Effects of terminal modification on the catalytic efficiency and thermostability of Brucella melitensis 7α-hydroxysteroid dehydrogenase
Effects of terminal modification on the catalytic efficiency and thermostability of Brucella melitensis 7α-hydroxysteroid dehydrogenase
复制标题
DOI：
10.1007/s43393-022-00124-5
复制
发表时间：
2022-08
期刊：
影响因子：
--
通讯作者：
Zhiyong Liu;Rongzhen Zhang;Wenchi Zhang;Yan Xu
中科院分区：
文献类型：
--
作者：
Zhiyong Liu;Rongzhen Zhang;Wenchi Zhang;Yan Xu
布氏杆菌7-羟基类固醇脱氢酶(Bm7α-α-hSDH)催化鹅去氧胆酸氧化生成7-氧石胆酸。在本工作中，我们考察了末端修饰(His-tag定位和末端截断)对其催化效率和热稳定性的影响。与C-末端标记的Bm7α-HSDH(C-Bm7α-HSDH)相比，N-Bm7α-HSDH的Kcat高3.6倍，Km低1.3倍，对鹅去氧胆酸的Kcat/Km值高7倍。圆二色谱表明，N-Bm7α-HSDH的熔融温度(46.13℃)比C-Bm7α-HSDH的熔融温度(49.13℃)低3.0℃。N-Bm7α-hSDH在4 h内合成7-氧代胆酸的产率最高，为96.7%，C-Bm7α-hsdh在10 h内的产率为96.4%，氨基酸截断和His-Tag切割实验证实C-末端残基在催化功能中起关键作用。分子动力学模拟进一步表明，C端His标记的修饰可以使底物结合区发生变形，从而扰乱酶-底物的相互作用和催化运动。然而，由于N-末端的组氨酸标签远离酶的活性部位，对催化效率几乎没有影响。本研究为羟基类固醇脱氢酶末端修饰对类固醇底物的识别和稳定，从而影响其催化功能提供了结构上的见解。
Brucella melitensis7α-hydroxysteroid dehydrogenase (Bm7α-HSDH) catalyzes the oxidation of chenodeoxycholic acid to 7-oxolithocholic acid. In this work, we investigated the effects of terminal modification (His-tags location and terminal truncation) on its catalytic efficiency and thermostability. Compared with C-terminal His-taggedBm7α-HSDH (C-Bm7α-HSDH), N-Bm7α-HSDH showed a 3.6-fold higherkcatand a 1.3-fold lowerKm, resulting in a 7.0-fold higherkcat/Kmvalue toward chenodeoxycholic acid. Circular dichroism spectroscopy indicated that the melting temperature of N-Bm7α-HSDH (46.13 °C) was 3.0 °C lower than that of C-Bm7α-HSDH (49.13 °C). N-Bm7α-HSDH produced 7-oxolithocholic acid in the highest yield of 96.7% in 4 h, whereas the C-Bm7α-HSDH gave 96.4% in 10 h. Moreover, amino acids truncation and His-tag cleave experiments confirmed the C-terminal residues played key roles in catalytic functions. Molecular dynamics simulations further indicated C-terminal His-tagged modification could deform the substrate-binding region to disrupt the enzyme–substrate interactions and catalytic motion. However, the N-terminal His-tag hardly affected the catalytic efficiency due to its location far from the active site of the enzyme. This study provides structural insights into the terminus modifications of hydroxysteroid dehydrogenase on steroid substrate recognition and stabilization, thus affecting its catalytic functions.