Literature summary for 5.4.99.9 extracted from

Da Fonseca, I.; Qureshi, I.A.; Mehra-Chaudhary, R.; Kizjakina, K.; Tanner, J.J.; Sobrado, P.
Contributions of unique active site residues of eukaryotic UDP-galactopyranose mutases to substrate recognition and active site dynamics (2014), Biochemistry, 53, 7794-7804 .

Application

Application	Comment	Organism
drug development	the enzyme is a cell surface virulence factor in protozoan parasites and absent in humans, it is therefore a good drug target	Mycobacterium tuberculosis
drug development	the enzyme is a cell surface virulence factor in protozoan parasites and absent in humans, it is therefore a good drug target	Aspergillus fumigatus
drug development	the enzyme is a cell surface virulence factor in protozoan parasites and absent in humans, it is therefore a good drug target	Trypanosoma cruzi

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified ligand-free recombinant oxidized mutant enzyme variants F66A, Y104A, Q107A, N207A, and Y317A, grown in sitting drops at room temperature with a reservoir solution containing 1.2-1.4 M ammonium sulfate and 0.1 M sodium acetate at pH 4.5, equal volumes of protein solution, containing 5-9 mg/ml enzyme in 125 mM NaCl, 25 mM HEPES, pH 7.5, and 1 mM Tris(3-hydroxypropyl)phosphine, and reservoir solution are mixed, and large yellow hexagonal crystals are obtained. Crystals of reduced, ligand-free AfUGM mutant enzymes and of reduced mutants AfUGMF66A, AfUGMN207A, and AfUGMY317A in complex with UDP are obtained by exchanging the mother liquor of the respective oxidized crystal with a reducing cryobuffer containing 1.6 M ammonium sulfate, 0.2 M sodium acetate pH 4.5, 25% ethylene glycol or glycerol, and 80 mM dithionite, with or without 200 mM UDP, AfUGM is fully reduced in solution by 80 mM dithionite, and the yellow color of the crystals is bleached upon soaking, consistent with reduction, X-ray diffraction structure determination and analysis at 2.05-2.30 A resolution	Aspergillus fumigatus

Crystallization (Comment)

Organism

purified ligand-free recombinant oxidized mutant enzyme variants F66A, Y104A, Q107A, N207A, and Y317A, grown in sitting drops at room temperature with a reservoir solution containing 1.2-1.4 M ammonium sulfate and 0.1 M sodium acetate at pH 4.5, equal volumes of protein solution, containing 5-9 mg/ml enzyme in 125 mM NaCl, 25 mM HEPES, pH 7.5, and 1 mM Tris(3-hydroxypropyl)phosphine, and reservoir solution are mixed, and large yellow hexagonal crystals are obtained. Crystals of reduced, ligand-free AfUGM mutant enzymes and of reduced mutants AfUGMF66A, AfUGMN207A, and AfUGMY317A in complex with UDP are obtained by exchanging the mother liquor of the respective oxidized crystal with a reducing cryobuffer containing 1.6 M ammonium sulfate, 0.2 M sodium acetate pH 4.5, 25% ethylene glycol or glycerol, and 80 mM dithionite, with or without 200 mM UDP, AfUGM is fully reduced in solution by 80 mM dithionite, and the yellow color of the crystals is bleached upon soaking, consistent with reduction, X-ray diffraction structure determination and analysis at 2.05-2.30 A resolution

Aspergillus fumigatus

Protein Variants

Protein Variants	Comment	Organism
F66A	site-directed mutagenesis, the mutant enzyme shows a high decrease in kcat/KM value compared to wild-type, but the mutation does not significantly affect the kinetics of enzyme activation by NADPH. Crystal structure determination of the enzyme ligand-free or in complex with UDP or UDP and substrate UDP-alpha-D-galactopyranose	Aspergillus fumigatus
H68A	site-directed mutagenesis, the mutant shows highly reduced catalytic efficiency compared to wild-type	Mycobacterium tuberculosis
additional information	kinetic parameters for the reduction of AfUGM mutant enzymes by NADPH compared to wild-type enzyme, overview	Aspergillus fumigatus
N201A	site-directed mutagenesis, the mutant shows highly reduced catalytic efficiency compared to wild-type	Trypanosoma cruzi
N207A	site-directed mutagenesis, the mutant enzyme shows a high decrease in kcat/KM value compared to wild-type, but the mutation does not significantly affect the kinetics of enzyme activation by NADPH. Crystal structure determination of the enzyme ligand-free or in complex with UDP	Aspergillus fumigatus
Q103A	site-directed mutagenesis, the mutant shows highly reduced catalytic efficiency compared to wild-type	Trypanosoma cruzi
Q107A	site-directed mutagenesis, the mutant enzyme shows a high decrease in kcat/KM value compared to wild-type, but the mutation does not significantly affect the kinetics of enzyme activation by NADPH. Crystal structure determination of the ligand-free enzyme	Aspergillus fumigatus
Y100A	site-directed mutagenesis, the mutant shows highly reduced catalytic efficiency compared to wild-type	Trypanosoma cruzi
Y104A	site-directed mutagenesis, the mutant enzyme shows a high decrease in kcat/KM value compared to wild-type, but the mutation does not significantly affect the kinetics of enzyme activation by NADPH. Crystal structure determination of the ligand-free enzyme	Aspergillus fumigatus
Y317A	site-directed mutagenesis, the mutant enzyme shows a high decrease in kcat/KM value compared to wild-type, but the mutation does not significantly affect the kinetics of enzyme activation by NADPH. Crystal structure determination of the enzyme ligand-free or in complex with UDP	Aspergillus fumigatus
Y317F	site-directed mutagenesis, the mutant shows highly reduced catalytic efficiency compared to wild-type	Trypanosoma cruzi

