4.2.1.108

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

4.2.1.108

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

4.2.1.108

C105A

site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme

Sphingopyxis alaskensis

4.2.1.108

C105S

site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme

Sphingopyxis alaskensis

4.2.1.108

D91A

site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme

Sphingopyxis alaskensis

4.2.1.108

D91E

site-directed mutagenesis, the mutant shows similar activity as the wild-type enzyme

Sphingopyxis alaskensis

4.2.1.108

E115A

site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme

Sphingopyxis alaskensis

4.2.1.108

E115D

site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme

Sphingopyxis alaskensis

4.2.1.108

E57A

site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme

Sphingopyxis alaskensis

4.2.1.108

E57D

site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme

Sphingopyxis alaskensis

4.2.1.108

F107A

site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme

Sphingopyxis alaskensis

4.2.1.108

F107W

site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme

Sphingopyxis alaskensis

4.2.1.108

F107Y

site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme

Sphingopyxis alaskensis

4.2.1.108

H117A

site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme

Sphingopyxis alaskensis

4.2.1.108

H51A

site-directed mutagenesis, the mutant shows similar activity as the wild-type enzyme

Sphingopyxis alaskensis

4.2.1.108

H55A

site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme

Sphingopyxis alaskensis

4.2.1.108

H93A

site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme

Sphingopyxis alaskensis

4.2.1.108

H93N

site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme

Sphingopyxis alaskensis

4.2.1.108

L87A

site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme

Sphingopyxis alaskensis

4.2.1.108

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

4.2.1.108

S23A

site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme

Sphingopyxis alaskensis

4.2.1.108

T40A

site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme

Sphingopyxis alaskensis

4.2.1.108

T41A

site-directed mutagenesis, the mutant shows similar activity as the wild-type enzyme

Sphingopyxis alaskensis

4.2.1.108

W21A

site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme

Sphingopyxis alaskensis

4.2.1.108

Y52A

site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme

Sphingopyxis alaskensis

4.2.1.108

Y85A

site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme

Sphingopyxis alaskensis

4.2.1.108

Y85F

site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme

Sphingopyxis alaskensis

4.2.1.108

Y85W

site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme

Sphingopyxis alaskensis

4.2.1.108

Michaelis-Menten-kinetics

Sphingopyxis alaskensis

4.2.1.108

4.9

(2S)-4-acetamido-2-aminobutanoate

pH 8.5, 15°C, recombinant enzyme

Sphingopyxis alaskensis

4.2.1.108

25.4

N-alpha-acetyl-L-2,4-diaminobutanoate

pH 8.5, 15°C, recombinant enzyme

Sphingopyxis alaskensis

4.2.1.108

Fe2+

required, activates 100fold at 1 mM

Sphingopyxis alaskensis

4.2.1.108

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

4.2.1.108

(2S)-4-acetamido-2-aminobutanoate

Sphingopyxis alaskensis

L-ectoine + H2O

?

4.2.1.108

(2S)-4-acetamido-2-aminobutanoate

Sphingopyxis alaskensis DSM 13593 / LMG 18877 / RB2256

L-ectoine + H2O

?

4.2.1.108

Sphingopyxis alaskensis

Q1GNW6

i.e. Sphingomonas alaskensis

4.2.1.108

Sphingopyxis alaskensis DSM 13593 / LMG 18877 / RB2256

Q1GNW6

i.e. Sphingomonas alaskensis

4.2.1.108

recombinant C-terminal Strep II-tagged enzyme from Escherichia coli by affinity chromatography and gel filtration to homogeneity

Sphingopyxis alaskensis

4.2.1.108

(2S)-4-acetamido-2-aminobutanoate

749081

Sphingopyxis alaskensis

L-ectoine + H2O

?

4.2.1.108

(2S)-4-acetamido-2-aminobutanoate

i.e. N-gamma-acetyl-L-2,4-diaminobutanoate

749081

Sphingopyxis alaskensis

L-ectoine + H2O

?

4.2.1.108

(2S)-4-acetamido-2-aminobutanoate

749081

Sphingopyxis alaskensis DSM 13593 / LMG 18877 / RB2256

L-ectoine + H2O

?

