SAGE-Hindawi Access to ResearchEnzyme ResearchVolume 2011, Article ID 415976, 13 pagesdoi:10.4061/2011/415976

Review Article

Biosynthesis of Galactofuranose in Kinetoplastids: NovelTherapeutic Targets for Treating Leishmaniasis and Chagas’Disease

Michelle Oppenheimer,1 Ana L. Valenciano,1, 2 and Pablo Sobrado1

1 Department of Biochemistry, Virginia Tech, Blacksburg, VA 24061, USA2 Instituto Tecnologico de Costa Rica, Cartago, Costa Rica

Correspondence should be addressed to Pablo Sobrado, psobrado@vt.edu

Received 15 December 2010; Revised 2 March 2011; Accepted 14 March 2011

Academic Editor: Ariel M. Silber

Copyright © 2011 Michelle Oppenheimer et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

Cell surface proteins of parasites play a role in pathogenesis by modulating mammalian cell recognition and cell adhesion duringinfection. β-Galactofuranose (Galf ) is an important component of glycoproteins and glycolipids found on the cell surface ofLeishmania spp. and Trypanosoma cruzi. β-Galf -containing glycans have been shown to be important in parasite-cell interactionand protection against oxidative stress. Here, we discuss the role of β-Galf in pathogenesis and recent studies on the Galf -biosynthetic enzymes: UDP-galactose 4′ epimerase (GalE), UDP-galactopyranose mutase (UGM), and UDP-galactofuranosyltransferase (UDP-GalfT). The central role in Galf formation, its unique chemical mechanism, and the absence of a homologousenzyme in humans identify UGM as the most attractive drug target in the β-Galf -biosynthetic pathway in protozoan parasites.

1. Galactofuranose1

Galactofuranose (β-Galf ) is the five-member ring isomer ofgalactose (Figure 1). This rare sugar was initially found inseveral human bacterial pathogens including Mycobacteriumtuberculosis, Escherichia coli, Salmonella typhimurium, andKlebsiella pneumoniae [1–4]. In M. tuberculosis, β-Galf isfound in the arabinogalactan layer where it links thepeptidoglycan and mycolic acid layers [1]. In E. coli andK. pneumoniae, it is present in the O antigen, while in S.typhimurium it is found in the T antigen [2–4]. In all ofthese organisms, the enzyme UDP-galactopyranose mutase(UGM) serves as the sole biosynthetic source of β-Galf as it isresponsible for converting UDP-Galp to UDP-Galf (Figures2 and 3) [5–10]. UDP-Galf serves as the precursor moleculeof β-Galf, which is attached to the various componentsof the cell surface by galactofuranosyl transferases (GalfTs)(Figure 2) [11, 12]. UGMs and GalfTs are not found inhumans, therefore, they have been examined as potentialdrug targets.

Deletion of the genes coding for UGM or GalfTs hasshown that these proteins are essential in M. tuberculo-sis, highlighting the important for Galf in bacteria [13].Studies have also been conducted to identify inhibitors forM. tuberculosis UGM [14–17]. These studies showed thatspecific inhibitors of M. tuberculosis UGM were able toprevent mycobacterium growth and, therefore, validated Galfbiosynthesis as a drug target against mycobacteria [14].

β-Galf has also been shown to be present in fungi[18–21]. In the human pathogen Aspergillus fumigatus, itis found in four components of the cell wall: galactoman-nan, glycoprotein oligosaccharides, glycophosphoinositol(GPI) anchored lipophosphogalactomannan (LPGM), andsphingolipids [18, 22]. Deletion of the UGM and theGalf transporter genes in Aspergillus resulted in attenuatedvirulence, increased temperature sensitivity, and thinner cellwalls [23, 24]. Galf is also present in protozoan parasites andis a virulence factor [25]. In Leishmania spp., it is present inthe lipophosphoglycan (LPG) and in glycoinositolphospho-lipids (GIPLs). In T. cruzi, Galf is found in the GIPLs andglycoprotein oligosaccharides [26, 27]. This paper focuses on

2 Enzyme Research

O

O

HO

HO HO

HO

HO

OH OH

OH

OH

OH

β-galactopyranose β-galactofuranose

Figure 1: Structures of β-Galactopyranose and β-Galactofuranose.

current knowledge about the biosynthetic pathway of β-Galfand its role in the pathogenesis of T. cruzi and Leishmaniaspp.

1.1. Overview of T. cruzi and Leishmania spp. T. cruzi isthe causative agent of Chagas’ disease, which often developssevere cardiac complications in patients with the chronicform of the disease [28]. In the T. cruzi life cycle, the parasiteundergoes three developmental stages as it is transmittedfrom the insect vector (triatomine bug) to mammals:trypomastigote (vector feces and mammalian bloodstream),epimastigote (vector midgut), and amastigote (mammaliansmooth muscle) [29]. Leishmania spp. are the causativeagents of leishmaniasis, which can manifest in three forms—visceral, cutaneous, or mucocutaneous—depending on thespecies [30]. In the Leishmania spp. lifecycle, there are twostages: the amastigote (mammalian host macrophages) andthe promastigote stage (vector (sand fly) midgut) [30].

