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Purification and Characterization of Chorismate Synthase from Euglena Gracilis' Plant Physiol. (1991) 97, 1271-1279 Received for publication May 14, 1991 0032-0889/91/97/1271 /09/$01 .00/0 Accepted July 16, 1991 Purification and Characterization of Chorismate Synthase from Euglena gracilis' Comparison with Chorismate Synthases of Plant and Microbial Origin Andreas Schaller, Manfred van Afferden2, Volker Windhofer3, Sven Bulow4, Gernot Abel, Jurg Schmid, and Nikolaus Amrhein* Institute of Plant Sciences, Swiss Federal Institute of Technology, Zurich, Switzerland (A.S., G.A., J.S., N.A.); and Lehrstuhl fur Pflanzenphysiologie, Ruhr-Universitat Bochum, Federal Republic of Germany (M.v.A., V. W., S.B.) ABSTRACT converting DAHP5 to EPSP reside in a single pentafunctional Chorismate synthase was purified 1200-fold from Euglena gra- polypeptide, the arom complex. In higher plants, two activities cilis. The molecular mass of the native enzyme is in the range of (3-dehydroquinate dehydratase and shikimate dehydrogen- 110 to 138 kilodaltons as judged by gel filtration. The molecular ase) form a bifunctional polypeptide, although the other five mass of the subunit was determined to be 41.7 kilodaltons by enzymes are monofunctional (reviewed in ref. 4). In Euglena sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Pu- gracilis, the organization of the prechorismate pathway ap- rified chorismate synthase is associated with an NADPH-depend- pears to resemble that found in fungi. The first and the last ent flavin mononucleotide reductase that provides in vivo the steps are catalyzed by single enzymes (DAHP-synthase and reduced flavin necessary for catalytic activity. In vitro, flavin chorismate synthase, respectively), whereas activities 2 to 6 reduction can be mediated by either dithionite or light. The en- form a complex the zyme obtained from E. gracilis was compared with chorismate large resembling fungal arom complex synthases purified from a higher plant (Corydalis sempervirens), (27). Euglena and fungi are strikingly similar in other bio- a bacterium (Escherichia co/i), and a fungus (Neurospora crassa). chemical pathways as well. Thus, both groups of organisms These four chorismate synthases were found to be very similar use the L-a-aminoadipate pathway for lysine biosynthesis, in terms of cofactor specificity, kinetic properties, isoelectric whereas bacteria, algae, and plants follow the diaminopime- points, and pH optima. All four enzymes react with polyclonal late route (30). On the basis of such findings, a close evolu- antisera directed against chorismate synthases from C. semper- tionary relationship was proposed for euglenoids and fungi. virens and E. coli. The closely associated flavin mononucleotide But aromatic biosynthesis shows several features that, among reductase that is present in chorismate synthase preparations all organisms studied so far, are unique for E. gracilis. An- from E. gracilis and N. crassa is the main difference between thranilate synthase, the first enzyme in the tryptophan those synthases and the monofunctional enzymes from C. sem- branch pervirens and E. coli. (Fig. 1), is monomeric in Euglena, whereas in all other an- thranilate synthases characterized so far the chorismate and glutamine binding sites reside on distinct polypeptide chains (16). The synthesis of tryptophan from anthranilate is cata- lyzed by a single protein in Euglena (17), but in all other known The three aromatic amino acids are synthesized via the examples tryptophan synthase is separable from the shikimate pathway in microorganisms and plants (Fig. 1). other activities of the tryptophan branch (17). Unlike other Although the reaction sequence in the prechorismate pathway organisms, E. gracilis possesses only a single chorismate mu- is identical in all organisms so there are tase isozyme, which is unusually large (31). In contrast to investigated far, microorganisms, L-arogenate is the considerable differences in enzyme organization, as well as last common precursor of regulation, between organisms ofdifferent taxonomic groups. both phenylalanine and tyrosine in Euglena (7), which also In Escherichia coli, the seven enzymatic activities of the appears to be the case for higher plants. prechorismate are associated Chorismate synthase is the only shikimate pathway enzyme pathway with monofunctional previously not in polypeptides. In contrast, in fungi the five enzyme activities detected E. gracilis, except indirectly (6), which remained to be characterized. A comparison of the Euglena enzyme with chorismate synthases of other species ' Supported at Ruhr-Universitat Bochum by a grant from the is particularly interesting considering the data mentioned Deutsche Forschungsgemeinschaft and at the Swiss Federal Institute of Technology by ETHZ grant 07592/41-1030.5. above. 