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Literature summary extracted from

  • Koski, M.K.; Hieta, R.; Hirsilae, M.; Roenkae, A.; Myllyharju, J.; Wierenga, R.K.
    The crystal structure of an algal prolyl 4-hydroxylase complexed with a proline-rich peptide reveals a novel buried tripeptide binding motif (2009), J. Biol. Chem., 284, 25290-25301.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.14.11.2 expression of the enzyme with a N-terminal His6-SUMO fusion protein in Escherichia coli Chlamydomonas reinhardtii

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
1.14.11.2 purifed recombinant detagged enzyme in ternary complex with Zn2+ and substrate (Ser-Pro)5 peptide, 3 mg/ml protein in 0.01 M Tris-HCl, 0.1 M NaCl, 0.1 M glycine, pH 7.8, 5 mM ZnSO4, 2 mM pyridine 2,4-dicarboxylate, and 2.5 mM (Ser-Pro)5, mixed with precipitant solution contains a 5% w/v polyethylene glycol mixture with equal amounts of PEG 400, PEG 1000, PEG 1500, PEG 4000, PEG 6000, PEG 8000, PEG MME 550, PEG MME 750, and PEG MME 5000 in 0.1 M acetate, pH 5.5, and 10 mM zinc acetate, 22°C, 1 week, X-ray diffraction structure determination and analysis at 1.98 A resolution Chlamydomonas reinhardtii

Protein Variants

EC Number Protein Variants Comment Organism
1.14.11.2 D149A a mutation at the tip of the betaII-betaIII loop, the mutant shows reduced activity and narrower substrate specificity due to altered substrate binding compared to the wild-type enzyme Chlamydomonas reinhardtii
1.14.11.2 D149N a mutation at the tip of the betaII-betaIII loop, the mutant shows reduced activity and narrower substrate specificity due to altered substrate binding compared to the wild-type enzyme Chlamydomonas reinhardtii
1.14.11.2 D81A a mutation at the tip of the beta3-beta4 loop, the mutant shows reduced activity and narrower substrate specificity due to altered substrate binding compared to the wild-type enzyme Chlamydomonas reinhardtii
1.14.11.2 G85A a mutation at the tip of the beta3-beta4 loop, the mutant shows reduced activity and narrower substrate specificity due to altered substrate binding compared to the wild-type enzyme Chlamydomonas reinhardtii
1.14.11.2 N152A a mutation at the tip of the betaII-betaIII loop, the mutant shows reduced activity and narrower substrate specificity due to altered substrate binding compared to the wild-type enzyme Chlamydomonas reinhardtii
1.14.11.2 S78T a mutation at the entrance and exit of the beta3-beta4 loop, the mutant shows slightly reduced activity compared to the wild-type enzyme Chlamydomonas reinhardtii
1.14.11.2 S78T/S87L a mutation at the entrance and exit of the beta3-beta4 loop, the mutant shows slightly reduced activity compared to the wild-type enzyme Chlamydomonas reinhardtii
1.14.11.2 S84A a mutation at the tip of the beta3-beta4 loop, the mutant shows reduced activity and narrower substrate specificity due to altered substrate binding compared to the wild-type enzyme Chlamydomonas reinhardtii
1.14.11.2 S87L a mutation at the entrance and exit of the beta3-beta4 loop, the mutant shows slightly reduced activity compared to the wild-type enzyme Chlamydomonas reinhardtii
1.14.11.2 Y140F a mutation at the betaI-betaII loop, the mutant shows reduced activity and narrower substrate specificity due to altered substrate binding compared to the wild-type enzyme Chlamydomonas reinhardtii

Inhibitors

EC Number Inhibitors Comment Organism Structure
1.14.11.2 Pyridine 2,4-dicarboxylate
-
 Chlamydomonas reinhardtii
1.14.11.2 Zn2+ Zn2+ is bound at each of the four active sites, binding structure, overview Chlamydomonas reinhardtii

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.14.11.2 0.11
-
 poly(L-proline) mutant S78T Chlamydomonas reinhardtii
1.14.11.2 0.14
-
 (Ser-Pro)5 mutant S78T/S87L Chlamydomonas reinhardtii
1.14.11.2 0.19
-
 (Ser-Pro)5 mutant S78T Chlamydomonas reinhardtii
1.14.11.2 0.26
-
 poly(L-proline) mutant S87L Chlamydomonas reinhardtii
1.14.11.2 0.29
-
 poly(L-proline) wild-type enzyme Chlamydomonas reinhardtii
1.14.11.2 0.34
-
 poly(L-proline) mutant Y140F Chlamydomonas reinhardtii
1.14.11.2 0.35
-
 poly(L-proline) mutant S78T/S87L Chlamydomonas reinhardtii
1.14.11.2 0.38
-
 (Ser-Pro)5 wild-type enzyme Chlamydomonas reinhardtii
1.14.11.2 0.56
-
 poly(L-proline) mutant D149N Chlamydomonas reinhardtii
1.14.11.2 0.63
-
 poly(L-proline) mutant D149A Chlamydomonas reinhardtii
1.14.11.2 0.77
-
 poly(L-proline) mutant S84A Chlamydomonas reinhardtii
1.14.11.2 0.78
-
 (Ser-Pro)5 mutant S87L Chlamydomonas reinhardtii
1.14.11.2 0.83
-
 (Pro-Pro-Gly)10 mutant S78T Chlamydomonas reinhardtii
1.14.11.2 0.88
-
 poly(L-proline) mutant D81A Chlamydomonas reinhardtii
1.14.11.2 0.93
-
 (Pro-Pro-Gly)10 wild-type enzyme Chlamydomonas reinhardtii
1.14.11.2 0.94
-
 poly(L-proline) mutant G85A Chlamydomonas reinhardtii
1.14.11.2 1.05
-
 (Pro-Pro-Gly)10 mutant S87L Chlamydomonas reinhardtii
1.14.11.2 1.27
-
 (Pro-Pro-Gly)10 mutant S78T/S87L Chlamydomonas reinhardtii
1.14.11.2 1.59
-
 poly(L-proline) mutant N152A Chlamydomonas reinhardtii