Current treatments are limited due to toxic side effectsand cost, therefore new drugs are needed [35–37]. Lifecycleprogression of both T. cruzi and Leishmania spp. is associatedwith changes in the carbohydrate composition on the cellsurface. These changes are important for mediating host-pathogen interaction and Galf levels and Galf -containing2glycans are shown to be modulated throughout the parasitelife cycles and are important for pathogenesis [26, 38–40].As Galf biosynthesis has been shown to be an attractive drugtarget for other pathogens, enzymes involved in this pathwaymay also prove to be ideal drug targets for the treatment ofChagas’ disease and leishmaniasis.

2. Biosynthesis of Galf in Kinetoplastids

The biosynthesis of Galf begins with the uptake andmetabolism of galactose (Gal). Gal is an epimer of glucosethat differs only by the orientation of the hydroxyl groupat the carbon 4 position. Gal is a component of lactose inmilk, is present in grains and beets, and can be utilizedfor energy after conversion to glucose (Glc). Gal is also amajor component of glycans, present in proteins and lipidsin most organisms, ranging from bacteria to mammals.The metabolism of Gal occurs via the Isselbacher or Leloirpathways (Figure 2). In the Leloir pathway, Gal is con-verted to glucose-6-phosphate (Glc-6-P), an intermediatein glycolysis (Figure 2(a)). After Gal is transported into thecytoplasm by hexose transporters it is phosphorylated by

galactokinase (GalK). Phosphorylation of Gal prevents itstransport out of the cell. Gal-1-phosphate (Gal-1-P) is thencoupled to uridyl diphosphate by galactose-1-phosphateuridyltransferase (GalPUT) yielding two products, UDP-Gal and Glc-1-phosphate (Glc-1-P). UDP-Gal is convertedto UDP-glucose (UDP-Glc) by UDP-glucose-4-epimerase(GalE). Glc-1-P is isomerized to Glc-6-P by phosphogluco-mutase (PGM) [41, 42]. In the Isselbacher pathway, Gal-1-Pcan be directly converted to UDP-Gal by the enzyme UDP-sugar-pyrophosphorylase (USP) (Figure 2(b)) [43]. Thesepathways contribute to the pool of UDP-Gal required for thebiosynthesis of the glycocalyx.

In Leishmania spp., galactose has been shown to beobtained from the environment by hexose transportersthrough radioactive labeling assays, and both the Leloir andIsselbacher pathways function to maintain proper levels ofUDP-Gal [44]. The Isselbacher pathway functions due tothe wide substrate specificity of USP in L. major, whichcan convert many sugars to the corresponding UDP-sugarincluding glucose, galactose, galacturonic acid, and arabinose[45]. The wide range of substrate specificity has beenexplored by crystallographic studies and has been attributedto a larger active site that can alter conformations of residuesinvolved with sugar binding and the flexibility of the sugar-binding loop [46]. Deletion of the USP gene in L. majorshowed that the protein is nonessential and demonstratesthat since the Leloir and Isselbacher pathways are redundant,proteins involved with the formation of UDP-Gal are notessential for Leishmania spp. survival [45, 47]. In T. cruziand Trypanosoma brucei, galactose cannot be obtained fromthe environment because it is not recognized by the hexosetransporters; therefore, these parasites rely on the action ofGalE from the Leloir pathway for the direct conversion ofUDP-Glc to UDP-Gal for galactose [41, 48, 49]. In both T.cruzi and L. major, UDP-Gal is converted to UDP-Galf byUGM (Figures 2(c) and 3) [7]. UDP-Galf is the substrate forseveral UDP-galactofuranosyl transferases, which decoratemany glycoproteins and glycolipids on the cell surface of T.cruzi and L. major.

2.1. Galactofuranose-Containing Proteins and Lipids. Galfis found in many major components of the glycocalyxof Leishmania spp. and T. cruzi. In Leishmania spp., andGalf is found in the lipophosphoglycan (LPG) and inglycoinositolphospholipids (GIPLs), while in T. cruzi, Galfis found in the GIPLs and glycoprotein oligosaccharides(Figure 4) [26, 27]. In this section, we will describe thestructure and role in pathogenesis of known Galf -containingglycoconjugates. 3

2.1.1. Lipophosphoglycan (LPG) from Leishmania. LPG fromLeishmania spp. has four components: a phosphoinositollipid, a core oligosaccharide, phosphoglycan (PG) repeatunits, and a cap (Figure 4(a)). β-Galf is found in the corestructure where it plays a role in connecting the PG repeatunits to the phospholipid [39, 50]. LPG has been found tobe important for adhesion to the sandfly midgut, resistanceto the human complement C3b, protection from oxidative

Enzyme Research 3

Galactose

Galactose-1-phosphate

Glucose-1-phosphate

Glucose-6-phosphate

UDP-glucose

UDP-galactose

UTP

Galactokinase(GK)

Phosphoglucomutase

(PGM)

epimerase(GalE)

Galactose-1-phosphate

(GalPUT)

UDP-sugar

(USP)

Glycolysis

Glycocalyx

UDP-galactofuranose

UDP-galactopyranose mutase(UGM)

