Structural Basis of Ligand Binding to UDP-Galactopyranose Mutase from <i>Mycobacterium tuberculosis</i> Using Substrate and Tetrafluorinated Substrate Analogues

Straaten, K.E. Van; Kuttiyatveetil, Jijin R. A.; Sevrain, Charlotte M.; Villaume, Sydney; Jiménez‐Barbero, Jesús; Linclau, Bruno; Vincent, Stéphane; Sanders, D.A.R.

doi:10.1021/ja511204p

Cited by 79 publications

(68 citation statements)

References 97 publications

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“…In this way, Y364 may prevent the inhibitor from binding CdUGM in the closed conformation. A similar inward-oriented position of the Y364 equivalent was observed in the closed structure of MtUGM 26 . Notably, no such conformational change of the corresponding residue (Y349) was detected in KpUGM (Figure 7C) 28 .…”

Section: Discussionsupporting

confidence: 75%

“…Many known inhibitors are UDP-Gal f analogs that bind UGM in vitro but have not been shown to function in cells. 19-26 Our group identified non-substrate analogue 2-aminothiazoles as some of the most potent UGM inhibitors described to date 14 (Figure 1). These compounds, however, exhibited some toxicity to mammalian cells and were difficult to optimize.…”

Section: Introductionmentioning

confidence: 99%

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Conformational Control of UDP-Galactopyranose Mutase Inhibition

et al. 2017

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UDP-galactopyranose mutase (Glf or UGM) catalyzes the formation of uridine 5′-diphosphate-α-D-galactofuranose (UDP-Galf) from UDP-galactopyranose (UDP-Galp). The enzyme is required for the production of Galf-containing glycans. UGM is absent in mammals, but members of the Corynebacterineae suborder require UGM for cell envelope biosynthesis. The need for UGM in some pathogens has prompted the search for inhibitors that could serve as antibiotic leads. Optimizing inhibitor potency, however, has been challenging. The UGM from Klebsiella pneumoniae (KpUGM), which is not required for viability, is more effectively impeded by small-molecule inhibitors than are essential UGMs from species such as Mycobacterium tuberculosis or Corynebacterium diphtheriae. Why KpUGM is more susceptible to inhibition than other orthologs is not clear. One potential source of difference is UGM ortholog conformation. We previously determined a structure of CdUGM bound to a triazolothiadiazine inhibitor in the open form, but it was unclear whether the small molecule inhibitor bound this form or to the closed form. By varying the terminal tag (CdUGM-His6 and GSG-CdUGM), we crystallized CdUGM to capture the enzyme in different conformations. These structures reveal a pocket in the active site that can be exploited to augment inhibitor affinity. Moreover, they suggest the inhibitor binds the open form of most prokaryotic UGMs but can bind the closed form of KpUGM. This model and the structures suggest strategies to optimize inhibitor potency by exploiting UGM conformational flexibility.

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Section: Discussionsupporting

confidence: 75%

Section: Introductionmentioning

confidence: 99%

Conformational Control of UDP-Galactopyranose Mutase Inhibition

et al. 2017

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“…It has been proposed that during the formation of UDP-Galf, a flavin adenine dinucleotide (FAD)-iminium ion intermediate is formed through nucleophilic addition that facilitates galactose ring opening and contraction (5,(12)(13)(14)(15)(16). Three dimensional structures for members of the UGM family have been determined by X-ray crystallography in free form and in complex with various substrates, as well as in association with both oxidized and reduced forms of FAD (12,13,(17)(18)(19). Notably, a covalent FAD-Galp intermediate involved in the UGM reaction has recently yielded to elucidation by X-ray crystallography shedding significant insight into UGM catalysis (20).…”

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confidence: 99%

Deciphering the sugar biosynthetic pathway and tailoring steps of nucleoside antibiotic A201A unveils a GDP- l -galactose mutase

Zhu

Chen

Song

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Galactose, a monosaccharide capable of assuming two possible configurational isomers (D-/L-), can exist as a six-membered ring, galactopyranose (Galp), or as a five-membered ring, galactofuranose (Galf). UDP-galactopyranose mutase (UGM) mediates the conversion of pyranose to furanose thereby providing a precursor for D-Galf. Moreover, UGM is critical to the virulence of numerous eukaryotic and prokaryotic human pathogens and thus represents an excellent antimicrobial drug target. However, the biosynthetic mechanism and relevant enzymes that drive L-Galf production have not yet been characterized. Herein we report that efforts to decipher the sugar biosynthetic pathway and tailoring steps en route to nucleoside antibiotic A201A led to the discovery of a GDP-L-galactose mutase, MtdL. Systematic inactivation of 18 of the 33 biosynthetic genes in the A201A cluster and elucidation of 10 congeners, coupled with feeding and in vitro biochemical experiments, enabled us to: (i) decipher the unique enzyme, GDP-Lgalactose mutase associated with production of two unique D-mannose-derived sugars, and (ii) assign two glycosyltransferases, four methyltransferases, and one desaturase that regiospecifically tailor the A201A scaffold and display relaxed substrate specificities. Taken together, these data provide important insight into the origin of L-Galf-containing natural product biosynthetic pathways with likely ramifications in other organisms and possible antimicrobial drug targeting strategies.nucleoside antibiotic A201A | biosynthesis | GDP-L-galactose mutase | methyltransferase | desaturase

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“…Sanders et al [6][7][8] showed from the crystal structure of UGM in E.coli that the diphosphate group of UDP-Galp forms ionic interactions with key amino acids R247 and R278 and that Y181, Y311 and Y346 are important for substrate binding through hydrogen bonding with the carbohydrate ring; W156 stacks against the ribose moiety. Schaefer et al 9,10 proposed that the ionic pyrophosphate group could be replaced by a nonionic group to increase bioavailability and facilitate penetration of cells and cell compartments to reach the target enzymes.…”

mentioning

confidence: 99%

“…The low activity may be due to the absence of the ionic charge in the diphosphate bridge that induces loop closure8,16,19 as well as the length of the polyhydroxylated chain, the Galp.and uracil moieties been unable to form optimal contacts in their respective subsites. Further optimization efforts through more refined molecular dynamicscalculations with the very recently published crystal structure of UGM from M. tuberculosis 8 should shed light on this issue and lead to the design of additional non-ionic substrate mimics.…”

mentioning

confidence: 99%