Cloning, expression, crystallization and preliminary X-ray data analysis of norcoclaurine synthase from Thalictrum flavum.
Norcoclaurine synthase (NCS) catalyzes the condensation of 3,4-dihydroxyphenylethylamine (dopamine) and 4-hydroxyphenylacetaldehyde (4-HPAA) as the first committed step in the biosynthesis of benzylisoquinoline alkaloids in plants. The protein was cloned, expressed and purified. Crystals were obtained at 294 K by the hanging-drop vapour-diffusion method using ammonium sulfate and sodium chloride as precipitant agents and diffract to better than 3.0 A resolution using a synchrotron-radiation source. The crystals belong to the trigonal space group P3(1)21, with unit-cell parameters a = b = 86.31, c = 118.36 A. A selenomethionine derivative was overexpressed, purified and crystallized in the same space group. A complete MAD data set was collected at 2.7 A resolution. The model is under construction.
Pasquo
Alessandra
A
ENEA Casaccia Research Centre, Dipartimento BIOTEC, Sezione Genetica e Genomica Vegetale, PO Box 2400, I-00100 Rome, Italy.
Bonamore
Alessandra
A
Franceschini
Stefano
S
Macone
Alberto
A
Boffi
Alberto
A
Ilari
Andrea
A
D019731:Carbon-Nitrogen Ligases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*; D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D018506:Gene Expression Regulation, Plant / Q000502:physiology*; D010940:Plant Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*; D031949:Thalictrum / Q000201:enzymology* / Q000235:genetics*
D010940:Plant Proteins; D019731:Carbon-Nitrogen Ligases; C450500:norcoclaurine synthase