Effects of terminal modification on the catalytic efficiency and thermostability of Brucella melitensis 7α-hydroxysteroid dehydrogenase

Abstract

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.