Effect of His6-tag Position on the Expression and Properties of Phenylacetone Monooxygenase from Thermobifida fusca

Parshin, P. D.; Pometun, A. A.; Martysuk, U. A.; Kleymenov, Sergey Y.; Atroshenko, D. L.; Pometun, E. V.; Савин, С. С.; Тишков, В. И.

doi:10.1134/s0006297920050065

Cited by 7 publications

(1 citation statement)

References 17 publications

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“…Due to the relatively small size, His-tag is generally assumed to have minimal or no influence on the function and structure of most proteins [8]. However, some studies suggested that the His-tags located at the C-or N-terminus of proteins could affect expression levels, purification yield, catalytic activity, stability, and crystallization [9][10][11]. Yeon et al reported C-terminal modification of 3-hydroxybutyrate dehydrogenase might deform the substrate-binding loop region of the active pocket, and disrupt the enzyme's catalytic motion.…”

Section: Introductionmentioning

confidence: 99%

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

Zhang

et al. 2022

Syst Microbiol and Biomanuf

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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 k cat and a 1.3-fold lower K m , resulting in a 7.0-fold higher k cat /K m 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.

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