Chemical Mechanism of UDP-Galactopyranose Mutase from Trypanosoma cruzi: A Potential Drug Target against Chagas' Disease

Oppenheimer, Michelle; Valenciano, Ana Lisa; Kizjakina, Karina; Qi, Jun; Sobrado, Pablo

doi:10.1371/journal.pone.0032918

Cited by 36 publications

(113 citation statements)

References 47 publications

Supporting

Mentioning

101

Contrasting

Order By: Relevance

“…1). This value is very similar to the values reported for Tc UGM and Af UGM, suggesting a similar environment around the flavin cofactor [14, 15]. …”

Section: Resultssupporting

confidence: 87%

“…It has been shown that in the reduced form, the FAD-N5 attacks the UDP-Gal p at the C1 position, leading to breaking of the anomeric bond and formation of an FAD-galactose adduct. This adduct is essential for sugar ring contraction [9, 14, 27, 31]. Recently, we showed that Tc UGM and Af UGM can be reduced by NADPH and to a lesser extent by NADH [14].…”

Section: Resultsmentioning

confidence: 99%

“…The extent of conversion was determined by comparing the areas under the substrate and product peaks. UDP-Gal f was synthesized using published protocols [14]. …”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

UDP-galactopyranose mutases from Leishmania species that cause visceral and cutaneous leishmaniasis

Fonseca

Kizjakina

Sobrado

2013

Archives of Biochemistry and Biophysics

Self Cite

View full text Add to dashboard Cite

Leishmaniasis is a vector-borne, neglected tropical disease caused by parasites from the genus Leishmania. Galactofuranose (Galf) is found on the cell surface of Leishmania parasites and is important for virulence. The flavoenzyme that catalyzes the isomerization of UDP-galactopyranose to UDP-Galf, UDP-galactopyranose mutase (UGM), is a validated drug target in protozoan parasites. UGMs from L. mexicana and L. infantum were recombinantly expressed, purified, and characterized. The isolated enzymes contained tightly bound flavin cofactor and were active only in the reduced form. NADPH is the preferred redox partner for both enzymes. A kcat value of 6 ± 0.4 s−1 and a Km value of 252 ± 42 μM were determined for L. infantum UGM. For L. mexicana UGM, these values were ~ 4-times lower. Binding of UDP-Galp is enhanced 10–20 fold in the reduced form of the enzymes. Changes in the spectra of the reduced flavin upon interaction with the substrate are consistent with formation of a flavin-iminium ion intermediate.

show abstract

“…1). This value is very similar to the values reported for Tc UGM and Af UGM, suggesting a similar environment around the flavin cofactor [14, 15]. …”

Section: Resultssupporting

confidence: 87%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

UDP-galactopyranose mutases from Leishmania species that cause visceral and cutaneous leishmaniasis

Fonseca

Kizjakina

Sobrado

2013

Archives of Biochemistry and Biophysics

Self Cite

View full text Add to dashboard Cite

show abstract

“…34,35 In addition, we provide biochemical data that the flavin cofactor of CeUGM participates in covalent catalysis (Figure 2), a result that is consistent with previous mechanistic studies of UGM homologs from other species. 36-38 These data prompted us to devise a homology model for CeUGM, and its validity is supported by the catalytic activities of CeUGM variants. Small molecule inhibitors developed against prokaryotic UGMs also block CeUGM.…”

