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Literature summary extracted from

  • 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 (2015), ACS Synth. Biol., 4, 383-392.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.14.11.56 expression in Escherichia coli Mesorhizobium loti

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
1.14.11.56 homology modeling based in the Rhizobium loti structure Mesorhizobium loti
1.14.11.56 in complex with in complex with Co2+, 2-oxoglutarate as cofactors, and L-proline or L-pipecolic acid. The active site is composed of a distorted jelly roll beta-sheet core, which is sandwiched by the N-terminal and C-terminal alpha-helical domains. Co2+ is coordinated by residues H106, H154, and D108 Mesorhizobium loti

Protein Variants

EC Number Protein Variants Comment Organism
1.14.11.56 V95A no increase in hydroxylation activity nor any improvement in the cis-5/cis-3 product ratio Mesorhizobium loti
1.14.11.56 V95A/V97A no increase in hydroxylation activity nor any improvement in the cis-5/cis-3 product ratio Mesorhizobium loti
1.14.11.56 V95W mutation significantly reduces the amount of cis-3-hydroxy-L-pipecolic acid (<5%) production compared with that of cis-5-hydroxy-L-pipecolic acid Mesorhizobium loti
1.14.11.56 V97A 2.5fold increase in production of cis-5-hydroxy-L-pipecolic acid, ratio of cis-5/cis-3 products is similar to wild-type Mesorhizobium loti
1.14.11.56 V97C 4fold increase in production of cis-5-hydroxy-L-pipecolic acid, ration of §-cis/5cis products is similar to wild-type Mesorhizobium loti
1.14.11.56 V97F the cis-5/cis-3 product ratio improves from 1.4 for the wild-type enzyme to 5.3, the total amount of product is similar to wild-type Mesorhizobium loti
1.14.11.56 V97F/V95W significantly improved regioselectivity toward cis-5-hydroxy-L-pipecolic acid Mesorhizobium loti
1.14.11.56 V97F/V95W/E114G significantly improved regioselectivity toward cis-5-hydroxy-L-pipecolic acid and high productivity Mesorhizobium loti
1.14.11.56 V97Y the cis-5/cis-3 product ratio improves from 1.4 for the wild-type enzyme to 9.0, the total amount of product decreases Mesorhizobium loti

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
1.14.11.56 Iron
-
 Mesorhizobium loti

Organism

EC Number Organism UniProt Comment Textmining
1.14.11.56 Mesorhizobium loti Q92LF6
-
-
1.14.11.56 Mesorhizobium loti Q989T9
-
-
1.14.11.56 Mesorhizobium loti 1021 Q92LF6
-
-
1.14.11.56 Mesorhizobium loti MAFF303099 Q989T9
-
-

Specific Activity [micromol/min/mg]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
1.14.11.56 0.1
-
 mutant V97F, product cis-3-hydroxy-L-pipecolic acid, pH 7.2, 30°C Mesorhizobium loti
1.14.11.56 0.29
-
 wild-type, product cis-5-hydroxy-L-pipecolic acid, pH 7.2, 30°C Mesorhizobium loti
1.14.11.56 0.34
-
 wild-type, product cis-3-hydroxy-L-pipecolic acid, pH 7.2, 30°C Mesorhizobium loti
1.14.11.56 0.79
-
 mutant V97F, product cis-5-hydroxy-L-pipecolic acid, pH 7.2, 30°C Mesorhizobium loti

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.14.11.56 L-pipecolic acid + 2-oxoglutarate + O2
-
 Mesorhizobium loti cis-5-hydroxy-L-pipecolic acid + cis-3-hydroxy-L-pipecolic acid + succinate + CO2 the ratio between cis-5/cis-3 isomers is about 1:7 ?
1.14.11.56 L-pipecolic acid + 2-oxoglutarate + O2
-
 Mesorhizobium loti cis-5-hydroxy-L-pipecolic acid + cis-3-hydroxy-L-pipecolic acid + succinate + CO2 the ratio between cis-5/cis-3 isomers is about 2:1 ?
1.14.11.56 L-pipecolic acid + 2-oxoglutarate + O2
-
 Mesorhizobium loti MAFF303099 cis-5-hydroxy-L-pipecolic acid + cis-3-hydroxy-L-pipecolic acid + succinate + CO2 the ratio between cis-5/cis-3 isomers is about 1:7 ?
1.14.11.56 L-pipecolic acid + 2-oxoglutarate + O2
-
 Mesorhizobium loti 1021 cis-5-hydroxy-L-pipecolic acid + cis-3-hydroxy-L-pipecolic acid + succinate + CO2 the ratio between cis-5/cis-3 isomers is about 2:1 ?
1.14.11.56 L-proline + 2-oxoglutarate + O2
-
 Mesorhizobium loti cis-4-hydroxy-L-proline + succinate + CO2
-
 ?
1.14.11.56 L-proline + 2-oxoglutarate + O2
-
 Mesorhizobium loti MAFF303099 cis-4-hydroxy-L-proline + succinate + CO2
-
 ?
1.14.11.56 L-proline + 2-oxoglutarate + O2
-
 Mesorhizobium loti 1021 cis-4-hydroxy-L-proline + succinate + CO2
-
 ?

Synonyms

EC Number Synonyms Comment Organism
1.14.11.56 cis-P4H
-
 Mesorhizobium loti
1.14.11.56 mlr6283
-
 Mesorhizobium loti
1.14.11.56 R03107
-
 Mesorhizobium loti