Literature summary for 1.14.11.2 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.

Search for more literature for 1.14.11.2

Cloned(Commentary)

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

Crystallization (Commentary)

Crystallization (Comment)	Organism
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

Crystallization (Comment)

Organism

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

Protein Variants	Comment	Organism
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
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
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
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
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
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
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
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
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
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

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

KM Value [mM]

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

Metals/Ions

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

Natural Substrates/ Products (Substrates)

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

Organism

Organism	UniProt	Comment	Textmining
Chlamydomonas reinhardtii	-	-	-

Purification (Commentary)

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

Reaction

Reaction	Comment	Organism	Reaction ID
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)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
(Pro-Pro-Gly)10 + O2	-	Chlamydomonas reinhardtii	?	-	?
(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	?	-	?
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	?	-	?
poly(L-proline) + O2	substrate MW is 5000 kDa	Chlamydomonas reinhardtii	?	-	?
procollagen L-proline + 2-oxoglutarate + O2	-	Chlamydomonas reinhardtii	procollagen trans-4-hydroxy-L-proline + succinate + CO2	-	?

Subunits

Subunits	Comment	Organism
monomer	-	Chlamydomonas reinhardtii

Synonyms

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

Turnover Number [1/s]

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

General Information

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