Engineering Glucosamine-6-Phosphate Synthase to Achieve Efficient One-Step Biosynthesis of Glucosamine,ACS Chemical Biology

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Engineering Glucosamine-6-Phosphate Synthase to Achieve Efficient One-Step Biosynthesis of Glucosamine
ACS Chemical Biology ( IF 3.5 ) Pub Date : 2024-05-09 , DOI: 10.1021/acschembio.4c00144
Zheng-Bing Guan ₁ , Xue-Ting Deng ₁ , Zi-Hao Zhang ₁ , Guo-Chao Xu ₁ , Wan-Li Cheng ₂ , Xiang-Ru Liao ₁ , Yu-Jie Cai ₁

Affiliation

As an important functional monosaccharide, glucosamine (GlcN) is widely used in fields such as medicine, food nutrition, and health care. Here, we report a distinct GlcN biosynthesis method that utilizes engineered Bacillus subtilis glucosamine-6-phosphate synthase (BsGlmS) to convert D-fructose to directly generate GlcN. The best variant obtained by using a combinatorial active-site saturation test/iterative saturation mutagenesis (CAST/ISM) strategy was a quadruple mutant S596D/V597G/S347H/G299Q (BsGlmS-BK19), which has a catalytic activity 1736-fold that of the wild type toward D-fructose. Upon using mutant BK19 as a whole-cell catalyst, D-fructose was converted into GlcN with 65.32% conversion in 6 h, whereas the wild type only attained a conversion rate of 0.31% under the same conditions. Molecular docking and molecular dynamics simulations were implemented to provide insights into the mechanism underlying the enhanced activity of BK19. Importantly, the BsGlmS-BK19 variant specifically catalyzes D-fructose without the need for phosphorylated substrates, representing a significant advancement in GlcN biosynthesis.

中文翻译：

改造葡萄糖胺-6-磷酸合成酶以实现葡萄糖胺的高效一步生物合成

氨基葡萄糖（GlcN）作为一种重要的功能性单糖，广泛应用于医药、食品营养、保健等领域。在这里，我们报告了一种独特的 GlcN 生物合成方法，该方法利用工程枯草芽孢杆菌葡萄糖胺-6-磷酸合酶 ( Bs GlmS) 将 D-果糖转化为直接生成 GlcN。使用组合活性位点饱和测试/迭代饱和诱变（CAST/ISM）策略获得的最佳变体是四重突变体S596D/V597G/S347H/G299Q（ Bs GlmS-BK19），其催化活性是该突变体的1736倍。野生型转向 D-果糖。使用突变体BK19作为全细胞催化剂时，D-果糖在6小时内转化为GlcN，转化率为65.32%，而野生型在相同条件下仅达到0.31%的转化率。通过分子对接和分子动力学模拟，深入了解 BK19 活性增强的机制。重要的是， Bs GlmS-BK19 变体无需磷酸化底物即可特异性催化 D-果糖，这代表了 GlcN 生物合成的重大进步。

更新日期：2024-05-09

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