AUTHOR=Mo Xuhua , Zhang Hui , Du Fengyu , Yang Song 

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

JOURNAL=Frontiers in Microbiology

VOLUME=11

YEAR=2020

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2020.610827

DOI=10.3389/fmicb.2020.610827

ISSN=1664-302X

ABSTRACT=<p>Nocamycins I and II, featured with a tetramic acid scaffold, were isolated from the broth of <italic>Saccharothrix syringae</italic> 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 <italic>NcmD</italic> deletion mutant strain <italic>S. syringae</italic> MoS-1005, which produced a new intermediate nocamycin F with a hydroxyl group at C-10 position. We then overexpressed <italic>NcmD</italic> in <italic>Escherichia coli</italic> BL21 (DE3), purified the His<sub>6</sub>-tagged protein NcmD to homogeneity and conducted <italic>in vitro</italic> enzymatic assays. NcmD showed preference to the cofactor NAD<sup>+</sup>, 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<sup>+</sup>. The <italic>K</italic><sub>m</sub> and <italic>k</italic><sub>cat</sub> values were 131 ± 13 μM and 65 ± 5 min<sup>−1</sup>, 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.</p>