Mg2+-Ion Dependence Revealed for a BAHD 13-O-β-Aminoacyltransferase from Taxus Plants

Abstract

A Taxus baccatin III:3-amino-3-phenylpropanoyltransferase (BAPT, Accession: AY082804) in clade 6 of the BAHD family catalyzed a Mg²⁺-dependent transfer of isoserines from their corresponding CoA thioesters. An advanced taxane baccatin III on the paclitaxel biosynthetic pathway in Taxus plants was incubated BAPT and phenylisoserine CoA or isobutenylisoserinyl CoA with and without MgCl₂. BAPT biocatalytically converted baccatin III to its 13-O-phenylisoserinyl and 3-(1',1'-dimethylvinyl)isoserinyl analogs, an activity that abrogated when Mg²⁺ ions were omitted. Baccatin III analogs that are precursors to new generation taxanes were also assayed with BAPT, the Mg²⁺ cofactor, and 3-(1',1'-dimethylvinyl)isoserinyl CoA to make paclitaxel derivatives at k_cat/K_M ranging between 27 and 234 s^–1 M^–1. Molecular dynamics simulations of the BAPT active site modeled on the crystal structure of a BAHD family member (PDB: 4G0B) suggest that Mg²⁺ causes BAPT to use an unconventional active site space compared to those of other BAHD catalysts, studied over the last 25 years, that use a conserved catalytic histidine residue that is glycine in BAPT. The simulated six-membered Mg²⁺–coordination complex includes an interaction that disrupts an intramolecular hydrogen bond between the C13-hydroxyl and the carbonyl oxygen of the C4-acetate of baccatin III. A simulation snapshot captured an active site conformation showing the liberated C13-hydroxyl of baccatin III poised for acylation by BAPT through a potential substrate-assisted mechanism.