Laboratory of Biochemistry, Wageningen University &Research, Stippeneng 4, 6708 WE Wageningen, The Netherlands.
Department of Biochemistry and Molecular Cell Biology and Institute for Biocomputation and Physics of Complex Systems, University of Zaragoza, Pedro Cerbuna 12, 50009, Zaragoza, Spain.
Sci Rep. 2017 Mar 3;7:43880. doi: 10.1038/srep43880.
Flavoenzymes are versatile biocatalysts containing either FAD or FMN as cofactor. FAD often binds to a Rossmann fold, while FMN prefers a TIM-barrel or flavodoxin-like fold. Proline dehydrogenase is denoted as an exception: it possesses a TIM barrel-like fold while binding FAD. Using a riboflavin auxotrophic Escherichia coli strain and maltose-binding protein as solubility tag, we produced the apoprotein of Thermus thermophilus ProDH (MBP-TtProDH). Remarkably, reconstitution with FAD or FMN revealed that MBP-TtProDH has no preference for either of the two prosthetic groups. Kinetic parameters of both holo forms are similar, as are the dissociation constants for FAD and FMN release. Furthermore, we show that the holo form of MBP-TtProDH, as produced in E. coli TOP10 cells, contains about three times more FMN than FAD. In line with this flavin content, the crystal structure of TtProDH variant ΔABC, which lacks helices αA, αB and αC, shows no electron density for an AMP moiety of the cofactor. To the best of our knowledge, this is the first example of a flavoenzyme that does not discriminate between FAD and FMN as cofactor. Therefore, classification of TtProDH as an FAD-binding enzyme should be reconsidered.
黄素酶是一类多功能的生物催化剂，包含黄素腺嘌呤二核苷酸（FAD）或黄素单核苷酸（FMN）作为辅因子。FAD 通常结合于 Rossmann 折叠结构，而 FMN 则偏好 TIM 桶或黄素蛋白样折叠结构。脯氨酸脱氢酶则是一个例外：它具有 TIM 桶样折叠结构，但结合的辅因子是 FAD。我们利用核黄素营养缺陷型大肠杆菌菌株和麦芽糖结合蛋白作为可溶性标签，生产了嗜热栖热菌 ProDH（MBP-TtProDH）的脱辅基蛋白。值得注意的是，与 FAD 或 FMN 的重新组装表明，MBP-TtProDH 对这两种辅因子没有偏好。两种全酶形式的动力学参数相似，FAD 和 FMN 释放的解离常数也相似。此外，我们表明，在大肠杆菌 TOP10 细胞中生产的 MBP-TtProDH 全酶形式，其 FMN 含量比 FAD 约多三倍。与这种黄素含量一致，缺乏螺旋 αA、αB 和 αC 的 TtProDH 变体 ΔABC 的晶体结构显示，辅因子的 AMP 部分没有电子密度。据我们所知，这是第一个不区分 FAD 和 FMN 作为辅因子的黄素酶的例子。因此，应重新考虑将 TtProDH 归类为 FAD 结合酶。
