BRENDA - Reference to 1.14.11.55; Id = 740868

structural comparison, molecular dynamics simulations, and site-directed mutagenesis suggest the positioning of the iron, ectoine, and 2-oxoglutarate ligands in close proximity to each other and with a spatial orientation that will allow the region-selective and stereo-specific hydroxylation of (4S)-ectoine to (4S,5S)-5-hydroxyectoine

Virgibacillus salexigens

A163C

residue is not involved in ligand binding

Virgibacillus salexigens

A163C/S244C

residues are not involved in ligand binding

Virgibacillus salexigens

D148A

loss of activity. Residue is involved in binding of Fe2+

Virgibacillus salexigens

D148E

loss of activity. Residue is involved in binding of Fe2+

Virgibacillus salexigens

F143A

loss of activity. Residue is involved in binding of 2-oxoglutarate

Virgibacillus salexigens

F143W

loss of activity. Residue is involved in binding of 2-oxoglutarate

Virgibacillus salexigens

F143Y

loss of activity. Residue is involved in binding of 2-oxoglutarate

Virgibacillus salexigens

F242A

loss of activity. Residue is involved in binding of ectoine

Virgibacillus salexigens

F242W

loss of activity. Residue is involved in binding of ectoine

Virgibacillus salexigens

F242Y

3fold increase in Km value

Virgibacillus salexigens

F263A

loss of activity. Residue is involved in binding of ectoine

Virgibacillus salexigens

F263W

loss of activity. Residue is involved in binding of ectoine

Virgibacillus salexigens

F263Y

30% increase in Km value

Virgibacillus salexigens

F95A

loss of activity. Residue is involved in binding of 2-oxoglutarate

Virgibacillus salexigens

H146A

loss of activity. Residue is involved in binding of Fe2+

Virgibacillus salexigens

H248A

loss of activity. Residue is involved in binding of Fe2+

Virgibacillus salexigens

N133A

loss of activity. Residue is involved in binding of 2-oxoglutarate

Virgibacillus salexigens

N261A

residue is not involved in ligand binding

Virgibacillus salexigens

P198A

activity similar to wild-type, residue of loop region

Virgibacillus salexigens

Q129A

loss of activity. Residue is involved in binding of ectoine

Virgibacillus salexigens

R131A

loss of activity. Residue is involved in binding of 2-oxoglutarate

Virgibacillus salexigens

R259A

loss of activity. Residue is involved in binding of 2-oxoglutarate

Virgibacillus salexigens

R259H

loss of activity. Residue is involved in binding of 2-oxoglutarate

Virgibacillus salexigens

R259K

loss of activity. Residue is involved in binding of 2-oxoglutarate

Virgibacillus salexigens

R259Q

loss of activity. Residue is involved in binding of 2-oxoglutarate

Virgibacillus salexigens

S165A

3fold increase in Km value

Virgibacillus salexigens

S167A

residue is not involved in ligand binding

Virgibacillus salexigens

S205A

activity similar to wild-type, residue of loop region

Virgibacillus salexigens

S244C

residue is not involved in ligand binding

Virgibacillus salexigens

S250A

loss of activity. Residue is involved in binding of 2-oxoglutarate

Virgibacillus salexigens

V265A

residue is not involved in ligand binding

Virgibacillus salexigens

V265L

residue is not involved in ligand binding

Virgibacillus salexigens

V265T

residue is not involved in ligand binding

Virgibacillus salexigens

W152A

loss of activity. Residue is involved in binding of ectoine

Virgibacillus salexigens

W152F

loss of activity. Residue is involved in binding of ectoine

Virgibacillus salexigens

W152Y

loss of activity. Residue is involved in binding of ectoine

Virgibacillus salexigens

5.9

-

ectoine

wild-type, pH 7.5, 32°C

Virgibacillus salexigens

6.8

-

ectoine

mutant S205A, pH 7.5, 32°C

Virgibacillus salexigens

7.1

-

ectoine

mutant P198A, pH 7.5, 32°C

Virgibacillus salexigens

8

-

ectoine

mutant F263Y, pH 7.5, 32°C

Virgibacillus salexigens

17.3

-

ectoine

mutant S165A, pH 7.5, 32°C

Virgibacillus salexigens

19.6

-

ectoine

mutant F242Y, pH 7.5, 32°C

Virgibacillus salexigens

Q2TDY4

-

ectoine + 2-oxoglutarate + O2

-

740868

Virgibacillus salexigens

5-hydroxyectoine + succinate + CO2

-

?

