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Title: First structure of full-length mammalian phenylalanine hydroxylase reveals the architecture of an autoinhibited tetramer

Abstract

Improved understanding of the relationship among structure, dynamics, and function for the enzyme phenylalanine hydroxylase (PAH) can lead to needed new therapies for phenylketonuria, the most common inborn error of amino acid metabolism. PAH is a multidomain homo-multimeric protein whose conformation and multimerization properties respond to allosteric activation by the substrate phenylalanine (Phe); the allosteric regulation is necessary to maintain Phe below neurotoxic levels. A recently introduced model for allosteric regulation of PAH involves major domain motions and architecturally distinct PAH tetramers [Jaffe EK, Stith L, Lawrence SH, Andrake M, Dunbrack RL, Jr (2013) Arch Biochem Biophys 530(2):73–82]. Herein, we present, to our knowledge, the first X-ray crystal structure for a full-length mammalian (rat) PAH in an autoinhibited conformation. Chromatographic isolation of a monodisperse tetrameric PAH, in the absence of Phe, facilitated determination of the 2.9 Å crystal structure. The structure of full-length PAH supersedes a composite homology model that had been used extensively to rationalize phenylketonuria genotype–phenotype relationships. Small-angle X-ray scattering (SAXS) confirms that this tetramer, which dominates in the absence of Phe, is different from a Phe-stabilized allosterically activated PAH tetramer. The lack of structural detail for activated PAH remains a barrier to complete understanding of phenylketonuria genotype–phenotypemore » relationships. Nevertheless, the use of SAXS and X-ray crystallography together to inspect PAH structure provides, to our knowledge, the first complete view of the enzyme in a tetrameric form that was not possible with prior partial crystal structures, and facilitates interpretation of a wealth of biochemical and structural data that was hitherto impossible to evaluate.« less

Authors:
; ; ; ; ; ; ;
Publication Date:
Research Org.:
Brookhaven National Lab. (BNL), Upton, NY (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES); USDOE Office of Science (SC), Biological and Environmental Research (BER); National Cancer Institute Comprehensive Cancer Center; National Center for Research Resources of the National Inst. of Health
OSTI Identifier:
1238204
Alternate Identifier(s):
OSTI ID: 1354369
Report Number(s):
BNL-112885-2016-JA
Journal ID: ISSN 0027-8424; /pnas/113/9/2394.atom
Grant/Contract Number:  
AC02-98CH10886; SC00112704; P41GM103473; P41RR012408; P30CA006927
Resource Type:
Published Article
Journal Name:
Proceedings of the National Academy of Sciences of the United States of America
Additional Journal Information:
Journal Name: Proceedings of the National Academy of Sciences of the United States of America Journal Volume: 113 Journal Issue: 9; Journal ID: ISSN 0027-8424
Publisher:
National Academy of Sciences
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; phenylalanine hydroxylase; phenylketonuria; X-ray crystallography; small-angle X-ray scattering; allosteric regulation

