Information on EC 5.1.3.14 - UDP-N-acetylglucosamine 2-epimerase (non-hydrolysing)

N-Acetyl-D-glucosamine

?

-

-

N-Acetyl-D-glucosamine 1-phosphate

?

-

-

UDP-GlcNAc

ManNAc + UDP

UDP-GlcNAc + H2O

ManNAc + UDP

-

biosynthesis of sialic acids

-

?

UDP-glucose

?

-

-

UDP-N-acetyl-alpha-D-glucosamine

UDP-N-acetyl-alpha-D-mannosamine

25 entries

UDP-N-acetyl-alpha-D-mannosamine

UDP-N-acetyl-alpha-D-glucosamine

8 entries

UDP-N-acetyl-D-glucosamine

?

7 entries

UDP-N-acetyl-D-glucosamine

UDP + N-acetyl-D-mannosamine

25 entries

UDP-N-acetyl-D-glucosamine

UDP-N-acetyl-D-mannosamine

UDP-N-acetyl-D-glucosamine + H2O

UDP + N-acetylmannosamine

5 entries

UDP-N-acetylgalactosamine

?

-

-

uridine 5'-diphospho-2-hydroxyacetamido-2-deoxy-alpha-D-mannopyranoside

?

uridine 5'-diphospho-2-propionamido-2-deoxy-alpha-D-mannopyranoside

?

Neisseria meningitidis serogroup A / serotype 4A

-

-

?

uridine 5'-diphospho-N-trifluoroaceto-2-deoxy-alpha-D-mannopyranoside

?

Neisseria meningitidis serogroup A / serotype 4A

-

-

?

additional information

?

-

30 entries

UDP-GlcNAc

ManNAc + UDP

UDP-GlcNAc + H2O

ManNAc + UDP

-

biosynthesis of sialic acids

-

?

UDP-N-acetyl-alpha-D-glucosamine

UDP-N-acetyl-alpha-D-mannosamine

14 entries

UDP-N-acetyl-D-glucosamine

?

7 entries

UDP-N-acetyl-D-glucosamine

UDP + N-acetyl-D-mannosamine

4 entries

UDP-N-acetyl-D-glucosamine + H2O

UDP + N-acetylmannosamine

4 entries

additional information

?

-

15 entries

Mg2+

-

increases activity

additional information

(2S)-2-[(4E)-4-[[5-(3,4-dichlorophenyl)furan-2-yl]methylidene]-5-oxo-2-thioxoimidazolidin-1-yl]-3-phenylpropanamide

(2S)-2-[(4E)-4-[[5-(3,4-dichlorophenyl)furan-2-yl]methylidene]-5-oxo-2-thioxoimidazolidin-1-yl]-3-phenylpropanoic acid

(2S)-2-[(4E)-4-[[5-(3,4-dichlorophenyl)furan-2-yl]methylidene]-5-oxo-2-thioxoimidazolidin-1-yl]butanoic acid

(2S)-2-[(4E)-4-[[5-(3,4-dichlorophenyl)thiophen-2-yl]methylidene]-5-oxo-2-thioxoimidazolidin-1-yl]-3-phenylpropanoic acid

(2S)-2-[(4E)-4-[[5-(3,5-dichlorophenyl)furan-2-yl]methylidene]-5-oxo-2-thioxoimidazolidin-1-yl]-3-phenylpropanoic acid

(2S)-2-[(4E)-4-[[5-(3-chloro-4-methoxyphenyl)furan-2-yl]methylidene]-5-oxo-2-thioxoimidazolidin-1-yl]-3-phenylpropanoic acid

(2S)-2-[(4E)-4-[[5-(3-chlorophenyl)furan-2-yl]methylidene]-5-oxo-2-thioxoimidazolidin-1-yl]-3-phenylpropanoic acid

(2S)-2-[(4E)-4-[[5-(4-bromo-3-chlorophenyl)furan-2-yl]methylidene]-5-oxo-2-thioxoimidazolidin-1-yl]-3-phenylpropanoic acid

(2S)-2-[(4E)-4-[[5-(4-bromo-3-chlorophenyl)thiophen-2-yl]methylidene]-5-oxo-2-thioxoimidazolidin-1-yl]-3-phenylpropanoic acid

(2S)-2-[(4E)-4-[[5-(4-bromophenyl)furan-2-yl]methylidene]-5-oxo-2-thioxoimidazolidin-1-yl]-3-phenylpropanoic acid

(2S)-2-[(4E)-4-[[5-(4-bromophenyl)thiophen-2-yl]methylidene]-5-oxo-2-thioxoimidazolidin-1-yl]-3-phenylpropanoic acid

(2S)-2-[(4E)-4-[[5-(4-chlorophenyl)furan-2-yl]methylidene]-5-oxo-2-thioxoimidazolidin-1-yl]-3-phenylpropanoic acid

(2S)-2-[(4E)-4-[[5-(4-chlorophenyl)thiophen-2-yl]methylidene]-5-oxo-2-thioxoimidazolidin-1-yl]-3-phenylpropanoic acid

