SimC7 Is a Novel NAD(P)H-Dependent Ketoreductase Essential for the Antibiotic Activity of the DNA Gyrase Inhibitor Simocyclinone

Abstract

Simocyclinone D8 (SD8) is a potent DNA gyrase inhibitor produced by Streptomyces antibioticus Tü6040. The simocyclinone (sim) biosynthetic gene cluster has been sequenced and a hypothetical biosynthetic pathway has been proposed. The tetraene linker in SD8 was suggested to be the product of a modular type I polyketide synthase working in trans with two monofunctional enzymes. One of these monofunctional enzymes, SimC7, was proposed to supply a dehydratase activity missing from two modules of the polyketide synthase. In this study, we report the function of SimC7. We isolated the entire ~ 72-kb sim cluster on a single phage artificial chromosome clone and produced simocyclinone heterologously in a Streptomyces coelicolor strain engineered for improved antibiotic production. Deletion of simC7 resulted in the production of a novel simocyclinone, 7-oxo-SD8, which unexpectedly carried a normal tetraene linker but was altered in the angucyclinone moiety. We demonstrate that SimC7 is an NAD(P)H-dependent ketoreductase that catalyzes the conversion of 7-oxo-SD8 into SD8. 7-oxo-SD8 was essentially inactive as a DNA gyrase inhibitor, and the reduction of the keto group by SimC7 was shown to be crucial for high-affinity binding to the enzyme. Thus, SimC7 is an angucyclinone ketoreductase that is essential for the biological activity of simocyclinone.

Substrate-Assisted Catalysis in Polyketide Reduction Proceeds via a Phenolate Intermediate

“…All constructs were verified by sequencing. Proteins were expressed in E. coli as N-terminally His-tagged fusions, purified by nickel-affinity chromatography and assayed by high-performance liquid chromatography as described previously ( Schäfer et al., 2015 ). The structural integrity of purified proteins was verified using circular dichroism.…”

“…SimC7 was originally annotated as a dehydratase and predicted to be involved in the biosynthesis of the tetraene linker of SD8 ( Trefzer et al., 2002 ). However, we recently showed that SimC7 is in fact an NAD(P)H-dependent ketoreductase that catalyzes the reduction of a carbonyl to a hydroxyl group at the C-7 position of the angucyclinone ( Schäfer et al., 2015 ). This enzymatic step is essential for antibiotic activity, converting the almost inactive 7-oxo-simocyclinone D8 (7-oxo-SD8; half maximal inhibitory concentration [IC 50 ] ∼50–100 μM) into the potent gyrase inhibitor SD8 (IC 50 ∼0.1–0.6 μM) ( Schäfer et al., 2015 ).…”

“…Based on the intermediates produced by S. antibioticus , it seems that the biosynthesis of SD8 starts with assembly of the angucyclinone, followed by the attachment of the deoxysugar, then the tetraene linker, and finally the aminocoumarin (i.e., SD8 is assembled from right to left in Figure 1 ) ( Schimana et al., 2001 ). Therefore, the physiological substrate of SimC7 is most likely a 7-oxo angucyclinone intermediate lacking the attached deoxysugar, tetraene linker, and aminocoumarin, an intermediate that is detectable only in ΔsimC7 mutants ( Schäfer et al., 2015 ). Despite this, the enzyme readily accepts as a substrate the full-length intermediate 7-oxo-SD8, the major product made by ΔsimC7 mutants ( Schäfer et al., 2015 ).…”

“…Therefore, the physiological substrate of SimC7 is most likely a 7-oxo angucyclinone intermediate lacking the attached deoxysugar, tetraene linker, and aminocoumarin, an intermediate that is detectable only in ΔsimC7 mutants ( Schäfer et al., 2015 ). Despite this, the enzyme readily accepts as a substrate the full-length intermediate 7-oxo-SD8, the major product made by ΔsimC7 mutants ( Schäfer et al., 2015 ).…”

Structural insights into simocyclinone as an antibiotic, effector ligand and substrate

“…8 ; Schäfer et al . 2015 ). This enzymatic step is essential for antibiotic activity, converting the almost inactive 7-oxo-SD8 (IC 50 ∼ 50–100 μM) into the potent gyrase inhibitor SD8 (IC 50 ∼ 0.1–0.6 μM) (Schäfer et al .…”