Information on EC 5.4.99.9 - UDP-galactopyranose mutase

649105

NAD(P)H

NADH

or NADPH required

NADPH

or NADH required

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(1R,3S,4R,7R,8S)-3-hydroxymethyl-2,6-dioxa-bicyclo-[2.2.2]-octane-7,8-diol

17% inhibition at 4 mM

706049

(2R,3S,4S,5S,2'R,3'S,4'S,5'S)-2,2'-butane-1,4-diylbis[5-[(1S)-1,2-dihydroxyethyl]-3,4-dihydroxypyrrolidinium] dichloride

(2S,3S,4S,5R)-2-[(1S)-1,2-dihydroxyethyl]-5-propylpyrrolidine-3,4-diol

(2S,3S,4S,5R)-2-[(1S)-1,2-dihydroxyethyl]-5-[(1E)-prop-1-en-1-yl]pyrrolidine-3,4-diol

(2Z)-2-(2-chloro-4-hydroxy-5-nitrobenzylidene)[1,3]thiazolo[3,2-a]benzimidazol-3(2H)-one

comparison with inhibition of Mycobacterium tuberculosis enzyme; dissociation constant 0.0053 mM, comparison with inhibition of Klebsiella pneumoniae enzyme

(4-chlorophenyl)-[1-(4-chlorophenyl)-3-hydroxy-5-methyl-1H-pyrazol-4-yl]-methanone

(4E)-4-(4-chloro-3-nitrobenzylidene)-1-(3,4-dichlorophenyl)pyrazolidine-3,5-dione

comparison with inhibition of Mycobacterium tuberculosis enzyme; dissociation constant 0.0049 mM, comparison with inhibition of Klebsiella pneumoniae enzyme

(Z)-N-((E)-5-(5-nitro-2-oxoindolin-3-ylidene)-4-oxothiazolidin-2-ylidene) benzenesulfonamide

727390

1(R)-1,4-dideoxy-1-C-3-[(ethyl)(uridin-5'-yl)phosphono]-2-propen-1-yl-1,4-imino-D-galactitol

25 mM, 52% residual activity

1(R)-1,4-dideoxy-1-C-3-[(uridin-5'-yl)phosphono]-2-propen-1-yl-1,4-imino-D-galactitol

2.5 mM, 43% residual activity

1(R)-1-C-allyl-1,4-dideoxy-1,4-imino-D-galactitol

25 mM, 61% residual activity

1,4-anhydro-beta-D-galactopyranose (1,5-anhydro-alpha-D-galactofuranose)

15% inhibition at 4 mM

706049

1,4-bis-[1(R)-1,4-dideoxy-1,4-imino-D-galactit-1-yl]-2-butene

2.5 mM, 50% residual activity

2-(([2-(4-bromophenyl)-1,3-dioxo-2,3-dihydro-1H-isoindol-5-yl]carbonyl)amino)benzoic acid

comparison with inhibition of Mycobacterium tuberculosis enzyme

2-(2-(4-bromophenyI)-1, 3-dioxoisoindolin-5-carboxamido) benzoic acid

727390

2-[(5E)-5-(3-bromobenzylidene)-4-oxo-2-thioxo-1,3-thiazolidin-3-yl]-3-phenylpropanoic acid

comparison with inhibition of Mycobacterium tuberculosis enzyme; dissociation constant 0.0094 mM, comparison with inhibition of Klebsiella pneumoniae enzyme

2-[(5E)-5-(4-bromobenzylidene)-2-thioxo-1,3-thiazolidin-3-yl]-3-phenylpropanoic acid

dissociation constant 0.0093 mM, comparison with inhibition of Klebsiella pneumoniae enzyme

2-[(5Z)-5-[(3-chlorophenyl)methylidene]-4-oxo-2-thioxo-1,3-thiazolidin-3-yl]-3-phenylpropanoic acid

692768

2-[5-(3-bromo-benzylidene)-4-oxo-2-thioxo-thiazolidin-3-yl]-3-phenyl-propionic acid