(2S)-2-[(4E)-4-[[5-(4-fluorophenyl)thiophen-2-yl]methylidene]-5-oxo-2-thioxoimidazolidin-1-yl]-3-phenylpropanoic acid

(2S)-2-[(4E)-4-[[5-(4-iodophenyl)furan-2-yl]methylidene]-5-oxo-2-thioxoimidazolidin-1-yl]-3-phenylpropanoic acid

(2S)-2-[(4E)-4-[[5-(4-methoxyphenyl)thiophen-2-yl]methylidene]-5-oxo-2-thioxoimidazolidin-1-yl]-3-phenylpropanoic acid

(2S)-2-[(4E)-5-oxo-2-thioxo-4-([5-[4-(trifluoromethoxy)phenyl]furan-2-yl]methylidene)imidazolidin-1-yl]-3-phenylpropanoic acid

(2S)-2-[(4E)-5-oxo-4-[(5-phenylfuran-2-yl)methylidene]-2-thioxoimidazolidin-1-yl]-3-phenylpropanoic acid

2',3'-dialdehydro-ADP

-

efficient inhibition is most likely due to the structural similarity to o-UDP and not to an allosteric effect via the ATP binding site

651305

2',3'-dialdehydro-UDP

-

binds to the active site of the enzyme

651305

2',3'-dialdehydro-UDP-alpha-D-N-acetylglucosamine

-

0.05 mM, 70% inhibition after 30 min. 0.25 mM, 90% inhibition. Covalently bound to amino acids in the active site causing an irreversible inhibition. Effective inhibitor may serve as a basis for the chemical synthesis of further inhibitors

651305

2-acetamidoglucal

3-acetamido-2,6-anhydro-3-deoxy-D-arabino-hept-2-enopyranosonate

-

Ca2+

Cd2+

-

CMP-N-acetylneuraminic acid

CMP-Neu5Ac

CMP-sialic acid

GNE/MNK is feedback inhibited by binding of the downstream product, CMP-sialic acid, in its allosteric site. The allosteric regulation by CMP-sialic acid involves residues D255, E260, R263, R266, K268, and N275

Co2+

Cs+

-

Cu2+

ethyl (2S)-2-[(4E)-4-[[5-(3,4-dichlorophenyl)furan-2-yl]methylidene]-5-oxo-2-thioxoimidazolidin-1-yl]-3-phenylpropanoate

Q9REV4

-

726555

ethyl (2S)-2-[(4E)-4-[[5-(3,4-dichlorophenyl)furan-2-yl]methylidene]-5-oxo-2-thioxoimidazolidin-1-yl]-3-phenylpropanoates

-

Fe3+

-

2257, 2277

Hg2+

-

Li+

-

Mg2+

Mn2+

N-[1-[(4E)-4-[[5-(3,4-dichlorophenyl)furan-2-yl]methylidene]-5-oxo-2-thioxoimidazolidin-1-yl]-2-phenylethyl]-1,1,1-trifluoromethanesulfonamide

N-[1-[(4E)-4-[[5-(3,4-dichlorophenyl)furan-2-yl]methylidene]-5-oxo-2-thioxoimidazolidin-1-yl]-2-phenylethyl]methanesulfonamide

N-[1-[(4E)-4-[[5-(3,4-dichlorophenyl)furan-2-yl]methylidene]-5-oxo-2-thioxoimidazolidin-1-yl]propyl]methanesulfonamide

NEM

-

Ni2+

Pb2+

-

PCMB

Selenite

-

SeO2

-

tunicamycin

UDP

UDPglucose

-

UMP

-

uridine 5'-(3-acetamido-3-deoxy-2-O-methyl-alpha-D-gluco-hept-2-ulopyranos-1-yl diphosphate)

-

uridine 5'-(3-acetamido-3-deoxy-2-O-methyl-alpha-D-manno-hept-2-ulopyranos-1-yl diphosphate)

-

weak

uridine 5'-[(Z)-2,6-anhydro-1-deoxy-D-galactohept-1-enitol-1-yl phosphono] phosphate

-

weak

uridine 5'-[(Z)-2,6-anhydro-1-deoxy-D-glucohept-1-enitol-1-yl phosphono] phosphate

-

uridine 5'-[(Z)-2,6-anhydro-1-deoxy-D-mannohept-1-enitol-1-yl phosphono] phosphate

-

uridine 5'-[(Z)-3-acetamido-2,6-anhydro-1,3-dideoxy-D-arabino-hept-1-enitol-1-yl phosphono] phosphate

-

uridine 5'-[(Z)-3-acetamido-2,6-anhydro-1,3-dideoxy-D-gluco-hept-1-enitol-1-yl phosphono] phosphate

-

UTP

-

VO3-

-

Zn2+

[1-[(4E)-4-[[5-(3,4-dichlorophenyl)furan-2-yl]methylidene]-5-oxo-2-thioxoimidazolidin-1-yl]-2-phenylethyl]cyanamide

additional information

10 entries

-

KCl

200 mM, twofold stimualtion

UDP-GlcNAc

UDP-N-acetyl-alpha-D-glucosamine

Neisseria meningitidis serogroup A /serotype 4A

activates the recombinant enzyme, UDP-GlcNAc is able to activate the epimerization process in a dose-dependent manner

