背景:硝基在许多有价值的物质中构成了重要的功能部分,如硝基咪唑,一类具有广谱活性的抗菌药物。合成硝基化合物的常规化学方法受到苛刻的条件,多个步骤,和环境问题。生物催化已成为克服这些缺点的有希望的替代方法。某些能够催化硝基形成的酶在自然界中逐渐被发现。然而,所报道的硝化酶具有有限的多样性和低的催化活性,阻碍了实际应用。
结果:表征了一种新型N-加氧酶SaRohS,该酶具有将2-氨基咪唑转化为偶氮霉素的更高催化能力。系统发育树分析显示,SaRohS属于血红素加氧酶样二铁加氧酶(HDOs)家族。SaRohS在pH5.5和25℃时表现出最佳活性,分别。该酶在pH值4.5~6.5和温度20~35℃范围内保持相对稳定的活性。在序列比对和结构分析之后,精心选择了几个有前途的氨基酸残基进行催化性能评估。定点突变表明苏氨酸75对于催化活性是必需的。双突变酶G95A/K115T表现出最高的催化效率,它比野生型高约5.8倍,比报告的来自北氮藻的N-加氧酶KaRohS高约22.3倍。通过分子对接和分子动力学研究了潜在的催化机理。最后,进行全细胞生物催化,2-氨基咪唑可以在14h内有效转化为偶氮霉素,反应转化率为42%。
结论:从蔗糖丝菌中筛选出一种有效的N-加氧酶,该酶将2-氨基咪唑催化成偶氮霉素。,对其进行了系统发育和酶学性质分析。通过定点突变,提高了催化能力,通过分子对接和动态模拟揭示了突变体酶活性增强的分子基础。此外,通过全细胞生物催化评估了该酶的应用潜力,证明它是一种有前途的替代方法,用于生产氮霉素。
BACKGROUND: The nitro group constitutes a significant functional moiety within numerous valuable substances, such as nitroimidazoles, a class of antimicrobial drugs exhibiting broad spectrum activity. Conventional chemical methods for synthesizing nitro compounds suffer from harsh conditions, multiple steps, and environmental issues. Biocatalysis has emerged as a promising alternative to overcome these drawbacks, with certain enzymes capable of catalyzing nitro group formation gradually being discovered in nature. Nevertheless, the practical application is hindered by the restricted diversity and low catalytic activity exhibited by the reported nitrifying enzymes.
RESULTS: A novel N-oxygenase SaRohS harboring higher catalytic capability of transformation 2-aminoimidazole to azomycin was characterized from Saccharothrix sp. Phylogenetic tree analysis revealed that SaRohS belongs to the heme-oxygenase-like diiron oxygenase (HDOs) family. SaRohS exhibited optimal activity at pH 5.5 and 25 ℃, respectively. The enzyme maintained relatively stable activity within the pH range of 4.5 to 6.5 and the temperature range of 20 ℃ to 35 ℃. Following sequence alignment and structural analysis, several promising amino acid residues were meticulously chosen for catalytic performance evaluation. Site-directed mutations showed that threonine 75 was essential for the catalytic activity. The dual mutant enzyme G95A/K115T exhibited the highest catalytic efficiency, which was approximately 5.8-fold higher than that of the wild-type and 22.3-fold higher than that of the reported N-oxygenase KaRohS from Kitasatospora azatica. The underlying catalytic mechanism was investigated through molecular docking and molecular dynamics. Finally, whole-cell biocatalysis was performed and 2-aminoimidazole could be effectively converted into azomycin with a reaction conversion rate of 42% within 14 h.
CONCLUSIONS: An efficient N-oxygenase that catalyzes 2-aminoimidazole to azomycin was screened form Saccharothrix sp., its phylogenetics and enzymatic properties were analyzed. Through site-directed mutation, enhancements in catalytic competence were achieved, and the molecular basis underlying the enhanced enzymatic activity of the mutants was revealed via molecular docking and dynamic simulation. Furthermore, the application potential of this enzyme was assessed through whole cell biocatalysis, demonstrating it as a promising alternative method for azomycin production.