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

Brucella melitensis 7α-hydroxysteroid dehydrogenase (Bm7α-HSDH) catalyzes the oxidation of chenodeoxycholic acid to 7-oxolithocholic acid. In this work, we investigated effects terminal modification (His-tags location and truncation) on its catalytic efficiency thermostability. Compared with C-terminal His-tagged Bm7α-HSDH (C-Bm7α-HSDH), N-Bm7α-HSDH showed a 3.6-fold higher kcat 1.3-fold lower Km, resulting in 7.0-fold kcat/Km value toward Circular dichroism spectroscopy indicated that melting temperature (46.13 °C) was 3.0 °C than C-Bm7α-HSDH (49.13 °C). produced highest yield 96.7% 4 h, whereas gave 96.4% 10 h. Moreover, amino acids truncation His-tag cleave experiments confirmed residues played key roles functions. Molecular dynamics simulations further could deform substrate-binding region disrupt enzyme–substrate interactions motion. However, N-terminal hardly affected due far from active site enzyme. This study provides structural insights into terminus modifications hydroxysteroid steroid substrate recognition stabilization, thus affecting