Purification and characterization of anthranilate synthase from Catharanthus roseus

Abstract

Anthranilate synthase (EC 4.1.3.27) has been purified from cell cultures of Catharanthus roseus by poly(ethy1ene glycol) precipitatiodfractionation and subsequent separation by anion exchange on Q-Sepharose, Orange A dye chromatography, Mono Q anion-exchange chromatography and Superose 6 gel filtration. By analogy to anthranilate synthases from other sources it does look like the enzyme is a tetramer composed of two large and two small subunits, with molecular mass 67 and 25.5 f 0.5 kDa, respectively. The molecular mass determined by gel filtration was 143 f 5 kDa. The enzyme had a PI of 5.1 determined by chromatofocusing. The pH optimum was between pH 7.5 and pH 8.3, but the type of buffer used affected the results. The enzyme could utilize NH,' as ammonium donor instead of glutamine. The enzyme showed normal Michaelis-Menten kinetics with respect to the substrates L-glutamine and chorismate, and the cofactor Mg", K,,, values for L-glutamine was determined to be 0.37f0.05 mM, for chorismate 67?3 pM, and for MgCl, 0.26 -C-0.03 mM respectively. Anthranilate synthase was inhibited by L-tryptophan, tryptamine and D-tryptophan (with L-tryptophan being the best inhibitor). The enzyme was allosterically regulated showing positive cooperativity of chorismate binding at higher concentrations of tryptophan. For a tryptophan concentration of 20pM the Hill coefficient was determined to be 2. The tryptophan binding sites showed positive cooperativity for higher concentrations of chorismate. The purified enzyme did not contain anthranilate-5-phosphoribosylpyrophosphate phosphoribosyltransferase activity and is thus not of the same type as the well characterized Salmonella typhimurium anthranilate synthase/phosphoribosyl pyrophosphate transferase bifunctional type.Anthranilate synthase (AS; EC 4.1.3.27) catalyzes the first reaction branching from the shikimate pathway toward the biosynthesis of tryptophan occurring in bacteria, fungi and plants [I] (Fig. 1).Tryptophan is a key precursor for the indole alkaloids, among which there are several pharmacologically important compounds [2], from Catharanthus roseus, for example, the two anticancer drugs vinblastine and vincristine. The availability of tryptophan might be a limiting factor in the production of indole alkaloids. In all plants and cell cultures where AS has been examined, the enzyme was found to be strongly feedback-inhibited by tryptophan ; in accordance with this, several studies have indicated that loss of feedback regulation of AS can lead to largely unregulated accumulation of tryptophan [3 -61.AS enzymes have been purified to homogeneity and characterized from a number of microorganisms. In all mi- crobial species studied, AS is composed of two nonidentical subunits [7-91. Component I (also called a-subunit) binds the substrate chorismate and catalyzes its aromatization; component I1 @-subunit) binds the other substrate glutamine and transfers an ammonia group from glutamine to component I (chorismate). This glutamine-dependent AS reaction requires both subunit...

Functional Expression of Arabidopsis thaliana Anthranilate Synthase Subunit I in Escherichia coli

“…The purified GST-ASA1 exhibited enzymatic properties similar to those observed for AS purified from C. roseus (Poulsen et al, 1993). The apparent KM for chorismate was about 180 p~ in the presence of 100 m~ ammonium chloride, in accordance with reported values (Table I).…”

“…The sensitive form was found to be predominant in normal cells, and the insensitive form was found in 5-methyltryptophan-resistant lines. In contrast, Poulsen et al (1993) found no evidence for a Trpinsensitive form of AS in C. roseus. However, they separated two sensitive forms of AS from the crude extract in an anionexchange column.…”

“…AS from plants has not been purified to homogeneity. The only extensively purified AS is from the cell cultures of Catharanthus roseus (Poulsen et al, 1993). After five purifi-* Corresponding author; fax 1-415-493-1073. cation steps, including four involving column chromatography, 54 pg of AS was obtained from 1 kg of cells, with a specific activity of 164 nmol min-' mg-*.…”

Metabolic engineering of the tryptophan and phenylalanine biosynthetic pathways in rice

“…Plants show a similarly complex regulation. Feedback regulation of both enzyme activities has been reported in various plants as well (Figure 2) (Poulsen et al 1993;Li and Last 1996;Goers and Jensen 1984;Mobley et al 1999). In addition, the expression of the genes encoding AS and CM is also regulated at the transcription stage.…”

Purification and cDNA cloning of anthranilate synthase from Ruta graveolens: modes of expression and properties of native and recombinant enzymes

“…Although Arabidopsis tha/iana cDNAs which can complement trpE (Niyogi and Fink, 1992) and trpG (Niyogi eta/., 1993) mutants of E. coil have been described, the structure and biochemistry of the plant enzymes themselves are still obscure. Studies with crude extracts from corn and pea (Hankins et aL, 1976) as well as with the partially purified enzyme from Catharanthus roseus (Poulsen et aL, 1993) have led to speculation that AS in these species possesses a monofunctional J3-subunit. But AS from plants is difficult to purify and, as a consequence, no direct evidence has yet been marshalled which conclusively demonstrates the function and size of AS~ in plants.…”