Stable preparations of tyrosine hydroxylase provide the solution structure of the full-length enzyme
| Autor: | Romain Meyer, Aurora Martinez, Lars Skjærven, Marte I. Flydal, Petri Kursula, Maria Teresa Bezem, Anne Baumann |
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| Jazyk: | angličtina |
| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Gene isoform Tyrosine 3-Monooxygenase Dopamine Plasma protein binding Crystal structure Article law.invention 03 medical and health sciences chemistry.chemical_compound Biosynthesis law Catalytic Domain Enzyme Stability Humans chemistry.chemical_classification Multidisciplinary Tyrosine hydroxylase Small-angle X-ray scattering 030104 developmental biology Enzyme chemistry Biochemistry Recombinant DNA ddc:000 Protein Binding |
| Zdroj: | Scientific Reports Scientific reports 6(1), 30390 (2016). doi:10.1038/srep30390 |
| ISSN: | 2045-2322 |
| DOI: | 10.1038/srep30390 |
| Popis: | Scientific reports 6(1), 30390(2016). doi:10.1038/srep30390 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 (D$_{max}$ = 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. Published by Nature Publishing Group, London |
| Databáze: | OpenAIRE |
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