TY - JOUR
T1 - Effects of terminal modification on the catalytic efficiency and thermostability of Brucella melitensis 7α-hydroxysteroid dehydrogenase
AU - Liu, Zhiyong
AU - Zhang, Rongzhen
AU - Zhang, Wenchi
AU - Xu, Yan
N1 - Funding Information:
This project was supported by the National Key research and Development Program of China (2018YFA0900302), the National Science Foundation of China (31970045), 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), and Top-notch Academic Programs Project of Jiangsu Higher Education Institutions.
Publisher Copyright:
© 2022, Jiangnan University.
PY - 2023/7
Y1 - 2023/7
N2 - 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 °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.
AB - 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 °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.
KW - 7α-hydroxysteroid dehydrogenase
KW - Catalytic efficiency
KW - Molecular dynamics simulations
KW - Terminal modification
KW - Thermostability
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U2 - 10.1007/s43393-022-00124-5
DO - 10.1007/s43393-022-00124-5
M3 - Article
AN - SCOPUS:85136569950
SN - 2662-7655
VL - 3
SP - 469
EP - 478
JO - Systems Microbiology and Biomanufacturing
JF - Systems Microbiology and Biomanufacturing
IS - 3
ER -