Design of a Distal Site Saturation Test-Iterative Parallel Mutagenesis for Engineering Hydroxysteroid Dehydrogenase DOI

Yuan Li,

Shufang Li, Shu Man Fu

et al.

Journal of Agricultural and Food Chemistry, Journal Year: 2025, Volume and Issue: unknown

Published: April 9, 2025

Distal protein engineering facilitates the efficient identification of novel modification sites and synergistic modulation enzyme functions to meet demands biocatalysts for industrial applications. Using hydroxysteroid dehydrogenase as a target protein, this study presents distal site saturation test-iterative parallel mutagenesis (DSST-IPM) strategy design high-performance enzymes. Twelve single-point mutations were identified improve stability-activity trade-off in site, targeting 34 residues. S176G Q245L exhibited significant melting temperature (Tm) increase 11.3 10.6 °C, respectively. Iterative screening yielded mutation7β-HSDH-M6b, which showed 13.3 °C higher Tm 5.92-fold catalytic activity (kcat/Km) than wild-type 7β-HSDH. Systematic analysis molecular dynamics simulations, quantum mechanical calculations, dynamic cross-correlation matrix (DCCM), mechanism behind enhanced performance M6b was elucidated. It uncovered that critical fourth shell could influence conformational during enzyme-catalyzed reaction, leading alterations inter-regional force interaction network. This thus offers an advanced framework improving efficiency highly active, thermostable

Language: Английский

Mining and engineering of pyrroline-5-carboxylate reductase for biocatalytic production of l-pipecolic acid with self-sufficient cofactor recycling DOI

S.-F. Zhu,

Binhao Wang,

Guochao Xu

et al.

Molecular Catalysis, Journal Year: 2025, Volume and Issue: 579, P. 115081 - 115081

Published: April 5, 2025

Language: Английский

Citations

0
Design of a Distal Site Saturation Test-Iterative Parallel Mutagenesis for Engineering Hydroxysteroid Dehydrogenase DOI

Yuan Li,

Shufang Li, Shu Man Fu

et al.

Journal of Agricultural and Food Chemistry, Journal Year: 2025, Volume and Issue: unknown

Published: April 9, 2025

Distal protein engineering facilitates the efficient identification of novel modification sites and synergistic modulation enzyme functions to meet demands biocatalysts for industrial applications. Using hydroxysteroid dehydrogenase as a target protein, this study presents distal site saturation test-iterative parallel mutagenesis (DSST-IPM) strategy design high-performance enzymes. Twelve single-point mutations were identified improve stability-activity trade-off in site, targeting 34 residues. S176G Q245L exhibited significant melting temperature (Tm) increase 11.3 10.6 °C, respectively. Iterative screening yielded mutation7β-HSDH-M6b, which showed 13.3 °C higher Tm 5.92-fold catalytic activity (kcat/Km) than wild-type 7β-HSDH. Systematic analysis molecular dynamics simulations, quantum mechanical calculations, dynamic cross-correlation matrix (DCCM), mechanism behind enhanced performance M6b was elucidated. It uncovered that critical fourth shell could influence conformational during enzyme-catalyzed reaction, leading alterations inter-regional force interaction network. This thus offers an advanced framework improving efficiency highly active, thermostable

Language: Английский

Citations

0