OsCM regulates rice defence system in response to UV light supplemented with drought stress.

Studies on plant responses to combined abiotic stresses are very limited, especially in major crop plants. The current study evaluated the response of chorismate mutase overexpressor (OxCM) rice line to combined UV light and drought stress. The experiments were conducted in pots in a growth chamber, and data were assessed for gene expression, antioxidant and hormone regulation, flavonoid accumulation, phenotypic variation, and amino acid accumulation. Wild-type (WT) rice had reduced the growth and vigour, while transgenic rice maintained growth and vigour under combined UV light and drought stress. ROS and lipid peroxidation analysis revealed that chorismate mutase (OsCM) reduced oxidative stress mediated by ROS scavenging and reduced lipid peroxidation. The combined stresses reduced biosynthesis of total flavonoids, kaempferol and quercetin in WT plants, but increased significantly in plants with OxCM. Phytohormone analysis showed that SA was reduced by 50% in WT and 73% in transgenic plants, while ABA was reduced by 22% in WT plants but increased to 129% in transgenic plants. Expression of chorismate mutase regulates phenylalanine biosynthesis, UV light and drought stress-responsive genes, e.g., phenylalanine ammonia lyase (OsPAL), dehydrin (OsDHN), dehydration-responsive element-binding (OsDREB), ras-related protein 7 (OsRab7), ultraviolet-B resistance 8 (OsUVR8), WRKY transcription factor 89 (OsWRKY89) and tryptophan synthase alpha chain (OsTSA). Moreover, OsCM also increases accumulation of free amino acids (aspartic acid, glutamic acid, leucine, tyrosine, phenylalanine and proline) and sodium (Na), potassium (K), and calcium (Ca) ions in response to the combined stresses. Together, these results suggest that chorismate mutase expression induces physiological, biochemical and molecular changes that enhance rice tolerance to combined UV light and drought stresses.

Evolution of allosteric regulation in chorismate mutases from early plants.

Plants, fungi, and bacteria synthesize the aromatic amino acids: l-phenylalanine, l-tyrosine, and l-tryptophan. Chorismate mutase catalyzes the branch point reaction of phenylalanine and tyrosine biosynthesis to generate prephenate. In Arabidopsis thaliana, there are two plastid-localized chorismate mutases that are allosterically regulated (AtCM1 and AtCM3) and one cytosolic isoform (AtCM2) that is unregulated. Previous analysis of plant chorismate mutases suggested that the enzymes from early plants (i.e. bryophytes/moss, lycophytes, and basal angiosperms) formed a clade distinct from the isoforms found in flowering plants; however, no biochemical information on these enzymes is available. To understand the evolution of allosteric regulation in plant chorismate mutases, we analyzed a basal lineage of plant enzymes homologous to AtCM1 based on sequence similarity. The chorismate mutases from the moss/bryophyte Physcomitrella patens (PpCM1 and PpCM2), the lycophyte Selaginella moellendorffii (SmCM), and the basal angiosperm Amborella trichopoda (AmtCM1 and AmtCM2) were characterized biochemically. Tryptophan was a positive effector for each of the five enzymes examined. Histidine was a weak positive effector for PpCM1 and AmtCM1. Neither tyrosine nor phenylalanine altered the activity of SmCM; however, tyrosine was a negative regulator of the other four enzymes. Phenylalanine down-regulates both moss enzymes and AmtCM2. The 2.0 ŠX-ray crystal structure of PpCM1 in complex with the tryptophan identified the allosteric effector site and reveals structural differences between the R- (more active) and T-state (less active) forms of plant chorismate mutases. Molecular insight into the basal plant chorismate mutases guides our understanding of the evolution of allosteric regulation in these enzymes.

[Cloning and bioinformatics analysis of chorismate mutase gene from Salvia miltiorrhiza].

Chorismate mutase catalyzes the conversion of chorismate to prephenate that is the first committed step in the biosynthesis of the aromatic amino acids phenylalanine and tyrosine. A chorismate mutase gene, designated SmCM1, was isolated from Salvia miltiorrhiza by using RT-PCR. The full length of SmCM1 cDNA consists of 948 nucleotides and has an open reading frame of 765 bp. The deduced amino acid sequence of SmCM1 has 255 amino acid residues which forms a 36.0 kD polypeptide with calculated pI of 6.41 as expected. The putative polypeptide contains a CM_2 super family function domain. Blast W results showed that SmCM1 had 70% of the similarity with Petunia x hybrid CM, 72% of the similarity with Arabidopsis thaliana CM, and 64% of similarity with Populus trichocarpa CM. The transcription level of SmCM1 in root, stem and leaf was analysed by realtime quantitative PCR. The results showed the expression level of the SmCM1 in leaf was highest, and lowest in root. Yeast extract and silver ion joint induction could markedly stimulate the increase of mRNA expression of SmCM1 and its upstream 3-deoxy-7- phosphoheptulonate synthase (DAHPS) and chorismate synthase (CS). It was 7.9, 5.5 and 9.8 times of control on 8 h after induction, respectively.

