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

Abstract

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.

N5 Is the New C4a: Biochemical Functionalization of Reduced Flavins at the N5 Position

“…However, it was later determined that UGM is only active when the flavin is in the reduced form (Zhang and Liu, 2000;Sanders et al, 2001;Goni et al, 2008;Oppenheimer et al, 2010Oppenheimer et al, , 2011Maaliki et al, 2020). Eukaryotic UGMs from A. fumigatus and the protozoa T. cruzi, L. Mexicana, and L. infantum utilize NADPH as a co-substrate and can both reinstate and stabilize the reduced form of the enzyme under aerobic conditions (Dhatwalia et al, 2012c;Oppenheimer et al, 2012;Fonseca et al, 2013). It was also demonstrated that once reduced, eukaryotic UGMs can complete an average of several hundred turnovers before oxidation/inactivation occurs.…”

“…It was also demonstrated that once reduced, eukaryotic UGMs can complete an average of several hundred turnovers before oxidation/inactivation occurs. This behavior occurs as a result of the rate of oxidation occurring 200 to 1500-fold slower than rate of re-reduction (Dhatwalia et al, 2012c;Fonseca et al, 2013;Tanner et al, 2014). Bacterial UGM can also utilize NADPH for enzyme reactivation but requires a large excess of NADPH and extended incubation times due to a 10,000-fold slower reduction rate compared to eukaryotic UGM, suggesting that alternative physiological reductants may be utilized by the bacterial forms in vivo (Barlow et al, 1999;Gusarov et al, 2008).…”

Multiple functionalities of reduced flavin in the non-redox reaction catalyzed by UDP-galactopyranose mutase

“…We have been working with eukaryotic UGMs (eUGM), which share low sequence identity to bUGMs (~ 15%) and are, on average, ~ 100- amino acid longer than bUGMs [37–40]. The presence of a covalent flavin-N5-adduct was demonstrated in eUGMs [41] and the structures of the free enzyme and in complex with UDP-Gal p in the oxidized and reduced state have been elucidated [42–45].…”

Leishmania cell wall as a potent target for antiparasitic drugs. A focus on the glycoconjugates

“…91,92 Many studies have been devoted to the search for inhibitors of both families of enzymes, but more extensively in prokaryotes, including Klebsiella pneumoniae 93,94 and Mycobacterium tuberculosis. 91,92 Many studies have been devoted to the search for inhibitors of both families of enzymes, but more extensively in prokaryotes, including Klebsiella pneumoniae 93,94 and Mycobacterium tuberculosis.…”

“…Regarding Leishmania, UDP-Galp mutases from L. major, L. infantum and L. mexicana species have been produced very recently. 91,92 Many studies have been devoted to the search for inhibitors of both families of enzymes, but more extensively in prokaryotes, including Klebsiella pneumoniae 93,94 and Mycobacterium tuberculosis. [95][96][97][98][99] Unfortunately, sequence identity between the prokaryotic mutase from Aspergillus fumigatus 100 and eukaryotic ones, such as Leishmania, is very low (14-18%).…”