Reconstruction of de novo verazine biosynthesis in Saccharomyces cerevisiae and computational analysis of cholesterol-binding geometry for 22R hydroxylation by VcCYP90B27 to dissect the cyclopamine biosynthetic pathway

Figure 1.

BY-SQ1 was engineered to biosynthesize verazine (6) from squalene through inducible expression of an engineered biosynthetic pathway. (Dr: Danio rerio; Gg: Gallus gallus; Vc: Veratrum californicum; Vv: Vitis vinifera).

Figure 2.

The characterization of cholesterol (1) in CEN-Chol and CEN-CPR strains by GC-MS. (a) TIC spectrum of each strain compared to the standard sample; (b) the structure of cholesterol (1) and its MS spectrum.

Figure 3.

The characterization of 22R-hydroxycholesterol (2) as well as 22R-26-dihydroxycholesterol (3) in different strains by GC-MS. (a), (b) EIC spectra of the four strains of CEN-V1, CEN-V2, CEN-V3, and CEN-Vera at m/z 173 and 99, respectively, and CEN-CPR as a control. (c), (d) MS spectra of 22R-hydroxycholesterol (2) and 22R-26-dihydroxycholesterol (3). (e), (f) Yields of 22R-hydroxycholesterol (2) and 22R-26-dihydroxycholesterol (3) in different strains. (** p < 0.01, **** p < 0.0001).

Figure 4.

The characterization of 22R-hydroxy-26-aminocholesterol (4) as well as verazine (6) in different strains by LC-MS in positive mode. (a), (b) EIC spectra of the four strains of CEN-V1, CEN-V2, CEN-V3, and CEN-Vera at m/z 418 and 398, respectively, and CEN-CPR as a control. (c), (d) MS spectra of 22-hydroxy-26-aminocholesterol (4) as well as verazine (6). (e), (f) Yields of 22-hydroxy-26-aminocholesterol (4) as well as verazine (6) in the different strains. (* p < 0.05).

Figure 5.

Conformational behavior and trajectory-based sampling of heme-proximal cholesterol-binding geometry in the VcCYP90B27-heme-cholesterol complex during a single 1,000-ns classical MD trajectory. (a) RMSD trajectories and probability-density distributions for the protein backbone (3.21 Å), binding site (1.99 Å), and cholesterol (1.74 Å). (b) Overlay of representative conformations from the later portion of the trajectory. (c) Per-residue RMSF profile, with an overall mean of 0.91 Å. (d) A three-dimensional surface representation colored by RMSF. (e) Relative positions of the two stereotopically distinct C22 hydrogens, designated pro-R and pro-S in the model, and the heme iron (Fe). (f) Trajectories and probability-density distributions of the Fe-to-pro-R H and Fe-to-pro-S H distances over 1,000 ns.