Cyclohexadienyl dehydrogenase from Pseudomonas stutzeri exemplifies a widespread type of tyrosine-pathway dehydrogenase in the TyrA protein family

The uni-domain cyclohexadienyl dehydrogenases are able to use the alternati ve intermediates of tyrosine biosynthesis, prephenate or L-arogenate, as su bstrates. Members of this TyrA protein family have been generally considere d to fall into two classes: sensitive or insensitive to feedback inhibition by L-tyrosine. A gene (tyrA(c)) encoding a cyclohexadienyl dehydrogenase f rom Pseudomonas stutzeri JM300 was cloned, sequenced, and expressed at a hi gh level in Escherichia call. This is the first molecular-genetic and bioch emical characterization of a purified protein representing the feedback-sen sitive type of cyclohexadienyl dehydrogenase. The catalytic-efficiency cons tant K-cat/K-m for prephenate (7.0 x 10(7) M/s) was much better than that o f L-arogenate (5.7 x 10(6) M/s). TprA(c) was sensitive to feedback inhibiti on by either L-tyrosine or 4-hydroxyphenylpyruvate, competitively with resp ect to either prephenate or L-arogenate and non-competitively with respect to NAD(+). A variety of related compounds were tested as inhibitors, and th e minimal inhibitor structure was found to require only the aromatic ring a nd a hydroxyl substituent. Analysis by multiple alignment was used to compa re 17 protein sequences representing TyrA family members having catalytic d omains that are independent or fused to other catalytic domains, that exhib it broad substrate specificity or narrow substrate specificity, and that po ssess or lack sensitivity to endproduct inhibitors. We propose that the ent ire TyrA protein family lacks a discrete allosteric domain and that inhibit ors act competitively at the catalytic site of different family members whi ch exhibit individuality in the range and extent of molecules recognized as substrate or inhibitor. (C) 2000 Elsevier Science Ireland Ltd. All rights reserved.