Section: Introductionmentioning

confidence: 99%

UDP-Galactopyranose Mutase in Nematodes

et al. 2013

View full text Add to dashboard Cite

Nematodes represent a diverse phylum of both free living and parasitic species. While the species Caenorhabditis elegans (C. elegans) is a valuable model organism, parasitic nematodes or helminths pose a serious threat to human health. Indeed, helminths cause many neglected tropical diseases that afflict humans. Nematode glycoconjugates have been implicated in evasive immunomodulation, a hallmark of nematode infections. One monosaccharide residue present in the glycoconjugates of several human pathogens is galactofuranose (Galf). This five-membered ring isomer of galactose has not been detected in mammals, making Galf metabolic enzymes attractive therapeutic targets. The only known pathway for biosynthetic incorporation of Galf into glycoconjugates depends upon generation of the glycosyl donor UDP-Galf by the flavoenzyme uridine 5’-diphosphate (UDP) galactopyranose mutase (UGM or Glf). A putative UGM encoding gene (glf-1) was recently identified in C. elegans. Because Galf has yet to be identified in any nematode glycan, we sought to assess the catalytic activity of the C. elegans glf-1 gene product (CeUGM). We found that CeUGM catalyzes the isomerization of UDP-Galf and UDP-galactopyranose (UDP-Galp). In the presence of enzyme, substrate, and a hydride source, a galactose–N5-FAD adduct was isolated, suggesting the CeUGM flavin adenine dinucleotide (FAD) cofactor serves as a nucleophile in covalent catalysis. Homology modeling and protein variants indicate that CeUGM possesses an active site similar to that of prokaryotic enzymes, despite the low sequence identity (~15%) between eukaryotic and prokaryotic UGM proteins. Even with the primary sequence differences, heterocyclic UGM inhibitors developed against prokaryotic proteins also inhibit CeUGM activity. We postulate that these inhibitors can serve as chemical probes of Galf in nematodes and as anthelmintic leads. Together, our data suggest that CeUGM facilitates the biosynthetic incorporation of Galf into nematode glycoconjugates through generation of the glycosyl donor UDP-Galf.

show abstract

“…7 Rather than serving as a redox center, the flavin cofactor in UGM is a nucleophile that attacks the anomeric carbon atom of the substrate (C1 in Figure 1A). 8–10 The role as nucleophile requires that the flavin be reduced (FADH − ) in the resting state of the enzyme.…”

Section: Introductionmentioning

confidence: 99%

Identification of the NAD(P)H Binding Site of Eukaryotic UDP-Galactopyranose Mutase

Dhatwalia

Singh²,

Solano

et al. 2012

J. Am. Chem. Soc.

Self Cite

View full text Add to dashboard Cite

UDP-galactopyranose mutase (UGM) plays an essential role in galactofuranose biosynthesis in microorganisms by catalyzing the conversion of UDP-galactopyranose to UDP-galactofuranose. The enzyme has gained attention recently as a promising target for the design of new antifungal, antitrypanosomal, and antileishmanial agents. Here we report the first crystal structure of UGM complexed with its redox partner NAD(P)H. Kinetic protein crystallography was used to obtain structures of oxidized Aspergillus fumigatus UGM (AfUGM) complexed with NADPH and NADH, as well as reduced AfUGM after dissociation of NADP+. NAD(P)H binds with the nicotinamide near the FAD isoalloxazine and the ADP moiety extending toward the mobile 200s active site flap. The nicotinamide riboside binding site overlaps that of the substrate galactopyranose moiety, thus NADPH and substrate binding are mutually exclusive. On the other hand, the pockets for the adenine of NADPH and uracil of the substrate are distinct and separated by only 6 Å, which raises the possibility of designing novel inhibitors that bind both sites. All twelve residues that contact NADP(H) are conserved among eukaryotic UGMs. Residues that form the AMP pocket are absent in bacterial UGMs, which suggests that eukaryotic and bacterial UGMs have different NADP(H) binding sites. The structures address the longstanding question of how UGM binds NAD(P)H and provide new opportunities for drug discovery.

show abstract

Chemical Mechanism of UDP-Galactopyranose Mutase from Trypanosoma cruzi: A Potential Drug Target against Chagas' Disease

Cited by 36 publications

References 47 publications

UDP-galactopyranose mutases from Leishmania species that cause visceral and cutaneous leishmaniasis

UDP-galactopyranose mutases from Leishmania species that cause visceral and cutaneous leishmaniasis

UDP-Galactopyranose Mutase in Nematodes

Identification of the NAD(P)H Binding Site of Eukaryotic UDP-Galactopyranose Mutase

Contact Info

Product

Resources

About