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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Blaster cassettes are of significant value in functional genomics, as they represent tools with which to inactivate duplicated or homologous genes in an individual organism. We have constructed a novel blaster module which allows repeated gene deletion in the filamentous fungus Aspergillus nidulans. Because bacterial resistance marker cassettes are employed as flanking repeats in direct orientation, the blaster cassette is suited for recombinogenic engineering by ET cloning in Escherichia coli. The functionality of the blaster module was demonstrated by deleting the chorismate mutase-encoding gene aroC of A. nidulans, followed by marker rescue based on mitotic recombination. The resulting aroCDelta strains are auxotrophic for phenylalanine but not tyrosine, and display a limited capacity for fruit body formation and ascosporogenesis, which depends on the phenylalanine/tyrosine supply. The data support the notion that amino acid status has a strong impact on cleistothecium development in A. nidulans.
Mol Genet Genomics 2003 Feb
PMID:Deletion of Aspergillus nidulans aroC using a novel blaster module that combines ET cloning and marker rescue. 1258 42

Parasitism genes from phytoparasitic nematodes are thought to be essential for nematode invasion of the host plant, to help the nematode establish feeding sites, and to aid nematodes in the suppression of host plant defenses. One gene that may play several roles in nematode parasitism is chorismate mutase (CM). This secreted enzyme is produced in the nematode's esophageal glands and appears to function within the plant cell to manipulate the plant's shikimate pathway, which controls plant cell growth, development, structure, and pathogen defense. Using degenerate polymerase chain reaction primers, we amplified and cloned a chorismate mutase (Hg-cm-1) from Heterodera glycines, the soybean cyst nematode (SCN), and showed it had CM activity. RNA in situ hybridization of Hg-cm-1 cDNA to SCN sections confirms that it is specifically expressed in the nematodes' esophageal glands. DNA gel blots of genomic DNA isolated from SCN inbred lines that have differing virulence on SCN resistant soybean show Hg-cm-1 is a member of a polymorphic gene family. Some Hg-cm family members predominate in SCN inbred lines that are virulent on certain SCN resistant soybean cultivars. The same polymorphisms and correlation with virulence are seen in the Hg-cm-1 expressed in the SCN second-stage juveniles. Based on the enzymatic activity of Hg-cm-1 and the observation that different forms of the mutase are expressed in virulent nematodes, we hypothesize that the Hg-cm-1 is a virulence gene, some forms of which allow SCN to parasitize certain resistant soybean plants.
Mol Plant Microbe Interact 2003 May
PMID:A chorismate mutase from the soybean cyst nematode Heterodera glycines shows polymorphisms that correlate with virulence. 1274 15

The yeast transcription factor Gcn4p contains two stretches of amino acid residues, NLS1 and NLS2, which are independently able to relocate the cytoplasmic protein chorismate mutase into the nucleus. Only NLS2 is conserved among fungi. A truncated version of CPCA (the counterpart of Gcn4p in Aspergillus nidulans), which lacks the conserved NLS, accumulates in the cytoplasm instead of the nucleus. Nuclear uptake mediated by the NLS1 of Gcn4p is impaired by defects in genes for several different karyopherins, whereas NLS2-dependent nuclear import specifically requires the alpha-importin Srp1p and the beta-importin Kap95p. Yeast strains that are defective in either of these two karyopherins are unable to respond to amino acid starvation. We have thus identified Gcn4p as a substrate for the Srp1p/Kap95p transport complex. Our data suggest that NLS2 is the essential and specific nuclear transport signal; NLS1 may play only an unspecific or accessory role.
Mol Genet Genomics 2004 Apr
PMID:Nuclear import of yeast Gcn4p requires karyopherins Srp1p and Kap95p. 1464

