title: 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.
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.
authors.affiliation: †Bioprocess Development Center, Kyowa Hakko Bio Co., Ltd., Tsukuba, Ibaraki 305-0841, Japan.
authors.forename: Kento
authors.initials: K
authors.lastname: Koketsu
authors.affiliation: ‡Graduate School of Life Science, University of Hyogo, Koto 3-2-1, Kamigori-cho, Ako-gun, Hyogo 678-1297, Japan.
authors.forename: Yasuhito
authors.initials: Y
authors.lastname: Shomura
authors.affiliation: †Bioprocess Development Center, Kyowa Hakko Bio Co., Ltd., Tsukuba, Ibaraki 305-0841, Japan.
authors.forename: Kei
authors.initials: K
authors.lastname: Moriwaki
authors.affiliation: †Bioprocess Development Center, Kyowa Hakko Bio Co., Ltd., Tsukuba, Ibaraki 305-0841, Japan.
authors.forename: Mikiro
authors.initials: M
authors.lastname: Hayashi
authors.affiliation: †Bioprocess Development Center, Kyowa Hakko Bio Co., Ltd., Tsukuba, Ibaraki 305-0841, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Mitsuhashi
authors.affiliation: §Department of Applied Chemistry, Faculty of Science and Engineering, Waseda University, Ohkubo 3-4-1, Shinjuku-ku, Tokyo 169-8555, Japan.
authors.forename: Ryotaro
authors.initials: R
authors.lastname: Hara
authors.affiliation: §Department of Applied Chemistry, Faculty of Science and Engineering, Waseda University, Ohkubo 3-4-1, Shinjuku-ku, Tokyo 169-8555, Japan.
authors.forename: Kuniki
authors.initials: K
authors.lastname: Kino
authors.affiliation: ‡Graduate School of Life Science, University of Hyogo, Koto 3-2-1, Kamigori-cho, Ako-gun, Hyogo 678-1297, Japan.
authors.forename: Yoshiki
authors.initials: Y
authors.lastname: Higuchi
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D018360:Crystallography, X-Ray; D006900:Hydroxylation; D059128:Mesorhizobium / Q000201:enzymology*; D010875:Pipecolic Acids / Q000737:chemistry / Q000378:metabolism; D064828:Prolyl Hydroxylases / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D016962:Sinorhizobium meliloti / Q000201:enzymology*
chemical_list: D001426:Bacterial Proteins; D010875:Pipecolic Acids; D064828:Prolyl Hydroxylases; C031345:pipecolic acid
keywords: hydroxy-l-pipecolic acid; hydroxylation; hydroxyproline; proline hydroxylase; protein engineering