UDP-N-acetyl-D-glucosamine

-

allosteric activation

UDP-N-acetylglucosamine

additional information

-

3.6

UDP-N-acetyl-alpha-D-glucosamine

Neisseria meningitidis serogroup A / serotype 4A

pH 8.5, 37°C, recombinant enzyme

5.2

UDP-N-acetyl-alpha-D-mannosamine

Neisseria meningitidis serogroup A / serotype 4A

pH 8.5, 37°C, recombinant enzyme

0.011 - 20.9

UDP-N-acetyl-D-glucosamine

18 entries

0.63

UDP-N-acetylglucosamine

-

0.22 - 1

UDP-N-acetylmannosamine

additional information

Hill coefficient of 2.7, positive cooperativity

-

31

UDP-N-acetyl-alpha-D-glucosamine

Neisseria meningitidis serogroup A / serotype 4A

pH 8.5, 37°C, recombinant enzyme

52

UDP-N-acetyl-alpha-D-mannosamine

Neisseria meningitidis serogroup A / serotype 4A

pH 8.5, 37°C, recombinant enzyme

0.021 - 7.9

UDP-N-acetyl-D-glucosamine

10 entries

10

UDP-N-acetyl-alpha-D-glucosamine

Neisseria meningitidis serogroup A / serotype 4A

pH 8.5, 37°C, recombinant enzyme

8.6

UDP-N-acetyl-alpha-D-mannosamine

Neisseria meningitidis serogroup A / serotype 4A

pH 8.5, 37°C, recombinant enzyme

14

2-acetamidoglucal

Neisseria meningitidis serogroup A / serotype 4A

pH 8.5, 37°C, recombinant enzyme

0.025

CMP-N-acetylneuraminic acid

-

0.8

UDP

Neisseria meningitidis serogroup A / serotype 4A

pH 8.5, 37°C, recombinant enzyme

0.29

-

0.6

-

0.7

-

0.878

-

10.9

-

enzyme expressed in Sf-900 insect cells and purified

663299

7.08

-

additional information

-

7 - 9

-

7.1

-

and a second optimum at pH 7.9

2259

7.5

-

assay at

7.5 - 8

-

7.7

-

7.9

-

and a second optimum at pH 7.1

8

8 - 8.5

-

8 - 9

6 - 10

-

6 - 11

6.5 - 8.6

-

6.5: about 30% of maximal activity, 8.6: about 65% of maximal activity

2259

30

37

50

-

-

-

-

-

Bacillus subtilis 168

-

UniProt

-

-

-

-

K1

-

-

-

Methanocaldococcus jannaschii DSM 2661

-

728782, 749249

Q58899

SwissProt

-

728782

Q58899

SwissProt

-

UniProt

Micrococcus luteus

-

2264, 2273

-

MC58 group B

-

Neisseria meningitidis serogroup A / serotype 4A

-

UniProt

Neisseria meningitidis serogroup A / serotype 4A Z2491

-

UniProt

Neisseria meningitidis serogroup A /serotype 4A

-

UniProt

Neisseria meningitidis serogroup A /serotype 4A Z2491

-

UniProt

MRSA

Staphylococcus epidermidis

UniProt

MRSE

A0A447XK48

UniProt

Staphylococcus epidermidis CLB26329

MRSE

Ehrlich ascites carcinoma cell

A0A447XK48

UniProt

-

HIV-infected. Downregulation of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase in hyposialylated HIV-infected T cells

661682

colon

4 entries

-

-

cell proliferation is directly correlated with GNE-expression and the cellular sialic acid concentration. Growth-related genes are differentially expressed in GNE-deficient embryonic stem cells compared to wild-type embryonic stem cells

703905

fibroblast

-

skin

2275

fibrosarcoma cell

-

enzyme activity is 18% of the activity found in normal livers

-

-

human epithel, HEK AD293

-

-

-

additional information

-

GNE2 and GNE3 display tissue-specific expression patterns

-

-

evolution

malfunction

metabolism

physiological function

16 entries

additional information

7 entries

3beo, download, 3D-view

Bacillus subtilis (strain 168)

5dld, download, 3D-view

SCOPe (5dld)

CATH (5dld)

A4JLG9

Burkholderia vietnamiensis (strain G4 / LMG 22486)

Escherichia coli (strain K12)

3eo3, download, 3D-view

3ot5, download, 3D-view

SCOPe (3ot5)

CATH (3ot5)

Q8Y4B4

Listeria monocytogenes serovar 1/2a (strain ATCC BAA-679 / EGD-e)

Methanocaldococcus jannaschii (strain ATCC 43067 / DSM 2661 / JAL-1 / JCM 10045 / NBRC 100440)

4hwg, download, 3D-view

SCOPe (4hwg)

CATH (4hwg)

Q1RIM5

Rickettsia bellii (strain RML369-C)

5enz, download, 3D-view

SCOPe (5enz)

CATH (5enz)

Q9REV4

1v4v, download, 3D-view

3dzc, download, 3D-view

SCOPe (3dzc)

CATH (3dzc)

