Literature summary for 5.4.99.9 extracted from

van Straaten, K.E.; Kuttiyatveetil, J.R.; Sevrain, C.M.; Villaume, S.A.; Jimenez-Barbero, J.; Linclau, B.; Vincent, S.P.; Sanders, D.A.
Structural basis of ligand binding to UDP-galactopyranose mutase from Mycobacterium tuberculosis using substrate and tetrafluorinated substrate analogues (2015), J. Am. Chem. Soc., 137, 1230-1244 .

Cloned(Commentary)

Cloned (Comment)	Organism
recombinant overexpression of active soluble His6-MBP-tagged MtUGM in Escherichia coli strain BL21(DE3) CodonPlus-RIL	Mycobacterium tuberculosis

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified detagged recombinant enzyme complexed with substrate UDP-Galp and inhibitors UDP, UDP-F4-Galf, and UDP-F4-Galp, hanging drop vapour diffusion method, mixing of 0.0012 ml of protein solution containing 6.5 mg/ml enzyme in 25 mM Tris, pH 7.5, 500 mM NaCl, and, added prior to crystallization, 20 mM sodium dithionite (final concentration), with 0.0012 ml of reservoir solution containing 20% PEG 3350, 0.1 M Bis-Tris pH 5.5, and additives, 1-4 weeks, X-ray diffraction structure determination and analysis at 2.25-2.60 A resolution	Mycobacterium tuberculosis

Crystallization (Comment)

Organism

purified detagged recombinant enzyme complexed with substrate UDP-Galp and inhibitors UDP, UDP-F4-Galf, and UDP-F4-Galp, hanging drop vapour diffusion method, mixing of 0.0012 ml of protein solution containing 6.5 mg/ml enzyme in 25 mM Tris, pH 7.5, 500 mM NaCl, and, added prior to crystallization, 20 mM sodium dithionite (final concentration), with 0.0012 ml of reservoir solution containing 20% PEG 3350, 0.1 M Bis-Tris pH 5.5, and additives, 1-4 weeks, X-ray diffraction structure determination and analysis at 2.25-2.60 A resolution

Mycobacterium tuberculosis

Protein Variants

Protein Variants	Comment	Organism
P306R	site-directed mutagenesis, the mutant shows similar kinetics compared to wild-type. Pro306 is located on the solvent-exposed loop (His300-Lys309) connecting the small helix nu3 and beta strand beta14 of the beta-sheet domain, over 25 A from the FAD. The Cdelta atom of Pro306 is 3.5 A from the main-chain oxygen of Thr53, located on the small sharp turn (Glu52-Gly56) connecting beta strands beta3 and beta4.The Pro306Arg mutation releases this clash and results in a 1 A shift in the position of the two solvent-exposed loops without affecting the position of side chains and interaction with the protein. Arg306 forms a salt bridge with the side chain of Asp308 and the main-chain oxygen of Gln54 and replaces a salt bridge formed by Lys309. The Lys309 side chain has rotated and forms a new salt bridge with Gln54. In addition, Lys309 is involved in crystal contacts with the side chain of Asp202. The Pro306Ala mutation therefore may be stabilizing the two loops and promoting the crystallization of MtUGM complex structures	Mycobacterium tuberculosis

Inhibitors

Inhibitors	Comment	Organism
additional information	dissociation constants for the inhibitors by saturation transfer difference (STD) NMR spectroscopy measurements	Mycobacterium tuberculosis
UDP	enzyme binding structure analysis and binding mode, overview	Mycobacterium tuberculosis
UDP-2,3-dideoxy-2,2,3,3-tetrafluoro-alpha-D-galactofuranose	UDP-F4-Galf, enzyme binding structure analysis and binding mode, overview	Mycobacterium tuberculosis
UDP-2,3-dideoxy-2,2,3,3-tetrafluoro-alpha-D-galactopyranose	UDP-F4-Galp, enzyme binding structure analysis and binding mode, overview	Mycobacterium tuberculosis
UDP-3-deoxy-3-fluoro-alpha-D-galactofuranose	-	Mycobacterium tuberculosis

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism	Structure
0.023	-	UDP-2,3-dideoxy-2,2,3,3-tetrafluoro-alpha-D-galactopyranose	pH 7.5, 37°C, recombinant enzyme	Mycobacterium tuberculosis

Natural Substrates/ Products (Substrates)

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

Organism

Organism	UniProt	Comment	Textmining
Mycobacterium tuberculosis	P9WIQ1	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant soluble His6-MBP-tagged MtUGM from Escherichia coli strain BL21(DE3) CodonPlus-RIL by amylose affinity chromatography, tag cleavage by TEV protease, and elimination of residual tag and tagged enzyme by nickel affinity chromatography	Mycobacterium tuberculosis

Reaction

Reaction	Comment	Organism	Reaction ID
UDP-alpha-D-galactopyranose = UDP-alpha-D-galactofuranose	enzyme-substrate binding structure and mechanism	Mycobacterium tuberculosis	a

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
additional information	analysis of the structural basis of ligand binding to UDP-galactopyranose mutase from Mycobacterium tuberculosis using substrate and tetrafluorinated substrate analogues, overview	Mycobacterium tuberculosis	?	-	?
UDP-2,3-dideoxy-2,2,3,3-tetrafluoro-alpha-D-galactopyranose	-	Mycobacterium tuberculosis	UDP-2,3-dideoxy-2,2,3,3-tetrafluoro-alpha-D-galactofuranose	-	r
UDP-alpha-D-galactopyranose	-	Mycobacterium tuberculosis	UDP-alpha-D-galactofuranose	-	r
UDP-alpha-D-galactopyranose	substrate binding structure of wild-type and mutant P306R, three-dimensional structure of the reduced MtUGM:UDP-Galp dimer, detailed overview. Substrate binding induces local changes in MtUGM active site	Mycobacterium tuberculosis	UDP-alpha-D-galactofuranose	-	r

Subunits

Subunits	Comment	Organism
dimer	-	Mycobacterium tuberculosis

Synonyms

Synonyms	Comment	Organism
MtUGM	-	Mycobacterium tuberculosis
UGM	-	Mycobacterium tuberculosis

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
37	-	assay at	Mycobacterium tuberculosis

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.5	-	assay at	Mycobacterium tuberculosis

Cofactor

Cofactor	Comment	Organism	Structure
FAD	enzyme interaction mechanism, overview	Mycobacterium tuberculosis

General Information

General Information	Comment	Organism
physiological function	UDP-galactopyranose mutase (UGM) is a flavin-containing enzyme that catalyzes the reversible conversion of UDP-galactopyranose (UDP-Galp) to UDP-galactofuranose (UDP-Galf) and plays a key role in the biosynthesis of the mycobacterial cell wall galactofuran. Substrate binding induces local changes in MtUGM active site	Mycobacterium tuberculosis