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Literature summary for 4.2.1.108 extracted from

  • Widderich, N.; Kobus, S.; Hoeppner, A.; Riclea, R.; Seubert, A.; Dickschat, J.S.; Heider, J.; Smits, S.H.; Bremer, E.
    Biochemistry and crystal structure of ectoine synthase a metal-containing member of the cupin superfamily (2016), PLoS ONE, 11, e0151285 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

Cloned (Comment) Organism
gene ectC, sequence comparisons, recombinant expression of a codon-optimized version of C-terminal Strep II-tagged enzyme in Escherichia coli strain BL21 from plasmid pNW12 under control of the tet promoter, which in turn is controlled by the TetR repressor, the genetic expression system can be induced by adding anhydrotetracycline, subcloning in Escherichia coli strain DH5alpha Sphingopyxis alaskensis

Crystallization (Commentary)

Crystallization (Comment) Organism
purified recombinnat Strep II-tagged enzyme, sitting drop vapour diffusion method, mixing of 100 nl of 11 mg/ml enzyme solution with 100 nl of reservoir solution containing 0.05 M, calcium acetate, 0.1 M sodium acetate, pH 4.5, and 40% v/v 1,2-propanediol, and equilibration against 0.05 ml of reservoir solution, or mixing of 0.001 ml of enzyme solution with 0.001 ml of reservoir solution containing 20% w/v PEG 6000, 0.9 M lithium chloride, and 0.1 M citric acid, pH 5.0, 3-10 weeks, X-ray diffraction structure determination and analysis at 1.2 A resolution, modelling Sphingopyxis alaskensis

Protein Variants

Protein Variants Comment Organism
C105A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Sphingopyxis alaskensis
C105S site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Sphingopyxis alaskensis
D91A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Sphingopyxis alaskensis
D91E site-directed mutagenesis, the mutant shows similar activity as the wild-type enzyme Sphingopyxis alaskensis
E115A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Sphingopyxis alaskensis
E115D site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Sphingopyxis alaskensis
E57A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Sphingopyxis alaskensis
E57D site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Sphingopyxis alaskensis
F107A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Sphingopyxis alaskensis
F107W site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Sphingopyxis alaskensis
F107Y site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Sphingopyxis alaskensis
H117A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Sphingopyxis alaskensis
H51A site-directed mutagenesis, the mutant shows similar activity as the wild-type enzyme Sphingopyxis alaskensis
H55A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Sphingopyxis alaskensis
H93A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Sphingopyxis alaskensis
H93N site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Sphingopyxis alaskensis
L87A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Sphingopyxis alaskensis
additional information structure-guided site-directed mutagenesis is used targeting amino acid residues that are evolutionarily highly conserved among the extended EctC protein family, including those forming the presumptive iron-binding site Sphingopyxis alaskensis
S23A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Sphingopyxis alaskensis
T40A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Sphingopyxis alaskensis
T41A site-directed mutagenesis, the mutant shows similar activity as the wild-type enzyme Sphingopyxis alaskensis
W21A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Sphingopyxis alaskensis
Y52A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Sphingopyxis alaskensis
Y85A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Sphingopyxis alaskensis
Y85F site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Sphingopyxis alaskensis
Y85W site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Sphingopyxis alaskensis

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
additional information Michaelis-Menten-kinetics Sphingopyxis alaskensis
4.9
-
(2S)-4-acetamido-2-aminobutanoate pH 8.5, 15°C, recombinant enzyme Sphingopyxis alaskensis
25.4
-
N-alpha-acetyl-L-2,4-diaminobutanoate pH 8.5, 15°C, recombinant enzyme Sphingopyxis alaskensis

Metals/Ions

Metals/Ions Comment Organism Structure
Fe2+ required, activates 100fold at 1 mM Sphingopyxis alaskensis
additional information determination of metal content of recombinant SaEctC protein by ICP-MS. Zn2+ or Co2+ can only weakly substitute for Fe2+. No activation with Ni2+, Mn2+, Cu2+, and Fe3+ at 1 mM Sphingopyxis alaskensis

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
(2S)-4-acetamido-2-aminobutanoate Sphingopyxis alaskensis
-
L-ectoine + H2O
-
?
(2S)-4-acetamido-2-aminobutanoate Sphingopyxis alaskensis DSM 13593 / LMG 18877 / RB2256
-
L-ectoine + H2O
-
?

