Expression of rat liver phenylalanine hydroxylase in insect cells and site-directed mutagenesis of putative non-heme iron-binding sites

• Published in: The Journal of biological chemistry Vol. 268; no. 11; pp. 8046 - 8052
• Main Authors: GIBBS, B. S, WOJCHOWSKI, D, BENKOVIC, S. J
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 15.04.1993
• Subjects: Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Animals; Base Sequence; Binding Sites; Biological and medical sciences; Cell Line; Cloning, Molecular; Electron Spin Resonance Spectroscopy; Electrophoresis, Polyacrylamide Gel; Enzymes and enzyme inhibitors; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Humans; Iron - metabolism; Kinetics; Liver - enzymology; Metalloproteins - genetics; Metalloproteins - isolation & purification; Metalloproteins - metabolism; Molecular Sequence Data; Molecular Weight; Moths; Mutagenesis, Site-Directed; Nonheme Iron Proteins; Oligodeoxyribonucleotides; Oxidoreductases; Phenylalanine Hydroxylase - genetics; Phenylalanine Hydroxylase - isolation & purification; Phenylalanine Hydroxylase - metabolism; Rats; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Sequence Homology, Amino Acid; Transfection; Vertebrata; Mammalia; Enzymatic activity; Phenylalanine 4-monooxygenase; Rat; Structure activity relation; Enzyme; Rodentia; Site directed mutagenesis; Iron; Binding site; Recombinant protein

Rat liver phenylalanine hydroxylase was expressed in both Escherichia coli and the Spodoptera frugiperda insect cell line, Sf9. Recombinant enzyme from E. coli was inactive and contained less than 0.1 iron atom/subunit. In contrast, recombinant enzyme expressed in Sf9 cells using a baculovirus vecto...