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 (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 | 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 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 | 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 | 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 | UniProt | Comment | Textmining |
---|---|---|---|
Sphingopyxis alaskensis | Q1GNW6 | i.e. Sphingomonas alaskensis | - |
Sphingopyxis alaskensis DSM 13593 / LMG 18877 / RB2256 | Q1GNW6 | i.e. Sphingomonas alaskensis | - |
Purification (Comment) | Organism |
---|---|
recombinant C-terminal Strep II-tagged enzyme from Escherichia coli by affinity chromatography and gel filtration to homogeneity | Sphingopyxis alaskensis |
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 | Comment | Organism |
---|---|---|
dimer | - |
Sphingopyxis alaskensis |
More | overall structure of the open and semi-closed crystal structures of SaEctC, overview | Sphingopyxis alaskensis |
Synonyms | Comment | Organism |
---|---|---|
(Sa)Ect | - |
Sphingopyxis alaskensis |
EctC | - |
Sphingopyxis alaskensis |
L-ectoine synthase | UniProt | Sphingopyxis alaskensis |
SaEctC | - |
Sphingopyxis alaskensis |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
15 | - |
recombinant enzyme | Sphingopyxis alaskensis |
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 Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
8.5 | - |
recombinant enzyme | Sphingopyxis alaskensis |
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 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 |