Q9KTI5

Vibrio cholerae serotype O1 (strain ATCC 39315 / El Tor Inaba N16961)

101000

gel filtration

330000

-

dynamic light-scattering

38000

-

2 * 38000, SDS-PAGE

39368

Bordetella pertussis

-

x * 39368, calculation from nucleotide sequence

649142

40000

42400

2 * 40000, SDS-PAGE, 2 * 42400, calculated, recombinant enzyme

450000

-

gel filtration

2280, 641739

500000

-

gel filtration

75000

-

6 * 75000, SDS-PAGE

76000

-

gel filtraton

79000

O35826

x * 79000, calculation from nucleotide sequence

2279

85000

-

6 * 85000, SDS-PAGE

?

dimer

hexamer

tetramer

additional information

6 entries

phosphoprotein

ternary complex between the UDP-GlcNAc 2-epimerase, its substrate UDP-GlcNAc and the reaction intermediate UDP, at 1.7 A resolution. Direct interactions between the substrate and UDP via two hydrogen bonds to the alpha- and beta-phosphates of the adjacent UDP molecule, and between the complex and highly conserved enzyme residues. The binding of UDP-GlcNAc is associated with conformational changes in the active site of the enzyme

-

UDP-GlcNAc 2-epimerase in complex with UDPGlcNAc and UDP, X-ray diffraction structure determination and analysis at 1.69 A resolution, molecular replacement using enzyme structure, PDB ID 3BEO, as search model

Bacillus subtilis

P39131

vapor diffusion method

Bordetella pertussis

-

649142

hanging drop vapor diffusion method, selenomethionyl enzyme, X-ray structure at 2.5 A of the enzyme with bound UDP

-

649940

sitting-drop vapor-diffusion method, crystal structures in open and closed conformations. A comparison of these crystal structures shows that upon UDP and UDPGlcNAc binding, the enzyme undergoes conformational changes involving a rigid-body movement of the C-terminal domain

-

728782

UDP-GlcNAc 2-epimerase in open and closed conformations, and UDP-GlcNAc 2-epimerase in complex with UDPGlcNAc and UDP, sitting drop vapor diffusion method, mixing of 0.001 ml of 5 mg/ml protein in 50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 5% glycerol, and 2 mM tris(2-carboxyethyl)phosphine, with 0.001 ml of reservoir solution containing 40 mM Tris-propane, 60 mM citrate, pH 4.1, and 16% PEG3350, and equilibration against 0.3 ml of reservoir solution at 20°C for 1 week, X-ray diffraction structure determination and analysis at 1.42-2.85 A resolution

-

purified enzyme MnaA, hanging drop vapor diffusion method, mixing of 31 mg/ml protein solution with crystallization solution containing 0.1 M Tris-HCl, pH 8.0, 0.1 M Na2SO4, 52% PEG 400, 22°C, method optimization, X-ray diffraction structure determination and analysis at 1.9 A resolution, molecular replacement using structure PDB ID 1F6D as template

H242A

-

dramatic decrease in catalytic efficiency

H44Q

-

dramatic decrease in catalytic efficiency

Q43A

-

dramatic decrease in catalytic efficiency

Q70A

-

dramatic decrease in catalytic efficiency

R210A

-

dramatic decrease in catalytic efficiency

C303V

-

exhibited almost no reduction in epimerase activity

C303X

-

the C303X protein does not display any enzymatic activity

D378Y

-

60% reduction of epimerase activity

D95N

-

about 18000 fold decrease in turnover number for UDP-N-acetyl-D-glucosamine, not possible to obtain accurate kinetic constants

E117Q

-

about 18000 fold decrease in turnover number for UDP-N-acetyl-D-glucosamine, not possible to obtain accurate kinetic constants

E131Q

-

about 18000 fold decrease in turnover number for UDP-N-acetyl-D-glucosamine, not possible to obtain accurate kinetic constants

H213N

-

30fold increase in Km-value and 50fold decrease in turnover-number for UDP-N-acetyl-D-glucosamine. Unlike the wild-type enzyme no inhibition is detected at UDP-concentrations up to 10 mM

I200F

-

exhibited almost no reduction in epimerase activity

K15A

-

more than 100fold increase in KM-value for UDP-N-acetyl-D-glucosamine

A630T

-

mutation in patients with distal myopathy with rimmed vacuoles, UDP-N-acetylglucosamine 2-epimerase activity of mutant enzyme is reduced to 70-80% of wild-type activity

A631V

-

a naturally occuring missense mutation in exon 11 of the GNE gene of a patient with hereditary inclusion body myopathy

C13S

C303V

a naturally occuirng missense mutation the epimerase part of the bifunctional enzyme

C303X

a naturally occuirng missense mutation the epimerase part of the bifunctional enzyme

D176V

D177C

-

mutation in patients with distal myopathy with rimmed vacuoles, UDP-N-acetylglucosamine 2-epimerase activity of mutant enzyme is reduced to less than 20% of wild-type

D225N

a naturally occuirng missense mutation the epimerase part of the bifunctional enzyme

D378Y

E2G

-

a naturally occuring missense mutation in exon 2 of the GNE gene of a patient with hereditary inclusion body myopathy