Citation Formats

Arturo, Emilia C., Gupta, Kushol, Héroux, Annie, Stith, Linda, Cross, Penelope J., Parker, Emily J., Loll, Patrick J., and Jaffe, Eileen K. First structure of full-length mammalian phenylalanine hydroxylase reveals the architecture of an autoinhibited tetramer. United States: N. p., 2016. Web. doi:10.1073/pnas.1516967113.
Arturo, Emilia C., Gupta, Kushol, Héroux, Annie, Stith, Linda, Cross, Penelope J., Parker, Emily J., Loll, Patrick J., & Jaffe, Eileen K. First structure of full-length mammalian phenylalanine hydroxylase reveals the architecture of an autoinhibited tetramer. United States. doi:10.1073/pnas.1516967113.
Arturo, Emilia C., Gupta, Kushol, Héroux, Annie, Stith, Linda, Cross, Penelope J., Parker, Emily J., Loll, Patrick J., and Jaffe, Eileen K. Tue . "First structure of full-length mammalian phenylalanine hydroxylase reveals the architecture of an autoinhibited tetramer". United States. doi:10.1073/pnas.1516967113.
@article{osti_1238204,
title = {First structure of full-length mammalian phenylalanine hydroxylase reveals the architecture of an autoinhibited tetramer},
author = {Arturo, Emilia C. and Gupta, Kushol and Héroux, Annie and Stith, Linda and Cross, Penelope J. and Parker, Emily J. and Loll, Patrick J. and Jaffe, Eileen K.},
abstractNote = {Improved understanding of the relationship among structure, dynamics, and function for the enzyme phenylalanine hydroxylase (PAH) can lead to needed new therapies for phenylketonuria, the most common inborn error of amino acid metabolism. PAH is a multidomain homo-multimeric protein whose conformation and multimerization properties respond to allosteric activation by the substrate phenylalanine (Phe); the allosteric regulation is necessary to maintain Phe below neurotoxic levels. A recently introduced model for allosteric regulation of PAH involves major domain motions and architecturally distinct PAH tetramers [Jaffe EK, Stith L, Lawrence SH, Andrake M, Dunbrack RL, Jr (2013) Arch Biochem Biophys 530(2):73–82]. Herein, we present, to our knowledge, the first X-ray crystal structure for a full-length mammalian (rat) PAH in an autoinhibited conformation. Chromatographic isolation of a monodisperse tetrameric PAH, in the absence of Phe, facilitated determination of the 2.9 Å crystal structure. The structure of full-length PAH supersedes a composite homology model that had been used extensively to rationalize phenylketonuria genotype–phenotype relationships. Small-angle X-ray scattering (SAXS) confirms that this tetramer, which dominates in the absence of Phe, is different from a Phe-stabilized allosterically activated PAH tetramer. The lack of structural detail for activated PAH remains a barrier to complete understanding of phenylketonuria genotype–phenotype relationships. Nevertheless, the use of SAXS and X-ray crystallography together to inspect PAH structure provides, to our knowledge, the first complete view of the enzyme in a tetrameric form that was not possible with prior partial crystal structures, and facilitates interpretation of a wealth of biochemical and structural data that was hitherto impossible to evaluate.},
doi = {10.1073/pnas.1516967113},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
number = 9,
volume = 113,
place = {United States},
year = {2016},
month = {2}
}

Journal Article:
Free Publicly Available Full Text
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DOI: 10.1073/pnas.1516967113

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Cited by: 9 works
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Works referenced in this record:

GNOM – a program package for small-angle scattering data processing
journal, October 1991


Phenylalanine hydroxylase: Function, structure, and regulation
journal, March 2013

  • Flydal, Marte I.; Martinez, Aurora
  • IUBMB Life, Vol. 65, Issue 4
  • DOI: 10.1002/iub.1150

Dynamic dissociating homo-oligomers and the control of protein function
journal, March 2012

  • Selwood, Trevor; Jaffe, Eileen K.
  • Archives of Biochemistry and Biophysics, Vol. 519, Issue 2
  • DOI: 10.1016/j.abb.2011.11.020

Architecture and Assembly of HIV Integrase Multimers in the Absence of DNA Substrates
journal, January 2013

  • Bojja, Ravi Shankar; Andrake, Mark D.; Merkel, George
  • Journal of Biological Chemistry, Vol. 288, Issue 10
  • DOI: 10.1074/jbc.M112.434431

Structural and stability effects of phosphorylation: Localized structural changes in phenylalanine hydroxylase
journal, May 2004

  • Miranda, Frederico Faria; Thórólfsson, Matthías; Teigen, Knut
  • Protein Science, Vol. 13, Issue 5
  • DOI: 10.1110/ps.03595904

Iterative model building, structure refinement and density modification with the PHENIX AutoBuild wizard
journal, December 2007

  • Terwilliger, Thomas C.; Grosse-Kunstleve, Ralf W.; Afonine, Pavel V.
  • Acta Crystallographica Section D Biological Crystallography, Vol. 64, Issue 1
  • DOI: 10.1107/S090744490705024X

CRYSOL – a Program to Evaluate X-ray Solution Scattering of Biological Macromolecules from Atomic Coordinates
journal, December 1995

  • Svergun, D.; Barberato, C.; Koch, M. H. J.
  • Journal of Applied Crystallography, Vol. 28, Issue 6
  • DOI: 10.1107/S0021889895007047

Cation-pi interactions in structural biology
journal, August 1999

  • Gallivan, J. P.; Dougherty, D. A.
  • Proceedings of the National Academy of Sciences, Vol. 96, Issue 17
  • DOI: 10.1073/pnas.96.17.9459

Phenylalanine Binding Is Linked to Dimerization of the Regulatory Domain of Phenylalanine Hydroxylase
journal, October 2014

