Abstract
Phenylalanine hydroxylase converts phenylalanine to tyrosine, a rate-limiting step in phenylalanine catabolism and protein and neurotransmitter biosynthesis. It is tightly regulated by the substrates phenylalanine and tetrahydrobiopterin and by phosphorylation. We present the crystal structures of dephosphorylated and phosphorylated forms of a dimeric enzyme with catalytic and regulatory properties of the wild-type protein. The structures reveal a catalytic domain flexibly linked to a regulatory domain. The latter consists of an N-terminal autoregulatory sequence (containing Ser 16, which is the site of phosphorylation) that extends over the active site pocket, and an α-β sandwich core that is, unexpectedly, structurally related to both pterin dehydratase and the regulatory domains of metabolic enzymes. Phosphorylation has no major structural effects in the absence of phenylalanine, suggesting that phenylalanine and phosphorylation act in concert to activate the enzyme through a combination of intrasteric and possibly allosteric mechanisms.
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Acknowledgements
We thank J. Rossjohn and A. Oakley for help with synchrotron data collection, F. Katsis for the synthesis of heavy-atom substituted pterins, M. Parker for discussions, A. Perrakis for help with the wARP procedure, J. Hunt for automated density modification and refinement scripts, R. Read and B. Hazes for a version of SIGMAA, J. Varghese, B. Vandonkelaar, M. Lawrence and P. Colman for the xenon soaking device, personnel at the Photon Factory and DESY synchrotrons for help with data collection, and T. Teh for comments on the manuscript. We apologize to those whose work or original publication could not be cited because of space limitations. This work was supported by Australian Research Council (B.K.), W.M. Keck Foundation (R.C.S.) and NHMRC (R.G.H.C, B.E.K.); B.K. is a Wellcome Senior Research Fellow in Medical Science in Australia.
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Kobe, B., Jennings, I., House, C. et al. Structural basis of autoregulation of phenylalanine hydroxylase. Nat Struct Mol Biol 6, 442–448 (1999). https://doi.org/10.1038/8247
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DOI: https://doi.org/10.1038/8247
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