Developing a highly efficient 4-hydroxyphenylacetate-3-hydroxylase for salvianic acid A synthesis by computer-aided molecular modification
Salvianic acid A (SAA) is a catechol compound known for its diverse physiochemical functions and has significant applications in the food and pharmaceutical industries. 4-Hydroxyphenylacetate-3-hydroxylase (4HPA3H) is a critical enzyme for SAA biosynthesis, and improving its activity towards p -hydroxyphenyllactate acid (4HPLA) is essential for highly efficient SAA production in stable biosynthetic pathways. To address this, the distal site and loops of the substrate pocket were modified to improve 4HPA3H catalytic activity towards 4HPLA using computer-aided molecular modification methods. As a result, we identified and mutated two critical sites in Ec HpaB, a 4HPA3H monooxygenase from Escherichia coli : T398, a substrate loop site, and M205, a distal site. The mutants M205F, T398S, and M205F/T398S enabled 2.51-, 2.07-, and 2.20-fold increases in catalytic efficiency ( k cat / K m towards 4HPLA), respectively. Molecular dynamics simulations showed that the overall decreased flexibility of the substrate pocket loop in Ec HpaB might help improve the enzyme's catalytic activity for 4HPLA. Owing to the T398 site in the substrate pocket coming from another monomer, molecular modification of Ec HpaB with multi-monomer interactions was also worthy of attention. These findings not only enhanced the biosynthetic efficiency of SAA but also provided insights into enzyme distal site modification and the engineering of multi-monomer enzymes.
危险品化学品经营许可证（带存储）