2-[[2-(4-bromo-phenyl)-1,3-dioxo-2,3-dihydro-1H-isoindole-5-carbonyl]-amino]-benzoic acid

2-[[4-(3,4-dichlorophenyl)-1,3-thiazol-2-yl]amino]-3-(3-iodophenyl)propanoic acid

692768

3-(4-iodophenyl)-2-[4-(3,4-dichlorophenyl)-thiazol-2-ylamino]-propionic acid

ethambutol

N-(4-[4-[8-([[3-carboxy-4-(6-hydroxy-3-oxo-3H-xanthen-9-yl)phenyl]carbamothioyl]amino)octyl]phenyl]-2,3-dihydro-1,3-thiazol-2-yl)-4-chlorophenylalanine

694527

N-[4-oxo-5-(2-oxo-1,2-dihydro-indol-3-ylidene)-thiazolidin-2-ylidene]-benzenesulfonamide

Quinine

Sodium borohydride

662936

Sodium cyanoborohydride

662936

UDP

4 entries

UDP-(1(1')E)-1_-fluoro-exo-glycal-D-galactofuranose

less than 10% inhibition at 1 mM. Time-dependent inactivation proceeds via two-electron processes

679224

UDP-(1(1')Z)-1'-fluoro-exo-glycal-D-galactofuranose

less than 10% inhibition at 1 mM. Time-dependent inactivation proceeds via two-electron processes

679224

UDP-C-alpha-D-galactofuranose

91% inhibition at 1 mM

UDP-C-alpha-D-galactopyranose

36% inhibition at 1 mM

UDP-C-beta-D-galactopyranose

8% inhibition at 1 mM

UDP-CH2-Galp

UDP-D-glucose

poor inhibitor of UGM

UDP-[1(1')Z]-exo-glycal-D-galactopyranose

42% inhibition at 1 mM

uridine diphosphate

uridine-5'-diphospho-(N-fluoresceinisothiocyano)hexanolamine

additional information

fluorinated substrate analogues under non-reducing conditions

661347

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5-Deazariboflavin

specific activity 29fold higher than of control without illumination

NAD(P)H

Sodium dithionite

20 mM

additional information

4 entries

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UDP-(6-deoxy-6-fluoro)-D-galactofuranose