(GalfT)

Galactose

Galactose-1-phosphate

Galactokinase(GK)

UDP-galactose

UDP-galactose

(a)

(b)

(c)

PPiuridyl transferasepyrophosphorylase

UDP-glucose-4′-

Galactofuranosyl transferases

Figure 2: Biosynthetic pathways of Galf. (a) In the Leloir pathway, Gal is transported to the cytoplasm where it is converted togalactose-1-phosphate by galactokinase. Galactose-1-phosphate uridyl transferase and UDP-Glc-4′-epimerase are involved in the synthesisof UDP-galactose. (b) Alternatively, galactose can be directly converted to UDP-galactose by the Isselbacher pathway by UDP-sugarpyrophosphorylase (USP). (c) UDP-Galactose is then converted to UDP-Galf by UDP-galactopyranose mutase (UGM), and UDP-Galf issubsequently added to the glycocalyx by Galactofuranosyl transferases (GalfT).

O

OH

HO

OOH

OH

NH

O

ONOO

OHHO

P

O

OP

OO

NH

O

ONOO

OHHO

P

O

OP

OO

OH

OH

HO

HO

UGM

UDP-galactopyranose UDP-galactofuranose

O−

O− O

−O−

Figure 3: Reaction catalyzed by UDP-Galactopyranose mutase (UGM).

Table 1: UDP-galactopyranose mutases.

Species Amino acids % identitya Oligomeric state Reference

E. coli 367 100 Dimer [32]

M. tuberculosis 399 44 Dimer [33]

L. major 491 15 Monomer b

T. cruzi 480 15 Monomer b

A. fumigatus 510 14 Tetramer [34]aIdentity to the E. coli enzymebOppenheimer and Sobrado unpublished results.

4 Enzyme Research

P = Phosphate

= β-galactopyranose

= α-glucose

= α-glucosamine

= α-mannose

= α-arabinose

= β-galactofuranose

= AEP, aminoethylphosphonic acid

= acetyl group Ac

OHO

P

P

P

P

P

P

P

P

P

P

P

P

P

P

Cap

LPG

Core

Inositol lipid

PG repeat units

X

X

L. major X = ,L. mexicana X= Y = H,

P Y

P

Ceramide

P

Ceramide

P

Ceramide

GIPLs of T. cruzi

(b)

OO

P

O

Z

L. major Z = H (1), (2), (3), (A)L. mexicana Z = (2),

OO

P

O

GIPL-A

Ac

Ser/Thr

Ac

Ser/Thr

Ac

Ser/Thr

Ac

Ser/Thr

O-linked glycans

T. cruzi strains

G and Tulahuen

= α-galactopyranose

LPPG

(3)

(a) (c) (d)

= myoinositol

GIPL-1–3, A

Figure 4: Structures of Galf -containing glycans of Leishmania spp. and T. cruzi. (a) Structure of LPG from Leishmania spp. (b) Structures ofGIPLs from T. cruzi, including the previously annotated LPPG and GIPL-A (c) Structures of GIPL-1-3, (A) from L. major and L. mexicana.(d) Selected subset of structures of O-linked glycans found in both T. cruzi strain G and Tuhulan.

stress, and prevention from phagosomal transient fusion[51–54].

2.1.2. Glycoinositolphospholipids (GIPLs). GIPLs are freeglycosylated phospholipids found in many kinetoplastids.Those found in Leishmania spp. and T. cruzi are considered

unique due to the presence of β-Galf (Figures 4(b) and 4(c))[26, 55–58]. GIPL structure is species and strain dependentand varies in expression levels throughout the life stagesof the parasite [59–62]. GIPLs from Leishmania spp. arethought to be precursor molecules for the synthesis of theLPG core structure [63]. L. major GIPL-1 has been shown

Enzyme Research 5

O

HO

HO

HO

O-UDP

OH

HN

O

O

R

HN

O

O

R

O

HO

HOHO

O-UDP

OHN

O

O

R

O

HO

HOHO

OH

O

O

RHO

HOHO

OH

N

O

O

R

O

OH

HO

HO

OH

HN

O

O

R

O

OH

HO

HO

OH

O-UDP

Sn1 or Sn2

Single-electron transfer

O-UDP O-UDP O-UDP21

3

654

HN

HN N HN HNHN HN

N− N−N− N−−

N−

+ −

O−

− − −

N+

Figure 5: Proposed chemical mechanism for UGMs. Nucleophilic attack by the reduced flavin (1) leads to a flavin-galactose adduct (2). Thisstep can either occur via an Sn1 or Sn2 reaction. Alternatively, the flavin can transfer one electron to a galactose oxocarbenium ion, forminga sugar and flavin radical that can also form the flavin-galactose adduct. Formation of a flavin iminium ion leads to sugar ring opening (4).Sugar ring contraction occurs by attack of the C4 hydroxyl to the C1-carbon (5). The final step is the bond formation to UDP (6).

to be involved in parasite-host interactions and is thought toplay an important role in establishing infection [61, 64].

