title: A multispecific quintet of aromatic aminotransferases that overlap different biochemical pathways in Pseudomonas aeruginosa.
abstract: Pseudomonas aeruginosa possesses dual enzymatic sequences to both L-phenylalanine and L-tyrosine, a biosynthetic arrangement further complicated by the presence of five aromatic aminotransferases. Each aminotransferase is capable of transamination in vitro with any of the three keto acid intermediates in the aromatic pathway (phenylpyruvate, 4-hydroxyphenylpyruvate, or prephenate). The fractional contribution of these aminotransferases to particular transamination reactions in vivo can best be approached through the systematic and sequential elimination of individual aminotransferase activities by mutation. A program of sequential mutagenesis has produced two aminotransferase-deficient mutations. The first mutation imposed a phenotype of bradytrophy for L-phenylalanine (doubling time of 2.4 h in minimal salts/glucose medium compared to a 1.0-h doubling time for wild type). This mutant completely lacked an enzyme denoted aminotransferase AT-2. A genetic background of aminotransferase AT-2 deficiency was used to select for a second mutation which produced a phenotype of multiple auxotrophy for L-phenylalanine, L-aspartate, and L-glutamate. The double mutant completely lacked activity for aromatic aminotransferase AT-1 in addition to the missing aminotransferase AT-2. Enzymes AT-1 (Mr = 64,000) and AT-2 (Mr = 50,000) were readily separated from one another by gel filtration and were individually characterized for pH optima, freeze-thaw stability, heat lability, and molecular weight. The phenotypic and enzymological characterizations of the aminotransferase mutants strongly support the primary in vivo role of enzyme AT-2 in L-phenylalanine and L-tyrosine biosynthesis, while enzyme AT-1 must primarily be engaged in L-aspartate and L-glutamate synthesis. The substrate specificities and possible in vivo functions for AT-3, AT-4, and AT-5 are also considered.
mesh_terms: D001219:Aspartate Aminotransferases / Q000378:metabolism; D051276:Leucine Transaminase; D009154:Mutation; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D000637:Transaminases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014444:Tyrosine Transaminase / Q000378:metabolism
chemical_list: D000637:Transaminases; C036402:glutamate aminotransferase; C022340:phenylalanine aminotransferase; D001219:Aspartate Aminotransferases; D014444:Tyrosine Transaminase; D051276:Leucine Transaminase