A single cyclohexadienyl dehydrogenase specifies the prephenate dehydrogenase and arogenate dehydrogenase components of the dual pathways to L-tyrosine in Pseudomonas aeruginosa.
Dual biosynthetic pathways diverge from prephenate to L-tyrosine in Pseudomonas aeruginosa, with 4-hydroxyphenylpyruvate and L-arogenate being the unique intermediates of these pathways. Prephenate dehydrogenase and arogenate dehydrogenase activities could not be separated throughout fractionation steps yielding a purification of more than 200-fold, and the ratio of activities was constant throughout purification. Thus, the enzyme is a cyclohexadienyl dehydrogenase. The native enzyme has a molecular weight of 150,000 and is a hexamer made up of identical 25,500 subunits. The enzyme is specific for NAD+ as an electron acceptor, and identical Km values of 0.25 mM were obtained for NAD+, regardless of whether activity was assayed as prephenate dehydrogenase or as arogenate dehydrogenase. Km values of 0.07 mM and 0.17 mM were calculated for prephenate and L-arogenate, respectively. Inhibition by L-tyrosine was noncompetitive with respect to NAD+, but was strictly competitive with respect to either prephenate or L-arogenate. With cyclohexadiene as variable substrate, similar Ki values for L-tyrosine of 0.06 mM (prephenate) and 0.05 mM (L-arogenate) were obtained. With NAD+ as the variable substrate, similar Ki values for L-tyrosine of 0.26 mM (prephenate) and 0.28 mM (L-arogenate), respectively, were calculated. This is the first characterization of a purified, monofunctional cyclohexadienyl dehydrogenase.
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Department of Microbiology and Cell Science, University of Florida, Gainesville 32611-0100.
Jensen
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D000600:Amino Acids, Dicarboxylic / Q000378:metabolism; D002384:Catalysis; D003509:Cyclohexanecarboxylic Acids / Q000378:metabolism; D053138:Cyclohexenes; D046911:Macromolecular Substances; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D010088:Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011303:Prephenate Dehydrogenase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D014443:Tyrosine / Q000031:analogs & derivatives / Q000096:biosynthesis* / Q000378:metabolism / Q000494:pharmacology
D000600:Amino Acids, Dicarboxylic; D003509:Cyclohexanecarboxylic Acids; D053138:Cyclohexenes; D046911:Macromolecular Substances; D009243:NAD; D014443:Tyrosine; C015478:pretyrosine; D010088:Oxidoreductases; D011303:Prephenate Dehydrogenase; C027017:cyclohexadienyl dehydrogenase; C005550:prephenic acid