An allosterically insensitive class of cyclohexadienyl dehydrogenase from Zymomonas mobilis.

The key enzyme of tyrosine biosynthesis in many Gram-negative prokaryotes is cyclohexadienyl dehydrogenase. The Zymomonas mobilis gene (tyrC) coding for this enzyme was cloned in Escherichia coli by complementation of a tyrosine auxotroph. The tyrC gene was 882 bp long, encoding a protein with a calculated molecular mass of 32086 Da. The Z. mobilis cyclohexadienyl dehydrogenase expressed in E. coli was purified to electrophoretic homogeneity. The subunit molecular mass of the purified enzyme was 32 kDa as determined by SDS/PAGE. The ratio of the activity of arogenate dehydrogenase to that of prephenate dehydrogenase (approximately 3:1) remained constant throughout purification, and the two activities were therefore inseparable. The genetic and biochemical data obtained demonstrated a single enzyme protein capable of catalyzing either of two reactions. Km values of 0.25 mM and 0.18 mM were obtained from prephenate and L-arogenate, respectively. The Km value obtained for NAD+ (0.09 mM) was the same regardless of whether the enzyme was assayed as arogenate dehydrogenase or as prephenate dehydrogenase. Unlike the corresponding enzyme of Pseudomonas aeruginosa or E. coli, the cyclohexadienyl dehydrogenase of Z. mobilis lacks sensitivity to feedback inhibition by L-tyrosine. A typical NAD(+)-binding domain was found to be located at the N-terminus of the protein. Although the deduced amino-acid sequence of the Z. mobilis cyclohexadienyl dehydrogenase showed relatively low identity (19-32%) with the prephenate dehydrogenases of Bacillus subtilis and Saccharomyces cerevisiae, as well as with the cyclohexadienyl dehydrogenase components of the bifunctional T-proteins of E. coli and Erwinia herbicola, a presumptive motif was identified which may correspond to critical residues of the binding site for cyclohexadienyl substrate molecules. Immediately upstream of tryC a portion of a gene was sequenced and found to exhibit clearcut homology of the deduced amino-acid sequence with the B. subtilis hisH gene product. Thus, the Zymomonas gene organization is reminiscent of the linkage of genes encoding a tryosine-pathway dehydrogenase and a histidine-pathway aminotransferase in B. subtilis.