Diverse bacteria contain DNA with sulfur incorporated stereo-specifically into their DNA backbone at specific sequences (phosphorothioation). We found that in vitro oxidation of phosphorothioate (PT) DNA by hydrogen peroxide (H 2 O 2 ) or peracetic acid has two possible outcomes: DNA backbone cleavage or sulfur removal resulting in restoration of normal DNA backbone. The physiological relevance of this redox reaction was investigated by challenging PT DNA hosting Salmonella enterica cells using H 2 O 2 . DNA phosphorothioation was found to correlate with increasing resistance to the growth inhibition by H 2 O 2 . Resistance to H 2 O 2 was abolished when each of the three dnd genes, required for phosphorothioation, was inactivated. In vivo , PT DNA is more resistant to the double-strand break damage caused by H 2 O 2 than PT-free DNA. Furthermore, sulfur on the modified DNA was consumed and the DNA was converted to PT-free state when the bacteria were incubated with H 2 O 2 . These findings are consistent with a hypothesis that phosphorothioation modification endows DNA with reducing chemical property, which protects the hosting bacteria against peroxide, explaining why this modification is maintained by diverse bacteria.

The role of the conserved active site tyrosine and serine residues in epimerization catalyzed by polyketide synthase ketoreductase (PKS KR) domains has been investigated. Both mutant and wild-type forms of epimerase-active KR domains, including the intrinsically redox-inactive EryKR30 and PicKR30 as well as redox-inactive mutants of EryKR1, were incubated with [2-2H]-(2R,3S)-2-methyl-3-hydroxypentanoyl-SACP ([2-2H]-2) and 0.05 equiv of NADP+ in the presence of the redox-active, epimerase-inactive EryKR6 domain. The residual epimerase activity of each mutant was determined by tandem equilibrium isotope exchange, in which the first-order, time-dependent washout of isotope from 2 was monitored by LC-MS-MS with quantitation of the deuterium content of the diagnostic pantetheinate ejection fragment (4). Replacement of the active site Tyr or Ser residues, alone or together, significantly reduced the observed epimerase activity of each KR domain with minimal effect on substrate binding. Our results demonstrate that the epimerase and reductase activities of PKS KR domains share a common active site, with both reactions utilizing the same pair of Tyr and Ser residues.

Streptonigrin (STN, 1) is a highly functionalized aminoquinone alkaloid with broad and potent antitumor activity. Here, we reported the biosynthetic gene cluster of STN identified by genome scanning of a STN producer Streptomyces flocculus CGMCC4.1223. This cluster consists of 48 genes determined by a series of gene inactivations. On the basis of the structures of intermediates and shunt products accumulated from five specific gene inactivation mutants and feeding experiments, the biosynthetic pathway was proposed, and the sequence of tailoring steps was preliminarily determined. In this pathway, a cryptic methylation of lavendamycin was genetically and biochemically characterized to be catalyzed by a leucine carboxyl methyltransferase StnF2. A [2Fe-2S](2+) cluster-containing aromatic ring dioxygenase StnB1/B2 system was biochemically characterized to catalyze a regiospecific cleavage of the N-C8' bond of the indole ring of the methyl ester of lavendamycin. This work provides opportunities to illuminate the enzymology of novel reactions involved in this pathway and to create, using genetic and chemo-enzymatic methods, new streptonigrinoid analogues as potential therapeutic agents.

Many modular polyketide synthases harbor one or more redox-inactive domains of unknown function that are highly homologous to ketoreductase (KR) domains. A newly developed tandem equilibrium isotope exchange (EIX) assay has now established that such “KR0” domains catalyze the biosynthetically essential epimerization of transient (2R)-2-methyl-3-ketoacyl-ACP intermediates to the corresponding (2S)-2-methyl-3-ketoacyl-ACP diastereomers. Incubation of [2-2H]-(2R,3S)-2-methyl-3-hydroxypentanoyl-SACP ([2-2H]-3b) with the EryKR30 domain from module 3 of the 6-deoxyerythronolide B synthase, and the redox-active, nonepimerizing EryKR6 domain and NADP+ resulted in time- and cofactor-dependent washout of deuterium from 3b, as a result of EryKR30-catalyzed epimerization of transiently generated [2-2H]-2-methyl-3-ketopentanoyl-ACP (4). Similar results were obtained with redox-inactive PicKR30 from module 3 of the picromycin synthase. Four redox-inactive mutants of epimerase-active EryKR1 were engineered by mutagenesis of the NADPH binding site of this enzyme. Tandem EIX established that these EryKR10 mutants retained the intrinsic epimerase activity of the parent EryKR1 domain. These results establish the intrinsic epimerase activity of redox-inactive KR0 domains, rule out any role for the NADPH cofactor in epimerization, and provide a general experimental basis for decoupling the epimerase and reductase activities of a large class of PKS domains.