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Literature summary for 1.14.11.56 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)

Cloned (Comment) Organism
recombinant expression of Strep-tagged wild-type enzyme in Escherichia coli strain W3110 Mesorhizobium loti
recombinant expression of wild-type and mutant enzymes in Escherichia coli strain W3110 Sinorhizobium meliloti

Crystallization (Commentary)

Crystallization (Comment) Organism
purified recombinant enzyme MlP4H in complex with Co2+, 2-oxoglutarate and L-Pro or L-Pip, the MlP4H protein used for crystallization includes an extra (Met)-Ser-Ala-Trp-Ser-His-Pro-Gln-Phe-Gly-Lys-Gly-Ala strep-tag II peptide at its N-terminus, and the original start codon of the wild-type is replaced by the underlined alanine, followed by the second codon of the wild-type. Crystallization of L-Pro complex crystals by sitting-drop vapor diffusion method mixing 0.001 ml of 28 mg/ml protein solution containing 2 mM CoCl2, 10 mM 2-oxoglutarate, and 20 mM L-Pro with reservoir solution containing 0.1 M bis-Tris propane, pH 8.5, 0.2 M sodium malonate, and 25% v/v PEG 3350, or of L-Pip complex crystals by sitting drop vapour diffusion method mixing 0.001 ml of 28 mg/ml protein solution containing 2 mM CoCl2, 10 mM ?2-oxoglutarate, and 20 mM L-Pip with 0.001 ml of reservoir solution containing 0.1 M CAPS, pH 10.5, 0.1 M lithium sulfate, and 1.8 M ammonium sulfate, all at 15°C, X-ray diffraction structure determination and analysis at 1.3-2.8 A resolution, by single-wavelength dispersion method with the bound Co2+ at the active site used as the anomalous scatter or by molecular replacement using the first L-Pro-bound structure as a search model Mesorhizobium loti

Protein Variants

Protein Variants Comment Organism
V95A site-directed mutagenesis, the mutant does not show any increase in hydroxylation activity nor any improvement in the cis-5/cis-3 ratio compared to the wild-type enzyme Sinorhizobium meliloti
V95A/V97A site-directed mutagenesis, the mutant does not show any increase in hydroxylation activity nor any improvement in the cis-5/cis-3 ratio compared to the wild-type enzyme Sinorhizobium meliloti
V97A site-directed mutagenesis, the mutant shows increased activity and production of cis-4-hydroxy-L-proline Sinorhizobium meliloti
V97C site-directed mutagenesis Sinorhizobium meliloti
V97F site-directed mutagenesis, the mutant shows improved regioselectivity of hydroxylation, the cis-5/cis-3 ratio improves from 1.4 for the wild-type enzyme to 5.3 for the mutant, the increase in activity is similar compared to mutant V97A, the V97F mutant demonstrates higher selectivity of C5-hydroxylation Sinorhizobium meliloti
V97F/V95W site-directed mutagenesis, the mutant shows improved regioselectivity and increased activity of hydroxylation Sinorhizobium meliloti
V97F/V95W/E114G site-directed mutagenesis, protein engineering of L-proline cis-4-hydroxylase based on the X-ray crystal structure leading to refined regio- and stereoselective hydroxylation of L-pipecolic acid, the engineered mutant enzyme produces 96% cis-5-hydroxypipecolate and 4% cis-3-hydroxypipecolate while the wild-type produces 60% cis-5-hydroxypipecolate and 40% cis-3-hydroxypipecolate. A structure homology model of the SmP4H triple mutant V97F/V95W/E114G is constructed based on the MlP4H crystal structure. addition of the E114G mutation improves the activity approximately 2fold compared to double mutant V97F/V95W. The triple mutant shows the highest growth and productivity of cis-5-hydroxy-L-pipecolate in a regioselective manner Sinorhizobium meliloti
V97Y site-directed mutagenesis, the mutant shows improved regioselectivity of hydroxylation, the cis-5/cis-3 ratio improves from 1.4 for the wild-type enzyme to 9.0 for the mutant, the increase in activity is decreased compared to mutant V97F Sinorhizobium meliloti

