Short-Chain Dehydrogenase NcmD Is Responsible for the C-10 Oxidation of Nocamycin F in Nocamycin Biosynthesis

Abstract

Nocamycins I and II, featured with a tetramic acid scaffold, were isolated from the broth of Saccharothrix syringae NRRL B-16468. The biosynthesis of nocamycin I require an intermediate bearing a hydroxyl group at the C-10 position. A short chain dehydrogenase/reductase NcmD was proposed to catalyze the conversion of the hydroxyl group to ketone at the C-10 position. By using the λ-RED recombination technology, we generated the NcmD deletion mutant strain S. syringae MoS-1005, which produced a new intermediate nocamycin F with a hydroxyl group at C-10 position. We then overexpressed NcmD in Escherichia coli BL21 (DE3), purified the His6-tagged protein NcmD to homogeneity and conducted in vitro enzymatic assays. NcmD showed preference to the cofactor NAD+, and it effectively catalyzed the conversion from nocamyin F to nocamycin G, harboring a ketone group at C-10 position. However, NcmD showed no catalytic activity toward nocamyin II. NcmD achieved maximum catalytic activity at 45°C and pH 8.5. The kinetics of NcmD toward nocamycin F was investigated at 45°C, pH 8.5 in the presence of 2 mM NAD+. The Km and kcat values were 131 ± 13 μM and 65 ± 5 min−1, respectively. In this study, we have characterized NcmD as a dehydrogenase, which is involved in forming the ketone group at the C-10 position of nocamycin F. The results provide new insights to the nocamycin biosynthetic pathway.

Biosynthesis of trans-AT PKS-Derived Shuangdaolides Featuring a trans-acting Enzyme for Online Epoxidation

“…Compounds 8 , 9 , and 14 were used for SdlQ biochemical experiments, respectively. After 3 h of incubation with SdlQ in the presence of NAD, SdlQ had transformed 14 into 11 (Figure A). The 11 is predicted to be generated from the intermediate 11a by spontaneous hemiketalization and dehydration, because they are interconverting .…”

Bacterial Associates of a Desert Specialist Fungus-Growing Ant Antagonize Competitors with a Nocamycin Analog

“…The complex oxidation patterns observed for the polycyclic ketal headgroups of the nocamycins and tirandamycins have inspired mechanistic studies to untangle the sequence of oxidative tailoring steps. 19,[26][27][28][29][30] Remarkably, a single cytochrome P450 enzyme, TamI, accomplishes three distinct oxidative steps as part of a cascade of oxidative reactions on the tirandamycin scaffold, resulting in installation of a hydroxyl group at C18, analogous to the C20 hydroxyl of nocamycin V. 28,29 From a phylogeny of cytochromes P450 from tirandamycin and nocamycin BGCs (Figure S5), we find that NmvR is homologous to TamI, which could indicate a role in installation of the C20 hydroxyl group. A complex interplay of oxidations is likely and future investigation of the biosynthesis of nocamycin V would contribute to our understanding of multifunctional enzymes and co-dependent oxidations.…”

Bacterial Associates of a Desert Specialist Fungus-Growing Ant Antagonize Competitors with a Nocamycin Analog

“…Because nocamycin V bears only a single additional oxidation relative to nocamycin Ithe C20 hydroxyl groupthe presence of not one but two additional oxidative enzymes was unexpected. The complex oxidation patterns observed for the polycyclic ketal headgroups of the nocamycins and tirandamycins have inspired mechanistic studies to untangle the sequence of oxidative tailoring steps. ,− Remarkably, a single cytochrome P450 enzyme, TamI, accomplishes three distinct oxidative steps as part of a cascade of oxidative reactions on the tirandamycin scaffold, resulting in the installation of a hydroxyl group at C18, analogous to the C20 hydroxyl of nocamycin V. , From a phylogeny of cytochromes P450 from tirandamycin and nocamycin BGCs (Figure S4), we find that NmvR is homologous to TamI, which could indicate a role in the installation of the C20 hydroxyl group. A complex interplay of oxidations is likely and future investigation of the biosynthesis of nocamycin V would contribute to our understanding of multifunctional enzymes and codependent oxidations.…”