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

B. S. Gibbs, D. Wojchowski, S. J. Benkovic

Research output: Contribution to journalArticle

43 Scopus citations

Abstract

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 K(m) for 6-methyltetrahydropterin was 39 μM (compared with 35 μM 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 ≤0.1 iron/subunit and were inactive. Thus, both His284 and His289 apparently are required for iron binding and hydroxylation activity.

Original languageEnglish (US)
Pages (from-to)8046-8052
Number of pages7
JournalJournal of Biological Chemistry
Volume268
Issue number11
StatePublished - Jan 1 1993

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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