1.14

UDP-2-amino-2-deoxy-D-galactopyranose

in 50 mM MOPS, 10 mM sodium dithionite, 2 mM MgCl2 pH 7.4, at 37°C

0.065

UDP-2-deoxy-2-fluoro-D-galactofuranose

0.203

UDP-2-deoxy-2-fluoro-D-galactopyranose

in 50 mM MOPS, 10 mM sodium dithionite, 2 mM MgCl2 pH 7.4, at 37°C

0.065

UDP-2-fluoro-galactofuranose

0.861

UDP-3-deoxy-3-fluoro-D-galactofuranose

0.28

UDP-3-deoxy-3-fluoro-D-galactopyranose

in 50 mM MOPS, 10 mM sodium dithionite, 2 mM MgCl2 pH 7.4, at 37°C

0.2

UDP-6-deoxy-6-fluoro-D-galactopyranose

in 50 mM MOPS, 10 mM sodium dithionite, 2 mM MgCl2 pH 7.4, at 37°C

3.15

UDP-6-deoxy-D-galactopyranose

in 50 mM MOPS, 10 mM sodium dithionite, 2 mM MgCl2 pH 7.4, at 37°C

0.022 - 0.607

UDP-alpha-D-galactopyranose

0.016 - 0.194

UDP-D-galactofuranose

5 entries

0.043 - 1.002

UDP-D-galactopyranose

8 entries

0.02 - 0.19

UDP-galactofuranose

0.022 - 0.055

UDP-galactopyranose

0.6

UDP-L-arabinofuranose

additional information

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7.4

UDP-(6-deoxy-6-fluoro)-D-galactofuranose

1.4

UDP-2-amino-2-deoxy-D-galactopyranose

in 50 mM MOPS, 10 mM sodium dithionite, 2 mM MgCl2 pH 7.4, at 37°C

0.033

UDP-2-deoxy-2-fluoro-D-galactofuranose

0.0007

UDP-2-deoxy-2-fluoro-D-galactopyranose

in 50 mM MOPS, 10 mM sodium dithionite, 2 mM MgCl2 pH 7.4, at 37°C

0.033

UDP-2-fluoro-galactofuranose

5.7

UDP-3-deoxy-3-fluoro-D-galactofuranose

0.9

UDP-3-deoxy-3-fluoro-D-galactopyranose

in 50 mM MOPS, 10 mM sodium dithionite, 2 mM MgCl2 pH 7.4, at 37°C

3.5

UDP-6-deoxy-6-fluoro-D-galactopyranose

in 50 mM MOPS, 10 mM sodium dithionite, 2 mM MgCl2 pH 7.4, at 37°C

42.2

UDP-6-deoxy-D-galactopyranose

in 50 mM MOPS, 10 mM sodium dithionite, 2 mM MgCl2 pH 7.4, at 37°C

0.0224 - 13.4

UDP-alpha-D-galactopyranose

1.5 - 72

UDP-D-galactofuranose

5 entries

0.13 - 35.3

UDP-D-galactopyranose

8 entries

UDP-galactofuranose

27 - 66

UDP-galactopyranose

UDP-L-arabinofuranose

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0.34

UDP-(6-deoxy-6-fluoro)-D-galactofuranose

0.41

UDP-2-fluoro-galactofuranose

0.00013 - 0.205

UDP-alpha-D-galactopyranose

57 - 650

UDP-D-galactofuranose

1230

UDP-galactofuranose

1180

UDP-galactopyranose

wild type enzyme, in 50 mM sodium phosphate buffer pH 7.0

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0.0016

2-[5-(3-bromo-benzylidene)-4-oxo-2-thioxo-thiazolidin-3-yl]-3-phenyl-propionic acid

0.0046

2-[[2-(4-bromo-phenyl)-1,3-dioxo-2,3-dihydro-1H-isoindole-5-carbonyl]-amino]-benzoic acid

0.017

N-[4-oxo-5-(2-oxo-1,2-dihydro-indol-3-ylidene)-thiazolidin-2-ylidene]-benzenesulfonamide

0.2

UDP

661347

0.0013

uridine-5'-diphospho-(N-fluoresceinisothiocyano)hexanolamine

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0.004 - 0.035

(2Z)-2-(2-chloro-4-hydroxy-5-nitrobenzylidene)[1,3]thiazolo[3,2-a]benzimidazol-3(2H)-one

0.044 - 0.062

(4-chlorophenyl)-[1-(4-chlorophenyl)-3-hydroxy-5-methyl-1H-pyrazol-4-yl]-methanone

0.004 - 0.041

(4E)-4-(4-chloro-3-nitrobenzylidene)-1-(3,4-dichlorophenyl)pyrazolidine-3,5-dione

0.0046

2-(([2-(4-bromophenyl)-1,3-dioxo-2,3-dihydro-1H-isoindol-5-yl]carbonyl)amino)benzoic acid

Klebsiella pneumoniae;

pH 7.0, 37°C

0.0016 - 0.065

2-[(5E)-5-(3-bromobenzylidene)-4-oxo-2-thioxo-1,3-thiazolidin-3-yl]-3-phenylpropanoic acid

0.0072 - 0.037

3-(4-iodophenyl)-2-[4-(3,4-dichlorophenyl)-thiazol-2-ylamino]-propionic acid

0.0035

N-(4-[4-[8-([[3-carboxy-4-(6-hydroxy-3-oxo-3H-xanthen-9-yl)phenyl]carbamothioyl]amino)octyl]phenyl]-2,3-dihydro-1,3-thiazol-2-yl)-4-chlorophenylalanine

Mycobacterium tuberculosis;

694527

0.411 - 0.495

UDP-CH2-Galp

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9 entries

7.5

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4mo2, download, 3D-view

SCOPe (4mo2)

CATH (4mo2)

