Abstract
Enzymatic regio- and stereoselective hydroxylation are valuable for the production of hydroxylated chiral ingredients. Proline hydroxylases are representative members of the nonheme Fe<sup>2+</sup>/α-ketoglutarate-dependent dioxygenase family. These enzymes catalyze the conversion of l-proline into hydroxy-l-prolines (Hyps). l-Proline cis-4-hydroxylases (cis-P4Hs) from Sinorhizobium meliloti and Mesorhizobium loti catalyze the hydroxylation of l-proline, generating cis-4-hydroxy-l-proline, as well as the hydroxylation of l-pipecolic acid (l-Pip), generating two regioisomers, cis-5-Hypip and cis-3-Hypip. To selectively produce cis-5-Hypip without simultaneous production of two isomers, protein engineering of cis-P4Hs is required. We therefore carried out protein engineering of cis-P4H to facilitate the conversion of the majority of l-Pip into the cis-5-Hypip isomer. We first solved the X-ray crystal structure of cis-P4H in complex with each of l-Pro and l-Pip. Then, we conducted three rounds of directed evolution and successfully created a cis-P4H triple mutant, V97F/V95W/E114G, demonstrating the desired regioselectivity toward cis-5-Hypip.
Original language | English |
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Pages (from-to) | 383-392 |
Number of pages | 10 |
Journal | ACS Synthetic Biology |
Volume | 4 |
Issue number | 4 |
DOIs | |
Publication status | Published - 2015 Apr 17 |
Keywords
- hydroxy- l -pipecolic acid
- hydroxylation
- hydroxyproline
- proline hydroxylase
- protein engineering
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology (miscellaneous)
- Biomedical Engineering