Stable preparations of tyrosine hydroxylase provide the solution structure of the full-length enzyme

• DOI: 10.1038/srep30390
• Publication Date: 2016-07-27T08:51:34Z
• Authors: Maria T. Bezem; Anne Baumann; Lars Skjærven; Romain Meyer; Petri Kursula; Aurora Martinez; Marte I. Flydal
• Keywords: Small-angle X-ray scattering; Maltose-binding protein; Enzyme Kinetics

Abstract Tyrosine hydroxylase (TH) catalyzes the rate-limiting step in biosynthesis of catecholamine neurotransmitters. TH is a highly complex enzyme at mechanistic, structural and regulatory levels preparation kinetically conformationally stable for characterization has been challenging. Here, we report on improved protocols purification recombinant human isoform 1 (TH1), which provide large amounts pure, stable, active TH1 with an intact N-terminus. purified through fusion His-tagged maltose-binding protein amylose resin was representative iron-bound functional enzyme, showing high activity stabilization by natural feedback inhibitor dopamine. ZZ domain TALON remarkably as it partially inhibited resin-derived cobalt. This more provided high-quality small-angle X-ray scattering (SAXS) data reliable models full-length tetrameric TH1. The SAXS-derived model reveals elongated conformation ( D max = 20 nm) TH1, different arrangement catalytic domains compared crystal structure truncated forms N-terminal region unstructured tail that hosts phosphorylation sites separated Ala-rich helical motif may have role regulation interacting binding partners.