Q0P8H5

Campylobacter jejuni subsp. jejuni serotype O:2 (strain ATCC 700819 / NCTC 11168);

Corynebacterium diphtheriae (strain ATCC 700971 / NCTC 13129 / Biotype gravis);

4 entries

Deinococcus radiodurans (strain ATCC 13939 / DSM 20539 / JCM 16871 / LMG 4051 / NBRC 15346 / NCIMB 9279 / R1 / VKM B-1422);

1i8t, download, 3D-view

SCOPe (1i8t)

CATH (1i8t)

P37747

Escherichia coli (strain K12);

7 entries

Klebsiella pneumoniae;

Mycobacterium smegmatis (strain ATCC 700084 / mc(2)155);

6 entries

Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv);

24 entries

Neosartorya fumigata;

Trypanosoma cruzi (strain CL Brener);

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43000

alpha2, 2 * 43000, crystallization

649105

44000

alpha2, 2 * 44000, calculated from translated peptide sequence

651570

45000

2 * 45000, SDS-PAGE

54960

Leishmania major

1 * 54960, calculated from amino acid sequence

714055

55000

57152

4 * 57152, calculated from amino acid sequence

57200

x * 57200, about, sequence calculation

72000

gel filtration

651570

92000

gel filtration

228600

calculated from amino acid sequence

275000

gel filtration

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x * 57200, about, sequence calculation

decamer

dimer

homodimer

homotetramer

4 * 55000, SDS-PAGE; 4 * 57152, calculated from amino acid sequence

monomer

tetramer

dimer-of-dimers tetramer in solution, the C-terminal helix of domain 1 and residue Arg133 in AfUGM form intersubunit hydrogen bonds in the AfUGM tetramer, while long side chains protruding from a helix of domain 2 likely prevent formation of the intersubunit 4-helix bundle that stabilizes the AfUGM tetramer

additional information

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crystal structure analysis, two crystal forms, hexagonal and triclinic

purified recombinant enzyme, sitting drop vapour diffusion method, mixing of 500 nl 15 mg/ml protein solution with 500 nl reservoir solution, containing 0.1 M HEPES, pH 7.5, 20% PEG 3350, 0.4 M ammonium sulfate, 4% formamide, and equilibration against 0.07 ml reservoir solution, for enzyme complex crystals, the enzyme is incubated for 10 min on ice with various substrate/inhibitor/reducing ligands 10 mM UDP-Galp, 10 mM UDP, 5 mM UDP-glucose, 5-20 mM sodium dithionite, respectively, before crystallization, 2 days, X-ray diffraction structure determination and analysis at 3.25 A resolution. Incorporation of selenomethionine is achieved, but the resulting crystals do not allow solution of the phase problem

wild-type and mutant enzyme in complex with substrate UDP-alpha-D-galactopyranose or with inhibitor UDP, microbatch method at room temperature, for the substrate complex crystals: 10 mg/ml protein in 50 mM Tris, pH 8.0, 5 mM DTT, with 10 mM UDP-alpha-D-galactopyranose, and 10 mM dithionite, for the inhibitor complex crystals: 10 mg/ml protein in 25 mM Tris malonate, pH 8.0, and 10 mM UDP, for mutant enzyme-substrate complexes: 10 mg/ml protein in 25 mM Tris malonate, pH 8.0, with 15 mM UDP-alpha-D-galactopyranose, mixing of equal volumes of protein solution (with or without ligand) and crystallization solution and overlaid with oil, X-ray diffraction structure determination and analysis at 2.3-3.15 A resolution

crystal structure analysis

in complex with UDP-CH2-Galp, hanging drop vapor diffusion method, using 0.1 M HEPES (pH 6.5), 0.2 M LiCl, and 28% (w/v) polyethylene glycol 6000

Q9RYF1;

715919

in complex with UDP-galactopyranose and UDP and uridine diphosphate, microbatch under oil method, using 0.1 M HEPES, pH 6.5, 0.2 M lithium chloride and 28% (w/v) PEG 6000

in complex with UDP-galactopyranose, microbatch-under-oil method, using 0.1 M HEPES pH 7.0, 0.2 M LiCl, and 20% (w/v) PEG 6000