GIPLs from T. cruzi include a class of phospholipidspreviously identified as lipopeptidophosphoglycans (LPPGs)[65–67]. The LPPGs were originally considered a separateclass from the GIPLs due to the presence of contaminatingamino acids during their purification; these amino acids havesince been identified as part of the NETNES [27, 68]. Theimportance of GIPLs in T. cruzi is revealed by studies thatshow that it plays a role in antigenicity, both with rabbitand human sera [40, 57]. The antigenicity is thought to beprimarily due to the terminal β-Galf residues either from theGIPLs or the O-linked mucins, as removal of β-Galf resultsin decreased levels of antigenicity [40, 57, 69]. It has also beenshown that GIPLs play a role in attachment of the parasite tothe luminal midgut of the vector Rhodnius prolixus [59]. T.cruzi modulates this interaction by altering GIPL expressionlevels during its life cycle, as epimastigotes have much higherexpression of GIPLs than trypomastigotes [59, 69, 70].

2.1.3. N-Linked Glycans. β-Galf is found in mannose N-linked oligosaccharides in several species of trypanosomatidflagellates including T. cruzi, Leptomonas samueli, Her-petomonas samuelpessoai, Crithidia fasciculate, and Crithidiaharmosa [40, 71–74]. The glycan structures have been solvedfor L. samueli, C. fasciculate, and C. harmosa and are shownto be species dependent [71, 73]. β-Galf units are found asterminal sugars linked to mannose residues in high mannosetype N-linked glycans [71, 73]. The role of N-linked glycanshas currently not been significantly explored for T. cruzi.

2.1.4. T. cruzi O-Linked Glycans and Mucins. T. cruzi mucinsare a family of GPI-linked glycoproteins with high levelsof O-linked glycosylation [75]. Several studies have beenconducted to determine the composition of the oligosaccha-rides bound to Thr and Ser residues in these glycoproteins

[76–80]. In T. cruzi, the O-glycans are not linked to N-acetylgalactosamine as in mammals and other organisms;instead, they are linked to N-acetylglucosamine [81]. It hasbeen demonstrated that these glycans vary highly amongT. cruzi strains, and β-Galf is a component of the glycanstructures of T. cruzi strains G, Tulahuen, and Dm28c;however, β-Galf is not found in T. cruzi strains CL-Brenerand Y (Figure 4(e)) [76–78, 82, 83]. These mucins playan important role in parasite-host interaction by bothprotecting against host defense mechanisms and ensuringtargeting of specific cells and tissues [75, 81].

3. Galactofuranose Is a Virulence Factor inKinetoplastids

It has been shown that incubation of L. major or T. cruziwith Galf -specific antibodies blocks parasite binding tomacrophages or mammalian cells, resulting in a 50–80%decrease in infection rates [64, 70, 84, 85]. It was furthershown that the antibody specifically bound to the β-Galf waspresent in GIPLs of T. cruzi and GIPL-1 of L. major [64, 70].This suggests that β-Galf and the GIPLs of T. cruzi andGIPL-1 of L. major play a role in cell adhesion and infection.Furthermore, it was shown that macrophages incubated withP-nitrophenol-β-Galf were infected 80% less by L. major, 4while macrophages incubated with P-nitrophenol-β-Galpsaw no decrease in infectivity [64]. Together, these resultsconfirm that β-Galf plays an important role in parasite-hostinteraction and suggest that β-Galf biosynthetic enzymes arepotential drug targets.

3.1. UDP-Glucose 4′-Epimerase (GalE). In T. cruzi, GalE isthe first protein required for Galf biosynthesis [86]. GalEis classified as a short-chain dehydrogenase/reductase (SDR)with a conserved Tyr-X-X-X-Lys motif and a characteristicRossmann fold structure for NAD(P)+ binding [42, 87]. GalE

6 Enzyme Research

1

G G G G G G HR L F V V S L A V T R V A A V L TM Q P M T A F D F F T I E R A Q L D K V L L E R R P H I N Y S E E P Q T G I E H K Y A F H S

G G G G G G HM Y I I V S A A E K K V A T I I T. . . . . . Y D L F V C N . L K L N K V L I E K R N H I N Y . . E D C E G I Q H K Y A F H N

G G G G G G HS I L I V A A G Q A Q I S R V I T. . . . M K K K F S V I R . L E K G H V H I D Q R D H I N Y D A D S E T N V M H V Y P F H D

G G G G G G HT I V I I A L A L E N L S L T V S. M A E L L P K P T G A V R T L G Y K W H Y E C N D T P L R S F D E N G . . F W D L G I F H

G G G G G G HA V V I I A L A L E N L S L L V S. . . . M S D K P T G A V R M L K H A F H Y D G G T V P L R S V D D K G . . F W D M G I F H

G G G G G G HS V L V I A L A L Q S I A V L V SM T H P D I V D P T G A K R N I D G P WM V D S N E T P L S T D T P E G . . F Y D V G I F H

PK D V T R R A N Q L Q I E AN R V W Y R Q F D . . . . F T D Y H V F M H G A Y Q F M G L G V S Q F F G K Y F T P E Q A R L A Q A A E I D T

PK D V V T S A K K T N I A KD Y I W Y N D L E . . . . F N R F N P L I Y D L F N L F N M N F H Q MW G . V K D P Q E A Q I N Q K K K Y G D

