Open AccessPKU mutation p.G46S prevents the stereospecific binding of l ‐phenylalanine to the dimer of human phenylalanine hydroxylase regulatory domain
Author(s) -
Leandro João,
Saraste Jaakko,
Leandro Paula,
Flatmark Torgeir
Publication year - 2017
Publication title -
febs open bio
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.718
H-Index - 31
ISSN - 2211-5463
DOI - 10.1002/2211-5463.12175
Subject(s) - phenylalanine hydroxylase , chemistry , phenylalanine , allosteric regulation , dimer , fusion protein , mutant , stereochemistry , stereospecificity , biochemistry , amino acid , enzyme , recombinant dna , catalysis , gene , organic chemistry
Mammalian phenylalanine hydroxylase (PAH) has a potential allosteric regulatory binding site for l ‐phenylalanine ( l ‐Phe), in addition to its catalytic site. This arrangement is supported by a crystal structure of a homodimeric truncated form of the regulatory domain of human PAH ( hPAH ‐ RD 1–118/19–118 ) [Patel D et al . (2016) Sci Rep doi: 10.1038/srep23748 ]. In this study, a fusion protein of the domain ( MBP ‐(pep Xa )‐ hPAH ‐ RD 1–120 ) was overexpressed and recovered in a metastable and soluble state, which allowed the isolation of a dimeric and a monomeric fusion protein. When cleaved from MBP , hPAH ‐ RD forms aggregates which are stereospecifically inhibited by l ‐Phe (> 95%) at low physiological concentrations. Aggregation of the cleaved dimer of the mutant form hPAH ‐G46S‐ RD was not inhibited by l ‐Phe, which is compatible with structurally/conformationally changed βαββαβ ACT domain folds in the mutant.