Purification and Characterization of Chorismate Synthase from Euglena gracilis

Abstract

Chorismate synthase was purified 1200-fold from Euglena gracilis. The molecular mass of the native enzyme is in the range of 110 to 138 kilodaltons as judged by gel filtration. The molecular mass of the subunit was determined to be 41.7 kilodaltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified chorismate synthase is associated with an NADPH-dependent flavin mononucleotide reductase that provides in vivo the reduced flavin necessary for catalytic activity. In vitro, flavin reduction can be mediated by either dithionite or light. The enzyme obtained from E. gracilis was compared with chorismate synthases purified from a higher plant (Corydalis sempervirens), a bacterium (Escherichia co/i), and a fungus (Neurospora crassa).These four chorismate synthases were found to be very similar in terms of cofactor specificity, kinetic properties, isoelectric points, and pH optima. All four enzymes react with polyclonal antisera directed against chorismate synthases from C. sempervirens and E. coli. The closely associated flavin mononucleotide reductase that is present in chorismate synthase preparations from E. gracilis and N. crassa is the main difference between those synthases and the monofunctional enzymes from C. sempervirens and E. coli.The three aromatic amino acids are synthesized via the shikimate pathway in microorganisms and plants (Fig. 1). Although the reaction sequence in the prechorismate pathway is identical in all organisms investigated so far, there are considerable differences in enzyme organization, as well as regulation, between organisms of different taxonomic groups. In Escherichia coli, the seven enzymatic activities of the prechorismate pathway are associated with monofunctional polypeptides. In contrast, in (reviewed in ref. 4). In Euglena gracilis, the organization of the prechorismate pathway appears to resemble that found in fungi. The first and the last steps are catalyzed by single enzymes (DAHP-synthase and chorismate synthase, respectively), whereas activities 2 to 6 form a large complex resembling the fungal arom complex (27). Euglena and fungi are strikingly similar in other biochemical pathways as well. Thus, both groups of organisms use the L-a-aminoadipate pathway for lysine biosynthesis, whereas bacteria, algae, and plants follow the diaminopimelate route (30). On the basis of such findings, a close evolutionary relationship was proposed for euglenoids and fungi. But aromatic biosynthesis shows several features that, among all organisms studied so far, are unique for E. gracilis. Anthranilate synthase, the first enzyme in the tryptophan branch ( Fig. 1), is monomeric in Euglena, whereas in all other anthranilate synthases characterized so far the chorismate and glutamine binding sites reside on distinct polypeptide chains (16). The synthesis of tryptophan from anthranilate is catalyzed by a single protein in Euglena (17), but in all other known examples tryptophan synthase is separable from the other activities of the tryptophan branch (17). Unlike ot...

Differential expression of tomato (Lycopersicon esculentum L.) genes encoding shikimate pathway isoenzymes. II. Chorismate synthase

“…microorganisms and plants) so far (for a review see [3]), there are considerable differences in enzyme organisation, as well as regulation, between organisms of different taxonomic groups. Chorismate synthases have been purified and characterized from the procaryotic microorganisms Escherichia coli [16,28] and Bacillus subtilis [9], the eucaryotic microorganisms Neurospora crassa [27,28] and Euglena gracilis [22], as well as from the higher plants Pisum sativum [17] and Corydalis sempervirens [23]. A comparative study of chorismate synthases from E. gracilis, C. sempervirens, E. coli and N. crassa showed that these enzymes were very similar in terms of cofactor specificity, kinetic and immunogenic properties, as well as their pH optima [22].…”

Escherichia coli Chorismate Synthase Catalyzes the Conversion of (6S)-6-Fluoro-5-enolpyruvylshikimate-3-phosphate to 6-Fluorochorismate

“…Reduced FMN is required for enzyme activity despite there being no overall reduction or oxidation in the conversion of EPSP to chorismate (15)(16)(17)(18)(19). The role of flavin is not yet clear but it appears to be directly involved in catalysis.…”

“…Homonuclear 1 H decoupling experiments confirmed the connectivities between the resonances associated with positions 2-CH through to 5-CH. The double doublet 19 F resonance of (6S)-6-fluoro-EPSP at Ϫ174.35 ppm disappeared (Fig. 3, inset) giving rise to a new quartet at Ϫ111.39 ppm (Fig.…”

“…Proposed mechanisms include an X-group mechanism, involving the nucleophilic attack of the C-1 position of the substrate (5) (Scheme 2d), and a cationic mechanism, involving the step-wise loss of phosphate and a proton (11, 12) (Scheme 2a). An allylic rearrangement of phosphate followed by a 1,2-elimination (13) has been discounted because the intermediate iso-EPSP is a competitive inhibitor rather than a substrate of the Neurospora crassa enzyme (14).Reduced FMN is required for enzyme activity despite there being no overall reduction or oxidation in the conversion of EPSP to chorismate (15)(16)(17)(18)(19). The role of flavin is not yet clear but it appears to be directly involved in catalysis.…”

Chorismate Synthase from the Hyperthermophile Thermotoga maritima Combines Thermostability and Increased Rigidity with Catalytic and Spectral Properties Similar to Mesophilic Counterparts

“…The chorismate synthases for which the reduced flavin has to be supplied exogenously are referred to as monofunctional, e.g. those from Escherichia coli and plants (3,(5)(6)(7)(8)(9), while chorismate synthases which possess the intrinsic ability to reduce the flavin at the expense of NADPH are referred to as being bifunctional, e.g. the Neurospora crassa enzyme (4,10,11).…”