G135V

a naturally occuirng missense mutation the epimerase part of the bifunctional enzyme

G135V/R246W

-

mutation in patients with hereditary inclusion body myopathy: G135V/R246W (GNE/GNE domain mutation), UDP-N-acetylglucosamine 2-epimerase activity is 38% of wild-type, N-acetylmannosamine kinase activity is 72% of wild-type

661840

G206S

a naturally occuirng missense mutation the epimerase part of the bifunctional enzyme

G708S

-

mutation in patients with distal myopathy with rimmed vacuoles, UDP-N-acetylglucosamine 2-epimerase activity of mutant enzyme is reduced to 50% of wild-type activity

G89R

a naturally occuirng missense mutation the epimerase part of the bifunctional enzyme

H132Q

I142T

-

a naturally occuring missense mutation in exon 3 of the GNE gene of a patient with hereditary inclusion body myopathy

I200F

a naturally occuirng missense mutation the epimerase part of the bifunctional enzyme

I241S

a naturally occuirng missense mutation the epimerase part of the bifunctional enzyme

I298T

-

a naturally occuring missense mutation in exon 5 of the GNE gene of a patient with hereditary inclusion body myopathy

I472T

-

mutation in patients with distal myopathy with rimmed vacuoles, UDP-N-acetylglucosamine 2-epimerase activity of mutant enzyme is reduced to 50% of wild-type activity

L379H

a naturally occuirng missense mutation the epimerase part of the bifunctional enzyme

L556S

-

a naturally occuring missense mutation in exon 10 of the GNE gene of a patient with hereditary inclusion body myopathy

M171V

a naturally occuirng missense mutation the epimerase part of the bifunctional enzyme

M29T

a naturally occuirng missense mutation the epimerase part of the bifunctional enzyme

M712T

M712T/M712T

-

M712T/M712T (MNK/MNK domain mutation), UDP-N-acetylglucosamine 2-epimerase activity is 83% of wild-type, N-acetylmannosamine kinase activity is 55% of wild-type

661840

P27S

a naturally occuirng missense mutation the epimerase part of the bifunctional enzyme

P283S

a naturally occuirng missense mutation the epimerase part of the bifunctional enzyme

P36L

a naturally occuirng missense mutation the epimerase part of the bifunctional enzyme

Q436X

-

a naturally occuring nonsense mutation in exon 8 of the GNE gene of a patient with hereditary inclusion body myopathy

R11W

a naturally occuirng missense mutation the epimerase part of the bifunctional enzyme

R129Q

a naturally occuirng missense mutation the epimerase part of the bifunctional enzyme

R162C

a naturally occuirng missense mutation the epimerase part of the bifunctional enzyme

R177C

a naturally occuirng missense mutation the epimerase part of the bifunctional enzyme

R202L

a naturally occuirng missense mutation the epimerase part of the bifunctional enzyme

R246Q

R246W

a naturally occuirng missense mutation the epimerase part of the bifunctional enzyme

R263L

a naturally occuirng missense mutation the epimerase part of the bifunctional enzyme

R266Q

R266W

R277C

a naturally occuirng missense mutation the epimerase part of the bifunctional enzyme

R306Q

a naturally occuirng missense mutation the epimerase part of the bifunctional enzyme

R335W

R71W

-

a naturally occuring missense mutation in exon 3 of the GNE gene of a patient with hereditary inclusion body myopathy

R8X

-

a naturally occuring nonsense mutation in exon 2 of the GNE gene of a patient with hereditary inclusion body myopathy

S615X

-

a naturally occuring nonsense mutation in exon 11 of the GNE gene of a patient with hereditary inclusion body myopathy

V216A

a naturally occuirng missense mutation the epimerase part of the bifunctional enzyme

V216A/A631V

-

V216A/A631V (GNE/MNK domain mutation), UDP-N-acetylglucosamine 2-epimerase activity is 48% of wild-type, N-acetylmannosamine kinase activity is 63% of wild-type

V331A

V367I

a naturally occuirng missense mutation the epimerase part of the bifunctional enzyme

V572L

-

mutation in patients with distal myopathy with rimmed vacuoles, UDP-N-acetylglucosamine 2-epimerase activity of mutant enzyme is reduced to 70-80% of wild-type activity

V696M

W204X

-

a naturally occuring nonsense mutation in exon 3 of the GNE gene of a patient with hereditary inclusion body myopathy

Y675H

-

a naturally occuring missense mutation in exon 12 of the GNE gene of a patient with hereditary inclusion body myopathy

D100N

-

no conversion of UDP-N-acetyl-D-glucosamine to UDP + N-acetyl-D-mannosamine

D131N

-

no conversion of UDP-N-acetyl-D-glucosamine to UDP + N-acetyl-D-mannosamine, acetamidoglucal is released from the active site during catalysis

E122Q

-

no conversion of UDP-N-acetyl-D-glucosamine to UDP + N-acetyl-D-mannosamine, acetamidoglucal is released from the active site during catalysis