structural comparison, molecular dynamics simulations, and site-directed mutagenesis suggest the positioning of the iron, ectoine, and 2-oxoglutarate ligands in close proximity to each other and with a spatial orientation that will allow the region-selective and stereo-specific hydroxylation of (4S)-ectoine to (4S,5S)-5-hydroxyectoine

Virgibacillus salexigens

A163C

residue is not involved in ligand binding

Virgibacillus salexigens

A163C/S244C

residues are not involved in ligand binding

Virgibacillus salexigens

D148A

loss of activity. Residue is involved in binding of Fe2+

Virgibacillus salexigens

D148E

loss of activity. Residue is involved in binding of Fe2+

Virgibacillus salexigens

F143A

loss of activity. Residue is involved in binding of 2-oxoglutarate

Virgibacillus salexigens

F143W

loss of activity. Residue is involved in binding of 2-oxoglutarate

Virgibacillus salexigens

F143Y

loss of activity. Residue is involved in binding of 2-oxoglutarate

Virgibacillus salexigens

F242A

loss of activity. Residue is involved in binding of ectoine

Virgibacillus salexigens

F242W

loss of activity. Residue is involved in binding of ectoine

Virgibacillus salexigens

F242Y

3fold increase in Km value

Virgibacillus salexigens

F263A

loss of activity. Residue is involved in binding of ectoine

Virgibacillus salexigens

F263W

loss of activity. Residue is involved in binding of ectoine

Virgibacillus salexigens

F263Y

30% increase in Km value

Virgibacillus salexigens

F95A

loss of activity. Residue is involved in binding of 2-oxoglutarate

Virgibacillus salexigens

H146A

loss of activity. Residue is involved in binding of Fe2+

Virgibacillus salexigens

H248A

loss of activity. Residue is involved in binding of Fe2+

Virgibacillus salexigens

N133A

loss of activity. Residue is involved in binding of 2-oxoglutarate

Virgibacillus salexigens

N261A

residue is not involved in ligand binding

Virgibacillus salexigens

P198A

activity similar to wild-type, residue of loop region

Virgibacillus salexigens

Q129A

loss of activity. Residue is involved in binding of ectoine

Virgibacillus salexigens

R131A

loss of activity. Residue is involved in binding of 2-oxoglutarate

Virgibacillus salexigens

R259A

loss of activity. Residue is involved in binding of 2-oxoglutarate

Virgibacillus salexigens

R259H

loss of activity. Residue is involved in binding of 2-oxoglutarate

Virgibacillus salexigens

R259K

loss of activity. Residue is involved in binding of 2-oxoglutarate

Virgibacillus salexigens

R259Q

loss of activity. Residue is involved in binding of 2-oxoglutarate

Virgibacillus salexigens

S165A

3fold increase in Km value

Virgibacillus salexigens

S167A

residue is not involved in ligand binding

Virgibacillus salexigens

S205A

activity similar to wild-type, residue of loop region

Virgibacillus salexigens

S244C

residue is not involved in ligand binding

Virgibacillus salexigens

S250A

loss of activity. Residue is involved in binding of 2-oxoglutarate

Virgibacillus salexigens

V265A

residue is not involved in ligand binding

Virgibacillus salexigens

V265L

residue is not involved in ligand binding

Virgibacillus salexigens

V265T

residue is not involved in ligand binding

Virgibacillus salexigens

W152A

loss of activity. Residue is involved in binding of ectoine

Virgibacillus salexigens

W152F

loss of activity. Residue is involved in binding of ectoine

Virgibacillus salexigens

W152Y

loss of activity. Residue is involved in binding of ectoine

Virgibacillus salexigens

5.9

-

ectoine

wild-type, pH 7.5, 32°C

Virgibacillus salexigens

6.8

-

ectoine

mutant S205A, pH 7.5, 32°C

Virgibacillus salexigens

7.1

-

ectoine

mutant P198A, pH 7.5, 32°C

Virgibacillus salexigens

8

-

ectoine

mutant F263Y, pH 7.5, 32°C

Virgibacillus salexigens

17.3

-

ectoine

mutant S165A, pH 7.5, 32°C

Virgibacillus salexigens

19.6

-

ectoine

mutant F242Y, pH 7.5, 32°C

Virgibacillus salexigens

ectoine + 2-oxoglutarate + O2

-

740868

Virgibacillus salexigens

5-hydroxyectoine + succinate + CO2

-

?