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
1.14.11.2 Fe2+ dependent on Chlamydomonas reinhardtii

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.14.11.2 procollagen L-proline + 2-oxoglutarate + O2 Chlamydomonas reinhardtii
-
 procollagen trans-4-hydroxy-L-proline + succinate + CO2
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
1.14.11.2 Chlamydomonas reinhardtii
-
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.14.11.2 recombinant enzyme with a N-terminal His6-SUMO fusion protein from Escherichia coli to homogeneity, the tag is cleaved off Chlamydomonas reinhardtii

Reaction

EC Number Reaction Comment Organism Reaction ID
1.14.11.2 procollagen L-proline + 2-oxoglutarate + O2 = procollagen trans-4-hydroxy-L-proline + succinate + CO2 active site and reaction mechanism, overview Chlamydomonas reinhardtii
a

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.14.11.2 (Pro-Pro-Gly)10 + O2
-
 Chlamydomonas reinhardtii ?
-
 ?
1.14.11.2 (Ser-Pro)5 + O2 the (Ser-Pro)5 peptide is found only in molecules A and C of the four polypeptides forming the enzyme Chlamydomonas reinhardtii ?
-
 ?
1.14.11.2 additional information substrate specificity, overview. The substrate is bound in a lefthanded (poly)-L-proline type II conformation in a tunnel shaped by two loops, mode of binding does not depend on stacking interactions of the proline sidechains with aromatic residues, substrate binding structure, modelling, overview. Major conformational changes of the two peptide binding loops are predicted to be a key feature of the catalytic cycle. These conformational changes are probably triggered by the conformational switch of Tyr140, as induced by the hydroxylation of the proline residue Chlamydomonas reinhardtii ?
-
 ?
1.14.11.2 poly(L-proline) + O2 substrate MW is 5000 kDa Chlamydomonas reinhardtii ?
-
 ?
1.14.11.2 procollagen L-proline + 2-oxoglutarate + O2
-
 Chlamydomonas reinhardtii procollagen trans-4-hydroxy-L-proline + succinate + CO2
-
 ?

Subunits

EC Number Subunits Comment Organism
1.14.11.2 monomer
-
 Chlamydomonas reinhardtii

Synonyms

EC Number Synonyms Comment Organism
1.14.11.2 More the enzyme belongs to the 2-oxoglutarate dioxygenase family Chlamydomonas reinhardtii
1.14.11.2 P4H
-
 Chlamydomonas reinhardtii
1.14.11.2 P4H-1
-
 Chlamydomonas reinhardtii
1.14.11.2 prolyl4-hydroxylase
-
 Chlamydomonas reinhardtii

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.14.11.2 2
-
 poly(L-proline) mutants S84A, D149A, and N152A Chlamydomonas reinhardtii
1.14.11.2 3
-
 poly(L-proline) mutant Y140F Chlamydomonas reinhardtii
1.14.11.2 4
-
 poly(L-proline) mutant G85A Chlamydomonas reinhardtii
1.14.11.2 4
-
 poly(L-proline) mutants S78T/S87L Chlamydomonas reinhardtii
1.14.11.2 5
-
 (Pro-Pro-Gly)10 mutant S78T/S87L Chlamydomonas reinhardtii
1.14.11.2 6
-
 poly(L-proline) mutant D149N Chlamydomonas reinhardtii
1.14.11.2 6
-
 (Ser-Pro)5 mutant S78T/S87L Chlamydomonas reinhardtii
1.14.11.2 6
-
 (Pro-Pro-Gly)10 mutant S87L Chlamydomonas reinhardtii
1.14.11.2 7
-
 (Pro-Pro-Gly)10 wild-type enzyme Chlamydomonas reinhardtii
1.14.11.2 7
-
 poly(L-proline) mutant D81A Chlamydomonas reinhardtii
1.14.11.2 8
-
 poly(L-proline) mutant S87L Chlamydomonas reinhardtii
1.14.11.2 9
-
 (Pro-Pro-Gly)10 mutant S78T Chlamydomonas reinhardtii
1.14.11.2 14
-
 (Ser-Pro)5 mutant S78T Chlamydomonas reinhardtii
1.14.11.2 18
-
 (Ser-Pro)5 mutant S87L Chlamydomonas reinhardtii
1.14.11.2 19
-
 (Ser-Pro)5 wild-type enzyme Chlamydomonas reinhardtii
1.14.11.2 20
-
 poly(L-proline) mutant S78T Chlamydomonas reinhardtii
1.14.11.2 30
-
 poly(L-proline) wild-type enzyme Chlamydomonas reinhardtii

General Information

EC Number General Information Comment Organism
1.14.11.2 physiological function plant and algal prolyl 4-hydroxylases are key enzymes in the synthesis of cell wall components Chlamydomonas reinhardtii