNA breaks are a programmed step in meiotic recombination. In mitotic yeast cells, the Chk1 protein couples DNA repair to the cell-cycle machinery. The Atm and Atr proteins are mitotic cell-cycle proteins that also associate with chromatin during meiotic prophase I. The genetic and regulatory interaction between Atm and mammalian Chk1 appears to be important for integrating DNA-damage repair with cell-cycle arrest.Results: We have identified structural homologs of yeast Chk1 in human and mouse. Chk1^Hu/Mo has protein kinase activity and is expressed in the testis. Chk1 accumulates in late zygotene and pachytene spermatocytes and is present along synapsed meiotic chromosomes. Chk1 localizes along the unsynapsed axes of X and Y chromosomes in pachytene spermatocytes. The association of Chk1 with meiotic chromosomes and levels of Chk1 protein depend upon a functional Atm gene product, but Chk1 is not dependent upon p53 for meiosis I functions. Mapping of CHK1 to human chromosomes indicates that the gene is located at 11q22–23, a region marked by frequent deletions and loss of heterozygosity in human tumors.Conclusions: The Atm-dependent presence of Chk1 in mouse cells and along meiotic chromosomes, and the late pachynema co-localization of Atr and Chk1 on the unsynapsed axes of the paired X and Y chromosomes, suggest that Chk1 acts as an integrator for Atm and Atr signals and may be involved in monitoring the processing of meiotic recombination. Furthermore, mapping of the CHK1 gene to a region of frequent loss of heterozygosity in human tumors at 11q22–23 indicates that the CHK1 gene is a candidate tumor suppressor gene.     

Crystal structure of brefeldin A esterase, a bacterial homolog of the mammalian hormone-sensitive lipase

Brefeldin A esterase (BFAE), a detoxifying enzyme isolated from Bacillus subtilis, hydrolyzes and inactivates BFA, a potent fungal inhibitor of intracellular vesicle-dependent secretory transport and poliovirus RNA replication. We have solved the crystal structure of BFAE and we discovered that the previously reported amino acid sequence was in serious error due to frame shifts in the cDNA sequence. The correct sequence, inferred from the experimentally phased electron density map, revealed that BFAE is a homolog of the mammalian hormone sensitive lipase (HSL). It is a canonical alpha/beta hydrolase with two insertions forming the substrate binding pocket. The enzyme contains a lipase-like catalytic triad, Ser 202, Asp 308 and His 338, consistent with mutational studies that implicate the homologous Ser 424, Asp 693 and His 723 in the catalytic triad in human HSL.     

A maize homolog of mammalian CENPC is a constitutive component of the inner kinetochore.

Genes for three maize homologs (CenpcA, CenpcB, and CenpcC) of the conserved kinetochore assembly protein known as centromere protein C (CENPC) have been identified. The C-terminal portion of maize CENPC shares similarity with mammalian CENPC and its yeast homolog Mif2p over a 23-amino acid region known as region I. Immunolocalization experiments combined with three-dimensional light microscopy demonstrated that CENPC is a component of the kinetochore throughout interphase, mitosis, and meiosis. It is shown that sister kinetochore separation occurs in two discrete phases during meiosis. A partial separation of sister kinetochores occurs in prometaphase I, and a complete separation occurs in prometaphase II. CENPC is absent on structures known as neocentromeres that, in maize, demonstrate poleward movement but lack other important features of centromeres/kinetochores. CENPC and a previously identified centromeric DNA sequence interact closely but do not strictly colocalize on meiotic chromosomes. These and other data indicate that CENPC occupies an inner domain of the maize kinetochore.     

Preparation, crystallization and preliminary X-ray analysis of threonine synthase from Streptococcus mutans

The thrC gene of Streptococcus mutans encodes threonine synthase, which is a potential target for drug design. To study the structure and function of the enzyme, the thrC gene was amplified from Streptococcus mutans genomic DNA and cloned into the expression vector pET28alpha. The protein was expressed in Escherichia coli in soluble form and purified to homogeneity. Crystals suitable for X-ray diffraction were obtained by hanging-drop vapor diffusion method. The crystal diffracted to 2.5 A and belonged to space group P3(1) or P3(2), with unit cell parameters a=b=60.39 A, c=118.62 A.     

Characterization of recombinant Arabidopsis thaliana threonine synthase.

Threonine synthase (TS) catalyses the last step in the biosynthesis of threonine, the pyridoxal 5'-phosphate dependent conversion of L-homoserine phosphate (HSerP) into L-threonine and inorganic phosphate. Recombinant Arabidopsis thaliana TS (aTS) was characterized to compare a higher plant TS with its counterparts from Escherichia coli and yeast. This comparison revealed several unique properties of aTS: (a) aTS is a regulatory enzyme whose activity was increased up to 85-fold by S-adenosyl-L-methionine (SAM) and specifically inhibited by AMP; (b) HSerP analogues shown previously to be potent inhibitors of E. coli TS failed to inhibit aTS; and (c) aTS was a dimer, while the E. coli and yeast enzymes are monomers. The N-terminal region of aTS is essential for its regulatory properties and protects against inhibition by HSerP analogues, as an aTS devoid of 77 N-terminal residues was neither activated by SAM nor inhibited by AMP, but was inhibited by HSerP analogues. The C-terminal region of aTS seems to be involved in dimer formation, as the N-terminally truncated aTS was also found to be a dimer. These conclusions are supported by a multiple amino-acid sequence alignment, which revealed the existence of two TS subfamilies. aTS was classified as a member of subfamily 1 and its N-terminus is at least 35 residues longer than those of any nonplant TS. Monomeric E. coli and yeast TS are members of subfamily 2, characterized by C-termini extending about 50 residues over those of subfamily 1 members. As a first step towards a better understanding of the properties of aTS, the enzyme was crystallized by the sitting drop vapour diffusion method. The crystals diffracted to beyond 0.28 nm resolution and belonged to the space group P222 (unit cell parameters: a = 6.16 nm, b = 10.54 nm, c = 14.63 nm, alpha = beta = gamma = 90 degrees).     

