Synthesis of arborane triterpenols by a bacterial oxidosqualene cyclase

Identification of key residues necessary for synthesis of pentacyclic arborinol versus tetracyclic sterols. (A) Partial amino acid sequence alignment of selected OSCs. Top numbers reflect positions in H. sapiens and bottom numbers reflect those in E. adriatica. Red boxes indicate positions that were changed by site-directed mutagenesis. (B) X-ray crystal structure of H. sapiens OSC (gray cartoon representation; PDB ID code 1W6K) bound to lanosterol (black stick representation with the C3–OH in red). Side chains of amino acids of interest are shown in stick representation in color as indicated (H. sapiens/M. alcaliphilum numbering). (C) GC-MS TIC of the alcohol-soluble fraction of a TLE (derivatized to TMS ethers) of E. coli oxidosqualene production strain overexpressing the M. alcaliphilum OSC W252S/H254Y/Y503V/N717Y variant, demonstrating partial conversion of an LAS to an EUS. Peaks labeled with Roman numerals have been identified by MS and/or NMR as follows: lanosterol (I), adriaticol (II), eudoraenol (III), parkeol (IV), boehmerol (V), and isoarborinol (VI). Structures of these lipids are shown in Fig. S4, and mass spectra are shown in Fig. S5.