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Literature summary extracted from

  • Barry, K.P.; Ngu, A.; Cohn, E.F.; Cote, J.M.; Burroughs, A.M.; Gerbino, J.P.; Taylor, E.A.
    Exploring allosteric activation of LigAB from Sphingobium sp. strain SYK-6 through kinetics, mutagenesis and computational studies (2015), Arch. Biochem. Biophys., 567, 35-45 .
    View publication on PubMed

Activating Compound

EC Number Activating Compound Comment Organism Structure
1.13.11.8 additional information allosteric rate enhancement in the presence of non-substrate protocatechuate-like aldehydes such as vanillin Sphingobium sp.
1.13.11.8 Vanillin heteroallosteric activation of LigAB by vanillin at an allosteric binding site in LigAB, regulatory feed-forward activation mechanism, kinetics, overview. Vanillin is the pre-pre-substrate of LigAB. Enhancement by vanillin is specific to protocatechuate. Glu86b is directly involved in a hydrogen bond network through the carboxylate with Ndelta of His127b, believed to be the catalytic base that performs the initial substrate deprotonation. The His127b methylene also contributes a small fraction to the identified allosteric binding pocket Sphingobium sp.

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.13.11.8 recombinant expression of His6-tagged wild-type and mutant enzymes in Escherichia coli strain DH5alpha Sphingobium sp.

Protein Variants

EC Number Protein Variants Comment Organism
1.13.11.8 A18W site-directed mutagenesis, mutation in the betaa-subunit, residue Ala18b is located opening of the putative allosteric pocket, the mutant displays reduced activity and altered rate enhancement by vanillin compared to the wild-type enzyme Sphingobium sp.
1.13.11.8 F103H site-directed mutagenesis, mutation in the alpha-subunit, residue Phe103a is located in the putative allosteric pocket, the mutant displays reduced activity and altered rate enhancement by vanillin compared to the wild-type enzyme Sphingobium sp.
1.13.11.8 F103L site-directed mutagenesis, mutation in the alpha-subunit, residue Phe103a is located in the putative allosteric pocket, the mutant displays reduced activity and altered rate enhancement by vanillin compared to the wild-type enzyme Sphingobium sp.
1.13.11.8 F103T site-directed mutagenesis, mutation in the alpha-subunit, residue Phe103a is located in the putative allosteric pocket, the mutant displays reduced activity and altered rate enhancement by vanillin compared to the wild-type enzyme Sphingobium sp.
1.13.11.8 F103V site-directed mutagenesis, mutation in the alpha-subunit, residue Phe103a is located in the putative allosteric pocket, the mutant displays reduced activity and altered rate enhancement by vanillin compared to the wild-type enzyme Sphingobium sp.

Inhibitors

EC Number Inhibitors Comment Organism Structure
1.13.11.8 protocatechuate substrate inhibition Sphingobium sp.

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.13.11.8 additional information
-
 additional information Michaelis-Menten steady-state kinetics measuring O2 consumption Sphingobium sp.
1.13.11.8 0.066
-
 protocatechuate pH 7.5, 25°C, recombinant mutant A18W Sphingobium sp.
1.13.11.8 0.081
-
 protocatechuate pH 7.5, 25°C, recombinant wild-type enzyme Sphingobium sp.
1.13.11.8 0.108
-
 protocatechuate pH 7.5, 25°C, recombinant mutant F103H Sphingobium sp.
1.13.11.8 0.13
-
 protocatechuate pH 7.5, 25°C, recombinant wild-type enzyme, with vanillin Sphingobium sp.
1.13.11.8 0.21
-
 protocatechuate pH 7.5, 25°C, recombinant mutant F103T Sphingobium sp.
1.13.11.8 0.26
-
 protocatechuate pH 7.5, 25°C, recombinant mutant F103V Sphingobium sp.
1.13.11.8 1.6
-
 protocatechuate pH 7.5, 25°C, recombinant mutant F103L Sphingobium sp.

