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 vecto...
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Published in | The Journal of biological chemistry Vol. 268; no. 11; pp. 8046 - 8052 |
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Main Authors | , , |
Format | Journal Article |
Language | English |
Published |
Bethesda, MD
American Society for Biochemistry and Molecular Biology
15.04.1993
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Subjects | |
Online Access | Get full text |
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Summary: | 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. |
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ISSN: | 0021-9258 1083-351X |
DOI: | 10.1016/S0021-9258(18)53061-1 |