Ile258Met mutation of Brucella melitensis 7α-hydroxysteroid dehydrogenase significantly enhances catalytic efficiency, cofactor affinity, and thermostability

Zhiyong Liu, Rongzhen Zhang, Wenchi Zhang, Yan Xu

Research output: Contribution to journalArticlepeer-review

Abstract

Abstract: NAD(H)-dependent 7α-hydroxysteroid dehydrogenase catalyzes the oxidation of chenodeoxycholic acid to 7-oxolithocholic acid. Here, we designed mutations of Ile258 adjacent to the catalytic pocket of Brucella melitensis 7α-hydroxysteroid dehydrogenase. The I258M variant gave a 4.7-fold higher kcat, but 4.5-fold lower KM, compared with the wild type, resulting in a 21.8-fold higher kcat/KM value for chenodeoxycholic acid oxidation. It presented a 2.0-fold lower KM value with NAD+, suggesting stronger binding to the cofactor. I258M produced 7-oxolithocholic acid in the highest yield of 92.3% in 2 h, whereas the wild-type gave 88.4% in 12 h. The I258M mutation increased the half-life from 20.8 to 31.1 h at 30 °C. Molecular dynamics simulations indicated increased interactions and a modified tunnel improved the catalytic efficiency, and enhanced rigidity at three regions around the ligand-binding pocket increased the enzyme thermostability. This is the first report about significantly improved catalytic efficiency, cofactor affinity, and enzyme thermostability through single site-mutation of Brucella melitensis 7α-hydroxysteroid dehydrogenase. Key points: • Sequence and structure analysis guided the site mutation design. • Thermostability, catalytic efficiency and 7-oxo-LCA production were determined. • MD simulation was performed to indicate the improvement by I258M mutation.

Original languageEnglish (US)
Pages (from-to)3573-3586
Number of pages14
JournalApplied Microbiology and Biotechnology
Volume105
Issue number9
DOIs
StatePublished - May 2021

Keywords

  • 7α-Hydroxysteroid dehydrogenase
  • Brucella melitensis
  • Chenodeoxycholic acid
  • Chiral catalysis
  • Protein engineering

ASJC Scopus subject areas

  • Biotechnology
  • Applied Microbiology and Biotechnology

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