Organism

Organism UniProt Comment Textmining
Sphingopyxis alaskensis Q1GNW6 i.e. Sphingomonas alaskensis
-
Sphingopyxis alaskensis DSM 13593 / LMG 18877 / RB2256 Q1GNW6 i.e. Sphingomonas alaskensis
-

Purification (Commentary)

Purification (Comment) Organism
recombinant C-terminal Strep II-tagged enzyme from Escherichia coli by affinity chromatography and gel filtration to homogeneity Sphingopyxis alaskensis

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
(2S)-4-acetamido-2-aminobutanoate
-
Sphingopyxis alaskensis L-ectoine + H2O
-
?
(2S)-4-acetamido-2-aminobutanoate i.e. N-gamma-acetyl-L-2,4-diaminobutanoate Sphingopyxis alaskensis L-ectoine + H2O
-
?
(2S)-4-acetamido-2-aminobutanoate
-
Sphingopyxis alaskensis DSM 13593 / LMG 18877 / RB2256 L-ectoine + H2O
-
?
(2S)-4-acetamido-2-aminobutanoate i.e. N-gamma-acetyl-L-2,4-diaminobutanoate Sphingopyxis alaskensis DSM 13593 / LMG 18877 / RB2256 L-ectoine + H2O
-
?
additional information EctC not only effectively converts its natural substrate N-gamma-acetyl-L-2,4-diaminobutyric acid into ectoine through a cyclocondensation reaction, but it can also use the isomer N-alpha-acetyl-L-2,4-diaminobutyric acid as its substrate, albeit with substantially reduced catalytic efficiency Sphingopyxis alaskensis ?
-
?
additional information EctC not only effectively converts its natural substrate N-gamma-acetyl-L-2,4-diaminobutyric acid into ectoine through a cyclocondensation reaction, but it can also use the isomer N-alpha-acetyl-L-2,4-diaminobutyric acid as its substrate, albeit with substantially reduced catalytic efficiency Sphingopyxis alaskensis DSM 13593 / LMG 18877 / RB2256 ?
-
?
N-alpha-acetyl-L-2,4-diaminobutanoate
-
Sphingopyxis alaskensis L-ectoine + H2O
-
?
N-alpha-acetyl-L-2,4-diaminobutanoate
-
Sphingopyxis alaskensis DSM 13593 / LMG 18877 / RB2256 L-ectoine + H2O
-
?

Subunits

Subunits Comment Organism
dimer
-
Sphingopyxis alaskensis
More overall structure of the open and semi-closed crystal structures of SaEctC, overview Sphingopyxis alaskensis

Synonyms

Synonyms Comment Organism
(Sa)Ect
-
Sphingopyxis alaskensis
EctC
-
Sphingopyxis alaskensis
L-ectoine synthase UniProt Sphingopyxis alaskensis
SaEctC
-
Sphingopyxis alaskensis

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
15
-
recombinant enzyme Sphingopyxis alaskensis

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.6
-
N-alpha-acetyl-L-2,4-diaminobutanoate pH 8.5, 15°C, recombinant enzyme Sphingopyxis alaskensis
7.2
-
(2S)-4-acetamido-2-aminobutanoate pH 8.5, 15°C, recombinant enzyme Sphingopyxis alaskensis

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
8.5
-
recombinant enzyme Sphingopyxis alaskensis

General Information

General Information Comment Organism
evolution the enzyme is a metal-containing member of the cupin superfamily. Cupins contain two conserved motifs: G(X)5HXH(X)3,4E(X)6G and G(X)5PXG(X)2H(X)3N (the letters in bold represent those residues that often coordinate the metal) Sphingopyxis alaskensis
metabolism synthesis of ectoine occurs from the intermediate metabolite L-aspartate-beta-semialdehyde and comprises the sequential activities of three enzymes: L-2,4-diaminobutyrate transaminase (EctB, EC 2.6.1.76), 2,4-diaminobutyrate acetyltransferase (EctA, EC 2.3.1.178), and ectoine synthase (EctC, EC 4.2.1.108) Sphingopyxis alaskensis

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
0.02
-
N-alpha-acetyl-L-2,4-diaminobutanoate pH 8.5, 15°C, recombinant enzyme Sphingopyxis alaskensis
1.47
-
(2S)-4-acetamido-2-aminobutanoate pH 8.5, 15°C, recombinant enzyme Sphingopyxis alaskensis