Structural highlights
Function
[P4H_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 PubMed
Enzymatic 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 Also
References
- ↑ Hara R, Kino K. Characterization of novel 2-oxoglutarate dependent dioxygenases converting L-proline to cis-4-hydroxy-l-proline. Biochem Biophys Res Commun. 2009 Feb 20;379(4):882-6. doi:, 10.1016/j.bbrc.2008.12.158. Epub 2009 Jan 6. PMID:19133227 doi:https://dx.doi.org/10.1016/j.bbrc.2008.12.158
- ↑ Koketsu K, Shomura Y, Moriwaki K, Hayashi M, Mitsuhashi S, Hara R, Kino K, Higuchi Y. 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. ACS Synth Biol. 2014 Sep 5. PMID:25171735 doi:https://dx.doi.org/10.1021/sb500247a