  • Zhang, Shengnan; Roberts, Kenneth M.; Fitzpatrick, Paul F.
  • Biochemistry, Vol. 53, Issue 42
  • DOI: 10.1021/bi501109s

Features and development of Coot
journal, March 2010

  • Emsley, P.; Lohkamp, B.; Scott, W. G.
  • Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 4
  • DOI: 10.1107/S0907444910007493

The Uppsala Electron-Density Server
journal, November 2004

  • Kleywegt, Gerard J.; Harris, Mark R.; Zou, Jin-yu
  • Acta Crystallographica Section D Biological Crystallography, Vol. 60, Issue 12
  • DOI: 10.1107/S0907444904013253

Linking genotypes database with locus-specific database and genotype–phenotype correlation in phenylketonuria
journal, June 2014

  • Wettstein, Sarah; Underhaug, Jarl; Perez, Belen
  • European Journal of Human Genetics, Vol. 23, Issue 3
  • DOI: 10.1038/ejhg.2014.114

A new model for allosteric regulation of phenylalanine hydroxylase: Implications for disease and therapeutics
journal, February 2013

  • Jaffe, Eileen K.; Stith, Linda; Lawrence, Sarah H.
  • Archives of Biochemistry and Biophysics, Vol. 530, Issue 2
  • DOI: 10.1016/j.abb.2012.12.017

Loss of Function in Phenylketonuria Is Caused by Impaired Molecular Motions and Conformational Instability
journal, July 2008

  • Gersting, Søren W.; Kemter, Kristina F.; Staudigl, Michael
  • The American Journal of Human Genetics, Vol. 83, Issue 1
  • DOI: 10.1016/j.ajhg.2008.05.013

Comprehensive macromolecular conformations mapped by quantitative SAXS analyses
journal, April 2013

  • Hura, Greg L.; Budworth, Helen; Dyer, Kevin N.
  • Nature Methods, Vol. 10, Issue 6
  • DOI: 10.1038/nmeth.2453

New developments in the ATSAS program package for small-angle scattering data analysis
journal, March 2012

  • Petoukhov, Maxim V.; Franke, Daniel; Shkumatov, Alexander V.
  • Journal of Applied Crystallography, Vol. 45, Issue 2
  • DOI: 10.1107/S0021889812007662

Towards automated crystallographic structure refinement with phenix.refine
journal, March 2012

  • Afonine, Pavel V.; Grosse-Kunstleve, Ralf W.; Echols, Nathaniel
  • Acta Crystallographica Section D Biological Crystallography, Vol. 68, Issue 4
  • DOI: 10.1107/S0907444912001308

Tyrosine Latching of a Regulatory Gate Affords Allosteric Control of Aromatic Amino Acid Biosynthesis
journal, January 2011

  • Cross, Penelope J.; Dobson, Renwick C. J.; Patchett, Mark L.
  • Journal of Biological Chemistry, Vol. 286, Issue 12
  • DOI: 10.1074/jbc.M110.209924

PHENIX: a comprehensive Python-based system for macromolecular structure solution
journal, January 2010

  • Adams, Paul D.; Afonine, Pavel V.; Bunkóczi, Gábor
  • Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 2, p. 213-221
  • DOI: 10.1107/S0907444909052925

Mechanism of Aromatic Amino Acid Hydroxylation
journal, December 2003


A low-background-intensity focusing small-angle X-ray scattering undulator beamline
journal, November 2013

  • Kirby, Nigel M.; Mudie, Stephen T.; Hawley, Adrian M.
  • Journal of Applied Crystallography, Vol. 46, Issue 6
  • DOI: 10.1107/S002188981302774X

ALAD Porphyria Is a Conformational Disease
journal, February 2007

  • Jaffe, Eileen K.; Stith, Linda
  • The American Journal of Human Genetics, Vol. 80, Issue 2
  • DOI: 10.1086/511444

Structural Rearrangement of Ebola Virus VP40 Begets Multiple Functions in the Virus Life Cycle
journal, August 2013


Activation of Phenylalanine Hydroxylase by Phenylalanine Does Not Require Binding in the Active Site
journal, December 2014

  • Roberts, Kenneth M.; Khan, Crystal A.; Hinck, Cynthia S.
  • Biochemistry, Vol. 53, Issue 49
  • DOI: 10.1021/bi501183x