2Present address: DMT Gesellschaft fuir Forschung und Prufung Chorismate synthase (EC 4.6.1.4) catalyzes the formation mbH, Essen, FRG. 'Present address: Institut fur physiologische Chemie, Ruhr Univ- 5Abbreviations: DAHP, 3-deoxy-D-arabino-heptulosonate 7-phos- ersitit Bochum, FRG. phate; EPSP, 5-enolpyruvylshikimate 3-phosphate; FMN, flavin mon- 4Present address: PALL Biomedizin GmbH, Dreieich, FRG. onucleotide; FAD, flavin adenine dinucleotide. 1271 1 272 SCHALLER ET AL. Plant Physiol. Vol. 97, 1991 E4P Q Cells were harvested after 7 d (late logarithmic phase of + ' DAHP 'DHQ ' DHS- growth) at a cell density of 2 x 106/mL. A cell suspension PEP culture of C. sempervirens was grown as described previously (28). The E. coli strain AB2849 carrying the plasmid pGM602 (33) was a gift of Dr. J.R. Coggins (University of Glasgow, UK). It was grown in 2YT medium in the presence of SHK ' S3P '- EPSP ' CHA ampicillin. (is \@1 1. DAHP-Synthase Materials 2. DHQ-Synthase PPA ANT 3. DHQ-Dehydratase All chemicals purchased from commercial sources were of 4. SHK-Dehydrogenase j1\ the highest purity available. Chorismate was isolated and 5. SHK-Kinase I purified according to the method of Gibson (15). Shikimate AGN 6. EPSP-Synthase 3-phosphate was prepared as in ref. 21 and EPSP was synthe- 7. CHA-Synthase sized from shikimate 3-phosphate as described in ref. 22. 8. CHA-Mutase I, Anthranilate synthase from Aerobacter aerogenes was purified 9. ANT-Synthase Pthe tyr trp following the procedure of Egan and Gibson (11) with the modifications described in ref. 28. Chorismate synthase was E4P, D-erythrose 4- Figure 1. Overview of the shikimate pathway. purified from C. sempervirens as described previously (28). phosphate; PEP, phosphoenolpyruvate; DHQ, 3-dehydroquinate; were from E. about 400- DHS, 3-dehydroshikimate; SHK, shikimate; S3P, shikimate 3-phos- Chorismate synthases purified coli phate; CHA, chorismate, PPA, prephenate; AGN, arogenate; ANT, fold and from N. crassa 260-fold essentially as described in anthranilate. ref. 33 with omission ofthe last chromatographic step. Specific enzyme activities were 48 and 6 nkat/mg, respectively. An antiserum was raised in rabbits against C. sempervirens chor- ismate synthase (28) and the immunoglobulin G fraction was of chorismate-the last common precursor for all three aro- purified on protein A beads (Pharmacia). The rabbit anti-E. matic amino acids-by 1,4-elimination of orthophosphate coli chorismate synthase serum was a gift of Dr. J.R. Coggins. from EPSP. The enzyme strictly requires a reduced flavin as cofactor, although no net change in redox state ofthe substrate is observed. Chorismate synthases have been purified and Assay of Chorismate Synthase Activity characterized from E. coli (25, 33), Neurospora crassa (32, All enzyme reactions were at 30°C unless otherwise stated. 33), and Bacillus subtilis (18). Although chorismate synthase Chorismate synthase activity can be assayed either by detec- activity was only recently discovered in an extract from a tion of the reaction products (chorismate or phosphate) or by higher plant (26), the enzyme has been isolated and charac- forward coupling to the reaction of anthranilate synthase and terized from a cell suspension culture of Corydalis sempervi- monitoring the anthranilate production. Throughout the en- rens in our laboratory (28). Furthermore, a cDNA coding for zyme purification, the discontinuous coupled assay was used, C. sempervirens chorismate synthase has been cloned and according to ref. 32 for E. gracilis and N. crassa, and accord- analyzed (A. Schaller, J. Schmid, U. Leibinger, N. Amrhein, ing to ref. 28 for C. sempervirens and E. coli. The coupled submitted for publication). Here we report the purification assay was not used for the characterization of chorismate and characterization of chorismate synthase from E. gracilis synthases, so as to exclude the influence of anthranilate syn- as a representative of lower photoautotrophic eukaryotes, thase on the results. For characterization of the E. gracilis together with a more detailed analysis of the properties of the chorismate synthase, the formation of chorismate was fol- C. sempervirens chorismate synthase. The results are discussed lowed at 274 nm using the assay system described in ref. 33. in the context of data obtained for chorismate synthases The assay mixture contained 50 mM triethanolamine/HCl, isolated from E. coli and N. crassa. We find that chorismate pH 8.0, 50 mm KCI, 2.5 mM MgCl2, 83 uM NADPH, 10 Mm synthase, unlike other shikimate pathway enzymes, is highly F-MN, and 80 uM EPSP. This assay is not compatible with similar in different organisms. The main difference appears chorismate synthases that lack an intrinsic flavin reductase to be the presence of a flavin reductase activity
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