The tsetse-transmitted protozoan parasite Trypanosoma brucei
causes African sleeping sickness in humans and nagana, a disease of cattle, in sub-Saharan Africa. The disease-causing bloodstream form of T. brucei
is rich in galactose-containing glycoproteins, including the protective variant surface glycoproteins (VSGs) that, depending on the variant, contain galactose (Gal) in glycosylphosphatidylinositol (GPI) anchor side chains and/or N-linked oligosaccharides (Mehlert et al.
). In addition, the parasite’s transferrin receptor, which is critical for the acquisition of iron from the host, and various invariant surface glycoproteins also contain Gal in the form of poly N
sugar chains containing Galβ1-4GlcNAc repeats (Nolan et al.
). Recently, ricin lectin affinity chromatography was used to isolate glycoproteins bearing terminal non-reducing βGal residues and these were found to contain a variety of Gal-containing N-linked oligosaccharides, including a family of novel giant structures that contain on average 54 N
-acetyllactosamine repeats. These ricin-binding glycoproteins are localized in the flagellar pocket and throughout the endosomal/lysosomal system of the parasite (Atrih et al.
). The insect-dwelling procyclic form of the parasite also expresses Gal-containing glycoconjugates, notably the surface procyclin glycoproteins (Treumann et al.
) and free GPI structures (Vassella et al.
; Lillico et al.
; Nagamune et al.
). Importantly, neither life-cycle stage can transport Gal across the plasma membrane (Tetaud et al.
) and for galactose metabolism both are dependent on the NADH-dependent oxidoreductase UDP-glucose-4′-epimerase (EC 18.104.22.168; GalE) encoded by the TbGalE
gene that interconverts UDP-Glc and UDP-Gal (Fig. 1; Roper et al.
). The same appears to be true of the related parasite T. cruzi
, the causal agent of Chagas’ disease in South and Central America (MacRae et al.
). The African trypanosome requires UDP-glucose-4′-epimerase activity for growth and survival in vitro
, providing genetic validation for this enzyme as a potential drug target against African trypanosomiasis (Roper et al.
(a) The epimerization catalyzed by TbGalE, interconverting UDP-Glc and UDP-Gal. The blue arrow indicates the Pβ—O anomeric bond about which rotation occurs during catalysis. (b) The chemical structure of UDP-FGal.
GalE is a short-chain dehydrogenase/reductase (SDR) (Holden et al.
). Despite displaying an enormous spread of substrate specificities that regulate diverse biological processes, SDRs possess conserved motifs that are important for aspects of the enzyme structure, the recognition, binding and orientation of cofactor (NADH or NADPH) and substrates together with catalysis (Oppermann et al.
; Filling et al.
; Shi & Lin, 2004
). Three amino acids are particularly important with respect to catalysis and two occur in a Tyr-XXX
-Lys motif (Holm et al.
). The tyrosine is the catalytic base in the enzyme mechanism and the lysine contributes to binding the cofactor nicotinamide ribose (Gourley et al.
). In addition, a serine or threonine is often associated with the catalytic tyrosine or with the substrate. In Tb
GalE the relevant residues are Ser142, Tyr173 and Lys177.
A mechanism for the Tb
GalE-catalyzed conversion of an equatorial hydroxyl substituent at C4 of glucose to an axial position in galactose can be described in distinct stages (Shaw et al.
). UDP-Glc first binds to the binary complex Tb
. The nicotinamide abstracts a hydride from the glucose C4 as Tyr173 acquires a proton from the O4′ hydroxyl to produce a 4-keto intermediate. For inversion to occur, hydride transfer from the reduced cofactor must be to the opposite side of the hexose, a feat only possible after a rotation of the 4-keto intermediate within the active site. NADH then transfers the hydride back to C4 with concomitant reprotonation of the O4 hydroxyl group by Tyr173 to produce UDP-Gal. Ser142 OG accepts a hydrogen bond from the main-chain amide of Ala144 and acts as a hydrogen-bond donor to the O4′ hydroxyl of substrate. This is an important contribution to enzyme reactivity since it ensures that the O4′ group on the substrate is committed to be a hydrogen-bond donor with the phenolic Tyr173 OH and facilitates the H-atom transfers from and to O4′ that occur.
A complete understanding of the specificity and reactivity of TbGalE is sought to support the search for new enzyme inhibitors of TbGalE. Here, we describe the structure of this essential enzyme in ternary complex with NAD+ and the substrate analogue UDP-4-deoxy-4-fluoro-α-d-galactose (UDP-FGal; Fig. 1
b). The fluorine substitutes for the 4′-hydroxyl group from which a proton is abstracted in the first step of the proposed mechanism. A detailed analysis of the active site and comparisons with the human enzyme (HsGalE) highlights differences between substrate/product binding and provides insight into the mechanism of the enzyme together with clues for inhibitor design.