PE N I A V R A N Q T A I E AN T V W Y N K H E . . . . MM P Y N V K T V G V F S L I N L H I N Q F F S K T C S P D E A R L A . K G D S T I

PQ D M V Q E V R R I V S A EY Y F D V D W A Q . . . G W N V L R S W W V G W V P Y F Q N N H R L P E Q D R K R C L D E L R H R . . . T Y T

PA D M I Q E V S A I E A A EY Y F D V N L A S . . . D W N T L R S W R C G W V P Y F Q S N H R L P P E V R D T C L K G I E E A R S V A A P

PK D L L Q I V Q Q I I A L TY Y F D C D E A P K E D D W Y T H R S Y R C G W V P Y F Q N N S M L P K E E Q V K C I D G M D A E A R . V A N

E G Y K WQ N L E A I S R L A F K Q L I R L VD A K L I P Y E V G T A Q T D P K E P A A N T P R Y T F D N R . . . . . . . . . . . . Y F S D

E G Y K WE N L E A I S E L A L K Q L I R I VV P Q L V D Y Q I G T E G R S A K E P A F I K P R F T F D N N . . . . . . . . . . . . Y F S D

E G Y K WQ T F E A L R K L A F K Q L L R L VD P Q F I E Y E F G T I G M Q P S E P A S I K P R F N Y D D N . . . . . . . . . . . . Y F N H

E G Y K WN N F E F T R E I I F R V M V E R AP P S Q F G A D M P N F A V P P C L S T E W E V P V D L E R I R R N I Q E N R D D L G W G P N

E G Y K WQ N F A V S R E I V F R V M V E R AK P Y H F G A E M P N F A V P L H L S T E W G V A V N V E R I R E N I Q L K R D D V G W G P N

E G Y K WK T F D I V R T I L F R V M L E R AK P W MM G A D M P N F A V P T T K Q C A W G V A P N L K A V T T N V I L G K T A G N W G P N

P G T DT T L A I V L R Q V V T LY E G L D Y A W Q N M A D H R . . . . . . . . . . . E R N T D W F D V G L R P G S P A A P Y G P R Y F D Y

P G T DR V I L V V L K S I I T IY Q G I G Y K L E K M E G . . . . . . . . . . . . . D K G I D F L K D D L A S . . K A H R Y G P Q Y F D Y

P G T DK K I L I V L E T V F S LF Q G M C Y Q M K S I N H E N . . . . . . . . . . . K D Q R E F I V D R H Y D . . . . . H Y G P A F Y G Y

P G T DA Q I I Q I I T S L I T FT F R F R G G I Y Q A K E K L P S E K L T F N S G F A A D A D A K T I F N G E V V S Y D Y S V P N L L R M

P G T DA K I V R T V T A L V T LT F R F S G G A Y K A W K M I P E A H K T L G P Q C V K N P I T K T L M N G E A V S Y D A S M P D L L L A

P G T DA A I V K T V T Q L V T VT F R F R G G G W I A A N T L P K E K T R F G E K G V K N A N N K T V L D G T T I G Y K K S M A F L A E A

G V P RV Q Y Y I EA E G . . . . . . . . . . . . . . R L G W R T L D F E E V L P I G D F T A M N . . . . . . . . . . . . . N D L D V T H F

G V P RT Q F Y I ER F G . . . . . . . . . . . . . . A L E Y R S L K F E E R H E F P N F N A I N . . . . . . . . . . . . . T D A N V T I H

G V P RK Q Y Y I EQ Y G . . . . . . . . . . . . . . R L G Y R T L D F K F I Y Q G . D Y C A M N . . . . . . . . . . . . . C S V D V T T H

G V P RK I F F A VT K G . . . . . . . . . . T G F K G Y D E W P A I A D M V Y S S T N V I G K G T P P P H L K T A C W L Y P E D T S Y T F

G V P RK I F F A IV A A G V E E D A E T A S A S A L K A P R L R E I A D M V Y S S T H I I G K G C P P P E M R T A C W L Y P E D G I Y T F

G V P RQ I F F A IM N D . . . . . . . . . . . . . . . . Q E L V G L T K L F Y S S T H V V G R G S R P E R I G D K C W L Y P E D N C Y T F

R FH H P E R . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D Y P T D K

K FH D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Y V E T K H

K FY S P . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . W E Q H D G

S YN S K Y N V P E G . . . . . . . . . . . . . . . . . . . . . . . . H W S L M L E V S E . S K Y K P V N H S T L I E D C I V G C L A S N L

S YR A D T N A P E G . . . . . . . . . . . . . . . . . . . . . . . . H W S I L L E V S Q N V L Y K P V N V D T I V E D C I A G L R T V T L

S YN S P Y N Q P E A S K K L P T M Q L A D G S R P Q S T E A K E G P Y W S I M L E V S E . S S M K P V N Q E T I L A D C I Q G L V N T E M

Y P G R YT V E R I L L A T R A E V G T Q M A LI M R Y S F A E D D D E P Y N T E A D R A Y A R K S T A S S K L F G L Y L D M H A I S A N M

