The low mutational flexibility of the EPSP synthase in Bacillus subtilis is due to a higher demand for shikimate pathway intermediates

Glyphosate (GS) inhibits the 5-enolpyruvyl-shikimate-3-phosphate (EPSP) synthase that is required for aromatic amino acid, folate and quinone biosynthesis in Bacillus subtilis and Escherichia coli. The inhibition of the EPSP synthase by GS depletes the cell of these metabolites, resulting in cell death. Here, we show that like the laboratory B. subtilis strains also environmental and undomesticated isolates adapt to GS by reducing herbicide uptake. Although B. subtilis possesses a GS-insensitive EPSP synthase, the enzyme is strongly inhibited by GS in the native environment. Moreover, the B. subtilis EPSP synthase mutant was only viable in rich medium containing menaquinone, indicating that the bacteria require a catalytically efficient EPSP synthase under nutrient-poor conditions. The dependency of B. subtilis on the EPSP synthase probably limits its evolvability. In contrast, E. coli rapidly acquires GS resistance by target modification. However, the evolution of a GS-resistant EPSP synthase under non-selective growth conditions indicates that GS resistance causes fitness costs. Therefore, in both model organisms, the proper function of the EPSP synthase is critical for the cellular viability. This study also revealed that the uptake systems for folate precursors, phenylalanine and tyrosine need to be identified and characterized in B. subtilis.     

Plant phenylacetaldehyde synthase is a bifunctional homotetrameric enzyme that catalyzes phenylalanine decarboxylation and oxidation

We have isolated and characterized Petunia hybrida cv. Mitchell phenylacetaldehyde synthase (PAAS), which catalyzes the formation of phenylacetaldehyde, a constituent of floral scent. PAAS is a cytosolic homotetrameric enzyme that belongs to group II pyridoxal 5'-phosphate-dependent amino-acid decarboxylases and shares extensive amino acid identity (approximately 65%) with plant L-tyrosine/3,4-dihydroxy-L-phenylalanine and L-tryptophan decarboxylases. It displays a strict specificity for phenylalanine with an apparent Km of 1.2 mM. PAAS is a bifunctional enzyme that catalyzes the unprecedented efficient coupling of phenylalanine decarboxylation to oxidation, generating phenylacetaldehyde, CO2, ammonia, and hydrogen peroxide in stoichiometric amounts.     

A unique reaction in a common pathway: mechanism and function of chorismate synthase in the shikimate pathway

Chorismate synthase, the seventh enzyme in the shikimate pathway, catalyzes the transformation of 5-enolpyruvylshikimate 3-phosphate to chorismate which is the last common precursor in the biosynthesis of numerous aromatic compounds in bacteria, fungi and plants. The enzyme has an absolute requirement for reduced FMN as a cofactor, although the 1,4-anti elimination of phosphate and the C(6proR)-hydrogen does not involve a net redox change. The role of the reduced FMN in catalysis has long been elusive. However, recent detailed kinetic and bioorganic approaches have fundamentally advanced our understanding of the mechanism of action, suggesting an initial electron transfer from tightly bound reduced flavin to the substrate, a process which results in C—O bond cleavage. Studies on chorismate synthases from bacteria, fungi and plants revealed that in these organisms the reduced FMN cofactor is made available in different ways to chorismate synthase: chorismate synthases in fungi – in contrast to those in bacteria and plants – carry a second enzymatic activity which enables them to reduce FMN at the expense of NADPH. Yet, as shown by the analysis of the corresponding genes, all chorismate synthases are derived from a common ancestor. However, several issues revolving around the origin of reduced FMN, as well as the possible regulation of the enzyme activity by means of the availability of reduced FMN, remain poorly understood. This review summarizes recent developments in the biochemical and genetic arena and identifies future aims in this field. Received: 22 June 1998 / Accepted: 7 August 1998     

The entry reaction of the plant shikimate pathway is subjected to highly complex metabolite-mediated regulation

The plant shikimate pathway directs bulk carbon flow toward biosynthesis of aromatic amino acids (AAAs, i.e. tyrosine, phenylalanine, and tryptophan) and numerous aromatic phytochemicals. The microbial shikimate pathway is feedback inhibited by AAAs at the first enzyme, 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DHS). However, AAAs generally do not inhibit DHS activities from plant extracts and how plants regulate the shikimate pathway remains elusive. Here, we characterized recombinant Arabidopsis thaliana DHSs (AthDHSs) and found that tyrosine and tryptophan inhibit AthDHS2, but not AthDHS1 or AthDHS3. Mixing AthDHS2 with AthDHS1 or 3 attenuated its inhibition. The AAA and phenylpropanoid pathway intermediates chorismate and caffeate, respectively, strongly inhibited all AthDHSs, while the arogenate intermediate counteracted the AthDHS1 or 3 inhibition by chorismate. AAAs inhibited DHS activity in young seedlings, where AthDHS2 is highly expressed, but not in mature leaves, where AthDHS1 is predominantly expressed. Arabidopsis dhs1 and dhs3 knockout mutants were hypersensitive to tyrosine and tryptophan, respectively, while dhs2 was resistant to tyrosine-mediated growth inhibition. dhs1 and dhs3 also had reduced anthocyanin accumulation under high light stress. These findings reveal the highly complex regulation of the entry reaction of the plant shikimate pathway and lay the foundation for efforts to control the production of AAAs and diverse aromatic natural products in plants.

