As part of the pksX gene cluster of Bacillus subtilis strain 168, pksS has been preliminarily annotated as a cytochrome P450 homolog that hydroxylates the polyketide product of this cluster, which was recently shown to be involved in the biosynthesis of bacillaene and dihydrobacillaene. Here we report that there is a frame-shift error in the reported sequence for pksS, and that we have successfully cloned, overexpressed, and purified the protein encoded by the corrected sequence. By utilizing electronic absorption spectrophotometry, we have observed that the ferrous CO complex of PksS absorbs maximally near 450 nm, which confirms the annotation that this protein is a cytochrome P450. We have also established a cell-free system derived from crude cytosolic R subtilis protein extracts which provides reductase activity essential to sustaining the putative catalytic cycle of PksS. Using LC-MS analysis we have collected data which suggests that the Substrate for PksS is dihydrobacillaene. (c) 2007 Elsevier Inc. All rights reserved.