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<p>1-deoxynojirimycin (1-DNJ) is an iminosugar biosynthesized from fructose-6-phosphate (F6P) by three biosynthetic enzymes such as aminotransferase (GabT1), phosphatase (Yktc1), and oxidoreductase (GutB1). Here, we expressed and purified GabT1, Yktc1, and GutB1 from <i>Bacillus velezensis</i> K26, whose genome sequence was previously analyzed, and subsequently characterized their biochemical properties and enzymatic roles in a one-pot in vitro reaction. In a one-pot reaction containing GabT1, Yktc1, and GutB1 with F6P as the substrate, LC-MS analysis revealed a major ion at <i>m/z</i> 162 corresponding to mannojirimycin (MJ)-dehydrate, whereas 1-deoxymannojirimycin and 1-DNJ were not detected. These results demonstrate that GabT1, Yktc1, and GutB1 function sequentially to catalyze the conversion of F6P into MJ-dehydrate under cell-free conditions, suggesting that additional enzymes, including an epimerase and a reductase, are required for the conversion of MJ to 1-DNJ. Overall, this work delineates the enzymatic sequence from F6P to MJ-dehydrate and provides direct biochemical validation of the initial 1-DNJ biosynthetic pathway, offering a basis for further studies on iminosugar biosynthesis.</p>

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Characterization of GabT1, Yktc1, and GutB1 of Bacillus velezensis K26 and analysis of enzymatic one-pot reaction

  • 期刊论文

  • 发表日期：

    2026年5月19日

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  • Hyunjin Lee,
  • Suhyeon Baek,
  • Sanggil Lee,
  • Sungho Jang,
  • Myung-Ji Seo

摘要

1-deoxynojirimycin (1-DNJ) is an iminosugar biosynthesized from fructose-6-phosphate (F6P) by three biosynthetic enzymes such as aminotransferase (GabT1), phosphatase (Yktc1), and oxidoreductase (GutB1). Here, we expressed and purified GabT1, Yktc1, and GutB1 from Bacillus velezensis K26, whose genome sequence was previously analyzed, and subsequently characterized their biochemical properties and enzymatic roles in a one-pot in vitro reaction. In a one-pot reaction containing GabT1, Yktc1, and GutB1 with F6P as the substrate, LC-MS analysis revealed a major ion at m/z 162 corresponding to mannojirimycin (MJ)-dehydrate, whereas 1-deoxymannojirimycin and 1-DNJ were not detected. These results demonstrate that GabT1, Yktc1, and GutB1 function sequentially to catalyze the conversion of F6P into MJ-dehydrate under cell-free conditions, suggesting that additional enzymes, including an epimerase and a reductase, are required for the conversion of MJ to 1-DNJ. Overall, this work delineates the enzymatic sequence from F6P to MJ-dehydrate and provides direct biochemical validation of the initial 1-DNJ biosynthetic pathway, offering a basis for further studies on iminosugar biosynthesis.