Engineering the cofactor binding site of 7α-hydroxysteroid dehydrogenase for improvement of catalytic activity, thermostability, and alteration of substrate preference

by Selleckchem | Feb 09 2024

Hydroxysteroid dehydrogenases (HSDHs) are crucial for bile acid metabolism and influence the size of the bile acid pool and gut microbiota composition. HSDHs with high activity, thermostability, and substrate selectivity are the basis for constructing engineered bacteria for disease treatment. In this study, we designed mutations at the cofactor binding site involving Thr15 and Arg16 residues of HSDH St-2-2. The T15A, R16A, and R16Q mutants exhibited 7.85-, 2.50-, and 4.35-fold higher catalytic activity than the wild type, respectively, while also displaying an altered substrate preference (from taurocholic acid (TCA) to taurochenodeoxycholic acid (TCDCA)). These mutants showed lower Km and higher kcat values, indicating stronger binding to the substrate and resulting in 3190-, 3123-, and 3093-fold higher kcat/Km values for TCDCA oxidation. Furthermore, the Tm values of the T15A, R16A, and R16Q mutants were found to increase by 4.3 °C, 6.0 °C, and 7.0 °C, respectively. Molecular structure analysis indicated that reshaped internal hydrogens and surface mutations could improve catalytic activity and thermostability, and altered interactions among the catalytic triad, cofactor binding sites, and substrates could change substrate preference. This work provides valuable insights into modifying substrate preference as well as enhancing the catalytic activity and thermostability of HSDHs by targeting the cofactor binding site.

Comments:

This study seems fascinating! The modifications in the cofactor binding site of the Hydroxysteroid dehydrogenase (HSDH) St-2-2 resulted in significant enhancements in catalytic activity, substrate selectivity, and thermostability. The mutations involving Thr15 and Arg16 residues led to the discovery of T15A, R16A, and R16Q mutants, which displayed substantial improvements in their enzymatic properties compared to the wild type.

The increased catalytic activity, lower Km values, and higher kcat values of these mutants suggest a stronger binding affinity to the substrate, especially for TCDCA oxidation, leading to significantly enhanced catalytic efficiency (as indicated by the substantially higher kcat/Km values).

Moreover, the rise in thermostability (measured by Tm values) of the mutants is impressive, indicating potential applications in various conditions where stability is crucial.

The molecular structure analysis revealing reshaped internal hydrogens and surface mutations contributing to improved catalytic activity and stability, along with altered interactions among catalytic triad, cofactor binding sites, and substrates, offers valuable insights into the manipulation of substrate preference and the enhancement of key enzymatic properties.

Overall, this work lays a solid foundation for the engineering of HSDHs with tailored substrate preferences, increased catalytic efficiency, and improved stability, potentially opening avenues for their application in disease treatment through engineered bacteria.