抄録
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 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.
| 元の言語 | English |
|---|---|
| ページ（範囲） | 383-392 |
| ページ数 | 10 |
| ジャーナル | ACS Synthetic Biology |
| 巻 | 4 |
| 発行部数 | 4 |
| DOI | |
| 出版物ステータス | Published - 2015 4 17 |
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ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology (miscellaneous)
- Biomedical Engineering
これを引用
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, Kento; Shomura, Yasuhito; Moriwaki, Kei; Hayashi, Mikiro; Mitsuhashi, Satoshi; Hara, Ryotaro; Kino, Kuniki; Higuchi, Yoshiki.
：: ACS Synthetic Biology, 巻 4, 番号 4, 17.04.2015, p. 383-392.研究成果: Article
}
TY - JOUR
T1 - 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
AU - Koketsu, Kento
AU - Shomura, Yasuhito
AU - Moriwaki, Kei
AU - Hayashi, Mikiro
AU - Mitsuhashi, Satoshi
AU - Hara, Ryotaro
AU - Kino, Kuniki
AU - Higuchi, Yoshiki
PY - 2015/4/17
Y1 - 2015/4/17
N2 - Enzymatic regio- and stereoselective hydroxylation are valuable for the production of hydroxylated chiral ingredients. Proline hydroxylases are representative members of the nonheme Fe2+/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.
AB - Enzymatic regio- and stereoselective hydroxylation are valuable for the production of hydroxylated chiral ingredients. Proline hydroxylases are representative members of the nonheme Fe2+/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.
KW - hydroxy- l -pipecolic acid
KW - hydroxylation
KW - hydroxyproline
KW - proline hydroxylase
KW - protein engineering
UR - http://www.scopus.com/inward/record.url?scp=84928151665&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84928151665&partnerID=8YFLogxK
U2 - 10.1021/sb500247a
DO - 10.1021/sb500247a
M3 - Article
C2 - 25171735
AN - SCOPUS:84928151665
VL - 4
SP - 383
EP - 392
JO - ACS Synthetic Biology
JF - ACS Synthetic Biology
SN - 2161-5063
IS - 4
ER -