Metals/Ions

Metals/Ions Comment Organism Structure
Co2+ exogenous Co2+ is coordinated by residues H106, H154, and D108. Co2+ is a mimic of the catalytic metal center because of its stability under aerobic conditions and its enzymatic inactivity under anaerobic conditions. The cis-face of the C4 carbon of L-Pro is properly oriented toward Co2+, which in nature exists as FeIV=O during the hydroxylation reaction, to generate the enantiopure cis-4-hydroxyproline Mesorhizobium loti
Fe2+ dependent on Mesorhizobium loti
Fe2+ dependent on Sinorhizobium meliloti

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
L-pipecolate + 2-oxoglutarate + O2 Mesorhizobium loti
-
cis-5-hydroxypipecolate + cis-3-hydroxypipecolate + succinate + CO2
-
?
L-pipecolate + 2-oxoglutarate + O2 Sinorhizobium meliloti
-
cis-5-hydroxypipecolate + cis-3-hydroxypipecolate + succinate + CO2
-
?
L-proline + 2-oxoglutarate + O2 Mesorhizobium loti
-
cis-4-hydroxy-L-proline + succinate + CO2
-
?
L-proline + 2-oxoglutarate + O2 Sinorhizobium meliloti
-
cis-4-hydroxy-L-proline + succinate + CO2
-
?

Organism

Organism UniProt Comment Textmining
Mesorhizobium loti Q989T9
-
-
Sinorhizobium meliloti Q92LF6
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant Strep-tagged wild-type enzyme from Escherichia coli strain W3110 extract by ultracentrifugation, affinity and anion exchange chrmatography, and gel filtration Mesorhizobium loti

Reaction

Reaction Comment Organism Reaction ID
L-proline + 2-oxoglutarate + O2 = cis-4-hydroxy-L-proline + succinate + CO2 proposed catalytic mechanism of cis-P4H with L-proline and L-pipecolate as substrates with 2-oxoglutarate and O2, overview Mesorhizobium loti
L-proline + 2-oxoglutarate + O2 = cis-4-hydroxy-L-proline + succinate + CO2 proposed catalytic mechanism of cis-P4H with L-proline and L-pipecolate as substrates with 2-oxoglutarate and O2, overview Sinorhizobium meliloti

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
L-pipecolate + 2-oxoglutarate + O2
-
Mesorhizobium loti cis-5-hydroxypipecolate + cis-3-hydroxypipecolate + succinate + CO2
-
?
L-pipecolate + 2-oxoglutarate + O2
-
Sinorhizobium meliloti cis-5-hydroxypipecolate + cis-3-hydroxypipecolate + succinate + CO2
-
?
L-proline + 2-oxoglutarate + O2
-
Mesorhizobium loti cis-4-hydroxy-L-proline + succinate + CO2
-
?
L-proline + 2-oxoglutarate + O2
-
Sinorhizobium meliloti cis-4-hydroxy-L-proline + succinate + CO2
-
?

Synonyms

Synonyms Comment Organism
cis-P4H
-
Mesorhizobium loti
cis-P4H
-
Sinorhizobium meliloti
MlP4H
-
Mesorhizobium loti
SmP4H
-
Sinorhizobium meliloti

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
30
-
assay at Mesorhizobium loti
30
-
assay at Sinorhizobium meliloti

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.2
-
assay at Mesorhizobium loti
7.2
-
assay at Sinorhizobium meliloti

General Information

General Information Comment Organism
evolution proline hydroxylases are representative members of the nonheme Fe2+/2-oxoglutarate-dependent dioxygenase family Mesorhizobium loti
evolution proline hydroxylases are representative members of the nonheme Fe2+/2-oxoglutarate-dependent dioxygenase family Sinorhizobium meliloti
additional information I95, I97, and E114 are active site residues, the active site was composed of a distorted jelly roll beta-sheet core, which is sandwiched by the N-terminal and C-terminal alpha-helical domains Mesorhizobium loti
additional information V95, V97, and G114 are active site residues, a structure homology model of the SmP4H triple mutant V97F/V95W/E114G is constructed based on the MlP4H crystal structure Sinorhizobium meliloti
physiological function the enzyme 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-hydroxypipecolate and cis-3-hydroxypipecolate in a 6:4 ratio Mesorhizobium loti
physiological function the enzyme 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-hydroxypipecolate and cis-3-hydroxypipecolate in a 6:4 ratio Sinorhizobium meliloti