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
0.032	-	UDP-alpha-D-galactofuranose	pH and temperature not specified in the publication, recombinant mutant Y100A	Trypanosoma cruzi
0.057	-	UDP-alpha-D-galactofuranose	pH and temperature not specified in the publication, recombinant mutant Y104A	Aspergillus fumigatus
0.078	-	UDP-alpha-D-galactofuranose	pH and temperature not specified in the publication, recombinant wild-type enzyme	Mycobacterium tuberculosis
0.11	-	UDP-alpha-D-galactofuranose	pH and temperature not specified in the publication, recombinant wild-type enzyme	Aspergillus fumigatus
0.14	-	UDP-alpha-D-galactofuranose	pH and temperature not specified in the publication, recombinant wild-type enzyme	Trypanosoma cruzi
0.166	-	UDP-alpha-D-galactofuranose	pH and temperature not specified in the publication, recombinant mutant N207A	Aspergillus fumigatus
0.334	-	UDP-alpha-D-galactofuranose	pH and temperature not specified in the publication, recombinant mutant Y317A	Trypanosoma cruzi
0.38	-	UDP-alpha-D-galactofuranose	pH and temperature not specified in the publication, recombinant mutant Y317A	Aspergillus fumigatus
0.478	-	UDP-alpha-D-galactofuranose	pH and temperature not specified in the publication, recombinant mutant Q103A	Trypanosoma cruzi
2.153	-	UDP-alpha-D-galactofuranose	pH and temperature not specified in the publication, recombinant mutant Q107A	Aspergillus fumigatus

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.
UDP-alpha-D-galactopyranose	Mycobacterium tuberculosis	-	UDP-alpha-D-galactofuranose	-	r
UDP-alpha-D-galactopyranose	Aspergillus fumigatus	-	UDP-alpha-D-galactofuranose	-	r
UDP-alpha-D-galactopyranose	Trypanosoma cruzi	-	UDP-alpha-D-galactofuranose	-	r

Organism

Organism	UniProt	Comment	Textmining
Aspergillus fumigatus	Q4W1X2	i.e. Neosartorya fumigata	-
Mycobacterium tuberculosis	-	-	-
Trypanosoma cruzi	Q5EEK1	-	-

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
additional information	active site and substrate binding structure, overview, the UDP exosite is located at a junction between domains 1 and 3	Aspergillus fumigatus	?	-	?
UDP-alpha-D-galactofuranose	-	Mycobacterium tuberculosis	UDP-alpha-D-galactopyranose	-	r
UDP-alpha-D-galactofuranose	-	Aspergillus fumigatus	UDP-alpha-D-galactopyranose	-	r
UDP-alpha-D-galactofuranose	-	Trypanosoma cruzi	UDP-alpha-D-galactopyranose	-	r
UDP-alpha-D-galactopyranose	-	Mycobacterium tuberculosis	UDP-alpha-D-galactofuranose	-	r
UDP-alpha-D-galactopyranose	-	Aspergillus fumigatus	UDP-alpha-D-galactofuranose	-	r
UDP-alpha-D-galactopyranose	-	Trypanosoma cruzi	UDP-alpha-D-galactofuranose	-	r

Synonyms

Synonyms	Comment	Organism
AfUGM	-	Aspergillus fumigatus
Glf	-	Mycobacterium tuberculosis
Glf	-	Trypanosoma cruzi
UGM	-	Aspergillus fumigatus

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
0.03	-	UDP-alpha-D-galactofuranose	pH and temperature not specified in the publication, recombinant mutant Y100A	Trypanosoma cruzi
0.13	-	UDP-alpha-D-galactofuranose	pH and temperature not specified in the publication, recombinant mutant N207A	Aspergillus fumigatus
0.14	-	UDP-alpha-D-galactofuranose	pH and temperature not specified in the publication, recombinant mutant Q103A	Trypanosoma cruzi
0.17	-	UDP-alpha-D-galactofuranose	pH and temperature not specified in the publication, recombinant mutant Y104A	Aspergillus fumigatus
0.28	-	UDP-alpha-D-galactofuranose	pH and temperature not specified in the publication, recombinant mutant Y317A	Aspergillus fumigatus
0.3	-	UDP-alpha-D-galactofuranose	pH and temperature not specified in the publication, recombinant mutant Y317A	Trypanosoma cruzi
4.7	-	UDP-alpha-D-galactofuranose	pH and temperature not specified in the publication, recombinant mutant Q107A	Aspergillus fumigatus
13.4	-	UDP-alpha-D-galactofuranose	pH and temperature not specified in the publication, recombinant wild-type enzyme	Trypanosoma cruzi
71	-	UDP-alpha-D-galactofuranose	pH and temperature not specified in the publication, recombinant wild-type enzyme	Mycobacterium tuberculosis
72	-	UDP-alpha-D-galactofuranose	pH and temperature not specified in the publication, recombinant wild-type enzyme	Aspergillus fumigatus