Neisseria meningitidis MC58 group B

D100N

-

no conversion of UDP-N-acetyl-D-glucosamine to UDP + N-acetyl-D-mannosamine

-

D131N

Neisseria meningitidis MC58 group B

-

no conversion of UDP-N-acetyl-D-glucosamine to UDP + N-acetyl-D-mannosamine, acetamidoglucal is released from the active site during catalysis

-

E122Q

Neisseria meningitidis MC58 group B

-

no conversion of UDP-N-acetyl-D-glucosamine to UDP + N-acetyl-D-mannosamine, acetamidoglucal is released from the active site during catalysis

-

D413K

-

enzyme with mutation in the putative kinase active site shows drastic loss in their kinase activity but retains their epimerase activity

D413N

-

enzyme with mutation in the putative kinase active site shows drastic loss in their kinase activity but retains their epimerase activity

DELTA1-234

-

mutant enzyme shows no N-epimerase activity

DELTA1-356

-

mutant enzyme shows no N-epimerase activity

DELTA1-39

-

mutant enzyme shows no N-epimerase activity

DELTA383-722

-

epimerase activity is 2% of wild-type enzyme

DELTA490-722

-

epimerase activity is 15% of wild-type enzyme

DELTA597-722

-

epimerase activity is 2% of wild-type enzyme

DELTA697-722

-

epimerase activity is about 70% of wild-type enzyme

DELTA717-722

-

epimerase activity is about 95% of wild-type enzyme

H110A

-

mutant enzyme shows a drastic loss of epimerase activity, oligomerization is significantly different from that of the wild-type enzyme,loss of epimerase activity can largely by attributed to incorrect protein folding

H132A

-

mutant enzyme shows a drastic loss of epimerase activity, oligomerization is significantly different from that of the wild-type enzyme, loss of epimerase activity can largely by attributed to incorrect protein folding

H155A

-

mutant enzyme forms mainly trimeric enzyme with small amounts of hexamer; mutant enzyme shows a drastic loss of epimerase activity, loss of epimerase activity can largely by attributed to incorrect protein folding

H157A

-

mutant enzyme forms mainly trimeric enzyme with small amounts of hexamer; mutant enzyme shows a drastic loss of epimerase activity, loss of epimerase activity can largely by attributed to incorrect protein folding

H45A

-

mutant enzyme shows a drastic loss of epimerase activity

R420M

-

enzyme with mutation in the putative kinase active site shows drastic loss in their kinase activity but retains their epimerase activity

P12L

site-diected mutagenesis

Y194X

site-directed mutagenesis

P12L

-

site-diected mutagenesis

-

Y194X

-

site-directed mutagenesis

-

additional information

13 entries

P95709

gene mnaA, genotyping and mutation identification. Mapping of MnaA LOF mutations into the MnaA crystal structure revealing key residues for substrate binding site stability and charge. To determine whether L638R mnaA LOF mutants are not identified in MRSA COL due to a functional redundancy between Cap5P and MnaA, a cap5P deletion mutant is constructed and the L638R studies are repeated. Under these conditions, in addition to identifying the expected tarG L638R mutations as well as tarO and tarA LOF mutations, multiple (n = 11) independent resistor isolates obtained uniquely possess distinct mutations that map to mnaA and are predicted to inactivate gene function as well as directly confer L638R drug resistance based on the absence of additional non-synonymous mutations in their genome following WGS analysis. While MRSA COL DELTAcap5P exhibits no wall teichoic acid (WTA) depletion phenotype and remains resistant to beta-lactams, MRSA COL mnaA, DELTAcap5P double mutants are completely devoid of WTA and are also highly sensitive to beta-lactams. MRSA COL mnaA, cap5P double mutants and MRSE mnaA single mutants reveal morphological phenotypes consistent with WTA depletion, including increased cell size heterogeneity and septation defects. Complementing DELTAcap5P mnaASa P12L and DELTAcap5P mnaASa Y194* with either cap5P or mnaASa reintroduced on an inducible plasmid restores WTA polymer levels, resistance to each of the beta-lactams tested, and wild-type sensitivity to L638; to determine whether L638R mnaA LOF mutants are not identified in MRSA COL due to a functional redundancy between Cap5P and MnaA, a cap5P deletion mutant is constructed and the L638R studies are repeated. Under these conditions, in addition to identifying the expected tarG L638R mutations as well as tarO and tarA LOF mutations, multiple (n = 11) independent resistor isolates obtained uniquely possess distinct mutations that map to mnaA and are predicted to inactivate gene function as well as directly confer L638R drug resistance based on the absence of additional non-synonymous mutations in their genome following WGS analysis. While MRSA COL DELTAcap5P exhibits no wall teichoic acid (WTA) depletion phenotype and remains resistant to beta-lactams, MRSA COL mnaA, DELTAcap5P double mutants are completely devoid of WTA and are also highly sensitive to beta-lactams. MRSA COL mnaA, cap5P double mutants and MRSE mnaA single mutants reveal morphological phenotypes consistent with WTA depletion, including increased cell size heterogeneity and septation defects