Y P G R YT V E L V L F K K R A E V A E K Q S LV T K Y P E W K V G D E P Y N D N K N M E Y E L S R . . . D K I F G L Y Y D M H V I A A Y Q

Y P G R YS V E R I L L E K L A E I G T R V A LC Y K Y S A C E E N D I P Y R Q M G E M A Y S L E N T N . . . T F V L Y L D M D T I E A K T

Y P G R YL L L S T L L E K Q L R I G A R Q S QP E D L V K W H Y R I E K G P F I G R N N A P E M S . . . . C Y S R F W E V G N D H F M G V

Y P G R YL R E S I L L E E Q L K I G A R Q S QP E D I V R W H H M E K K G P F V G R N E V P V R D Y . . . Q Y S R F W E V A N D H L M G V

Y P G R YL K E S T A L T Q L L K I G S R Q S LP T D I V T Y H R R F D H G P T L E R E G I P K Q D . . . . D W S R F W E V G N D H F M G V

MtUGM N AY D V L P H L R D G V P L L Q D G A . . . . . . . . . . . . . . . . . . . . . . . . .

EcUGM N SV K I M T D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

KpUGM V NA E Y L S L T D N Q P M P V F T V S V G . . . . . . . . . . . . . . . . . . . . . . .

TcUGM I VE A D H L G L A T E E T T V A N P G R V N G T R A T T H F G L L Q K D M . . . . . . .

LmUGM V IE A G H F . Y G T D E D T V H K P E K V N T R R G E M R C T W S S T A S . . . . . . .

AfUGM

MtUGM

EcUGM

KpUGM

TcUGM

LmUGM

AfUGM

MtUGM

EcUGM

KpUGM

TcUGM

LmUGM

AfUGM

MtUGM

EcUGM

KpUGM

TcUGM

LmUGM

AfUGM

MtUGM

EcUGM

KpUGM

TcUGM

LmUGM

AfUGM

MtUGM

EcUGM

KpUGM

TcUGM

LmUGM

AfUGM

MtUGM

EcUGM

KpUGM

TcUGM

LmUGM

AfUGM

MtUGM

EcUGM

KpUGM

TcUGM

LmUGM

AfUGM

V IE A D N V . N G A V E L T L N Y P D F V N G R Q N T E R R L V D G A Q V F A K S K A Q

*

^ ^ ^ ^ ^ ^^

*

*

^ ^ ^

*

* *^ ^

10 20 30 40 50 60 70

310 320

380 390

330 340 350 360 370

80 90 100 110 120 130

140 150 160 170 180 190

200 210 220 230 240 250

260

300

270 280 290

Figure 6: Multiple sequence alignment of UDP-galactopyranose mutases. Conserved amino acids found in the active site of bacterial UGMare marked with an asterisk, and those involved in flavin binding are marked with arrowheads. Mt: M. tuberculosis, Ec: E. coli; Kp, K.pneumoniae; Tc: T. cruzi; Lm: L. major; Af: A. fumigates. The program ClustalW was used to generate the alignment and Espript 2.2 to createthe figure [31].

Enzyme Research 7

1. . . . . . . . . . . . . . . . . . . . MA P R RWHHN C R RMA S F V R I G L Y T L L F LMG Y I V P L I I F Y N R S G T E T F E D T P R P G E R F I S D E. . . . . . . . . . . . . . . . . . . . MA S A R A L Y V R R RMR P F V R AWL Y T L L F LMG Y L G P L I I F Y R R S R E E T F T D I A R P G E V F I S D E. . . . . . . . . . . . . . . . . . . . MA P L R S VH Y R R RMA K L V R I G L Y T L L F LMG Y F V P L I I F Y N R S G T D T F V D T S R P G E A V I S D E

ML PWP C L Q R GMR R V T R S L K NG A T S L FWK G C F I F L L I S F F L F ML I YMY T C L E V E P L F GDD S AHD V E V D P T R A D Y I HC V G E RML PWL C L Q R DMH R V T R G L K T E A T S F FWK G C F I F L L I S F F L F V L I Y I Y T C L E V E P L F GDD P AHG F G V G P TMA E Y I HC V G E R. . . . . . . . . . MR R V T R I L K T E A T S L FWK R C F I L L L T L N L L S MLMV F F F G . . F E S L L DDD P A Y G F G V D P T R A D Y I R C V G E R. . . . . . . . . . . . . . . . . . . . . . . . . . . . . ML Y L S S N F N L L S MLM F F F L G . . . S S S Y L V T I P RMVWCWS DQG G L Y P L R R G K. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

ML PWL C L Q R GMR G V T R I L K T E A T S P FWK K C F I F L L T L N F L S MLMV F F F G . . V E F L L GDD P A Y G F G V D P T R A D Y I HC V G E R. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MLM F F F F G . . V E F I L R DD P A Y G F G V G P TMA E Y I R C V G E R. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MA D C I HC V G E R

