title: Prephenate dehydratase of the actinomycete Amycolatopsis methanolica: purification and characterization of wild-type and deregulated mutant proteins.
abstract: Prephenate dehydratase (PDT) is a key regulatory enzyme in L-phenylalanine biosynthesis in the Gram-positive bacterium Amycolatopsis methanolica. The PDT protein was purified to homogeneity (1957-fold) from wild-type cells with a final yield of 6.5%. It was characterized as a 150 kDa homotetrameric protein with a subunit size of 34 kDa. The first 35 N-terminal amino acids were identified, revealing highest similarity to the PDT proteins from Corynebacterium glutamicum and Bacillus subtilis. Kinetic studies showed that the A. methanolica PDT is allosterically inhibited by phenylalanine and activated by tyrosine. Phenylalanine caused an increase in the S0.5 for prephenate and a decrease in the Vmax. Tyrosine caused a decrease in the S0.5 for prephenate and an increase in the Vmax. Spontaneous o-fluoro- and p-fluoro-DL-phenylalanine-resistant mutants of A. methanolica were isolated. Kinetic studies with the partially purified PDT proteins of strains pFPhe32 and oFPhe84 showed that these mutant proteins had become (partly) insensitive to both phenylalanine inhibition and tyrosine activation.
authors.affiliation: Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Haren, The Netherlands.
authors.forename: G J
authors.initials: GJ
authors.lastname: Euverink
authors.forename: D J
authors.initials: DJ
authors.lastname: Wolters
authors.forename: L
authors.initials: L
authors.lastname: Dijkhuizen
mesh_terms: D039903:Actinobacteria / Q000201:enzymology*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D007700:Kinetics; D008969:Molecular Sequence Data; D010649:Phenylalanine / Q000494:pharmacology; D011302:Prephenate Dehydratase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014443:Tyrosine / Q000494:pharmacology
chemical_list: D001426:Bacterial Proteins; D014443:Tyrosine; D010649:Phenylalanine; D011302:Prephenate Dehydratase