Characterization of Monofunctional Chorismate Mutase/Prephenate Dehydrogenase Enzymes Obtained via Mutagenesis of Recombinant Plasmids in vitro

Abstract

Mutagenesis in vitro has been used to obtain mutant forms of the bifunctional enzyme, chorismate mutase/ prephenate dehydrogenase. Plasmid D N A containing the genes that code for the enzyme was treated with hydroxylamine and the resulting products were used to transform strains of Eschericliia coli. Two types of mutant were isolated. One contained enzyme which was niutase-positive, dehydrogenase-negative while the other did not exhibit either activity. Kinetic and physical analysis of one of the purified monofunctional enzymes showed that the loss of dehydrogenase activity was due to modification of the binding site for NAD. The results open the way for molecular studies of structure-function relationships with this bifunctional enzyme. A separate bifunctional enzyme chorismate mutase/prephenate dehydrogenase (EC 1.3.1.12), which is coded for by the tyrA gene, catalyses both the conversion of chorismate to prephenate, in the presence of NAD, and of prephenate to 4-hydroxyphenylpyruvate. These reactions form part of the tyrosine biosynthetic pathway. Chorismate mutase/prephenate dehydrogenase from both E. coli and Aerobacter aerogenes has been extensively studied. However, the kinetic approaches to the determination of the number of catalytic sites that are involved in the conversions of chorismate to prephenate and of prephenate to hydroxyphenylpyruvate have not yielded definitive answers [6,7]. Because of the successful isolation of monofunctional mutant forms of chorismate mutasef prephenate dehydrogenase from S. typhimurium [2,8] it seemed that more clear-cut results might be obtained by using the genetic approach for studies on the E. coli enzyme. T o facilitate the subsequent purification of mutant forms of chorismate mutasefprephenate dehydrogenase, mutagenesis was performed in vitro on a multicopy recombinant plasmid which carries the tyrA gene and which is responsible for markedly elevated yields of chorismate mutasefprephenate dehydrogenase [9]. This paper reports the isolation and subsequent characterization of chorismate mutase/prephenate dehydrogenase obtained from six mutants and evidence is presented to show Enzymes. Chorismate mutase, prephenate dehydrogenase (EC ~-1.3.1.12); chorismate mutase, prephenate dehydratase (EC 4.2.1.51). In the phenylalanine biosynthetic pathway in

Imbalance of tyrosine by modulating TyrA arogenate dehydrogenases impacts growth and development of Arabidopsis thaliana

“…Many microbes use the 4‐hydroxylphenylpyruvate pathway, in which prephenate is first oxidatively decarboxylated to 4‐hydroxylphenylpyruvate by TyrA prephenate dehydrogenase followed by transamination to tyrosine (Bentley, ). For example, Escherichia coli has a prephenate‐specific TyrA enzyme, whose deficiency results in tyrosine auxotroph (Davidson et al ., ; Rood et al ., ; Turnbull et al ., ). Prior studies found that most plants have prephenate aminotransferase activity and enzymes, which transaminate prephenate into arogenate (Graindorge et al ., ; Dal Cin et al ., ; Maeda et al ., ).…”