4p7x
L-pipecolic acid-bound L-proline cis-4-hydroxylaseL-pipecolic acid-bound L-proline cis-4-hydroxylase
Structural highlights
FunctionP4H_RHILO Dioxygenase that catalyzes the 2-oxoglutarate-dependent selective hydroxylation of free L-proline to cis-4-hydroxy-L-proline (cis-4-Hyp).[1] Publication Abstract from PubMedEnzymatic regio- and stereoselective hydroxylation are valuable for the production of hydroxylated chiral ingredients. Proline hydroxylases are representative members of the nonheme Fe2+/alpha-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. Refined Regio- and Stereoselective Hydroxylation of l-Pipecolic Acid by Protein Engineering of l-Proline cis-4-Hydroxylase Based on the X-ray Crystal Structure.,Koketsu K, Shomura Y, Moriwaki K, Hayashi M, Mitsuhashi S, Hara R, Kino K, Higuchi Y ACS Synth Biol. 2014 Sep 5. PMID:25171735[2] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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