Expression of rat liver phenylalanine hydroxylase in insect cells and site-directed mutagenesis of putative non-heme iron-binding sites.
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 vector was active and identical in several properties to phenylalanine hydroxylase from rat liver: the Km for 6-methyltetrahydropterin was 39 microM (compared with 35 microM for the rat liver enzyme), 1 atom of iron was "associated" per enzyme subunit, and electron paramagnetic resonance spectra showed that iron was distributed within two distinct environments. Putative iron-binding sites of phenylalanine hydroxylase were studied by mutating either histidine 284 or 289 to serine and expressing these mutant enzymes (PAH-H284S and PAH-H289S) in Sf9 cells. Mutants were expressed at levels similar to wild-type PAH, but contained < or = 0.1 iron/subunit and were inactive. Thus, both His284 and His289 apparently are required for iron binding and hydroxylation activity.
Department of Chemistry, Pennsylvania State University, University Park 16802.
B S
BS
Gibbs
D
D
Wojchowski
S J
SJ
Benkovic
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D003001:Cloning, Molecular; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007501:Iron / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008667:Metalloproteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009036:Moths; D016297:Mutagenesis, Site-Directed; D019159:Nonheme Iron Proteins; D009838:Oligodeoxyribonucleotides; D010651:Phenylalanine Hydroxylase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014162:Transfection
D008667:Metalloproteins; D019159:Nonheme Iron Proteins; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D007501:Iron; D010651:Phenylalanine Hydroxylase