Rational Design of Meso-Diaminopimelate Dehydrogenase with Enhanced Reductive Amination Activity for Efficient Production of D-p-Hydroxyphenylglycine

d-p-hydroxyphenylglycine (d-HPG) is an important intermediate in the pharmaceutical industry. In this study, a tri-enzyme cascade for the production of d-HPG from l-HPG was designed. However, the amination activity of Prevotella timonensis meso-diaminopimelate dehydrogenase (PtDAPDH) toward 4-hydroxyphenylglyoxylate (HPGA) was identified as the rate-limiting step. To overcome this issue, the crystal structure of PtDAPDH was solved, and a binding pocket and conformation remodeling strategy was developed to improve the catalytic activity toward HPGA. The best variant obtained, PtDAPDH(M4), exhibited a catalytic efficiency (k(cat)/K-m) that was 26.75-fold higher than that of the wild type. This improvement was due to the enlarged substrate-binding pocket and enhanced hydrogen bond networks around the active center; meanwhile, the increased number of interdomain residue interactions drove the conformation distribution toward the closed state. Under optimal transformation conditions, PtDAPDH(M4) produced 19.8 g/L d-HPG from 40 g/L racemate DL-HPG in a 3 L fermenter within 10 h, with 49.5% conversion and >99% enantiomeric excess. Our study provides an efficient three-enzyme cascade pathway for the industrial production of d-HPG from racemate DL-HPG.

Automated summary

In this study, a tri-enzyme cascade pathway was designed for the efficient industrial production of d-HPG from racemate DL-HPG. The rate-limiting step, amination activity of PtDAPDH toward HPGA, was improved by solving the crystal structure of PtDAPDH and developing a binding pocket and conformation remodeling strategy. The best variant, PtDAPDH(M4), showed a catalytic efficiency 26.75-fold higher than the wild type, leading to the synthesis of 19.8 g/L d-HPG with high conversion and enantiomeric excess.