RmpM is a putative peptidoglycan binding protein from Neisseria meningitidis that has been shown to interact with integral outer membrane proteins such as porins and TonB-dependent transporters. Here we report the 1.9 A crystal structure of the C-terminal domain of RmpM. The 150-residue domain adopts a betaalphabetaalphabetabeta fold, as first identified in Bacillus subtilis chorismate mutase. The C-terminal RmpM domain is homologous to the periplasmic, C-terminal domain of Escherichia coli OmpA; these domains are thought to be responsible for non-covalent interactions with peptidoglycan. From the structure of the OmpA-like domain of RmpM, we suggest a putative peptidoglycan binding site and identify residues that may be essential for binding. Both the crystal structure and solution experiments indicate that RmpM may exist as a dimer. This would promote more efficient peptidoglycan binding, by allowing RmpM to interact simultaneously with two glycan chains through its C-terminal, OmpA-like binding domain, while its (structurally uncharacterized) N-terminal domain could stabilize oligomers of porins and TonB-dependent transporters in the outer membrane.
Mol Microbiol 2004 Feb
PMID:Structure of the OmpA-like domain of RmpM from Neisseria meningitidis. 1476 78

A genetic linkage map of the soybean cyst nematode (SCN) Heterodera glycines was constructed using a population of F2 individuals obtained from matings between two highly inbred SCN lines, TN16 and TN20. The AFLP fingerprinting technique was used to genotype 63 F2 progeny with two restriction enzyme combinations (EcoRI/MseI and PstI/TaqI) and 38 primer combinations. The same F2 population was also genotyped for Hg-cm-1 (H. glycines chorismate mutase-1), a putative virulence gene, using real-time quantitative PCR. Some of the markers were found to be distributed non-randomly. Even so, of the 230 markers analyzed, 131 could be mapped onto ten linkage groups at a minimum LOD of 3.0, for a total map distance of 539 cM. The Hg-cm-1 locus mapped to linkage group III together with 16 other markers. The size of the H. glycines genome was estimated to be in the range of 630-743 cM, indicating that the current map represents 73-86% of the genome, with a marker density of one per 4.5 cM, and a physical/genetic distance ratio of between 124 kb/cM and 147 kb/cM. This genetic map will be of great assistance in mapping H. glycines markers to genes of interest, such as nematode virulence genes and genes that control aspects of nematode parasitism.
Mol Genet Genomics 2005 May
PMID:A genetic linkage map of the soybean cyst nematode Heterodera glycines. 1590 93

The soybean cyst nematode Heterodera glycines is the most destructive pathogen of soybean in the Unites States. Diversity in the parasitic ability of the nematode allows it to reproduce on nematode-resistant soybean. H. glycines chorismate mutase-1 (Hg-CM-1) is a nematode enzyme with the potential to suppress host plant defense compounds; therefore, it has the potential to enhance the parasitic ability of nematodes expressing the gene. Hg-cm-1 is a member of a gene family where two alleles, Hg-cm-1A and Hg-cm-1B, have been identified. Analysis of the Hg-cm-1 gene copy number revealed that there are multiple copies of Hg-cm-1 alleles in the H. glycines genome. H. glycines inbred lines were crossed to ultimately generate three F2 populations of second-stage juveniles (J2s) segregating for Hg-cm-1A and Hg-cm-1B. Segregation of Hg-cm-1A and 1B approximated a 1:2:1 ratio, which suggested that Hg-cm-1 is organized in a cluster of genes that segregate roughly as a single locus. The F2 H. glycines J2 populations were used to infect nematode-resistant (Hartwig, PI88788, and PI90763) and susceptible (Lee 74) soybean plants. H. glycines grown on Hartwig, Lee 74, and PI90763 showed allelic frequencies similar to Hg-cm-1A/B, but nematodes grown on PI88788 contained predominately Hg-cm-1A allele as a result of a statistically significant drop of Hg-cm-1B in the population. This result suggests that specific Hg-cm-1 alleles, or a closely linked gene, may aid H. glycines in adapting to particular soybean hosts.
Mol Plant Microbe Interact 2005 Jun
PMID:Selection of Heterodera glycines chorismate mutase-1 alleles on nematode-resistant soybean. 1598 29

