Characterization of the 2,6-Dimethylphenol Monooxygenase MpdAB and Evaluation of Its Potential in Vitamin E Precursor Synthesis
Characterization of the 2,6-Dimethylphenol Monooxygenase MpdAB and Evaluation of Its Potential in Vitamin E Precursor Synthesis
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DOI：
10.1128/aem.00110-22
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发表时间：
2022-04
影响因子：
4.4
通讯作者：
Junbin Ji;Minggen Cheng;Xin Yan
中科院分区：
文献类型：
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作者：
Junbin Ji;Minggen Cheng;Xin Yan
尽管二甲基苯酚六种异构体的微生物降解已被广泛研究，但 2,6-DMP 降解的遗传和生化机制仍不清楚。这项研究鉴定了 M. neoaurum B5-4 中负责 2,6-DMP 分解代谢途径第一步的基因。摘要 2,6-二甲基苯酚（2,6-DMP）是一种广泛使用的化学中间体，其残留在环境中经常被检测到，对一些水生生物构成威胁。微生物降解是消除自然界中2,6-DMP的有效方法。然而，2,6-DMP代谢的遗传和生化机制仍不清楚。新金分枝杆菌 B5-4 是我们之前研究中分离出的一种 2,6-DMP 降解细菌。在这里，筛选了菌株B5-4的2,6-DMP降解缺陷突变体。比较基因组、转录组、基因破坏和遗传互补数据表明，mpdA 和 mpdB 负责 M. neoaurum B5-4 中 2,6-DMP 降解的第一步。 MpdAB 被预测为双组分黄素依赖性单加氧酶系统，与结核分枝杆菌 CDC1551 的 HsaAB 具有 32% 和 36% 的同一性。暴露于 2,6-DMP 后，mpdA 和 mpdB 的转录显着增加。核磁共振分析表明，以NADH和黄素腺嘌呤二核苷酸（FAD）为辅因子，纯化的6×His-MpdA和6×His-MpdB对位2,6-DMP和2,3,6-三甲基苯酚（2,3,6-TMP）进行羟基化。 2,6-DMP 和 2,3,6-TMP 的 MpdAB 的表观 Km 值分别为 0.12 ± 0.01 和 0.17 ± 0.01 mM，相应的 kcat/Km 值分别为 4.02 和 2.84 s−1 mM−1。由于对羟基化 2,3,6-TMP 是维生素 E 合成的主要前体，因此使用全细胞催化初步评估了 MpdAB 在维生素 E 合成中的潜力。 MpdA 的低表达水平和 2,3,6-TMP 细胞毒性限制了全细胞催化的效率。总之，这项研究揭示了 2,6-DMP 生物降解第一步的遗传和生化基础，并为维生素 E 合成提供了候选酶。重要性 尽管二甲基苯酚六种异构体的微生物降解已被广泛研究，但 2,6-DMP 降解的遗传和生化机制仍不清楚。这项研究鉴定了 M. neoaurum B5-4 中负责 2,6-DMP 分解代谢途径第一步的基因。此外，MpdAB 还催化 2,3,6-TMP 转化为 2,3,5-三甲基氢醌 (2,3,5-TMHQ)，这是维生素 E 合成的关键步骤。总体而言，这项研究为 2,6-DMP 污染的生物修复和开发合成维生素 E 的绿色方法提供了候选酶。
Although the microbial degradation of the six isomers of dimethylphenol has been extensively studied, the genetic and biochemical mechanisms of 2,6-DMP degradation remain unclear. This study identified the genes responsible for the initial step in the 2,6-DMP catabolic pathway in M. neoaurum B5-4. ABSTRACT 2,6-Dimethylphenol (2,6-DMP) is a widely used chemical intermediate whose residue has been frequently detected in the environment, posing a threat to some aquatic organisms. Microbial degradation is an effective method to eliminate 2,6-DMP in nature. However, the genetic and biochemical mechanisms of 2,6-DMP metabolism remain unknown. Mycobacterium neoaurum B5-4 is a 2,6-DMP-degrading bacterium isolated in our previous study. Here, a 2,6-DMP degradation-deficient mutant of strain B5-4 was screened. Comparative genomic, transcriptomic, gene disruption, and genetic complementation data indicated that mpdA and mpdB are responsible for the initial step of 2,6-DMP degradation in M. neoaurum B5-4. MpdAB was predicted to be a two-component flavin-dependent monooxygenase system, which shows 32% and 36% identities with HsaAB from Mycobacterium tuberculosis CDC1551. The transcription of mpdA and mpdB was substantially increased upon exposure to 2,6-DMP. Nuclear magnetic resonance analysis showed that purified 6×His-MpdA and 6×His-MpdB hydroxylated 2,6-DMP and 2,3,6-trimethylphenol (2,3,6-TMP) at the para-position using NADH and flavin adenine dinucleotide (FAD) as cofactors. The apparent Km values of MpdAB for 2,6-DMP and 2,3,6-TMP were 0.12 ± 0.01 and 0.17 ± 0.01 mM, respectively, and the corresponding kcat/Km values were 4.02 and 2.84 s−1 mM−1, respectively. Since para-hydroxylated 2,3,6-TMP is a major precursor for vitamin E synthesis, the potential of MpdAB in vitamin E synthesis was preliminarily evaluated using whole-cell catalysis. Low expression levels of MpdA and 2,3,6-TMP cytotoxicity limited the efficiency of whole-cell catalysis. Together, this study reveals the genetic and biochemical basis for the initial step of 2,6-DMP biodegradation and provides candidate enzymes for vitamin E synthesis. IMPORTANCE Although the microbial degradation of the six isomers of dimethylphenol has been extensively studied, the genetic and biochemical mechanisms of 2,6-DMP degradation remain unclear. This study identified the genes responsible for the initial step in the 2,6-DMP catabolic pathway in M. neoaurum B5-4. Moreover, MpdAB also catalyzed the transformation of 2,3,6-TMP to 2,3,5-trimethylhydroquinone (2,3,5-TMHQ), a crucial step in vitamin E synthesis. Overall, this study provides candidate enzymes for both the bioremediation of 2,6-DMP contamination and the development of a green method to synthesize vitamin E.