DHS-16 acts as a 3-hydroxysteroid dehydrogenase in DA biosynthesis.

<p>(A) LC/MS/MS analysis of lipid extracts from DHS-16 and control microsomes incubated with the proposed substrate lathosterol, shown quantitatively in (B). Significantly more lathosterone is detected in incubations of DHS-16 microsomes with lathosterol than in incubations with empty vehicle ethanol controls. DHS-16 microsomes also do not produce lathosterone when incubated with cholesterol, demonstrating that specific products are made depending upon the substrate provided (<i>N</i>≥3, M ± SD; **<i>p</i><0.005). (C) LC/MS/MS analysis of 4-cholesten-3-one levels in lipid extracts from DHS-16 and control microsomes incubated with cholesterol, shown quantitatively in (D). Concentrations of 4-cholesten-3-one in incubations of DHS-16 microsomes with cholesterol are significantly greater than that seen in control microsomes or incubations with lathosterol (<i>N</i> = 3, M ± SD; *<i>p</i><0.05). (E) Rescue of the Daf-c phenotype of <i>dhs-16(tm1890)</i> mutants at 27°C is seen when fed lipid extracts of DHS-16 microsomes incubated with the proposed substrate lathosterol. Rescue is not seen with extracts from DHS-16 microsomes incubated with ethanol vehicle alone or with cholesterol, and extracts from empty vector pCMV control microsomes do not rescue in any condition (estimated concentrations of 300 nM in plates; <i>N</i> = 3, M ± SEM; *<i>p</i><0.05). (F) GC/MS/MS analysis of sterol levels in N2 wild-type, <i>dhs-16(tm1890)</i>, <i>hsd-1(mg433)</i>, and <i>dhs-16;hsd-1</i> double mutants reveals that <i>hsd-1</i> is not required for 4-cholesten-3-one production, as previously proposed, suggesting HSD-1 may act in an alternative parallel pathway. In addition, although the <i>dhs-16;hsd-1</i> double mutants did not contain measurable lathosterone levels, Δ⁷-DA levels were not reduced, suggesting that lathosterone is not required for its production and that alternate pathways must exist that are independent of <i>dhs-16</i> (<i>N</i>≥6, M ± SEM; **below detection limit, *<i>p</i><0.05).