Characterization of PksS in Bacillus subtilis

UNCG Author/Contributor (non-UNCG co-authors, if there are any, appear on document)
Stephanie Anne Antolak (Creator)
Institution
The University of North Carolina at Greensboro (UNCG )
Web Site: http://library.uncg.edu/
Advisor
Jason J Reddick

Abstract: "Cytochrome P450s are a common type of the polyketide tailoring enzymes that are responsible for the hydroxylation of polyketides. PksS, a likely tailoring enzyme, is found within the gene cluster pksX, from Bacillus subtilis. The gene cluster was found to synthesize bacillaene, a polyketide with antibiotic activity. Sequence homology has classified pksS as a cytochrome P450. We have hypothesized that PksS is responsible for the hydroxylation of bacillaene, dihydrobacillaene, or both. After several attempts to clone, overexpress, and purify pksS, we determined a sequence error within the published sequence. Following the correction of this error, the next in-frame stop codon was located. Using this newly identified sequence, PksS was overexpressed successfully and assayed using a spectrophotometric cytochrome P450 assay that detects a carbon-monoxide complex with the ferrous form of the P450. Spectra obtained definitively showed that PksS is a cytochrome P450. Several steps have been taken to characterize the role PksS serves in the biosynthesis of polyketides by B. subtilis. High-pressure liquid chromatography, coupled to mass spectrometry has preliminarily shown that the substrate for PksS is dihydrobacillaene. Other attempts to construct mutations of pksS have been performed, but experiments with the mutant strains did not yield conclusive information."--Abstract from author supplied metadata.

Additional Information

Publication
Thesis
Language: English
Date: 2007
Keywords
Cytochrome P450, polyketide, enzymes, hydroxylation, polyketides, PksS, gene, pksX, Bacillus subtilis, synthesize, bacillaene, polyketide, antibiotic
Subjects
Polyketides
Cytochrome P-450
Bacillus subtilis