title: Superstoichiometric binding of L-Phe to phenylalanine hydroxylase from Caenorhabditis elegans: evolutionary implications.
abstract: Phenylalanine hydroxylase (PAH) catalyzes the hydroxylation of L-Phe to L-Tyr. Dysfunctional PAH results in phenylketonuria and mammalian PAH is therefore highly regulated and displays positive cooperativity for L-Phe (Hill coefficient (h)=2). L-Phe does not bind to the regulatory ACT domain in full-length tetrameric human PAH and cooperativity is elicited by homotropic binding to the catalytic site (Thórólfsson et al. in Biochemistry 41:7573-7585, 2002). PAH from Caenorhabditis elegans (cePAH) is devoid of cooperativity for L-Phe (h=0.9), and, as shown in this work, structural analysis reveal an additional L-Phe binding site at the regulatory domain of full-length cePAH. This site involves the GA(S)L/ISRP motifs, which are also found in ACT domains of other L-Phe binding proteins, such as prephenate dehydratase. Isothermal titration calorimetry further demonstrated 2 binding sites per subunit for cePAH versus ~1 for hPAH. Steric occlusion of the regulatory site, notably by residues Lys215/Tyr216 from the adjacent catalytic domain, appears to hinder regulatory binding in full-length hPAH. Accordingly, the humanized mutant Q215K/N216Y of cePAH binds ~1.4 L-Phe/subunit. This mutant also displays high catalytic activity and certain positive cooperativity for L-Phe (h=1.4). Our results support that the acquisition of positive cooperativity in mammalian forms of PAH is accompanied by a closure of the regulatory L: -Phe binding site. Concomitantly, the function of the regulatory ACT domain appears to be adapted from amino acid binding to serving the communication of conformational changes among catalytic subunits.
authors.affiliation: Department of Biomedicine, University of Bergen, Jonas Lies vei 91, 5009, Bergen, Norway.
authors.forename: Marte I
authors.initials: MI
authors.lastname: Flydal
authors.forename: Tonje C
authors.initials: TC
authors.lastname: Mohn
authors.forename: Angel L
authors.initials: AL
authors.lastname: Pey
authors.forename: Jessica
authors.initials: J
authors.lastname: Siltberg-Liberles
authors.forename: Knut
authors.initials: K
authors.lastname: Teigen
authors.forename: Aurora
authors.initials: A
authors.lastname: Martinez
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D055162:Biocatalysis; D017173:Caenorhabditis elegans / Q000201:enzymology*; D002151:Calorimetry; D002942:Circular Dichroism; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010649:Phenylalanine / Q000737:chemistry*; D010651:Phenylalanine Hydroxylase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013237:Stereoisomerism
chemical_list: D010649:Phenylalanine; D010651:Phenylalanine Hydroxylase