Improved catalytic efficiency of P450 OleP for converting lithocholic acid into murideoxycholic and ursodeoxycholic acids through semi-rational and rational design.
Engineered P450 OleP mutants show enhanced catalytic efficiency for synthesizing therapeutic bile acids from lithocholic acid, but further research is needed to assess industrial applicability.
Where it sits
this study against the rest of the nad+ (nicotinamide adenine dinucleotide) corpusSummary and findings
The study engineered P450 OleP to enhance its catalytic efficiency in converting lithocholic acid to murideoxycholic and ursodeoxycholic acids. The S240A mutant improved the MDCA conversion rate from 32.5% to 98.3%. The F84Q/S240A/V291G/E89N mutant increased the UDCA conversion rate from 6.5% to 12.1%.
Abstract
Cytochrome P450 enzymes are promising for synthesizing therapeutically beneficial bile acids murideoxycholic acid (MDCA) and ursodeoxycholic acid (UDCA) from lithocholic acid (LCA). However, the catalytic efficiency of P450 enzymes remains inadequate for large-scale production. Here, we address this limitation by engineering P450 OleP, achieving high activity for the efficient synthesis of MDCA and UDCA. Firstly, an improved high-throughput detection method for UDCA was developed to accelerate the screening process for mutants with improved UDCA synthesis capabilities. Next, the mutant S240A was generated through semi-rational design guided by the crystal structure of the OleP-LCA complex, resulting in an improvement in the MDCA conversion rate from 32.5% to 98.3% using 0.5 g L-1 of LCA as the substrate. Furthermore, the crystal structure of the S240A mutant was resolved and analyzed. Subsequently, the mutant F84Q/S240A/V291G/E89 N was developed through rational design, improving the UDCA conversion rate from 6.5% to 12.1% with 0.5 g L-1 of LCA as the substrate. Finally, molecular dynamics simulations were employed to elucidate the mechanisms underlying the enhanced catalytic activity of the S240A and F84Q/S240A/V291G/E89 N mutants. This study provides optimized P450 enzyme mutants for the synthesis of MDCA and UDCA and offers significant insights into the catalytic mechanisms underlying the production of hydroxylated, LCA-derived high-value-added compounds.
Background
Cytochrome P450 enzymes are valuable for synthesizing bile acids like murideoxycholic acid (MDCA) and ursodeoxycholic acid (UDCA), which have therapeutic benefits. However, their catalytic efficiency is insufficient for industrial-scale production. This study aims to enhance the catalytic efficiency of P450 OleP to improve the synthesis of these acids from lithocholic acid (LCA).
Methods
The study employed semi-rational and rational design approaches to engineer P450 OleP. The S240A mutant was generated using semi-rational design based on the crystal structure of the OleP-LCA complex. A high-throughput detection method for UDCA was developed to facilitate mutant screening. The F84Q/S240A/V291G/E89N mutant was created through rational design. Molecular dynamics simulations were used to understand the catalytic mechanisms of the mutants.
Results
The S240A mutant increased the MDCA conversion rate from 32.5% to 98.3% using 0.5 g L^-1 of LCA as the substrate. The F84Q/S240A/V291G/E89N mutant improved the UDCA conversion rate from 6.5% to 12.1% under the same conditions. The study also resolved and analyzed the crystal structure of the S240A mutant.
Interpretation
The engineered P450 OleP mutants demonstrated significantly improved catalytic efficiency for converting LCA to MDCA and UDCA. While the improvements are statistically significant, the clinical or industrial relevance remains uncertain without further in vivo or large-scale production data. The study provides a foundation for future research into enzyme optimization for bile acid synthesis.
Key findings
- MDCA conversion rate improved from 32.5% to 98.3% with S240A mutant.
- UDCA conversion rate increased from 6.5% to 12.1% with F84Q/S240A/V291G/E89N mutant.
- 0.5 g L^-1 of LCA used as substrate.
- High-throughput detection method for UDCA developed.
- Molecular dynamics simulations elucidated enhanced catalytic mechanisms.
Limitations
- No clinical or in vivo data.
- Limited to enzyme engineering.
- Uncertain industrial scalability.
- Results based on molecular dynamics simulations.