4.2.1.108

(2S)-4-acetamido-2-aminobutanoate

i.e. N-gamma-acetyl-L-2,4-diaminobutanoate

749081

Sphingopyxis alaskensis DSM 13593 / LMG 18877 / RB2256

L-ectoine + H2O

?

4.2.1.108

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

749081

Sphingopyxis alaskensis

?

4.2.1.108

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

749081

Sphingopyxis alaskensis DSM 13593 / LMG 18877 / RB2256

?

4.2.1.108

N-alpha-acetyl-L-2,4-diaminobutanoate

749081

Sphingopyxis alaskensis

L-ectoine + H2O

?

4.2.1.108

N-alpha-acetyl-L-2,4-diaminobutanoate

749081

Sphingopyxis alaskensis DSM 13593 / LMG 18877 / RB2256

L-ectoine + H2O

?

4.2.1.108

dimer

Sphingopyxis alaskensis

4.2.1.108

More

overall structure of the open and semi-closed crystal structures of SaEctC, overview

Sphingopyxis alaskensis

4.2.1.108

(Sa)Ect

Sphingopyxis alaskensis

4.2.1.108

EctC

Sphingopyxis alaskensis

4.2.1.108

L-ectoine synthase

UniProt

Sphingopyxis alaskensis

4.2.1.108

SaEctC

Sphingopyxis alaskensis

4.2.1.108

15

recombinant enzyme

Sphingopyxis alaskensis

4.2.1.108

0.6

N-alpha-acetyl-L-2,4-diaminobutanoate

pH 8.5, 15°C, recombinant enzyme

Sphingopyxis alaskensis

4.2.1.108

7.2

(2S)-4-acetamido-2-aminobutanoate

pH 8.5, 15°C, recombinant enzyme

Sphingopyxis alaskensis

4.2.1.108

8.5

recombinant enzyme

Sphingopyxis alaskensis

4.2.1.108

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

4.2.1.108

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

4.2.1.108

C105A

site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme

Sphingopyxis alaskensis

4.2.1.108

C105S

site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme

Sphingopyxis alaskensis

4.2.1.108

D91A

site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme

Sphingopyxis alaskensis

4.2.1.108

D91E

site-directed mutagenesis, the mutant shows similar activity as the wild-type enzyme

Sphingopyxis alaskensis

4.2.1.108

E115A

site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme

Sphingopyxis alaskensis

4.2.1.108

E115D

site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme

Sphingopyxis alaskensis

4.2.1.108

E57A

site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme

Sphingopyxis alaskensis

4.2.1.108

E57D

site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme

Sphingopyxis alaskensis

4.2.1.108

F107A

site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme

Sphingopyxis alaskensis

4.2.1.108

F107W

site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme

Sphingopyxis alaskensis

4.2.1.108

F107Y

site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme

Sphingopyxis alaskensis

4.2.1.108

H117A

site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme

Sphingopyxis alaskensis

4.2.1.108

H51A

site-directed mutagenesis, the mutant shows similar activity as the wild-type enzyme

Sphingopyxis alaskensis

4.2.1.108

H55A

site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme

Sphingopyxis alaskensis

4.2.1.108

H93A

site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme

Sphingopyxis alaskensis

4.2.1.108

H93N

site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme

Sphingopyxis alaskensis

4.2.1.108

L87A

site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme

Sphingopyxis alaskensis

4.2.1.108

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

4.2.1.108

S23A

site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme

Sphingopyxis alaskensis

4.2.1.108

T40A

site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme

Sphingopyxis alaskensis

4.2.1.108

T41A

site-directed mutagenesis, the mutant shows similar activity as the wild-type enzyme