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.13.11.8 protocatechuate + O2 Sphingobium sp.
-
 4-carboxy-2-hydroxymuconate semialdehyde
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
1.13.11.8 Sphingobium sp. G2IQQ4 AND G2IQQ3 alpha- and beta-chain of LigAB; formerly Sphingomonas paucimobilis SYK-6
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.13.11.8 recombinant His6-tagged wild-type and mutant enzymes anaerobically from Escherichia coli strain DH5alpha by nickel affinity chromatography, tag cleavage anaerobically by thrombin cleavage Sphingobium sp.

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.13.11.8 additional information LigAB has the broadest substrate utilization profile Sphingobium sp. ?
-
 ?
1.13.11.8 protocatechuate + O2
-
 Sphingobium sp. 4-carboxy-2-hydroxymuconate semialdehyde
-
 ?

Synonyms

EC Number Synonyms Comment Organism
1.13.11.8 LigAB
-
 Sphingobium sp.

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.13.11.8 25
-
 assay at Sphingobium sp.

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.13.11.8 33
-
 protocatechuate pH 7.5, 25°C, recombinant mutant F103L Sphingobium sp.
1.13.11.8 89
-
 protocatechuate pH 7.5, 25°C, recombinant mutant F103V Sphingobium sp.
1.13.11.8 110
-
 protocatechuate pH 7.5, 25°C, recombinant mutant F103H Sphingobium sp.
1.13.11.8 112
-
 protocatechuate pH 7.5, 25°C, recombinant mutant F103T Sphingobium sp.
1.13.11.8 122
-
 protocatechuate pH 7.5, 25°C, recombinant mutant A18W Sphingobium sp.
1.13.11.8 178
-
 protocatechuate pH 7.5, 25°C, recombinant wild-type enzyme Sphingobium sp.
1.13.11.8 230
-
 protocatechuate pH 7.5, 25°C, recombinant wild-type enzyme, with vanillin Sphingobium sp.

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.13.11.8 7.5
-
 assay at Sphingobium sp.

Ki Value [mM]

EC Number Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
1.13.11.8 17
-
 protocatechuate pH 7.5, 25°C, recombinant wild-type enzyme, with vanillin Sphingobium sp.
1.13.11.8 18
-
 protocatechuate pH 7.5, 25°C, recombinant wild-type enzyme Sphingobium sp.

General Information

EC Number General Information Comment Organism
1.13.11.8 evolution protocatechuate 4,5-dioxygenase (LigAB) from Sphingobium sp. strain SYK-6 is the defining member of the type II extradiol dioxygenase superfamily, a.k.a. PCA dioxygenase superfamily or PCADSF, computational docking and sequence comparisons of the enzyme with PCA dioxygenase superfamily LigAB enzymes Sphingobium sp.
1.13.11.8 physiological function the enzyme plays a key aromatic ring-opening role in the metabolism of several lignin derived aromatic compounds Sphingobium sp.

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
1.13.11.8 21
-
 protocatechuate pH 7.5, 25°C, recombinant mutant F103L Sphingobium sp.
1.13.11.8 340
-
 protocatechuate pH 7.5, 25°C, recombinant mutant F103V Sphingobium sp.
1.13.11.8 530
-
 protocatechuate pH 7.5, 25°C, recombinant mutant F103T Sphingobium sp.
1.13.11.8 1100
-
 protocatechuate pH 7.5, 25°C, recombinant mutant F103H Sphingobium sp.
1.13.11.8 1800
-
 protocatechuate pH 7.5, 25°C, recombinant wild-type enzyme, with vanillin Sphingobium sp.
1.13.11.8 1900
-
 protocatechuate pH 7.5, 25°C, recombinant mutant A18W Sphingobium sp.
1.13.11.8 2200
-
 protocatechuate pH 7.5, 25°C, recombinant wild-type enzyme Sphingobium sp.