Enzymology of l-Tyrosine Biosynthesis in Mung Bean (Vigna radiata [L.] Wilczek)

Abstract

The enzymes of the 4-hydroxyphenylpyruvate (prephenate dehydrogen-ase and 4-hydroxyphenylpyruvate aminotransferase) and pretyrosine (pre-phenate aminotransferase and pretyrosine dehydrogenase) pathways of L-tyrosine biosynthesis were partially purifled from mung bean (Vigna radiata IL.1 Wilczek) seedlings. NADP-dependent prephenate dehydrogenase and pretyrosine dehydrogenase activities coeluted from ion exchange, adsorp-tion, and gel-ftration columns, suggesting that a single protein (52,000 daltons) catalyzes both reactions. The ratio of the activities of partially purified prephenate to pretyrosine dehydrogenase was constant during al purification steps as weU as after partial inactivation caused by p-hydrox-ymercuribenzoic acid or heat. The activity of prephenate dehydrogenase, but not of pretyrosine dehydrogenase, was inhibited by L-tyrosine at nonsaturating levels of substrate. The K. values for prephenate and pretyrosine were similar, but the specific activity with prephenate was 2.9 times greater than with pretyrosine. Two peaks of aromatic aminotransferase activity utilizing L-glutamate or L-aspartate as amino donors and 4-hydroxyphenylpyruvate, phenylpyr-uvate, and/or prephenate as keto acid substrates were eluted from DEAE-cellulose. Of the three keto acid substrates, 4-hydroxyphenylpyruvate was preferentially utilized by 4-hydroxyphenylpyruvate aminotransferase whereas prephenate was best utilized by prephenate aminotransferase. The identity of a product of prephenate aminotransferase as pretyrosine following reaction with prephenate was established by thin layer chromatography of the dansyl-derivative. The shikimate pathway of aromatic amino acid biosynthesis begins with the condensation of erythrose-4-P and phosphoenol-pyruvate and culminates in the formation of L-tryptophan, L-tyrosine, and L-phenylalanine. Although many of the pathway reactions have been studied in higher plants (for reviews, see 2 and 23), the enzymology of the distal branches of the shikimate pathway, leading from prephenate to L-tyrosine and L-phenylal-anine, has neither been fully investigated nor evaluated for the presence of biosynthetic routes to L-phenylalanine or L-tyrosine that proceed via pretyrosine. Previous studies showed the following. Labeled tyrosine or phenylalanine was formed when young plant shoots were fed radioactive 4-hydroxyphenylpyruvate or phenylpyruvate (12). Cell-free extracts of plants catalyzed the following enzymic conversions: 4-hydroxyphenylpyruvate to tyrosine; phenylpyruvate to ' This study represents part of a thesis presented by Judith L. Rubin to the Graduate School of the State University of New York at Binghamton in partial fulfillment of the requirements for the M.A. degree. The study was supported in part by Department of Energy Contract EP-78-S-02-4967. phenylalanine; and prephenate to 4-hydroxyphenylpyruvate, ty-rosine, and phenylalanine (13). Prephenate dehydrogenase and an aromatic aminotransferase (utilizing tyrosine to form 4-hydroxy-phenylpyruvate) were partially purified and studie...

The aromatic amino acid pathway branches at L-arogenate in Euglena gracilis.

“…In E. gracilis arogenate dehydrogenase is specific for NADP. Shoots of mung bean ( Vigna radiata) also posess a NADP-specific arogenate dehydrogenase (31). This contrasts with arogenate dehydrogenase of corn, which is specific for NAD (G. S. Byng, R. J. Whitaker, C. E. Flick, and R. A. Jensen, Phytochemistry, in press) L-Arogenate is formed via transamination of prephenate.…”

“…In Pseudomonas aeruginosa enzymes of both the phenylpyruvate route and the alternative arogenate route have been characterized (27,28 (25). A mixed-substrate assay (31) b Position of enzyme in the pathway corresponds to the numbering shown in Fig. 10.…”

5‐Enolpyruvylshikimate‐3‐phosphate synthase of Klebsiella pneumoniae

“…Inhibition by glyphosate, competitive with PEP, has by now been established for EPSP synthases from K. pneumoniae (this paper and [9], E. coli [S], N . crasea [lo], and pea seedlings [ll], as well as cultured higher plant cells [6,12]. The nature of the inhibition by glyphosate with respect to the second substrate in the forward reaction, S3P, is less clear.…”

“…In agreement with this hypothesis we observed an inhibition of the synthesis of chorismate-derived primary and secondary metabolites, as well as a massive accumulation of shikimic acid, in plants exposed to glyphosate [4 -61. As the biosynthesis of chorismate proceeds via the sequence shikimate+shikimate 3-phosphate (S3P)+5-enolpyruvylshikimate 3-phosphate (EPSP)-+chorismate [7], the inhibition of EPSP synthase by glyphosate [2] offered a satisfactory explanation for these observations. The potent inhibition of EPSP synthase by glyphosate has been confirmed for the enzymes of Escherichia coli [8] [6] and Nicotiana silvestris [12], as well as a large number of other plant species and bacteria (Amrhein and coworkers, unpublished). Furthermore, resistance to glyphosate in microorganisms and a plant tissue culture was found to be correlated with either an increased production of glyphosate-sensitive EPSP synthase [8,13,14] or the production of a glyphosate-insensitive EPSP synthase [15,16].…”

RNAi Suppression of Arogenate Dehydratase1 Reveals That Phenylalanine Is Synthesized Predominantly via the Arogenate Pathway in Petunia Petals

“…Indeed, ADT from tobacco (Nicotiana sylvestris), spinach (Spinacia oleracea), Sorghum bicolor, and rice (Jung et al, 1986;Siehl and Conn, 1988;Yamada et al, 2008) and DAHPS from mung bean (Vigna radiata) and spinach (Rubin and Jensen, 1979;Doong et al, 1993) were shown to be inhibited in vitro by Phe and arogenate, respectively. Although this model still awaits genetic proof, our data are consistent with this hypothesis.…”