New semi-synthetic aminoglycoside antibiotics generally use chemical modifications to avoid inactivity from pathogens. One of the most used modifications is 3′,4′-di-deoxygenation, which imitates the structure of gentamicin. However, the mechanism of di-deoxygenation has not been clearly elucidated. Here, we report that the bifunctional enzyme, GenB4, catalyzes the last step of gentamicin 3′,4′-di-deoxygenation via reduction and transamination activities. Following disruption of genB4 in wild-type M. echinospora, its products accumulated in 6′-deamino-6′-oxoverdamicin (1), verdamicin C2a (2), and its epimer, verdamicin C2 (3). Following disruption of genB4 in M. echinospora ΔgenK, its products accumulated in sisomicin (4) and 6′-N-methylsisomicin (5, G-52). Following in vitro catalytic reactions, GenB4 transformed sisomicin (4) to gentamicin C1a (9) and transformed verdamicin C2a (2) and its epimer, verdamicin C2 (3), to gentamicin C2a (11) and gentamicin C2 (12), respectively. This finding indicated that in addition to its transamination activity, GenB4 exhibits specific 4′,5′ double-bond reducing activity and is responsible for the last step of gentamicin 3′,4′-di-deoxygenation. Taken together, we propose three new intermediates that may refine and supplement the specific biosynthetic pathway of gentamicin C components and lay the foundation for the complete elucidation of di-deoxygenation mechanisms.

中文翻译：

---

双功能酶GenB4通过还原和氨基转移活性催化庆大霉素3'，4'-二脱氧的最后一步。

新的半合成氨基糖苷类抗生素通常使用化学修饰以避免病原体的失活。最常用的修饰之一是3'，4'-二脱氧，它模仿庆大霉素的结构。然而，二脱氧的机理尚未清楚阐明。在这里，我们报道双功能酶GenB4通过还原和氨基转移活性催化庆大霉素3'，4'-二脱氧的最后一步。genB4在野生型棘球孢菌中被破坏后，其产物积聚在6'-脱氨基-6'-oxoverdamicin（1），verdamicin C2a（2）和其差向异构体verdamicin C2（3）中。在棘孢棘孢菌ΔgenK中的genB4破坏后，其产物积聚在sisomicin（4）和6'-N-methylsisomicin（5，G-52）中。经过体外催化反应，GenB4将sisomicin（4）转化为庆大霉素C1a（9），将verdamicin C2a（2）及其差向异构体verdamicin C2（3）分别转化为庆大霉素C2a（11）和庆大霉素C2（12）。该发现表明，GenB4除了其转氨活性外，还具有特异性的4'，5'双键还原活性，并负责庆大霉素3'，4'-二脱氧的最后一步。综上所述，我们提出了三种新的中间体，它们可以改进和补充庆大霉素C组分的特定生物合成途径，并为彻底阐明双脱氧机理奠定基础。5'具有双键还原活性，是庆大霉素3'，4'-二脱氧的最后一步。综上所述，我们提出了三种新的中间体，它们可以改进和补充庆大霉素C组分的特定生物合成途径，并为彻底阐明双脱氧机理奠定基础。5'具有双键还原活性，是庆大霉素3'，4'-二脱氧的最后一步。综上所述，我们提出了三种新的中间体，它们可以改进和补充庆大霉素C组分的特定生物合成途径，并为彻底阐明双脱氧机理奠定基础。