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Abstract

Uridine diphosphogalactofuranose (UDP-Galf ) is the precursor of the d-galactofuranose (Galf ) residues found in bacterial and parasitic cell walls, including those of many pathogens, such as Mycobacterium tuberculosis and Trypanosoma cruzi. UDP-Galf is made from UDP-galactopyranose (UDP-Galp) by the enzyme UDP-galactopyranose mutase (mutase). The mutase enzyme is essential for the viability of mycobacteria and is not found in humans, making it a viable therapeutic target. The mechanism by which mutase achieves the unprecedented ring contraction of a nonreducing sugar is unclear. We have solved the crystal structure of Escherichia coli mutase to 2.4 A resolution. The novel structure shows that the flavin nucleotide is located in a cleft lined with conserved residues. Site-directed mutagenesis studies indicate that this cleft contains the active site, with the sugar ring of the substrate UDP-galactose adjacent to the exposed isoalloxazine ring of FAD. Assay results establish that the enzyme is active only when flavin is reduced. We conclude that mutase most likely functions by transient reduction of substrate.

Investigation of Binding of UDP-Galf and UDP-[3-F]Galf to UDP-galactopyranose Mutase by STD-NMR Spectroscopy, Molecular Dynamics, and CORCEMA-ST Calculations

“…Purified UGM from K. pneumoniae was obtained as previously described by Sanders et al 10 UDP-Galf 1 was kindly provided by Dr. T. L Lowary at the University of Alberta. UDP-[3-F] Galf 4 was synthesized according to the method of Zhang et al 15 All chemicals, unless noted otherwise, were purchased from Aldrich or Sigma and used without further purification.…”

“…9,10 The mechanism of action of UGM is not yet completely resolved, and is still the subject of active investigation. [10][11][12][13] Analogues of the natural substrates have been synthesized and the activities of these compounds have been used to infer the mechanism of reaction.…”

“…9,10 The mechanism of action of UGM is not yet completely resolved, and is still the subject of active investigation. [10][11][12][13] Analogues of the natural substrates have been synthesized and the activities of these compounds have been used to infer the mechanism of reaction. [14][15][16][17][18] Among these compounds, synthetic C2-and C3-fluorinated UDP-Galf 1 analogues 15 and UDP-Galp 2 analogues 14 functioned as substrates for the reduced UGM, thereby discounting a previous mechanism involving the generation of a 2,3-enediol intermediate via redox recycling of the FAD coenzyme to facilitate the ring cleavage.…”

“…10 Thus far, five crystal structures of UGM from Escherichia coli, Mycobacterium tuberculosis, and Klebsiella pneumoniae have been solved. 10,11 The structures of all three UGMs are essentially identical, with the only significant differences occurring in the positioning of a mobile loop located next to the active site. 11 The novel structure shows that the flavin nucleotide is located in domain 1 with the re face of the isoalloxazine ring open to a cleft lined with conserved residues.…”

Bases of biocontrol: Sequence predicts synthesis and mode of action of agrocin 84, the Trojan Horse antibiotic that controls crown gall

“…The broad distribution among prokaryotes of aldohexofuranosyl moieties in complex carbohydrates has only recently become appreciated, and there is some interest in how these furanosyl forms are produced and incorporated into other compounds (30). Galactofuranose can be derived enzymatically from galactopyranose by UDP-galactopyranose mutase (epimerase) (31). AgnC2 contains a domain that exhibits significant relatedness to WcaG, a nucleoside-diphosphate sugar epimerase.…”