Characterization of a Radical <i>S</i>‑Adenosyl‑l‑methionine Epimerase, NeoN, in the Last Step of Neomycin B Biosynthesis

The last step of neo­mycin biosynthesis is the epimer­ization at C-5‴ of neo­mycin C to give neo­mycin B. A candidate enzyme responsible for the epimer­ization was a putative radical <i>S</i>-adenosyl-l-methionine (SAM) enzyme, NeoN, which is uniquely encoded in the neo­mycin biosynthetic gene cluster and remained an unassigned protein in the neo­mycin biosynthesis. The reconstituted and reduced NeoN showed the expected epimer­ization activity in the presence of SAM. In the epimer­ization, 1 equiv of SAM was consumed to convert neo­mycin C into neo­mycin B. The site of neo­mycin C reactive toward epimer­ization was clearly confirmed to be C-5‴ by detecting the incorporation of a deuterium atom from the deuterium oxide-based buffer solution. Further, alanine scanning of the NeoN cysteine residues revealed that C249 is a critical amino acid residue that provides a hydrogen atom to complete the epimer­ization. Furthermore, electron paramagnetic resonance analysis of the C249A variant in the presence of SAM and neo­mycin C revealed that a radical intermediate is generated at the C-5‴ of neo­mycin C. Therefore, the present study clearly illustrates that the epimer­ization of neo­mycin C to neo­mycin B is catalyzed by a unique radical SAM epimerase NeoN with a radical reaction mechanism

Characteristics of alveolar macrophages from murine models of OVA-induced allergic airway inflammation and LPS-induced acute airway inflammation

<div><p>ABSTRACT</p><p><i>Background</i>: Macrophages include the classically activated pro-inflammatory M1 macrophages (M1s) and alternatively activated anti-inflammatory M2 macrophages (M2s). The M1s are activated by both interferon-γ and Toll-like receptor ligands, including lipopolysaccharide (LPS), and have potent pro-inflammatory activity. In contrast, Th2 cytokines activate the M2s, which are involved in the immune response to parasites, promotion of tissue remodeling, and immune regulatory functions. Although alveolar macrophages (AMs) play an essential role in the pulmonary immune system, little is known about their phenotypes. <i>Methods</i>: Quantitative reverse transcription polymerase chain reaction and flow cytometry were used to define the characteristics of alveolar macrophages derived from untreated naïve mice and from murine models of both ovalbumin (OVA)-induced allergic airway inflammation and LPS-induced acute airway inflammation. AMs were co-cultured with CD4⁺ T cells and were pulsed with tritiated thymidine to assess proliferative responses. <i>Results</i>: We characterized in detail murine AMs and found that these cells were not completely consistent with the current M1 versus M2-polarization model. OVA-induced allergic and LPS-induced acute airway inflammation promoted the polarization of AMs towards the current M2-skewed and M1-skewed phenotypes, respectively. Moreover, our data also show that CD11c⁺ CD11b⁺ AMs from the LPS-treated mice play a regulatory role in antigen-specific T-cell proliferation in vitro. <i>Conclusions</i>: These characteristics of AMs depend on the incoming pathogens they encounter and on the phase of inflammation and do not correspond to the current M1 versus M2-polarization model. These findings may facilitate an understanding of their contributions to the pulmonary immune system in airway inflammation.</p></div