1 research outputs found
Refined Regio- and Stereoselective Hydroxylation of l‑Pipecolic Acid by Protein Engineering of l‑Proline <i>cis</i>-4-Hydroxylase Based on the X‑ray Crystal Structure
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 <i>cis</i>-4-hydroxylases (<i>cis</i>-P4Hs) from <i>Sinorhizobium meliloti</i> and <i>Mesorhizobium loti</i> catalyze the hydroxylation of l-proline, generating <i>cis</i>-4-hydroxy-l-proline, as well as the hydroxylation
of l-pipecolic acid (l-Pip), generating two regioisomers, <i>cis</i>-5-Hypip and <i>cis</i>-3-Hypip. To selectively
produce <i>cis</i>-5-Hypip without simultaneous production
of two isomers, protein engineering of <i>cis</i>-P4Hs is
required. We therefore carried out protein engineering of <i>cis</i>-P4H to facilitate the conversion of the majority of l-Pip into the <i>cis</i>-5-Hypip isomer. We first
solved the X-ray crystal structure of <i>cis</i>-P4H in
complex with each of l-Pro and l-Pip. Then, we conducted
three rounds of directed evolution and successfully created a <i>cis</i>-P4H triple mutant, V97F/V95W/E114G, demonstrating the
desired regioselectivity toward <i>cis</i>-5-Hypip