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Superstoichiometric binding of L-Phe to phenylalanine hydroxylase from Caenorhabditis elegans: evolutionary implications

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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.

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Abbreviations

AAAH:

Aromatic amino acid hydroxylases

BH4 :

(6R)-L-erythro-5,6,7,8-tetrahydrobiopterin

cePAH:

Phenylalanine hydroxylase from C. elegans

hPAH:

Human phenylalanine hydroxylase

ITC:

Isothermal titration calorimetry

MD:

Molecular dynamics

PAH:

Phenylalanine hydroxylase

PDT:

Prephenate dehydratase

TH:

Tyrosine hydroxylase

T m :

Midpoint melting temperature

TPH:

Tryptophan hydroxylase

wt:

Wild type

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Acknowledgments

The expert assistance of Randi M. Svebak and Ali J. Muñoz Sepulveda is greatly appreciated. We are grateful to Anne Wiemhoefer and Thies Nolte for preliminary DSC experiments. This work received support from the Research Council of Norway and Meltzer L. Høyskolefond.

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Author notes

  1. Angel L. Pey

    Present address: Departamento de Quimica Fisica, Facultad de Ciencias, University of Granada, Av. Fuentenueva s/n, 18071, Granada, Spain

  2. Jessica Siltberg-Liberles

    Present address: Department of Molecular Biology, University of Wyoming, 1000 E. University Ave., Laramie, WY, 82071, USA

Authors and Affiliations

  1. Department of Biomedicine, University of Bergen, Jonas Lies vei 91, 5009, Bergen, Norway

    Marte I. Flydal, Tonje C. Mohn, Angel L. Pey, Jessica Siltberg-Liberles, Knut Teigen & Aurora Martinez

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  1. Marte I. Flydal
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Correspondence to Aurora Martinez.

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Flydal, M.I., Mohn, T.C., Pey, A.L. et al. Superstoichiometric binding of L-Phe to phenylalanine hydroxylase from Caenorhabditis elegans: evolutionary implications. Amino Acids 39, 1463–1475 (2010). https://doi.org/10.1007/s00726-010-0611-6

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