Sphingopyxis alaskensis

4.2.1.108

W21A

site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme

Sphingopyxis alaskensis

4.2.1.108

Y52A

site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme

Sphingopyxis alaskensis

4.2.1.108

Y85A

site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme

Sphingopyxis alaskensis

4.2.1.108

Y85F

site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme

Sphingopyxis alaskensis

4.2.1.108

Y85W

site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme

Sphingopyxis alaskensis

4.2.1.108

Michaelis-Menten-kinetics

Sphingopyxis alaskensis

4.2.1.108

4.9

(2S)-4-acetamido-2-aminobutanoate

pH 8.5, 15°C, recombinant enzyme

Sphingopyxis alaskensis

4.2.1.108

25.4

N-alpha-acetyl-L-2,4-diaminobutanoate

pH 8.5, 15°C, recombinant enzyme

Sphingopyxis alaskensis

4.2.1.108

Fe2+

required, activates 100fold at 1 mM

Sphingopyxis alaskensis

4.2.1.108

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

4.2.1.108

(2S)-4-acetamido-2-aminobutanoate

Sphingopyxis alaskensis

L-ectoine + H2O

?

4.2.1.108

(2S)-4-acetamido-2-aminobutanoate

Sphingopyxis alaskensis DSM 13593 / LMG 18877 / RB2256

L-ectoine + H2O

?

4.2.1.108

recombinant C-terminal Strep II-tagged enzyme from Escherichia coli by affinity chromatography and gel filtration to homogeneity

Sphingopyxis alaskensis

4.2.1.108

(2S)-4-acetamido-2-aminobutanoate

749081

Sphingopyxis alaskensis

L-ectoine + H2O

?

4.2.1.108

(2S)-4-acetamido-2-aminobutanoate

i.e. N-gamma-acetyl-L-2,4-diaminobutanoate

749081

Sphingopyxis alaskensis

L-ectoine + H2O

?

4.2.1.108

(2S)-4-acetamido-2-aminobutanoate

749081

Sphingopyxis alaskensis DSM 13593 / LMG 18877 / RB2256

L-ectoine + H2O

?

4.2.1.108

(2S)-4-acetamido-2-aminobutanoate

i.e. N-gamma-acetyl-L-2,4-diaminobutanoate

749081

Sphingopyxis alaskensis DSM 13593 / LMG 18877 / RB2256

L-ectoine + H2O

?

4.2.1.108

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

749081

Sphingopyxis alaskensis

?

4.2.1.108

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

749081

Sphingopyxis alaskensis DSM 13593 / LMG 18877 / RB2256

?

4.2.1.108

N-alpha-acetyl-L-2,4-diaminobutanoate

749081

Sphingopyxis alaskensis

L-ectoine + H2O

?

4.2.1.108

N-alpha-acetyl-L-2,4-diaminobutanoate

749081

Sphingopyxis alaskensis DSM 13593 / LMG 18877 / RB2256

L-ectoine + H2O

?

4.2.1.108

dimer

Sphingopyxis alaskensis

4.2.1.108

More

overall structure of the open and semi-closed crystal structures of SaEctC, overview

Sphingopyxis alaskensis

4.2.1.108

15

recombinant enzyme

Sphingopyxis alaskensis

4.2.1.108

0.6

N-alpha-acetyl-L-2,4-diaminobutanoate

pH 8.5, 15°C, recombinant enzyme

Sphingopyxis alaskensis

4.2.1.108

7.2

(2S)-4-acetamido-2-aminobutanoate

pH 8.5, 15°C, recombinant enzyme

Sphingopyxis alaskensis

4.2.1.108

8.5

recombinant enzyme

Sphingopyxis alaskensis

4.2.1.108

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

4.2.1.108

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

4.2.1.108

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

4.2.1.108

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

4.2.1.108

0.02

N-alpha-acetyl-L-2,4-diaminobutanoate

pH 8.5, 15°C, recombinant enzyme

Sphingopyxis alaskensis

4.2.1.108

1.47

(2S)-4-acetamido-2-aminobutanoate

pH 8.5, 15°C, recombinant enzyme

Sphingopyxis alaskensis

4.2.1.108

0.02

N-alpha-acetyl-L-2,4-diaminobutanoate

pH 8.5, 15°C, recombinant enzyme

Sphingopyxis alaskensis

4.2.1.108

1.47

(2S)-4-acetamido-2-aminobutanoate

pH 8.5, 15°C, recombinant enzyme

Sphingopyxis alaskensis