Literature summary for 3.7.1.7 extracted from

Yang, Y.; Ko, T.P.; Liu, L.; Li, J.; Huang, C.H.; Chen, J.; Guo, R.T.; Du, G.
Roles of tryptophan residue and disulfide bond in the variable lid region of oxidized polyvinyl alcohol hydrolase (2014), Biochem. Biophys. Res. Commun., 452, 509-514.

Cloned(Commentary)

Cloned (Comment)	Organism
gene oph, recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)	Sphingopyxis sp.
recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21 trxB (DE3)	Pseudomonas sp.

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified recombinant detagged wild-type enzyme and mutants S172A and S172C, free and in complexes with PEG, butyrate, or DMSO, respectively, by sitting drop method using 30% PEG 5000 MME, 0.1 M MES pH 6.5. and 0.2 M (NH4)2SO4 for the wild-type, and 0.1 M tri-sodium citrate, pH 5.6, 30% w/v PEG 4000, and 0.3% w/v n-octyl-beta-D-glucoside for the mutants, X-ray diffraction structure determination and analysis at 1.49-2.10 A resolution, molecular replacement	Pseudomonas sp.

Crystallization (Comment)

Organism

purified recombinant detagged wild-type enzyme and mutants S172A and S172C, free and in complexes with PEG, butyrate, or DMSO, respectively, by sitting drop method using 30% PEG 5000 MME, 0.1 M MES pH 6.5. and 0.2 M (NH4)2SO4 for the wild-type, and 0.1 M tri-sodium citrate, pH 5.6, 30% w/v PEG 4000, and 0.3% w/v n-octyl-beta-D-glucoside for the mutants, X-ray diffraction structure determination and analysis at 1.49-2.10 A resolution, molecular replacement

Pseudomonas sp.

Protein Variants

Protein Variants	Comment	Organism
C241A/C248A	site-directed mutagenesis, the mutant shows unaltered activity compared to the wild-type enzyme	Sphingopyxis sp.
additional information	the mutants show reduced kcat/Km for 4-nitrophenyl acetate, indicating the importance of Trp255 in sequestering the active site from solvent. The significantly lower activity for 4-nitrophenyl butyrate can be a result of product inhibition. The mutant activity is retained with 4-nitrophenyl caprylate and 4-nitrophenyl laurate as the substrates, reflecting the amphipathic nature of the cleft	Pseudomonas sp.
S172A	site-directed mutagenesis, the mutant shows 20% activity compared to the wild-type enzyme	Pseudomonas sp.
S172C	site-directed mutagenesis	Pseudomonas sp.
W255A	site-directed mutagenesis	Pseudomonas sp.
W255F	site-directed mutagenesis	Pseudomonas sp.
W255Y	site-directed mutagenesis	Pseudomonas sp.

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism	Structure
additional information	-	additional information	Michaelis-Menten kinetics of the wild-type enzyme with 4-nitrophenyl fatty acid (C2-C16) substrates, overview	Sphingopyxis sp.
additional information	-	additional information	Michaelis-Menten kinetics of wild-type and mutant enzymes with 4-nitrophenyl fatty acid (C2-C16) substrates, overview	Pseudomonas sp.

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.
additional information	Pseudomonas sp.	the enzyme catalyzes the cleavage of C-C bond in beta-diketone	?	-	?
additional information	Sphingopyxis sp.	the enzyme catalyzes the cleavage of C-C bond in beta-diketone	?	-	?
additional information	Pseudomonas sp. VM15C	the enzyme catalyzes the cleavage of C-C bond in beta-diketone	?	-	?
oxidized polyvinyl alcohol + H2O	Pseudomonas sp.	-	?	-	?
oxidized polyvinyl alcohol + H2O	Sphingopyxis sp.	-	?	-	?
oxidized polyvinyl alcohol + H2O	Pseudomonas sp. VM15C	-	?	-	?

