Abstract.Homogentisate is the precursor in the biosynthesis of ~-tocopherol and plastoquinone-9 in chloroplasts. It is formed of 4-hydroxyphenylpyruvate of the shikimate pathway by the 4-hydroxyphenylpyruvate dioxygenase. In experiments with spinach the dioxygenase was shown to be localized predominatedly in the chloroplasts. Envelope membranes exhibit the highest specific activity, however, because of the high stromal portion of chloroplasts, 60-80% of the total activity is housed in the stroma. The incorporation of 4-hydroxyphenylpyruvate into 2-methyl-6-phytylquinol as the first intermediate in the tocopherol synthesis by the two-step-reaction: 4-Hydroxyphenylpyruvate ~ Homogentisate Phytyl-PP 2-Methyl-6-phytylquinol was demonstrated by using envelope membranes. Homogentisate originates directly from 4-hydroxyphenylpyruvate of the shikimate pathway. Additionally, a bypass exists in chloroplasts which forms 4-hydroxyphenylpyruvate from tyrosine by an L-amino-acid oxidase of the thylakoids and in peroxisomes by a transaminase reaction. Former results about the dioxygenase in peroxisomes were verified.
The stroma of chloroplasts is probably the sole site of the shikimate pathway enzymes shikimate oxidoreductase/dehydroquinate hydrolyase (SORase/DHQase) in spinach leaves. (a) The (Fig. 1). We have used an essentially different approach to provide evidence that-in spinach leaves-two crucial enzymic activities SORase and DHQase of the shikimate pathway are restricted to the soluble chloroplast extract. It was proposed that the activities of SORase and DHQase in higher plants were on a single polypeptide (20). This bifunctional protein catalyzes the conversion ofdehydroquinate into dehydroshikimate(DHQase activity) and sequentially the synthesis of shikimate from dehydroshikimate (SORase activity). The two enzymic activities were found exclusively in the stroma ofspinach chloroplasts as demonstrated in this paper and the preceding abstract (13). To understand the structural/functional relationships ofthe multiple forms requires the ability to isolate them in a purified state.We have improved the previously reported purification procedure (12) to achieve the apparent homogeneity and stability which was necessary for the characterization of SORase/ DHQase. The purification procedure of a shikimate pathway enzyme from the chloroplast stroma is described here for the first time. Furthermore we investigated the regulatory properties of these enzymes.
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