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Query: UNIPROT:P06889 (Mol)
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Recent experimental advances have shown that enzymes are flexible molecules, and point to a direct link between dynamics and catalysis. Movements span a wide time range, from nano- to milli-seconds. In this paper we introduce two aspects of enzyme flexibility that are treated with two appropriate techniques. First, transition path sampling is used to obtain an unbiased picture of the transition state ensemble in chorismate mutase, as well as its local flexibility and the energy flow during the chemical step. Second, we consider the binding and release of substrates in L-rhamnulose-1-phosphate aldolase. We have calculated the normal modes of the enzyme with the elastic network model. The lowest frequency modes generate active site deformations that change the coordination number of the catalytic zinc ion. The coordination lability of zinc allows the binding and release of substrates. Substitution of zinc by magnesium blocks the exchange of ligands.
J Mol Model 2008 Aug
PMID:A dynamic view of enzyme catalysis. 1832 11

Escherichia coli NikR is a homotetrameric Ni(2+)- and DNA-binding protein that functions as a transcriptional repressor of the NikABCDE nickel permease. The protein is composed of two distinct domains. The N-terminal 50 amino acids of each chain forms part of the dimeric ribbon-helix-helix (RHH) domains, a well-studied DNA-binding fold. The 83-residue C-terminal nickel-binding domain forms an ACT (aspartokinase, chorismate mutase, and TyrA) fold and contains the tetrameric interface. In this study, we have utilized an equilibrium molecular dynamics simulation in order to explore the conformational dynamics of the NikR tetramer and determine important residue interactions within and between the RHH and ACT domains to gain insight into the effects of Ni(2+) on DNA-binding activity. The molecular simulation data were analyzed using two different correlation measures based on fluctuations in atomic position and noncovalent contacts together with a clustering algorithm to define groups of residues with similar correlation patterns for both types of correlation measure. Based on these analyses, we have defined a series of residue interrelationships that describe an allosteric communication pathway between the Ni(2+)- and DNA-binding sites, which are separated by 40 A. Several of the residues identified by our analyses have been previously shown experimentally to be important for NikR function. An additional subset of the identified residues structurally connects the experimentally implicated residues and may help coordinate the allosteric communication between the ACT and RHH domains.
J Mol Biol 2008 May 16
PMID:Molecular dynamics simulation of the Escherichia coli NikR protein: equilibrium conformational fluctuations reveal interdomain allosteric communication pathways. 1843 69

An engineered monomeric chorismate mutase (mMjCM) has been found to combine high catalytic activity with the characteristics of a molten globule. To gain insight into the dramatic structural changes that accompany binding of a transition-state analog, we examined mMjCM by isothermal calorimetry and compared it with its dimeric parent protein, MjCM (CM from Methanococcus jannaschii), a thermostable and conventionally folded enzyme. As expected for a ligand-induced ordering process, there is a large entropic penalty for binding to the monomer relative to the dimer (-TDeltaDeltaS=5.1+/-0.5 kcal/mol, at 20 degrees C). However, this unfavorable entropy term is largely offset by enthalpic gains (DeltaDeltaH=-3.5+/-0.4 kcal/mol), presumably arising from tightening of non-covalent interactions throughout the monomeric complex. Stopped-flow kinetic measurements further reveal that the catalytic molten globule binds and releases ligands significantly faster than its natural counterpart, demonstrating that partial structural disorder can speed up molecular recognition. These results illustrate how structural plasticity may strongly perturb the thermodynamics and kinetics of transition-state recognition while negligibly affecting catalytic efficiency.
J Mol Biol 2008 Oct 17
PMID:Kinetics and thermodynamics of ligand binding to a molten globular enzyme and its native counterpart. 1868 Jul 48

Chorismate mutase (CM) secreted from the stylet of plant-parasitic nematodes plays an important role in plant parasitism. We isolated and characterized a new nematode CM gene (Gr-cm-1) from the potato cyst nematode, Globodera rostochiensis. The Gr-cm-1 gene was found to exist in the nematode genome as a single-copy gene that has two different alleles, Gr-cm-1A and Gr-cm-1B, both of which could give rise to two different mRNA transcripts of Gr-cm-1 and Gr-cm-1-IRII. In situ mRNA hybridization showed that the Gr-cm-1 gene was exclusively expressed within the subventral oesophageal gland cells of the nematode. Gr-cm-1 was demonstrated to encode a functional CM (GR-CM-1) potentially having a dimeric structure as the secreted bacterial *AroQ CMs. Gr-cm-1-IRII, generated by retention of intron 2 of the Gr-cm-1 pre-mRNA through alternative splicing (AS), would encode a truncated protein (GR-CM-1t) lacking the CM domain with no CM activity. The quantitative real-time reverse transcription-PCR assay revealed that splicing of the Gr-cm-1 gene was developmentally regulated; Gr-cm-1 was up-regulated whereas Gr-cm-1-IRII was down-regulated in early nematode parasitic stages compared to the preparasitic juvenile stage. Low-temperature SDS-PAGE analysis revealed that GR-CM-1 could form homodimers when expressed in Escherichia coli and the dimerization domain was retained in the truncated GR-CM-1t protein. The specific interaction between the two proteins was demonstrated in yeast. Our data suggested that the novel splice variant might function as a dominant negative isoform through heterodimerization with the full-length GR-CM-1 protein and that AS may represent an important mechanism for regulating CM activity during nematode parasitism.
Mol Biochem Parasitol 2008 Nov
PMID:Alternative splicing: a novel mechanism of regulation identified in the chorismate mutase gene of the potato cyst nematode Globodera rostochiensis. 1878 75

