Mg<sup>2+</sup>-Ion\nDependence Revealed for a BAHD\n13‑<i>O</i>‑β-Aminoacyltransferase from <i>Taxus</i> Plants

A <i>Taxus</i> baccatin III:3-amino-3-phenylpropanoyltransferase\n(BAPT, Accession: AY082804) in clade 6 of the BAHD family catalyzed\na Mg<sup>2+</sup>-dependent transfer of isoserines from their corresponding\nCoA thioesters. An advanced taxane baccatin III on the paclitaxel\nbiosynthetic pathway in <i>Taxus</i> plants was incubated\nBAPT and phenylisoserine CoA or isobutenylisoserinyl CoA with and\nwithout MgCl<sub>2</sub>. BAPT biocatalytically converted baccatin\nIII to its 13-<i>O</i>-phenylisoserinyl and 3-(1',1'-dimethylvinyl)­isoserinyl\nanalogs, an activity that abrogated when Mg<sup>2+</sup> ions were\nomitted. Baccatin III analogs that are precursors to new generation\ntaxanes were also assayed with BAPT, the Mg<sup>2+</sup> cofactor,\nand 3-(1',1'-dimethylvinyl)­isoserinyl CoA to make paclitaxel\nderivatives\nat <i>k</i><sub>cat</sub>/<i>K</i><sub>M</sub> ranging between 27 and 234 s<sup>–1</sup> M<sup>–1</sup>. Molecular dynamics simulations of the BAPT active site modeled\non the crystal structure of a BAHD family member (PDB: 4G0B) suggest that Mg<sup>2+</sup> causes BAPT to use an unconventional active site space compared\nto those of other BAHD catalysts, studied over the last 25 years,\nthat use a conserved catalytic histidine residue that is glycine in\nBAPT. The simulated six-membered Mg<sup>2+</sup>–coordination\ncomplex includes an interaction that disrupts an intramolecular hydrogen\nbond between the C13-hydroxyl and the carbonyl oxygen of the C4-acetate\nof baccatin III. A simulation snapshot captured an active site conformation\nshowing the liberated C13-hydroxyl of baccatin III poised for acylation\nby BAPT through a potential substrate-assisted mechanism.