The effector-regulated allosteric mechanism of yeast chorismate mutase (YCM) was studied by normal mode analysis and targeted molecular dynamics. The normal mode analysis shows that the conformational change between YCM in the R state and in the T state can be represented by a relatively small number of low-frequency modes. This suggests that the transition is coded in the structure and is likely to have a low energetic barrier. Quantitative comparisons (i.e. frequencies) between the low-frequency modes of YCM with and without effectors (modeled structures) reveal that the binding of Trp increases the global flexibility, whereas Tyr decreases global flexibility. The targeted molecular dynamics simulation of substrate analog release from the YCM active site suggests that a series of residues are critical for orienting and "recruiting" the substrate. The simulation led to the switching of a series of substrate-release-coupled salt-bridge partners in the ligand-binding domain; similar changes occur in the transition between YCM R-state and T-state crystal structures. Thus, the normal mode analysis and targeted molecular dynamics results provide evidence that the effectors regulate YCM activity by influencing the global flexibility. The change in flexibility is coupled to the binding of substrate to the T state and release of the product from the R state, respectively.
J Mol Biol 2006 Feb 10
PMID:The allosteric mechanism of yeast chorismate mutase: a dynamic analysis. 1633 51

The presence of exported chorismate mutases produced by certain organisms such as Mycobacterium tuberculosis has been shown to correlate with their pathogenicity. As such, these proteins comprise a new group of promising selective drug targets. Here, we report the high-resolution crystal structure of the secreted dimeric chorismate mutase from M. tuberculosis (*MtCM; encoded by Rv1885c), which represents the first 3D-structure of a member of this chorismate mutase family, termed the AroQ(gamma) subclass. Structures are presented both for the unliganded enzyme and for a complex with a transition state analog. The protomer fold resembles the structurally characterized (dimeric) Escherichia coli chorismate mutase domain, but exhibits a new topology, with helix H4 of *MtCM carrying the catalytic site residue missing in the shortened helix H1. Furthermore, the structure of each *MtCM protomer is significantly more compact and only harbors one active site pocket, which is formed entirely by one polypeptide chain. Apart from the structural model, we present evidence as to how the substrate may enter the active site.
J Mol Biol 2006 Apr 14
PMID:1.6 A crystal structure of the secreted chorismate mutase from Mycobacterium tuberculosis: novel fold topology revealed. 1649 27

Iminodisuccinate (IDS) epimerase catalyzes the epimerisation of R,R-, S,S- and R,S- iminodisuccinate, one step in the biodegradation of the chelating agent iminodisuccinate by Agrobacterium tumefaciens BY6. The enzyme is a member of the MmgE/PrpD protein family, a diverse and little characterized class of proteins of prokaryotic and eukaryotic origin. IDS epimerase does not show significant overall amino acid sequence similarity to any other protein of known three-dimensional structure. The crystal structure of this novel epimerase has been determined by multi-wavelength diffraction to 1.5 A resolution using selenomethionine-substituted enzyme. In the crystal, the enzyme forms a homo-dimer, and the subunit consists of two domains. The larger domain, not consecutive in sequence and comprising residues Met1-Lys266 and Leu400-Pro446, forms a novel all alpha-helical fold with a central six-helical bundle. The second, smaller domain folds into an alpha+beta domain, related in topology to chorismate mutase by a circular permutation. IDS epimerase is thus not related in three-dimensional structure to other known epimerases. The fold of the IDS epimerase is representative for the whole MmgE/PrpD family. The putative active site is located at the interface between the two domains of the subunit, and is characterized by a positively charged surface, consistent with the binding of a highly negatively charged substrate such as iminodisuccinate. Docking experiments suggest a two-base mechanism for the epimerisation reaction.
J Mol Biol 2006 Sep 22
PMID:Three-dimensional structure of iminodisuccinate epimerase defines the fold of the MmgE/PrpD protein family. 1693 91

Although protein dynamics has been recognized as a potentially important contributor to enzyme catalysis, structural disorder is generally considered to reduce catalytic efficiency. This widely held assumption has recently been challenged by the finding that an engineered chorismate mutase combines high catalytic activity with the properties of a molten globule, a loosely packed and highly dynamic conformational ensemble. Taking advantage of the ordering observed upon ligand binding, we have now used NMR spectroscopy to characterize this enzyme in complex with a transition-state analog. The complex adopts a helix-bundle structure, as designed, but retains unprecedented flexibility on the millisecond timescale across its entire length. Moreover, pre-steady-state kinetics data show that binding occurs by an induced-fit mechanism on the same timescale as the enzymatic reaction, linking global conformational plasticity with efficient catalysis.
Nat Struct Mol Biol 2007 Dec
PMID:Structure and dynamics of a molten globular enzyme. 1799 4


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