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Enzymatic Synthesis of D-pipecolic Acid by Engineering the Substrate Specificity of Trypanosoma cruzi Proline Racemase and Its Molecular Docking Study
- Byun, Sungmin, Park, Hyun June, Joo, Jeong Chan, Kim, Yong Hwan
- Biotechnology and bioprocess engineering 2019 v.24 no.1 pp. 215-222
- Trypanosoma cruzi, active sites, biosynthesis, computer simulation, drugs, enantiomers, energy, engineering, enzyme activity, hydrophobicity, mutants, optical isomerism, pipecolic acid, proline, proline racemase, proteins, substrate specificity
- Pipecolic acid is an unnatural amino acid mostly used for pharmaceutical purposes. Pipecolic acid has two types of enantiomers with different roles in the synthesis of drugs. The development of efficient catalytic methods for the production of enantiopure pipecolic acid is currently a crucial topic of research. Few chemo- or biosynthetic methods have been proposed for the synthesis of pure enantiomers; however, enzymatic conversion of the chirality of pipecolic acid has not been demonstrated because no pipecolic acid racemase has been reported yet. In this work, we attempted to engineer pipecolic acid racemase activity into Trypanosoma cruzi proline racemase (TcPRAC) for the enzymatic synthesis of D-pipecolic acid from L-pipecolic acid. For the binding of pipecolic acid (C6 ring) into the active site of TcPRAC, which was optimized for the original substrate proline (C5 ring), four bulky aromatic residues (Phe102, Phe120, Phe220, and Phe 290) of TcPRAC were mutated to smaller hydrophobic residues. Among the mutants, six single-point mutants (F102A, F102I, F102L, F102V, F290L, and F290V) exhibited significant racemase activity against L-pipecolic acid. The most efficient variant, F102V, showed 74% racemization. Molecular docking simulations revealed that lowering the binding energy of L-pipecolic acid to the active site was important for achieving high racemization activity of TcPRAC mutant proteins.