Non-specific biosynthesis of hopane triterpenes by a cell-free system from Acetobacter pasteurianum

1. A cell-free system from the bacterium Acetobacter pasteurianum was incubated with [12-3H]squalene; diploptene and diplopterol, hopanoids normally present in the bacterium, were labelled. Their radioactivity was confirmed by purification using thin-layer chromatography, synthesis of derivatives and recrystallization to constant specific activity. This demonstrates the direct cyclization of squalene into diploptene and diplopterol, catalysed by a squalene cyclase activity in A. pasteurianum. 2. The same cell-free system transformed (RS)-2,3-epoxy-2,3-dihydro-[12,13-3H]squalene into labelled 3 alpha-hydroxyhop-22(29)-ene, 3 beta-hydroxyhop-22(29)-ene, hopane-3 alpha,22-diol and hopane-3 beta,22-diol. Their radioactivity was similarly confirmed. This bacterial homogenate is thus capable of cyclizing an unnatural substrate, 2,3-epoxy-squalene, into 3-hydroxyhopanoids normally absent in the bacterium. 3. The 3 alpha-hydroxy and 3 beta-hydroxyhopanoids could have been enzymatically interconverted via the 3-oxo compound. Synthetic racemic (RS)-2,3-epoxy-2,3-dihydro-[3-3H]squalene was incubated and gave rise to 3-3H-labelled 3 alpha and 3 beta-hydroxyhopanoids. This excludes an isomerization via a 3-oxo compound which would give unlabelled 3-hydroxyhopanoids. 4. In conclusion, the cyclase of A. pasteurianum accepts the replacement of the normal substrate, squalene, by the corresponding epoxide. Furthermore it is not selective in the stereochemistry of the epoxide and cyclizes both enantiomers, contrary to the epoxysqualene cyclase of eukaryotes.