741719

Tao

Ectoine and 5-hydroxyectoine ...

Virgibacillus halodenitrificans, Virgibacillus halodenitrificans PDB-F2

Appl. Microbiol. Biotechnol.

100

6779-6789

2016

-

2

-

2

-

2

-

1

-

2

-

2

-

1

-

1

-

742383

Widderich

Strangers in the archaeal wor ...

Nitrosopumilus maritimus

Environ. Microbiol.

18

1227-1248

2016

-

1

-

1

-

7

-

1

-

1

-

1

-

1

-

1

-

1

-

741466

Yang

Crystallization and X-ray diff ...

Bacillus pseudofirmus, Bacillus pseudofirmus OF4

Wei Sheng Wu Xue Bao

55

1468-1474

2015

-

1

-

5

-

1

-

1

-

1

-

739813

Hoeppner

Overexpression, crystallizatio ...

Sphingopyxis alaskensis

Acta Crystallogr. Sect. F

70

493-496

2014

-

1

-

1

-

2

-

1

-

1

-

1

-

740729

Hoeppner

Crystal structure of the ectoi ...

Sphingopyxis alaskensis, Sphingopyxis alaskensis DSM 13593

J. Biol. Chem.

289

29570-29583

2014

-

1

7

-

2

-

2

-

5

-

2

1

-

1

7

-

2

-

2

-

2

1

-

740868

Widderich

Molecular dynamics simulations ...

Virgibacillus salexigens

J. Mol. Biol.

426

586-600

2014

-

1

36

-

6

-

3

-

1

-

1

36

-

6

-

1

-

741304

Widderich

Biochemical properties of ecto ...

Acidiphilium cryptum, Alkalilimnicola ehrlichii, Halomonas elongata, Halomonas elongata DSM 2581, Paenibacillus lautus, Pseudomonas stutzeri, Pseudomonas stutzeri A1501, Sphingopyxis alaskensis, Sphingopyxis alaskensis DSM 13593, Virgibacillus salexigens

PLoS ONE

9

e93809

2014

-

1

-

7

14

-

21

7

20

-

17

-

20

7

8

7

-

7

-

7

-

1

-

7

-

14

-

21

7

20

-

20

7

-

7

-

7

-

7

743087

Widderich

Molecular dynamics simulation ...

Virgibacillus salexigens

J. Mol. Biol.

426

586-600

2014

-

1

-

36

-

14

-

1

-

1

-

3

-

1

-

1

-

1

-

36

-

14

-

1

-

1

-

1

-

740784

Eilert

Synthesis and release of the b ...

Halomonas elongata, Halomonas elongata DSM 2581

J. Biotechnol.

167

85-93

2013

-

1

-

3

-

1

-

1

-

741265

Reuter

Synthesis of 5-hydroxyectoine ...

Virgibacillus salexigens

PLoS ONE

5

e10647

2010

-

1

-

5

-

1

-

1

-

739899

Bursy

Synthesis and uptake of the co ...

Streptomyces coelicolor, Streptomyces coelicolor A3(2)

Appl. Environ. Microbiol.

74

7286-7296

2008

-

1

2

-

1

-

43

-

1

-

1

-

4

1

-

1

-

1

-

1

-

2

-

1

-

1

-

1

-

4

1

-

1

-

1

-

740684

Bursy

Osmotically induced synthesis ...

Virgibacillus salexigens

J. Biol. Chem.

282

31147-31155

2007

-

1

2

-

1

3

-

6

-

1

-

1

-

2

1

-

1

-

1

-

1

-

2

-

1

3

-

1

-

1

-

2

1

-

1

-

1

-

1

-

739896

Prabhu

Functional expression of the e ...

Streptomyces anulatus

Appl. Environ. Microbiol.

70

3130-3132

2004

-

1

-

3

-

1

-

1

-

1

-

1

-

Molecular dynamics simulations and structure-guided mutagenesis provide insight into the architecture of the catalytic core of the ectoine hydroxylase

Data extracted from this reference: