Delineation of the catalytic core of phenylalanine hydroxylase and identification of glutamate 286 as a critical residue for pterin function.
Rat phenylalanine hydroxylase was expressed in Escherichia coli. High level expression was achieved when the transformed E. coli were incubated at 27 degrees C for 24 h. A series of truncated fragments were expressed. The smallest fragment that gave an active soluble protein was from Leu142 to Phe410. This fragment corresponds closely to the region where there is highest homology between the three aromatic amino acid hydroxylases. The circular dichroism spectra of the phenylalanine hydroxylase catalytic core suggested that it contains around 50% alpha-helix. The core fragment is monomeric in dilute solutions but self-associates at higher concentrations. The E. coli expression system was used to generate a number of mutations in phenylalanine hydroxylase from position 264 to 290. This region had been previously shown to be important for pterin binding. Characterization of the mutant phenylalanine hydroxylase molecules identified Glu286 as an amino acid critical for pterin function in phenylalanine hydroxylase.
Dickson
P W
PW
Russell Grimwade Laboratory of Protein Engineering, Russell Grimwade School of Biochemistry, University of Melbourne, Parkville, Victoria, Australia.
Jennings
I G
IG
Cotton
R G
RG
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002384:Catalysis; D002942:Circular Dichroism; D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005971:Glutamates / Q000378:metabolism*; D018698:Glutamic Acid; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D010446:Peptide Fragments / Q000302:isolation & purification; D010651:Phenylalanine Hydroxylase / Q000737:chemistry / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011622:Pterins / Q000737:chemistry* / Q000378:metabolism; D051381:Rats; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D005971:Glutamates; D010446:Peptide Fragments; D011622:Pterins; D018698:Glutamic Acid; D010651:Phenylalanine Hydroxylase