4P7X

L-pipecolic acid-bound L-proline cis-4-hydroxylase

Summary for 4P7X

• Entry DOI: 10.2210/pdb4p7x/pdb
• Descriptor: L-proline cis-4-hydroxylase, COBALT (II) ION, 2-OXOGLUTARIC ACID, ... (7 entities in total)
• Functional Keywords: hydroxylase, proline, oxidoreductase
• Biological source: Rhizobium loti
• Total number of polymer chains: 1
• Total formula weight: 34748.08

Authors

Shomura, Y.,Koketsu, K.,Moriwaki, K.,Hayashi, M.,Mitsuhashi, S.,Hara, R.,Kino, K.,Higuchi, Y. (deposition date: 2014-03-28, release date: 2014-09-17, Last modification date: 2023-12-27)

Primary citation

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, 4:383-392, 2015

PubMed Abstract: Enzymatic regio- and stereoselective hydroxylation are valuable for the production of hydroxylated chiral ingredients. Proline hydroxylases are representative members of the nonheme Fe(2+)/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.

PubMed: 25171735
DOI: 10.1021/sb500247a

Experimental method

X-RAY DIFFRACTION (1.3 Å)