H P N I G Y VN G R A L V P F T N D V F P L T L EI E A S A E L NF V A Q I I A A M E V S Q AH I R Q Q L E I R E I V A E K T L K VH H S W R G D F G R R D I NH P N I G Y VN G R A L V P F T N D V F P L T L EV E A A A E L QF I E Q V I A A I E V S Q AHA K R Q R E L M E V A A E R T L R VH H S W R D P F N I R D A NH P N I G Y VN G R A L V P F T N D V F P L T L EI E A A A E L S F V A Q V I A A I E V A Q T K E K E Q L E I S L V I A E R T L E VH H S W R G A F G R R D A NH P N I G Y VN G R A L V P F T N D V F P L T L ES E S A A I L S L R E E V V S S L N L R D VH EM R D A A M E A V A N E S E Y S VR L P F M D P L H R D A P PH P N I G Y VN G R A L V P F T N D V F P L T L ES E S A A I L S L R E E V V S S L N L R D VH EM R D A A M E A V A N E S E Y S VR L P F M D P L H R D A P PH P N I G Y VN G R A L V P F T N D V F P L T L ES E S A A I L S L R E E V V S S L N L R D VH EM R D A A M E A V A N E S E Y S VR L P F E D P L H R D A P PH P N I G Y VN G R A L V P F T N D V F P L T L ES E S A D I S S L R E E V V S S L S L R D VHGM R D A A M E A A A N R S E Y S VR L P F M D P L H R D S P PH P N I G Y VN G R A L V P F T N D V F P L T L ES K S A A I L S L R E E V V G S L N L R D VH EM R D A A M E A V A N E S E Y S VR L P F M D P L H R D A P PH P N I G Y VN G R A L V P F T N D V F P L T L ES E S A A I S S L R E E V V S S L S L R D VH EM R D A A M E A A A N E S E Y S VR L P F K D S L H R D S P PH P N I G Y VN G R A L V P F T N D V F P L T L ES E S A A I S S L R E E V V S S L N L R D VH EM R D A A M E A V A N E S E Y S VR L P F M D P P H R D A P PH P N I G Y VN G R A L V P F T N D V F P L T L ES E S A A I L S L R E E V V S S L S L R D VH EM R D A A M E A V A N E S E Y S VR L P F E D P L H R D A P PH P N I G Y VN G R A L V P F T N D V F P L T L ES E S A A I L S L R E E V V S S F N L R D VH EM R D A A M E A MA N E S E Y S IR L P F K D P L H R D A P PH P N I G Y VN G R A L V P F T N D V F P L T L ES E S A A I L S L R E E V V S L L N L R D VH EM R D A A M E A MA N E S E Y S VR L P F M D P L H C D A P P

S L P R F G F K L R L A I T F D E PF A F R I V T A S S I R M E QMK K Q A Y T E A T F S A L G F N F Y A YP D P R R S H P . . . Y T P P D A I F DH . F M A GS L P R F G F K L R L A I T F D E PF A Y E V V S A S S I R M E EMK R Q A Y T E A T F S A V G F N F C A YP DQ R R S L P . . . Y T A P D T V Y D S . F M A GS L P R F G F K L R L A I T F D E PF A F S I I T A S S I R M Q EMK K Q A Y T E A T F S A V G F N Y Y A YP D P R H S Y P . . . Y T P P D T I Y D Y . F M A GS L P R F G F K L R L A I T F D E PL R V T L S T S A L I R A K E R E K A S Y A S T S V T S V G Y N F Y A YL R R G L R VND S R D Y S A KH H C Y Y S C F M VS L P R F G F K L R L A I T F D E PL R V T L S T S A L I R A K E R A KM S Y A T A S V T S V G Y N F Y A YL R R G L R V K DN R D Y S A KH H C Y Y G C F M VS L P R F G F K L R L A I T F D E PL R V T L S T S A L I R A K E R E K A S Y A T T S V T S V G Y N F Y A YL R R G L R VNG S R D Y TV KH N C Y Y G C F I VS L P R F G F K L R L A I T F D E PL R V T L S T S A L I R A K E R E K A S Y A S A S V T G V E Y N F Y A YL R R G L R VNDG R D Y S V KH H C Y Y S C F I VS L P R F G F K L R L A I T F D E PL K V T L S T S A L I R A K E R E K A S Y A S A S V T S V G Y N F Y A YL R R G L R VND S R D Y S A KH H C Y Y S C F M VS L P R F G F K L R L A I T F D E PL R V T L S T S A L V R A K E R E K A S Y A S A S V T S V G Y N F Y DCL RWG L R V K DG R D Y S V KH H C Y Y S C F I VS L P R F G F K L R L A I T F D E PV R V T L S T S A L I R A R E R E TM S C A S A S V T S V G Y N F Y A YL R R G L R VNDG R D Y S A KH H C Y Y S F F V MS L P R F G F K L R L A I T F D E PL R V T L S T S A L I R A K E R E K A S Y A S T S V T S V G Y N F Y A YL R R G L R V K DD R D Y S A KH H C Y Y S C F M VS L P R F G F K L R L A I T F D E PL K V T L S T S A L I R A K E R E K A S Y A S T S V T S V G Y N F Y A YL R R G L H V K DN R D Y S V KH H C Y Y G C F M VS L P R F G F K L R L A I T F D E PL K V T L S T S A L I C A K E R E K A S Y A T T S V T S V G Y S F Y A YL R R G L H V K DN R D Y S T KH H C Y C S C F I V