In this article, we present the cloning of Hscm1, a gene for chorismate mutase (CM) from the beet cyst nematode Heterodera schachtii. CM is a key branch-point enzyme of the shikimate pathway, and secondary metabolites that arise from this pathway control developmental programmes and defence responses of the plant. By manipulating the plant's endogenous shikimate pathway, the nematode can influence the plant physiology for its own benefit. Hscm1 is a member of the CM gene family and is expressed during the pre-parasitic and parasitic stages of the nematode's life cycle. In situ mRNA hybridization reveals an expression pattern specific to the subventral and dorsal pharyngeal glands. The predicted protein has a signal peptide for secretion in addition to two domains. The N-terminal domain of the mature protein, which is only found in cyst nematodes, contains six conserved cysteine residues, which may reflect the importance of disulphide bond formation for protein stabilization. The C-terminal domain holds a single catalytic site and has similarity to secreted CMs of pathogenic bacteria, classifying HsCM1 as an AroQ(gamma) enzyme. The presumed catalytic residues are discussed in detail, and genetic complementation experiments indicate that the C-terminal domain is essential for enzyme activity. Finally, we show how the modular design of the protein is mirrored in the genomic sequence by the intron/exon organization, suggesting exon shuffling as a mechanism for the evolutionary assembly of this protein.
Mol Plant Pathol 2009 Mar
PMID:Structural and functional investigation of a secreted chorismate mutase from the plant-parasitic nematode Heterodera schachtii in the context of related enzymes from diverse origins. 1923 68

Plant processes resulting from primary or secondary metabolism have been hypothesized to contribute to defense against microbial attack. Barley chorismate synthase (HvCS), anthranilate synthase alpha subunit 2 (HvASa2), and chorismate mutase 1 (HvCM1) occupy pivotal branch points downstream of the shikimate pathway leading to the synthesis of aromatic amino acids. Here, we provide functional evidence that these genes contribute to penetration resistance to Blumeria graminis f. sp. hordei, the causal agent of powdery mildew disease. Single-cell transient-induced gene silencing of HvCS and HvCM1 in mildew resistance locus a (Mla) compromised cells resulted in increased susceptibility. Correspondingly, overexpression of HvCS, HvASa2, and HvCM1 in lines carrying mildew resistance locus o (Mlo), a negative regulator of penetration resistance, significantly decreased susceptibility. Barley stripe mosaic virus-induced gene silencing of HvCS, HvASa2, and HvCM1 significantly increased B. graminis f. sp. hordei penetration into epidermal cells, followed by formation of haustoria and secondary hyphae. However, sporulation of B. graminis f. sp. hordei was not detected on the silenced host plants up to 3 weeks after inoculation. Taken together, these results establish a previously unrecognized role for the influence of HvCS, HvASa2, and HvCM1 on penetration resistance and on the rate of B. graminis f. sp. hordei development in Mla-mediated, barley-powdery mildew interactions.
Mol Plant Microbe Interact 2009 Mar
PMID:Functional contribution of chorismate synthase, anthranilate synthase, and chorismate mutase to penetration resistance in barley-powdery mildew interactions. 1924 25

Aspartate kinases (AKs) can be divided in two subhomology divisions, AKalpha and AKbeta, depending on the presence of an extra sequence of about 60 amino acids, which is found only in the N-terminus of all AKalpha's. To date, the structures of AKalpha failed to provide a role for this additional N-terminal sequence. In this study, the structure of the AKbeta from the Cyanobacteria Synechocystis reveals that this supplementary sequence is linked to the dimerization mode of AKs. Its absence in AKbeta leads to the dimerization by the catalytic domain instead of involving the ACT domains [Pfam 01842; small regulatory domains initially found in AK, chorismate mutase and TyrA (prephenate dehydrogenase)] as observed in AKalpha. Thus, the structural analysis of the Synechocystis AKbeta revealed a dimer with a novel architecture. The four ACT domains of each monomer interact together and do not make any contact with those of the second monomer. The enzyme is inhibited synergistically by threonine and lysine with the binding of threonine first. The interaction between ACT1 and ACT4 or between ACT2 and ACT3 generates a threonine binding site and a lysine binding site at each interface, making a total of eight regulatory sites per dimer and allowing a fine-tuning of the AK activity by the end products, threonine and lysine.
J Mol Biol 2010 Jun 04
PMID:A new mode of dimerization of allosteric enzymes with ACT domains revealed by the crystal structure of the aspartate kinase from Cyanobacteria. 2039 76

