Brucella melitensis 7α-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 loca...Brucella melitensis 7α-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-tagged Bm7α-HSDH(C-Bm7α-HSDH),N-Bm7α-HSDH showed a 3.6-fold higher kcat and a 1.3-fold lower Km,resulting in a 7.0-fold higher kcat/Km value toward chenodeoxycholic acid.Circular dichroism spectroscopy indicated that the melting temperature of N-Bm7α-HSDH(46.13℃)was 3.0℃lower than that of C-Bm7α-HSDH(49.13℃).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.展开更多
基金the National Key research and Development Program of China(2018YFA0900302)the National Science Foundation of China(31970045)+2 种基金the National First-class Discipline Program of Light Industry Technology and Engineering(LITE2018-12)the Program of Introducing Talents of Discipline to Universities(111-2-06)Top-notch Academic Programs Project of Jiangsu Higher Education Institutions.
文摘Brucella melitensis 7α-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-tagged Bm7α-HSDH(C-Bm7α-HSDH),N-Bm7α-HSDH showed a 3.6-fold higher kcat and a 1.3-fold lower Km,resulting in a 7.0-fold higher kcat/Km value toward chenodeoxycholic acid.Circular dichroism spectroscopy indicated that the melting temperature of N-Bm7α-HSDH(46.13℃)was 3.0℃lower than that of C-Bm7α-HSDH(49.13℃).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.