Characterization of Arabidopsis 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase enzymes and mutants revealed highly complex metabolite-mediated feedback regulation of the plant shikimate pathway.     

Gene trap mutagenesis-based forward genetic approach reveals that the tumor suppressor OVCA1 is a component of the biosynthetic pathway of diphthamide on elongation factor 2

OVCA1 is a tumor suppressor identified by positional cloning from chromosome 17p13.3, a hot spot for chromosomal aberration in breast and ovarian cancers. It has been shown that expression of OVCA1 is reduced in some tumors and that it regulates cell proliferation, embryonic development, and tumorigenesis. However, the biochemical function of OVCA1 has remained unknown. Recently, we isolated a novel mutant resistant to diphtheria toxin and Pseudomonas exotoxin A from the gene trap insertional mutants library of Chinese hamster ovary cells. In this mutant, the Ovca1 gene was disrupted by gene trap mutagenesis, and this disruption well correlated with the toxin-resistant phenotype. We demonstrated direct evidence that the tumor suppressor OVCA1 is a component of the biosynthetic pathway of diphthamide on elongation factor 2, the target of bacterial ADP-ribosylating toxins. A functional genetic approach utilizing the random gene trap mutants library of mammalian cells should become a useful strategy to identify the genes responsible for specific phenotypes.     

The Mycobacterium tuberculosis shikimate pathway genes: Evolutionary relationship between biosynthetic and catabolic 3-dehydroquinases

Summary The Mycobacterium tuberculosis shikimate pathway genes designated aroB and aroQ encoding 3-dehydroquinate synthase and 3-dehydroquinase, respectively were isolated by molecular cloning and their nucleotide sequences determined. The deduced dehydroquinate synthase amino acid sequence from M. tuberculosis showed high similarity to those of equivalent enzymes from prokaryotes and filamentous fungi. Surprisingly, the deduced M. tuberculosis 3-dehydroquinase amino acid sequence showed no similarity to other characterised prokaryotic biosynthetic 3-dehydroquinases (bDHQases). A high degree of similarity was observed, however, to the fungal catabolic 3-dehydroquinases (cDHQases) which are active in the quinic acid utilisation pathway and are isozymes of the fungal bDHQases. This finding indicates a common ancestral origin for genes encoding the catabolic dehydroquinases of fungi and the biosynthetic dehydroquinases present in some prokaryotes. Deletion of genes encoding shikimate pathway enzymes represents a possible approach to generation of rationally attenuated strains of M. tuberculosis for use as live vaccines.     

DSF-家族群体感应信号生物合成途径与调控机制研究进展

群体感应是微生物间相互交流的一种重要机制.Diffusible Signaling Factor(DSF)-家族群体感应信号分子存在于多种革兰氏阴性菌中,调控细菌的致病性和适应性.本文首先介绍DSF-家族群体感应信号的结构多样性与保守性、生物合成途径和两类调控机制.DSF家族群体感应信号属于一类长链不饱和脂肪酸,碳水化...     

Regulation of the isofunctional genes ubiD and ubiX of the ubiquinone biosynthetic pathway of Escherichia coli

The expressions of the isofunctional genes ubiD and ubiX of the ubiquinone biosynthetic pathway of Escherichia coli were compared under a variety of growth conditions and in several genetic backgrounds. LacZ operon fusions were constructed and were inserted in single copies into strain MC4100 and into its fnr, arcA or hemA carrying derivatives. During aerobic growth the expressions of both ubiD and ubiX depended on the carbon source: succinate>glycerol>glucose. Mutations in fnr, arcA or hemA increased the expressions of both genes. During anaerobic growth in LB medium glucose strongly inhibited the expression of ubiD but not of ubiX.     

Inhibitory diffusible factor 45 bifunctional activity. As a cell growth inhibitor and as an insulin-like growth factor I-binding protein