Direct evidence for a phenylalanine site in the regulatory domain of phenylalanine hydroxylase
journal, January 2011

  • Li, Jun; Ilangovan, Udayar; Daubner, S. Colette
  • Archives of Biochemistry and Biophysics, Vol. 505, Issue 2
  • DOI: 10.1016/j.abb.2010.10.009

Iterative-build OMIT maps: map improvement by iterative model building and refinement without model bias
journal, April 2008

  • Terwilliger, Thomas C.; Grosse-Kunstleve, Ralf W.; Afonine, Pavel V.
  • Acta Crystallographica Section D Biological Crystallography, Vol. 64, Issue 5
  • DOI: 10.1107/S0907444908004319

Kinetic Mechanism of Phenylalanine Hydroxylase: Intrinsic Binding and Rate Constants from Single-Turnover Experiments
journal, January 2013

  • Roberts, Kenneth M.; Pavon, Jorge Alex; Fitzpatrick, Paul F.
  • Biochemistry, Vol. 52, Issue 6
  • DOI: 10.1021/bi301675e

The Structural Basis of Phenylketonuria
journal, October 1999

  • Erlandsen, Heidi; Stevens, Raymond C.
  • Molecular Genetics and Metabolism, Vol. 68, Issue 2
  • DOI: 10.1006/mgme.1999.2922

Improving physical realism, stereochemistry, and side-chain accuracy in homology modeling: Four approaches that performed well in CASP8
journal, January 2009

  • Krieger, Elmar; Joo, Keehyoung; Lee, Jinwoo
  • Proteins: Structure, Function, and Bioinformatics, Vol. 77, Issue S9
  • DOI: 10.1002/prot.22570

Single Amino Acid Mutations Alter the Distribution of Human Porphobilinogen Synthase Quaternary Structure Isoforms (Morpheeins)
journal, December 2005

  • Tang, Lei; Breinig, Sabine; Stith, Linda
  • Journal of Biological Chemistry, Vol. 281, Issue 10
  • DOI: 10.1074/jbc.M511134200

FEM: feature-enhanced map
journal, February 2015

  • Afonine, Pavel V.; Moriarty, Nigel W.; Mustyakimov, Marat
  • Acta Crystallographica Section D Biological Crystallography, Vol. 71, Issue 3
  • DOI: 10.1107/S1399004714028132

Control of tetrapyrrole biosynthesis by alternate quaternary forms of porphobilinogen synthase
journal, August 2003

  • Breinig, Sabine; Kervinen, Jukka; Stith, Linda
  • Nature Structural & Molecular Biology, Vol. 10, Issue 9
  • DOI: 10.1038/nsb963

Validation of metal-binding sites in macromolecular structures with the CheckMyMetal web server
journal, December 2013

  • Zheng, Heping; Chordia, Mahendra D.; Cooper, David R.
  • Nature Protocols, Vol. 9, Issue 1
  • DOI: 10.1038/nprot.2013.172

The Solution Structure of the Regulatory Domain of Tyrosine Hydroxylase
journal, April 2014

  • Zhang, Shengnan; Huang, Tao; Ilangovan, Udayar
  • Journal of Molecular Biology, Vol. 426, Issue 7
  • DOI: 10.1016/j.jmb.2013.12.015

Integration, scaling, space-group assignment and post-refinement
journal, January 2010

  • Kabsch, Wolfgang
  • Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 2, p. 133-144
  • DOI: 10.1107/S0907444909047374

Allostery and the dynamic oligomerization of porphobilinogen synthase
journal, March 2012

  • Jaffe, Eileen K.; Lawrence, Sarah H.
  • Archives of Biochemistry and Biophysics, Vol. 519, Issue 2
  • DOI: 10.1016/j.abb.2011.10.010

Allosteric regulation of phenylalanine hydroxylase
journal, March 2012


PAHdb: A locus-specific knowledgebase
journal, January 2000


Computational study of missense mutations in phenylalanine hydroxylase
journal, March 2015

  • Réblová, Kamila; Kulhánek, Petr; Fajkusová, Lenka
  • Journal of Molecular Modeling, Vol. 21, Issue 4
  • DOI: 10.1007/s00894-015-2620-6

A Flexible Loop in Tyrosine Hydroxylase Controls Coupling of Amino Acid Hydroxylation to Tetrahydropterin Oxidation
journal, June 2006

