title: Stable preparations of tyrosine hydroxylase provide the solution structure of the full-length enzyme.
abstract: Tyrosine hydroxylase (TH) catalyzes the rate-limiting step in the biosynthesis of catecholamine neurotransmitters. TH is a highly complex enzyme at mechanistic, structural, and regulatory levels, and the preparation of kinetically and conformationally stable enzyme for structural characterization has been challenging. Here, we report on improved protocols for purification of recombinant human TH isoform 1 (TH1), which provide large amounts of pure, stable, active TH1 with an intact N-terminus. TH1 purified through fusion with a His-tagged maltose-binding protein on amylose resin was representative of the iron-bound functional enzyme, showing high activity and stabilization by the natural feedback inhibitor dopamine. TH1 purified through fusion with a His-tagged ZZ domain on TALON is remarkably stable, as it was partially inhibited by resin-derived cobalt. This more stable enzyme preparation provided high-quality small-angle X-ray scattering (SAXS) data and reliable structural models of full-length tetrameric TH1. The SAXS-derived model reveals an elongated conformation (Dmax = 20 nm) for TH1, different arrangement of the catalytic domains compared with the crystal structure of truncated forms, and an N-terminal region with an unstructured tail that hosts the phosphorylation sites and a separated Ala-rich helical motif that may have a role in regulation of TH by interacting with binding partners.
authors.affiliation: Department of Biomedicine, University of Bergen, Bergen, Norway.
authors.forename: Maria T
authors.initials: MT
authors.lastname: Bezem
authors.affiliation: Department of Biomedicine, University of Bergen, Bergen, Norway.|K.G. Jebsen Centre for Neuropsychiatric Disorders, University of Bergen, Bergen, Norway.|Kronstad District Psychiatric Centre, Haukeland University Hospital Bergen, Norway.
authors.forename: Anne
authors.initials: A
authors.lastname: Baumann
authors.affiliation: Department of Biomedicine, University of Bergen, Bergen, Norway.
authors.forename: Lars
authors.initials: L
authors.lastname: Skjærven
authors.affiliation: Centre for Geobiology and Department of Earth Science, University of Bergen, Bergen, Norway and GFZ German Research Centre for Geosciences, Section 3.3, Earth Surface Geochemistry, Telegrafenberg, Potsdam, Germany.
authors.forename: Romain
authors.initials: R
authors.lastname: Meyer
authors.affiliation: Department of Biomedicine, University of Bergen, Bergen, Norway.
authors.forename: Petri
authors.initials: P
authors.lastname: Kursula
authors.affiliation: Department of Biomedicine, University of Bergen, Bergen, Norway.|K.G. Jebsen Centre for Neuropsychiatric Disorders, University of Bergen, Bergen, Norway.
authors.forename: Aurora
authors.initials: A
authors.lastname: Martinez
authors.affiliation: Department of Biomedicine, University of Bergen, Bergen, Norway.|Department of Neurology, Haukeland University Hospital, Bergen, Norway.
authors.forename: Marte I
authors.initials: MI
authors.lastname: Flydal
mesh_terms: D020134:Catalytic Domain; D004298:Dopamine / Q000494:pharmacology; D004795:Enzyme Stability; D006801:Humans; D011485:Protein Binding; D014446:Tyrosine 3-Monooxygenase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism
chemical_list: D014446:Tyrosine 3-Monooxygenase; D004298:Dopamine