Sequence and structure-guided discovery of a novel NADH-dependent 7β-hydroxysteroid dehydrogenase for efficient biosynthesis of ursodeoxycholic acid

Bin Huang; Kai Yang; Charles Amanze; Zhen Yan; Hongbo Zhou; Xueduan Liu; Guanzhou Qiu; Weimin Zeng

doi:10.1016/j.bioorg.2022.106340

Sequence and structure-guided discovery of a novel NADH-dependent 7β-hydroxysteroid dehydrogenase for efficient biosynthesis of ursodeoxycholic acid

Bioorg Chem. 2023 Feb:131:106340. doi: 10.1016/j.bioorg.2022.106340. Epub 2022 Dec 24.

Authors

Bin Huang¹, Kai Yang¹, Charles Amanze¹, Zhen Yan¹, Hongbo Zhou¹, Xueduan Liu¹, Guanzhou Qiu¹, Weimin Zeng²

Affiliations

¹ School of Minerals Processing and Bioengineering, Central South University, Hunan 410083, China.
² School of Minerals Processing and Bioengineering, Central South University, Hunan 410083, China. Electronic address: zengweimin1024@126.com.

PMID: 36586301
DOI: 10.1016/j.bioorg.2022.106340

Abstract

7β-Hydroxysteroid dehydrogenases (7β-HSDHs) have attracted increasing attention due to their crucial roles in the biosynthesis of ursodeoxycholic acid (UDCA). However, most published 7β-HSDHs are strictly NADPH-dependent oxidoreductases with poor activity and low productivity. Compared with NADPH, NADH is more stable and cheaper, making it the more popular cofactor for industrial applications of dehydrogenases. Herein, by using a sequence and structure-guided genome mining approach based on the structural information of conserved cofactor-binding motifs, we uncovered a novel NADH-dependent 7β-HSDH (Cle7β-HSDH). The Cle7β-HSDH was overexpressed, purified, and characterized. It exhibited high specific activity (9.6 U/mg), good pH stability and thermostability, significant methanol tolerance, and showed excellent catalytic efficiencies (k_cat/K_m) towards 7-oxo-lithocholic acid (7-oxo-LCA) and NADH (70.8 mM^-1s^-1 and 31.8 mM^-1s^-1, respectively). Molecular docking and mutational analyses revealed that Asp42 could play a considerable role in NADH binding and recognition. Coupling with a glucose dehydrogenase for NADH regeneration, up to 20 mM 7-oxo-LCA could be completely transformed to UDCA within 90 min by Cle7β-HSDH. This study provides an efficient approach for mining promising enzymes from genomic databases for cost-effective biotechnological applications.

Keywords: 7β-hydroxysteroid dehydrogenase; AlphaFold 2; Cost-effective; Genome mining; NADH-dependent; Ursodeoxycholic acid.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Hydroxysteroid Dehydrogenases* / chemistry
Hydroxysteroid Dehydrogenases* / metabolism
Molecular Docking Simulation
NAD* / chemistry
NADP / chemistry
Ursodeoxycholic Acid* / biosynthesis

Substances

7 beta-hydroxysteroid dehydrogenase
Hydroxysteroid Dehydrogenases
NAD
NADP
Ursodeoxycholic Acid