Q9REV4

gene mnaA, genotyping and mutation identification. Mapping of MnaA LOF mutations into the MnaA crystal structure revealing key residues for substrate binding site stability and charge. To determine whether L638R mnaA LOF mutants are not identified in MRSA COL due to a functional redundancy between Cap5P and MnaA, a cap5P deletion mutant is constructed and the L638R studies are repeated. Under these conditions, in addition to identifying the expected tarG L638R mutations as well as tarO and tarA LOF mutations, multiple (n = 11) independent resistor isolates obtained uniquely possess distinct mutations that map to mnaA and are predicted to inactivate gene function as well as directly confer L638R drug resistance based on the absence of additional non-synonymous mutations in their genome following WGS analysis. While MRSA COL DELTAcap5P exhibits no wall teichoic acid (WTA) depletion phenotype and remains resistant to beta-lactams, MRSA COL mnaA, DELTAcap5P double mutants are completely devoid of WTA and are also highly sensitive to beta-lactams. MRSA COL mnaA, cap5P double mutants and MRSE mnaA single mutants reveal morphological phenotypes consistent with WTA depletion, including increased cell size heterogeneity and septation defects. Complementing DELTAcap5P mnaASa P12L and DELTAcap5P mnaASa Y194* with either cap5P or mnaASa reintroduced on an inducible plasmid restores WTA polymer levels, resistance to each of the beta-lactams tested, and wild-type sensitivity to L638; to determine whether L638R mnaA LOF mutants are not identified in MRSA COL due to a functional redundancy between Cap5P and MnaA, a cap5P deletion mutant is constructed and the L638R studies are repeated. Under these conditions, in addition to identifying the expected tarG L638R mutations as well as tarO and tarA LOF mutations, multiple (n = 11) independent resistor isolates obtained uniquely possess distinct mutations that map to mnaA and are predicted to inactivate gene function as well as directly confer L638R drug resistance based on the absence of additional non-synonymous mutations in their genome following WGS analysis. While MRSA COL DELTAcap5P exhibits no wall teichoic acid (WTA) depletion phenotype and remains resistant to beta-lactams, MRSA COL mnaA, DELTAcap5P double mutants are completely devoid of WTA and are also highly sensitive to beta-lactams. MRSA COL mnaA, cap5P double mutants and MRSE mnaA single mutants reveal morphological phenotypes consistent with WTA depletion, including increased cell size heterogeneity and septation defects

-

gene mnaA, genotyping and mutation identification. Mapping of MnaA LOF mutations into the MnaA crystal structure revealing key residues for substrate binding site stability and charge. To determine whether L638R mnaA LOF mutants are not identified in MRSA COL due to a functional redundancy between Cap5P and MnaA, a cap5P deletion mutant is constructed and the L638R studies are repeated. Under these conditions, in addition to identifying the expected tarG L638R mutations as well as tarO and tarA LOF mutations, multiple (n = 11) independent resistor isolates obtained uniquely possess distinct mutations that map to mnaA and are predicted to inactivate gene function as well as directly confer L638R drug resistance based on the absence of additional non-synonymous mutations in their genome following WGS analysis. While MRSA COL DELTAcap5P exhibits no wall teichoic acid (WTA) depletion phenotype and remains resistant to beta-lactams, MRSA COL mnaA, DELTAcap5P double mutants are completely devoid of WTA and are also highly sensitive to beta-lactams. MRSA COL mnaA, cap5P double mutants and MRSE mnaA single mutants reveal morphological phenotypes consistent with WTA depletion, including increased cell size heterogeneity and septation defects. Complementing DELTAcap5P mnaASa P12L and DELTAcap5P mnaASa Y194* with either cap5P or mnaASa reintroduced on an inducible plasmid restores WTA polymer levels, resistance to each of the beta-lactams tested, and wild-type sensitivity to L638; to determine whether L638R mnaA LOF mutants are not identified in MRSA COL due to a functional redundancy between Cap5P and MnaA, a cap5P deletion mutant is constructed and the L638R studies are repeated. Under these conditions, in addition to identifying the expected tarG L638R mutations as well as tarO and tarA LOF mutations, multiple (n = 11) independent resistor isolates obtained uniquely possess distinct mutations that map to mnaA and are predicted to inactivate gene function as well as directly confer L638R drug resistance based on the absence of additional non-synonymous mutations in their genome following WGS analysis. While MRSA COL DELTAcap5P exhibits no wall teichoic acid (WTA) depletion phenotype and remains resistant to beta-lactams, MRSA COL mnaA, DELTAcap5P double mutants are completely devoid of WTA and are also highly sensitive to beta-lactams. MRSA COL mnaA, cap5P double mutants and MRSE mnaA single mutants reveal morphological phenotypes consistent with WTA depletion, including increased cell size heterogeneity and septation defects

-

DTT protects against inactivation at 4 C

-

the purified enzyme is 80% pure, no further purification because it is unstable to freezing and has limited stability upon storage at 4°C

-

651592

UDP and UDP-N-acetylglucosamine protect the enzyme from inactivation by aging

-

-10°C, overnight, in a saturated (NH4)2SO4 solution, approximately 20% loss of activity