Cloning and characterization of a mammalian pseudouridine synthase

This report describes the cloning and characterization of a pseudouridine (psi) synthase from mouse that we have named mouse pseudouridine synthase 1 (mpus1p). The cDNA is approximately 1.5 kb and when used as a probe on a Northern blot of mouse RNA from tissues and cultured cells, several bands were detected. The open reading frame is 393 amino acids and has 35% identity over its length with yeast psi synthase 1 (pus1p). The recombinant protein was expressed in Escherichia coli and the purified protein converted specific uridines to psi in a number of tRNA substrates. The positions modified in stoichiometric amounts in vitro were 27/28 in the anticodon stem and also positions 34 and 36 in the anticodon of an intron containing tRNA. A human cDNA was also cloned and the smaller open reading frame (348 amino acids) was 92% identical over its length with mpus1p but is shorter by 45 amino acids at the amino terminus. The expressed recombinant human protein has no activity on any of the tRNA substrates, most probably the result of the truncated open reading frame.     

Identification of a mammalian mitochondrial homolog of ribosomal protein S7

Bovine mitochondrial small subunit ribosomal proteins were separated by two-dimensional electrophoresis. The region containing the most basic protein(s) was excised and the protein(s) present subjected to in-gel digestion with trypsin. Electrospray tandem mass spectrometry was used to provide sequence information on some of the peptide products. Searches of the human EST database using the sequence of the longest peptide analyzed indicated that this peptide was from the mammalian mitochondrial homolog of prokaryotic ribosomal protein S7 (MRP S7(human)). MRP S7(human) is a 28-kDa protein with a pI of 10. Significant homology to bacterial S7 is observed especially in the C-terminal half of the protein. Surprisingly, MRP S7(human) shows less homology to the corresponding mitochondrial proteins from plants and fungi than to bacterial S7.     

Identification of an Arabidopsis thaliana mutant accumulating threonine resulting from mutation in a new dihydrodipicolinate synthase gene

A novel Arabidopsis DHDPS gene named DHDPS2 was found through identification of a mutant by promoter trapping. The mutation promotes a reduction of growth resulting from combination of a defect in lysine biosynthesis and accumulation of a toxic level of threonine or derived products. The mutant also modifies the amino acid composition issuing from the pyruvate and aspartate pathways, affecting mainly the root compartment. These data are in accordance with the expression of DHDPS2 in the root apex as visualized by expression of the GUS reporter gene. This suggests that a large proportion of the amino acids derived from pyruvate and aspartate are synthesized in this organ.     

Characterization of mammalian eIF2A and identification of the yeast homolog

To begin the physical characterization of eukaryotic initiation factor (eIF) 2A, a translation initiation factor that binds Met-tRNA(i), tryptic peptides from rabbit reticulocyte eIF2A were analyzed to obtain amino acid sequence information. Sequences for 8 peptides were matched to three different expressed sequence tag clones. The sequence predicted for eIF2A is 585 amino acids. Matching of the cDNA sequence to the human genome revealed that the eIF2A mRNA is made up of 15 or 16 exons, and the gene is contained on chromosome 3. A homolog in Saccharomyces cerevisiae was identified, YGR054W, which is a non-essential gene. Hemagglutinin-tagged yeast eIF2A localizes on both 40 S and 80 S ribosomes. A knockout of both eIF2A and eIF5B yielded a "synthetically sick" yeast strain with a severe slow growth phenotype. The phenotype of this double mutant and the biochemical localization suggest that eIF2A participates in translation initiation. eIF2A does not appear to participate in re-initiation as the DeltaeIF2A strain shows the same level of GCN4 induction with amino acid starvation as seen in wild type yeast. The lack of any apparent phenotype in the DeltaeIF2A strain suggests that eIF2A functions in a minor pathway, perhaps internal initiation or in the translation of a small number of specific mRNAs.     

Evolution of mammalian genome organization inferred from comparative gene mapping

Comparative genome analyses, including chromosome painting in over 40 diverse mammalian species, ordered gene maps from several representatives of different mammalian and vertebrate orders, and large-scale sequencing of the human and mouse genomes are beginning to provide insight into the rates and patterns of chromosomal evolution on a whole-genome scale, as well as into the forces that have sculpted the genomes of extant mammalian species.     

Vasa homolog genes in mammalian germ cell development

Many vasa homologue genes to Drosophila vasa have been isolated in various animal species. They provide specific molecular probes to analyze the establishment and the differentiation of germ cell lineage. In mammals, the expression of VASA protein becomes detectable in PGCs at the late migrating stage. Interestingly, during spermatogenesis the intracellular localization of VASA protein is closely associated with the chromatoid body.