701535

crystal structure analysis

FAD is located in a cleft lined with conserved residues

P37747;

vapour-diffusion method in hanging drops, orthorhombic, space group P21 with a: 71.12 A, b: 58.42 A, c: 96.38 A, beta: 96.38°

649105

crystal structure analysis

in complex with UDP-D-glucose

705152

in the (active) reduced state, with FAD, crystal structure at 2.2 A resolution, with FADH-, crystal structure at 2.25 A resolution

crystal structure analysis

in the oxidized state, with FAD, crystal structure at 2.25 A resolution

substrate-bound enzyme in oxidized and reduced forms

P9WIQ1;

702351

crystal structure analysis

crystal structure analysis, molecular dynamics simulations and modeling, overview

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HQ20 column chromatography and HP-20 column chromatography

HQ20 column chromatography and HP-20 hydrophobic interaction chromatography column

701535

nickel immobilized affinity chromatography and DEAE column chromatography

nickel immobilized metal affinity chromatography, DEAE column chromatography, and purification with HaloLink resin

Leishmania major

714055

Protino Ni-IDA 1000 column chromatography

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

727937

recombinant GST-tagged enzyme from Escherichia coli strain BL21 (DE3) pLysS or B893 (DE3) pLysS by glutathione affinity chroatography and gel filtration

the recombinant enzyme

the recombinant enzyme from Escherichia coli

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expressed in Escherichia coli

expressed in Escherichia coli BL21T1 and BL21(DE3) cells

Leishmania major

714055

expressed in Escherichia coli BL21T1 cells

expressed in Escherichia coli C43 cells

expressed in Escherichia coli DH5alpha cell

Aspergillus niger

692507

expressed in Escherichia coli strain strain MFF1

Campylobacter jejuni

704695

expressed in Escherichia coli Tuner cells

expression in Escherichia coli

5 entries

expression in Escherichia coli (BL21(DE3))

expression in Escherichia coli (ER2566)

662936

expression in Escherichia coli with a His-tag

expression in Escherichia coli, His-tagged protein

expression with His-tag in Escherichai coli

gene glf, overexpression of C-terminally His6-tagged enzyme in Escherichia coi strain BL21 StarTM (DE3)

727937

overexpression in Escherichia coli

recombinant expression of GST-tagged enzyme in Escherichia coli strain BL21 (DE3) pLysS, and in strain B893 (DE3) pLysS for the selnomethionine-labeled enzyme

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R182A

site-directed mutagenesis, the mutant is almost inactive

R182K

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

R327A

site-directed mutagenesis, inactive mutant

R327K

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

D351A

mutation of conserved residue of putative active site cleft. 4% of wild-type activity

E301A

mutation of conserved residue of putative active site cleft. 21% of wild-type activity

R174A

no catalytic activity

R280A

mutation of conserved residue of putative active site cleft. No catalytic activity

W156A

lower activity than wild-type enzyme

W156Y

lower activity than wild-type enzyme

W160A

W70F/W290F

Y151F

lower activity than wild-type enzyme

Y155F

7% of wild-type activity

Y181F

lower activity than wild-type enzyme

Y185F

11% of wild-type activity

Y311F

lower activity than wild-type enzyme

Y314F

5% of wild-type activity

Y346F

lower activity than wild-type enzyme

Y349F

6% of wild-type activity

R176A

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

R327A

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

Y317F

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

Y395F

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

Y429F

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

additional information

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analysis

high-throughput fluorescence polarization assay for indentification of inhibitors of enzyme and homologues

drug development

medicine

the UDP-galactopyranose mutase is an appealing adjunct therapeutic target in combination with other drugs against Aspergillus fumigatus

692130

synthesis

production of uridine 5'-(trihydrogen diphosphate) P'-alpha-D-galactofuranosyl ester, a precursor required for the formation of the lipopolysaccharide O-antigen of Klebsiella pneumoniae serotype O1