Organism

Organism	UniProt	Comment	Textmining
Pseudomonas sp.	Q9LCQ7	-	-
Pseudomonas sp. VM15C	Q9LCQ7	-	-
Sphingopyxis sp.	Q588Z2	gene oph	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21 trxB (DE3) by nickel affinity chromatography and digestion by TEV protease to remove the His-tagged thioredoxin, followed by a second nickel affinity chromatography, and anion exchange chromatography and ultrafiltration	Pseudomonas sp.
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography	Sphingopyxis sp.

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
additional information	the enzyme catalyzes the cleavage of C-C bond in beta-diketone	Pseudomonas sp.	?	-	?
additional information	the enzyme catalyzes the cleavage of C-C bond in beta-diketone	Sphingopyxis sp.	?	-	?
additional information	the enzyme shows lipase activity with 4-nitrophenyl esters as the substrates. The wild-type enzyme shows increased Km and decreased kcat/Km with the acyl chain length	Sphingopyxis sp.	?	-	?
additional information	the enzyme shows lipase activity with 4-nitrophenyl esters as the substrates. The wild-type enzyme shows increased Km and decreased kcat/Km with the acyl chain length, and the mutants show reduced kcat/Km for 4-nitrophenyl acetate, indicating the importance of Trp255 in sequestering the active site from solvent. The significantly lower activity for 4-nitrophenyl butyrate can be a result of product inhibition	Pseudomonas sp.	?	-	?
additional information	the enzyme catalyzes the cleavage of C-C bond in beta-diketone	Pseudomonas sp. VM15C	?	-	?
additional information	the enzyme shows lipase activity with 4-nitrophenyl esters as the substrates. The wild-type enzyme shows increased Km and decreased kcat/Km with the acyl chain length, and the mutants show reduced kcat/Km for 4-nitrophenyl acetate, indicating the importance of Trp255 in sequestering the active site from solvent. The significantly lower activity for 4-nitrophenyl butyrate can be a result of product inhibition	Pseudomonas sp. VM15C	?	-	?
oxidized polyvinyl alcohol + H2O	-	Pseudomonas sp.	?	-	?
oxidized polyvinyl alcohol + H2O	-	Sphingopyxis sp.	?	-	?
oxidized polyvinyl alcohol + H2O	-	Pseudomonas sp. VM15C	?	-	?

Synonyms

Synonyms	Comment	Organism
OPH	-	Pseudomonas sp.
OPH	-	Sphingopyxis sp.
oxidized polyvinyl alcohol hydrolase	-	Pseudomonas sp.
oxidized polyvinyl alcohol hydrolase	-	Sphingopyxis sp.
oxidized PVA hydrolase	-	Pseudomonas sp.
oxidized PVA hydrolase	-	Sphingopyxis sp.
pOPH	-	Pseudomonas sp.
sOPH	-	Sphingopyxis sp.

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
37	-	assay at	Pseudomonas sp.
37	-	assay at	Sphingopyxis sp.

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
8	-	assay at	Pseudomonas sp.
8	-	assay at	Sphingopyxis sp.

General Information

General Information	Comment	Organism
evolution	the enzyme belongs to the alpha/beta-hydrolase family and contains a unique lid region that covers the active site	Pseudomonas sp.
evolution	the enzyme belongs to the alpha/beta-hydrolase family and contains a unique lid region that covers the active site	Sphingopyxis sp.
additional information	roles of tryptophan residue and disulfide bond in the variable lid region of oxidized polyvinyl alcohol hydrolase, the lid is the most variable region of the enzyme. The disulfide bond formation of Cys257/267 is important for the activity of pOPH	Pseudomonas sp.
additional information	roles of tryptophan residue and disulfide bond in the variable lid region of oxidized polyvinyl alcohol hydrolase, the lid is the most variable region of the enzyme. The disulfide bond formation of Cys257/267 is not essential for sOPH, which has a shorter lid structure	Sphingopyxis sp.
physiological function	the enzyme is involved in degradation of polyvinyl alcohol	Pseudomonas sp.
physiological function	the enzyme is involved in degradation of polyvinyl alcohol	Sphingopyxis sp.