Cofactor

Cofactor	Comment	Organism
FAD	-	Mycobacterium tuberculosis
FAD	-	Aspergillus fumigatus
FAD	-	Trypanosoma cruzi
additional information	UGMs are active only in the reduced form	Mycobacterium tuberculosis
additional information	UGMs are active only in the reduced form	Trypanosoma cruzi
additional information	UGMs are active only in the reduced form. Kinetic parameters for the reduction of AfUGM mutant enzymes by NADPH compared to wild-type enzyme, overview	Aspergillus fumigatus
NADPH	-	Mycobacterium tuberculosis
NADPH	-	Aspergillus fumigatus
NADPH	-	Trypanosoma cruzi

General Information

General Information	Comment	Organism
malfunction	kinetic parameters for the reduction of AfUGM mutant enzymes by NADPH compared to wild-type enzyme, overview. The active site mutants show high losses of catalytic efficiency compared to the wild-type	Aspergillus fumigatus
malfunction	the active site mutant shows high losses of catalytic efficiency compared to the wild-type enzyme	Mycobacterium tuberculosis
malfunction	the active site mutants show high losses of catalytic efficiency compared to the wild-type	Trypanosoma cruzi
additional information	residues F66, Y104, Q107, N207, and Y317 are important for promoting the transition state conformation for UDP-galactofuranose formation. The active site residues are conserved in eukaryotic UGMs but are absent or different in bacterial UGMs	Aspergillus fumigatus
additional information	residues H66, Y100, Q103, N201, and Y317 are important for promoting the transition state conformation for UDP-galactofuranose formation. The active site residues are conserved in eukaryotic UGMs but are absent or different in bacterial UGMs	Trypanosoma cruzi
additional information	residues H68 is important for promoting the transition state conformation for UDP-galactofuranose formation	Mycobacterium tuberculosis
physiological function	UDP-galactopyranose mutase (UGM) catalyzes the interconversion between UDP-galactopyranose and UDPgalactofuranose. Galactofuranose is found in bacterial and fungal cell walls and is a cell surface virulence factor in protozoan parasites. UGMs are active only in the reduced form	Mycobacterium tuberculosis
physiological function	UDP-galactopyranose mutase (UGM) catalyzes the interconversion between UDP-galactopyranose and UDPgalactofuranose. Galactofuranose is found in bacterial and fungal cell walls and is a cell surface virulence factor in protozoan parasites. UGMs are active only in the reduced form	Aspergillus fumigatus
physiological function	UDP-galactopyranose mutase (UGM) catalyzes the interconversion between UDP-galactopyranose and UDPgalactofuranose. Galactofuranose is found in bacterial and fungal cell walls and is a cell surface virulence factor in protozoan parasites. UGMs are active only in the reduced form	Trypanosoma cruzi

kcat/KM [mM/s]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism
0.06	-	UDP-alpha-D-galactofuranose	pH and temperature not specified in the publication, recombinant mutant N201A	Trypanosoma cruzi
0.29	-	UDP-alpha-D-galactofuranose	pH and temperature not specified in the publication, recombinant mutant Q103A	Trypanosoma cruzi
0.7	-	UDP-alpha-D-galactofuranose	pH and temperature not specified in the publication, recombinant mutant Y317A	Aspergillus fumigatus
0.8	-	UDP-alpha-D-galactofuranose	pH and temperature not specified in the publication, recombinant mutant N207A	Aspergillus fumigatus
0.9	-	UDP-alpha-D-galactofuranose	pH and temperature not specified in the publication, recombinant mutant Y317A	Trypanosoma cruzi
0.94	-	UDP-alpha-D-galactofuranose	pH and temperature not specified in the publication, recombinant mutant Y100A	Trypanosoma cruzi
2	-	UDP-alpha-D-galactofuranose	pH and temperature not specified in the publication, recombinant mutant Q107A	Aspergillus fumigatus
3	-	UDP-alpha-D-galactofuranose	pH and temperature not specified in the publication, recombinant mutant Y104A	Aspergillus fumigatus
3.5	-	UDP-alpha-D-galactofuranose	pH and temperature not specified in the publication, recombinant mutant F66A	Aspergillus fumigatus
95.7	-	UDP-alpha-D-galactofuranose	pH and temperature not specified in the publication, recombinant wild-type enzyme	Trypanosoma cruzi
654.5	-	UDP-alpha-D-galactofuranose	pH and temperature not specified in the publication, recombinant wild-type enzyme	Aspergillus fumigatus
1048.6	-	UDP-alpha-D-galactofuranose	pH and temperature not specified in the publication, recombinant wild-type enzyme	Mycobacterium tuberculosis