-

2259

-18°C, stable for several months

-70°C, stable for several months

-

2280

2°C, several days, maintains well over 50% of its activity

-

4°C, 80-90% loss of activity after 60 h

-

2258

4°C, in presence of 0.1 mM UDP and 1 mM DTT, 3 days, 20% loss of activity

-

4°C, purified enzyme expressed in insect cells, stable

-

663299

4°C, purified enzyme is 80% pure, no further purification because it is unstable to freezing and has limited stability upon storage at 4°C

-

651592

-

6 entries

bifunctional enzyme EC 5.1.3.14/EC 2.7.1.60

-

2280

enzyme expressed in Escherichia coli and enzyme expressed in insect cells

-

partial

-

recombinant C-terminally His6-tagged enzyme NmSacA from Escherichia coli strain BL21(DE3) by nickel affinity chromatography

Neisseria meningitidis serogroup A /serotype 4A

recombinant C-terminally His6-tagged NmSacA from Escherichia coli strain BL21(DE3) by nickel affinity chromatography

Neisseria meningitidis serogroup A / serotype 4A

recombinant enzyme expressed in Spodoptera frugiperda cells using a baculovirus expression system

-

651592

recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity and anion exchange chromatography, gel filtration, and ultrafiltration

-

recombinant His6-tagged enzyme from Escherichia coli strain BL21(DE3)

Bacillus subtilis

P39131

recombinant wild-type and mutant enzymes, His-tagged proteins

-

bifunctional enzyme EC 5.1.3.14/EC 2.7.1.60, expressed in COS7 cells

O35826

2279

cloning of the neuC gene into an intein expression vector to facilitate purification

-

651736

DNA sequencing of the GNE coding region of 64 symptomatic patients with autosomal recessive hereditary inclusion body myopathy, genotyping-phenotyping of patients from different ethnics, overview

-

expression in Arabidopsis thaliana

-

689622

expression in Chinese Hamster Ovary (CHO)

-

673699

expression in Escherichia coli

expression in Escherichia coli, C-terminal His-tag

expression in insect cells

-

expression in Insect Cells and supercompetent InvalphaF' Escherichia coli cells (Invitrogen)

-

expression in Sf9 insect cells, wild-type enzyme and mutant enzymes DELTA1-39, DELTA1-234, DELTA1-356, DELTA383-722, DELTA490-722, DELTA597-722, DELTA697-722, DELTA717-722

-

expression of M712T mutant enzyme in Sf9 insect cells

-

661735

expression of wild-type and mutant enzymes (C13S, H132Q, D176V, D177C, V331A, I472T, G708S, A631V, V572L and A630T) in COS-7 cells

-

functional expression in Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris or insect cells. In all cell types, the expressed enzyme displayes both epimerase and kinase activities. In Escherichia coli up to 2 mg protein/l cell culture is expressed in yeast cells only 0.4 mg/ml, in insect cells up to 100 mg/L. In all three cell systems, insoluble protein aggregates are also observed. Expression and purification of the UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase in Sf-900 cells can yield the miligram amount of protein required for structural characterization of the enzyme. The easier expression in Escherichia coli and yeast provides sufficient quantities for enzymatic and kinetic characterization

-

663299

gene gneY, DNA and amino acid sequence determination and analysis, genetic structure, sequence comparisons; gene gneYZ, DNA and amino acid sequence determination and analysis, genetic structure, sequence comparisons

Q81K32, Q81X16

748089

gene mnaA, genotyping and mutation identification

gene sacA, encoded in the serogroup A cps operon, sequence comparisons, recombinant overexpression of C-terminally His6-tagged recombinant NmSacA in Escherichia coli strain BL21(DE3)

Neisseria meningitidis serogroup A / serotype 4A

gene sacA, recombinant expression of C-terminally His6-tagged enzyme NmSacA in Escherichia coli strain BL21(DE3)

Neisseria meningitidis serogroup A /serotype 4A

gene wecB, sequence comparisons, recombinant expression of C-terminally His6-tagged enzyme in Escherichia coli strain BL21(DE3)

Bacillus subtilis

P39131

gene wecB, sequence comparisons, recombinant expression of His-tagged enzyme in Escherichia coli strain BL21(DE3)

-

high level overexpression of the active enzyme is established by using the baculovirus/Sf9 system

-

overexpression in Escherichia coli

Bordetella pertussis

-

649142

overexpression in Escherichia coli BL21(DE3), wild-type enzyme and mutant enzymes D100N, E122Q and D131N

-

proteome expression analysis in wild-type and GNE-deficient embryonic stem cells, overview. GNE overexpression in HEK cells

-

703905

splice variant hGNE2, expression in insect and mammalian cells. Splice variant hGNE3, expression in Escherichia coli

-

692536

splice variants mGNE1, mGNE2, mGNE3

-

692536

wild-type and mutant enzymes expressed in Escherichia coli

-

biotechnology

-

production of erythropoietin (EPO) in Chinese Hamster Ovary (CHO) cells

673699

drug development

6 entries

medicine

-

UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase interacts with the non-muscle form of alpha-actinin, alpha-actinin-1, in mature skeletal muscle cells. No significant difference in the binding of alpha-actinin-1 with either wild-type or mutant enzyme, and therefore no conclusions wether and how the interaction is relevant to the muscle-restricted pathology of hereditary inclusion body myopathy

694815

synthesis