SUMMARY Parasitism genes encoding secretory proteins expressed in the oesophageal glands of phytoparasitic nematodes play critical roles in nematode invasion of host plants, establishment of feeding sites and suppression of host defences. Two chorismate mutase (CM) genes potentially having a role in one or more of these processes were identified from a Meloidogyne incognita oesophageal gland-cell subtractive cDNA library. These M. incognita enzymes (designated as MI-CM-1 and MI-CM-2) with amino-terminal signal peptides, were significantly similar to chorismate mutases in M. javanica and bacteria. The complementation of an Escherichia coli CM-deficient mutant by the expression of Mi-cm-1 or Mi-cm-2 confirmed their CM activity. In-situ mRNA hybridization showed that the transcripts of Mi-cm-1 and Mi-cm-2 accumulated specifically in the two subventral oesophageal gland cells of M. incognita. RT-PCR analysis confirmed that their transcript abundances were high in the early parasitic juvenile stages, and low (Mi-cm-1) or undetectable (Mi-cm-2) in later parasitic stages of the nematode. Southern blot analysis revealed that these CM genes were members of a small multigene family in Meloidogyne species. The widespread presence of CMs in the specialized sedentary endoparasitic nematode species suggests that this multifunctional enzyme may be a key factor in modulating plant parasitism.
Mol Plant Pathol 2005 Jan 01
PMID:Two chorismate mutase genes from the root-knot nematode Meloidogyne incognita. 2056 35

SUMMARY Some plant endoparasitic nematodes are biotrophic and induce remarkable changes in their hosts in order to ensure a continuous supply of food. Proteins secreted from oesophageal gland cells have been implicated in this pathogenic process. A potentially secreted chorismate mutase has been isolated from the potato cyst nematode Globodera pallida. The gene encoding this protein is expressed in the subventral oesophageal gland cells of the nematode, and the mRNA derived from this gene is only present in the early parasitic stages. Sequence analysis of this gene shows that, like other genes involved in the host-parasite interaction of plant parasitic nematodes, it is likely to have been acquired by horizontal gene transfer from bacteria. The presence of a signal peptide in the deduced amino acid sequence of the G. pallida chorismate mutase and its expression in the subventral oesophageal gland cells suggest that it is secreted from the nematode, pointing to a role for the protein in the host-parasite interaction. The shikimate pathway, of which chorismate mutase is normally a part, is not found in animals but is present in plants and bacteria. In plants it gives rise to a variety of compounds which are important in amino acid synthesis and defence signalling pathways, as well as auxins, which have been implicated in the early development of nematode feeding sites. The potential roles of a nematode chorismate mutase are discussed.
Mol Plant Pathol 2003 Jan 01
PMID:Characterization of a chorismate mutase from the potato cyst nematode Globodera pallida. 2056 61

To study interactions between plants and plant-parasitic nematodes, several omics studies have nowadays become extremely useful. Since most data available so far is derived from sedentary nematodes, we decided to improve the knowledge on migratory nematodes by studying the transcriptome of the nematode Pratylenchus coffeae through generating expressed sequence tags (ESTs) on a 454 sequencing platform. In this manuscript we present the generation, assembly and annotation of over 325,000 reads from P. coffeae. After assembling these reads, 56,325 contigs and singletons with an average length of 353bp were selected for further analyses. Homology searches revealed that 25% of these sequences had significant matches to the Swiss-prot/trEMBL database and 29% had significant matches in nematode ESTs. Over 10,000 sequences were successfully annotated, corresponding to over 6000 unique Gene Ontology identifiers and 5000 KEGG orthologues. Different approaches led to the identification of different sequences putatively involved in the parasitism process. Several plant cell wall modifying enzymes were identified, including an arabinogalactan galactosidase, so far identified in cyst nematodes only. Additionally, some new putative cell wall modifying enzymes are present belonging to GHF5 and GHF16, although further functional studies are needed to determine the true role of these proteins. Furthermore, a homologue to a chorismate mutase was found, suggesting that this parasitism gene has a wider occurrence in plant-parasitic nematodes than previously assumed. Finally, the dataset was searched for orthologues against the Meloidogyne genomes and genes involved in the RNAi pathway. In conclusion, the generated transcriptome data of P. coffeae will be very useful in the future for several projects: (1) evolutionary studies of specific gene families, such as the plant cell wall modifying enzymes, (2) the identification and functional analysis of candidate effector genes, (3) the development of new control strategies, e.g. by finding new targets for RNAi and (4) the annotation of the upcoming genome sequence.
Mol Biochem Parasitol
PMID:Analysis of the transcriptome of the root lesion nematode Pratylenchus coffeae generated by 454 sequencing technology. 2151 48


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