From medium conditioned by 3T3 cells, we had previously purified to apparent homogeneity a novel inhibitory diffusible factor of 45 kDa (IDF45), and then determined the amino-terminal sequence. IDF45 prevented reversibly the growth of chick embryo fibroblast (CEF). In these cells, DNA synthesis stimulated by 1% serum was 50% inhibited in the presence of 45 ng/ml (1 nM) IDF45. In the present article, we show that, in CEF, DNA synthesis stimulated by IGF-I was 100% inhibited in the presence of purified IDF45. Furthermore, the 45-kDa protein (IDF45) was, after Western blotting, able to bind IGF-I. The inhibitory effect of IDF45 upon serum stimulation did not seem to be the result of its inhibitory activity upon IGF-I stimulation, since stimulation by IGF-I and serum were additive. Moreover, it was possible to dissociate the two inhibitory effects: when added to v-src transformed CEF, IDF45 was able to 100% inhibit stimulation induced by IGF-I and was unable to significantly decrease stimulation induced by serum, as was previously observed. Taken together, our results strongly suggest that IDF45 has two distinct functions, one of which was to bind IGF-I and the other to inhibit serum stimulation. Indeed, it was impossible to separate the two functions when IDF45 was purified by cation exchange fast protein liquid chromatography, a method very different from reverse-phase fast protein liquid chromatography previously used for purification to apparent homogeneity of IDF45. On the other hand, if the IGF binding activity and inhibitory activity effect upon serum stimulation were carried by two different proteins, the presence of IGF-I (in conditions where most of the 45-kDa proteins were bound to IGF-I) should not have affected the activity of the molecule inhibiting serum stimulation. However, we observed the contrary: when IDF45 was bound to IGF-I, it lost its inhibitory effect upon stimulation induced by serum. This suggests that the two activities occurred on the same protein and that IDF45 is a bifunctional protein.     

The second enzyme in pyrrolnitrin biosynthetic pathway is related to the heme-dependent dioxygenase superfamily

Pyrrolnitrin is a commonly used and clinically effective treatment for fungal infections and provides the structural basis for the more widely used fludioxinil. The pyrrolnitrin biosynthetic pathway consists of four chemical steps, the second of which is the rearrangement of 7-chloro-tryptophan by the enzyme PrnB, a reaction that is so far unprecedented in biochemistry. When expressed in Pseudomonas fluorescens, PrnB is red in color due to the fact that it contains 1 mol of heme b per mole of protein. The crystal structure unexpectedly establishes PrnB as a member of the heme-dependent dioxygenase superfamily with significant structural but not sequence homology to the two-domain indoleamine 2,3-dioxygenase enzyme (IDO). The heme-binding domain is also structurally similar to that of tryptophan 2,3-dioxygenase (TDO). Here we report the binary complex structures of PrnB with d- and l-tryptophan and d- and l-7-chloro-tryptophan. The structures identify a common hydrophobic pocket for the indole ring but exhibit unusual heme ligation and substrate binding when compared with that observed in the TDO crystal structures. Our solution studies support the heme ligation observed in the crystal structures. Purification of the hexahistidine-tagged PrnB yields homogeneous protein that only displays in vitro activity with 7-chloro-l-tryptophan after reactivation with crude extract from the host strain, suggesting that an as yet unknown cofactor is required for activity. Mutation of the proximal heme ligand results, not surprisingly, in inactive enzyme. Redox titrations show that PrnB displays a significantly different reduction potential to that of IDO or TDO, indicating possible differences in the PrnB catalytic cycle. This is confirmed by the absence of tryptophan dioxygenase activity in PrnB, although a stable oxyferrous adduct (which is the first intermediate in the TDO/IDO catalytic cycle) can be generated. We propose that PrnB shares a key catalytic step with TDO and IDO, generation of a tryptophan hydroperoxide intermediate, although this species suffers a different fate in PrnB, leading to the eventual formation of the product, monodechloroaminopyrrolnitrin.     

The argos gene encodes a diffusible factor that regulates cell fate decisions in the Drosophila eye.

The argos gene encodes a protein that is required for viability and that regulates the determination of cells in the Drosophila eye. A developmental analysis of argos mutant eyes indicates that the mystery cells, which are usually nonneuronal, are transformed into extra photoreceptors, and that supernumerary cone cells and pigment cells are also recruited. Clonal analysis indicates that argos acts nonautonomously and can diffuse over the range of several cell diameters. Conceptual translation of the argos gene suggests that it encodes a secreted protein.     

Conservation of NADPH utilization by chorismate synthase and its implications for the evolution of the shikimate pathway

The shikimate pathway is essential for the biosynthesis of aromatic compounds. The seventh and last step is catalysed by chorismate synthase, which has an absolute requirement for reduced FMN in its active site. There are two classes of this enzyme, which are distinguished according to the origin of the reduced cofactor. Monofunctional chorismate synthases sequester it from the cellular environment whereas bifunctional enzymes can generate reduced FMN at the expense of NADPH. These bifunctional enzymes are found in fungi and the ciliated protozoan Euglena gracilis while all bacterial and plant enzymes are monofunctional. In this study, we introduce an in vivo screen, which is based on a chorismate synthase-deficient Saccharomyces cerevisiae strain, allowing the classification of hitherto uncharacterized chorismate synthases. This analysis revealed that bifunctionality is present in the enzymes of protozoan species. In contrast, all bacterial and plant enzymes tested are monofunctional. In addition, we demonstrate that a monofunctional chorismate synthase confers prototrophy in conjunction with a NADPH : FMN oxidoreductase indicating that bifunctionality is required due to the lack of free reduced FMN in fungal and possibly protozoan species. Interestingly, the distribution of bifunctional chorismate synthase concurs with the presence of a pentafunctional enzyme complex.