High-throughput virtual screening of proteins using GRID molecular interaction fields.

A new computational algorithm for protein binding sites characterization and comparison has been developed, which uses a common reference framework of the projected ligand-space four-point pharmacophore fingerprints, includes cavity shape, and can be used with diverse proteins as no structural alignment is required. Protein binding sites are first described using GRID molecular interaction fields (GRID-MIFs), and the FLAP (fingerprints for ligands and proteins) method is then used to encode and compare this information. The discriminating power of the algorithm and its applicability for large-scale protein analysis was validated by analyzing various scenarios: clustering of kinase protein families in a relevant manner, predicting ligand activity across related targets, and protein-protein virtual screening. In all cases the results showed the effectiveness of the GRID-FLAP method and its potential use in applications such as identifying selectivity targets and tools/hits for new targets via the identification of other proteins with pharmacophorically similar binding sites.

Alternative splicing: a novel mechanism of regulation identified in the chorismate mutase gene of the potato cyst nematode Globodera rostochiensis.

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.

Design, synthesis, and evaluation of aza inhibitors of chorismate mutase.

A series of aza inhibitors (4-9) of chorismate mutase (E.C. 5.4.99.5) was designed, prepared, and evaluated against the enzyme by monitoring the direct inhibition of the chorismate, 1, to prephenate, 2, conversion. None of these aza inhibitors displayed tighter binding to the enzyme than the native substrate chorismate or greater inhibitory action than the previously reported ether analogue, 3. Furthermore, no time-dependent loss of enzyme activity was observed in the presence of the two potentially reactive aza inhibitors (7 and 9). These results in conjunction with inhibition data from a broader series of chorismate mutase inhibitors allowed a novel proposal for the mechanistic role of chorismate mutase to be developed. This proposed mechanism was computationally verified and correlated with crystallographic studies of various chorismate mutases.

Selective stabilization of the chorismate mutase transition state by a positively charged hydrogen bond donor.

Citrulline was incorporated via chemical semisynthesis at position 90 in the active site of the AroH chorismate mutase from Bacillus subtilis. The wild-type arginine at this position makes hydrogen-bonding interactions with the ether oxygen of chorismate. Replacement of the positively charged guanidinium group with the isosteric but neutral urea has a dramatic effect on the ability of the enzyme to convert chorismate into prephenate. The Arg90Cit variant exhibits a >104-fold decrease in the catalytic rate constant kcat with a 2.7-fold increase in the Michaelis constant Km. In contrast, its affinity for a conformationally constrained inhibitor molecule that effectively mimics the geometry but not the dissociative character of the transition state is only reduced by a factor of approximately 6. These results show that an active site merely complementary to the reactive conformation of chorismate is insufficient for catalysis of the mutase reaction. Instead, electrostatic stabilization of the polarized transition state by provision of a cationic hydrogen bond donor proximal to the oxygen in the breaking C-O bond is essential for high catalytic efficiency.

A selective inhibitor of Escherichia coli prephenate dehydratase.

To identify selective prephenate dehydratase (PDT) inhibitors, a series of substituted biphenic acid derivatives was synthesized using the Ullmann reaction. Screening experiments identified 18 as a promising new PDT inhibitor.

Isolation of a cDNA from tomato coding for an unregulated, cytosolic chorismate mutase.

A cDNA coding for chorismate mutase was isolated from tomato by complementing a chorismate mutase-deficient Escherichia coli strain with a cDNA library. Southern blot analysis suggests the existence of a single gene of this chorismate mutase type per haploid tomato genome. The abundance of the corresponding transcripts was highest in roots, lower in stems and cotyledons, and even lower in flowers and leaves. The activity of the protein expressed in E. coli was not regulated by the three aromatic amino acids. Characteristics of the sequence and of the enzymatic activity suggest that the identified cDNA encodes a cytosolic, unregulated CM-2 type chorismate mutase.

Chorismate mutase of Chlamydomonas reinhardi. Partial purification and some properties.

Chorismate mutase (chorismate pyruvatemutase, EC 5.4.99.5) was extracted from Chlamydomonas reinhardi by sonication. Fractionation of crude sonic extracts with (NH4)2SO4 and by DEAE-cellulose and Sephadex gel chromatography indicated a single peak of chorismate mutase activity with molecular weight 61 000. The Michaelis constant for 20-fold purified enzyme was 0.46 mM. Prephenate dehydrogenase (EC 1.3.1.9) and prephenate dehydratase (EC 4.2.1.40) activities were not detected in our crude or partially purified preparations of chorismate mutase. Tyrosine (1.25 mM) inhibited chorismate mutase activity by approx. 85% in crude and partially purified preparations. Phenylalanine (1.25 mM) inhibited 20%. Tryptophan (1.25 mM) by itself had no detectable effect on chorismate mutase activity but it completely reversed inhibition by tyrosine and phenylalanine. No repression of chorismate mutase was observed when the minimal growth medium was supplemented with aromatic end products.