EC Number | Cloned (Comment) | Organism |
---|---|---|
1.14.11.56 | recombinant expression of Strep-tagged wild-type enzyme in Escherichia coli strain W3110 | Mesorhizobium loti |
1.14.11.56 | recombinant expression of wild-type and mutant enzymes in Escherichia coli strain W3110 | Sinorhizobium meliloti |
EC Number | Crystallization (Comment) | Organism |
---|---|---|
1.14.11.56 | 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 |
EC Number | Protein Variants | Comment | Organism |
---|---|---|---|
1.14.11.56 | 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 |
1.14.11.56 | 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 |
1.14.11.56 | V97A | site-directed mutagenesis, the mutant shows increased activity and production of cis-4-hydroxy-L-proline | Sinorhizobium meliloti |
1.14.11.56 | V97C | site-directed mutagenesis | Sinorhizobium meliloti |
1.14.11.56 | 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 |
1.14.11.56 | V97F/V95W | site-directed mutagenesis, the mutant shows improved regioselectivity and increased activity of hydroxylation | Sinorhizobium meliloti |
1.14.11.56 | 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 |
1.14.11.56 | 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 |
EC Number | Metals/Ions | Comment | Organism | Structure |
---|---|---|---|---|
1.14.11.56 | 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 | |
1.14.11.56 | Fe2+ | dependent on | Mesorhizobium loti | |
1.14.11.56 | Fe2+ | dependent on | Sinorhizobium meliloti |
EC Number | Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|---|
1.14.11.56 | L-pipecolate + 2-oxoglutarate + O2 | Mesorhizobium loti | - |
cis-5-hydroxypipecolate + cis-3-hydroxypipecolate + succinate + CO2 | - |
? | |
1.14.11.56 | L-pipecolate + 2-oxoglutarate + O2 | Sinorhizobium meliloti | - |
cis-5-hydroxypipecolate + cis-3-hydroxypipecolate + succinate + CO2 | - |
? | |
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 | Sinorhizobium meliloti | - |
cis-4-hydroxy-L-proline + succinate + CO2 | - |
? |
EC Number | Organism | UniProt | Comment | Textmining |
---|---|---|---|---|
1.14.11.56 | Mesorhizobium loti | Q989T9 | - |
- |
1.14.11.56 | Sinorhizobium meliloti | Q92LF6 | - |
- |
EC Number | Purification (Comment) | Organism |
---|---|---|
1.14.11.56 | recombinant Strep-tagged wild-type enzyme from Escherichia coli strain W3110 extract by ultracentrifugation, affinity and anion exchange chrmatography, and gel filtration | Mesorhizobium loti |
EC Number | Reaction | Comment | Organism | Reaction ID |
---|---|---|---|---|
1.14.11.56 | 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 | |
1.14.11.56 | 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 |
EC Number | Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|---|
1.14.11.56 | L-pipecolate + 2-oxoglutarate + O2 | - |
Mesorhizobium loti | cis-5-hydroxypipecolate + cis-3-hydroxypipecolate + succinate + CO2 | - |
? | |
1.14.11.56 | L-pipecolate + 2-oxoglutarate + O2 | - |
Sinorhizobium meliloti | cis-5-hydroxypipecolate + cis-3-hydroxypipecolate + succinate + CO2 | - |
? | |
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 | - |
Sinorhizobium meliloti | cis-4-hydroxy-L-proline + succinate + CO2 | - |
? |
EC Number | Synonyms | Comment | Organism |
---|---|---|---|
1.14.11.56 | cis-P4H | - |
Mesorhizobium loti |
1.14.11.56 | cis-P4H | - |
Sinorhizobium meliloti |
1.14.11.56 | MlP4H | - |
Mesorhizobium loti |
1.14.11.56 | SmP4H | - |
Sinorhizobium meliloti |
EC Number | Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|---|
1.14.11.56 | 30 | - |
assay at | Mesorhizobium loti |
1.14.11.56 | 30 | - |
assay at | Sinorhizobium meliloti |
EC Number | pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|---|
1.14.11.56 | 7.2 | - |
assay at | Mesorhizobium loti |
1.14.11.56 | 7.2 | - |
assay at | Sinorhizobium meliloti |
EC Number | General Information | Comment | Organism |
---|---|---|---|
1.14.11.56 | evolution | proline hydroxylases are representative members of the nonheme Fe2+/2-oxoglutarate-dependent dioxygenase family | Mesorhizobium loti |
1.14.11.56 | evolution | proline hydroxylases are representative members of the nonheme Fe2+/2-oxoglutarate-dependent dioxygenase family | Sinorhizobium meliloti |
1.14.11.56 | 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 |
1.14.11.56 | 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 |
1.14.11.56 | 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 |
1.14.11.56 | 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 |