  • Colette Daubner, S.; McGinnis, James Thomas; Gardner, Meredith
  • Journal of Molecular Biology, Vol. 359, Issue 2
  • DOI: 10.1016/j.jmb.2006.03.016

Structure of Tetrameric Human Phenylalanine Hydroxylase and Its Implications for Phenylketonuria
journal, July 1998

  • Fusetti, Fabrizia; Erlandsen, Heidi; Flatmark, Torgeir
  • Journal of Biological Chemistry, Vol. 273, Issue 27
  • DOI: 10.1074/jbc.273.27.16962

Phaser crystallographic software
journal, July 2007

  • McCoy, Airlie J.; Grosse-Kunstleve, Ralf W.; Adams, Paul D.
  • Journal of Applied Crystallography, Vol. 40, Issue 4
  • DOI: 10.1107/S0021889807021206

Leucine zippers and coiled-coils in the aromatic amino acid hydroxylases
journal, January 1991


Crystal structure of the catalytic domain of human phenylalanine hydroxylase reveals the structural basis for phenylketonuria
journal, December 1997

  • Erlandsen, Heidi; Fusetti, Fabrizia; Martinez, Aurora
  • Nature Structural Biology, Vol. 4, Issue 12
  • DOI: 10.1038/nsb1297-995

    Works referencing / citing this record:

    Genetics of Phenylketonuria: Then and Now
    journal, March 2016


    Toward mechanistic models for genotype–phenotype correlations in phenylketonuria using protein stability calculations
    journal, January 2019

    • Scheller, Rasmus; Stein, Amelie; Nielsen, Sofie V.
    • Human Mutation, Vol. 40, Issue 4
    • DOI: 10.1002/humu.23707

    Mutational and phenotypic spectrum of phenylalanine hydroxylase deficiency in Zhejiang Province, China
    journal, November 2018


    Structure of full-length wild-type human phenylalanine hydroxylase by small angle X-ray scattering reveals substrate-induced conformational stability
    journal, September 2019


    Stable preparations of tyrosine hydroxylase provide the solution structure of the full-length enzyme
    journal, July 2016

    • Bezem, Maria T.; Baumann, Anne; Skjærven, Lars
    • Scientific Reports, Vol. 6, Issue 1
    • DOI: 10.1038/srep30390

    Structure of full-length human phenylalanine hydroxylase in complex with tetrahydrobiopterin
    journal, May 2019

    • Flydal, Marte Innselset; Alcorlo-Pagés, Martín; Johannessen, Fredrik Gullaksen
    • Proceedings of the National Academy of Sciences, Vol. 116, Issue 23
    • DOI: 10.1073/pnas.1902639116

    Simulations of the regulatory ACT domain of human phenylalanine hydroxylase (PAH) unveil its mechanism of phenylalanine binding
    journal, October 2018

    • Ge, Yunhui; Borne, Elias; Stewart, Shannon
    • Journal of Biological Chemistry, Vol. 293, Issue 51
    • DOI: 10.1074/jbc.ra118.004909

    The phenylketonuria-associated substitution R68S converts phenylalanine hydroxylase to a constitutively active enzyme but reduces its stability
    journal, January 2019

    • Khan, Crystal A.; Meisburger, Steve P.; Ando, Nozomi
    • Journal of Biological Chemistry, Vol. 294, Issue 12
    • DOI: 10.1074/jbc.ra118.006477

    Biophysical characterization of full-length human phenylalanine hydroxylase provides a deeper understanding of its quaternary structure equilibrium
    journal, May 2019

    • Arturo, Emilia C.; Gupta, Kushol; Hansen, Michael R.
    • Journal of Biological Chemistry, Vol. 294, Issue 26
    • DOI: 10.1074/jbc.ra119.008294

    The quaternary structure of human tyrosine hydroxylase: effects of dystonia‐associated missense variants on oligomeric state and enzyme activity
    journal, December 2018

    • Szigetvari, Peter D.; Muruganandam, Gopinath; Kallio, Juha P.
    • Journal of Neurochemistry, Vol. 148, Issue 2
    • DOI: 10.1111/jnc.14624

    Structural and Functional Impact of Seven Missense Variants of Phenylalanine Hydroxylase
    journal, June 2019

    • Pecimonova, Martina; Kluckova, Daniela; Csicsay, Frantisek
    • Genes, Vol. 10, Issue 6
    • DOI: 10.3390/genes10060459