E D D Y R G G K H R Y KWM MN A L YR Y S E Y V F N N L NV G D T A F QNV F MN E R R AH W K F R Q A G S A K N R G V F K Y V K W N L F L Q H F P DD V T IE D D Y R G G K H R Y KWV MK A L YK Y SM Y V F N N L N A D S T A F Q S M F MN E R R NH W S M R E A G A A R N R G I F R Y M R E Y L F L E R F S V E G T LE D D Y R G G K H R Y KWL MA A L YK Y S E Y V Y N N L K V D S T V F Q S M F MN Q R R AH W K F R E A E A A R N R G I F K Y I K G H P F L Q R F P D S V T LE D D Y R G G K H R Y KWS L R L L FQ R V L F V R S N I R L S E Q L L D A L Y VK L N A MV L E N Y G S . . . . . . . . . . E P W R S MT Q P K N . . . . G LE D D Y R G G K H R Y KWS L R L L FQ R V L F V R N N I R L S E Q L L D A L Y VK L N V MV L E N Y S Y . . . . . . . . . . E P W R S MT Q P M K . . . . G LE D D Y R G G K H R Y KWS L R L L FQ R V L F V R N N I R L S N K V L D A L Y VR L N A MF L E N Y S Y . . . . . . . . . . E P W R S S A D A M N . . . . R PE D D Y R G G K H R Y KWS L R L P FQ R V L F V R S N I R F S N KM L D A L Y VK L N A MV L E N C G S . . . . . . . . . . E P W R S MT K P M G . . . . P QE D D Y R G G K H R Y KWS L R L L FQ R V S F V R S S I R L S N E V L D A L Y VR L N A MV L E N Y G S . . . . . . . . . . E P W R S S A D A M N . . . . R PE D D Y R G G K H R Y KWS L R L L FR Q V L F V R S N I R L S E Q L L D A L Y VK L N A MV L G N Y G S . . . . . . . . . . E P W R S MT Q P M N . . . . G LE D D Y R G G K H R Y KWS L R L L FR Q V L F V R S N I R L S E Q L L D A L Y VK L N A MV L G N Y G S . . . . . . . . . . E P W R S MT Q P M N . . . . G LE D D Y R G G K H R Y KWG L R L L FQ R V L F A R S N I R L S N K V L D A L Y VR L N A MV L E N Y S Y . . . . . . . . . . E P W R S S A D A M N . . . . R PE D D Y R G G K H R Y KWS L R L L FQ R V S F V R S N I R F S N KM L D A L Y VR L N A MV L E N Y G S . . . . . . . . . . E P W R S S A K P M N . . . . R PE D D Y R G G K H R Y KWS L R L L FQ R V S F V R S N I R F S E Q L L D A L Y VK L N V MV L E N Y G S . . . . . . . . . . E P W R S MT Q P V N . . . . G L

G P D WV D R I G Y LY R P F T I V M L A E Q R S Q MR R T K L VG V R R R P L N W I E S CH D Y K P S D F A D P S . . . . . . . . . . . . . . . . . . . .G P D WV D R I G Y LY Q P F T I V T L P Q L R S E MR S T E L AE T G R K P L N Y I E G C R S Y K Y N Q F S Y V S . . . . . . . . . . . . . . . . . . . .G P D WV D R I G Y LY P P F T I V M L T E R R F E I R Y MK L VE I GN K P L N W V E N CH D Y K P N D F A R S R . . . . . . . . . . . . . . . . . . . .G P D WV D R I G Y LK A P Y MV L V K E A I Q R A E I V R R L RC C DG Y K E D R Y HD R R P S I D D Y P V T K G R . . . . . . . . . . . . . . . . . . .G P D WV D R I G Y LK A P Y MV L V K E A I Q R A E I V R S L TC C NG Y K E N R Y HD R R P S I D D Y P F T K G R . . . . . . . . . . . . . . . . . . .G P D WV D R I G Y LR A P Y MV L V K E A I Q R A E E V R T L RC C G G Y K E D R Y HD QG P K V E D H P V T K G S . . . . . . . . . . . . . . . . . . .G P D WV D R I G Y LR A P Y MV L V K E A I Q R A E E V R T L TC S G G C K E D R Y HD QG P K V E D H P F T K G R . . . . . . . . . . . . . . . . . . .G P D WV D R I G Y LR A P Y MV A V K E A I Q R A E E V R T L RC S G G Y K G D W Y HD QG P R V E D Y P V T K G R C T G A S R K F Y V F I I L F Y F S LG P D WV D R I G Y LK A P Y MV A V K E T I Q R V E K V R S W RC C NG Y K E D R H HD QG P K D E E Y L S R R E DD S A T N T S F A F L F C F L . . . .G P D WV D R I G Y LK A P Y M I L V K E T I Q R V E K V R R L RC C DG Y K E D R H HD QG P K D E D H P L T K G R . . . . . . . . . . . . . . . . . . .G P D WV D R I G Y LR A P Y MV L V K E A I Q R A E E V R T L RC C G G Y K E D R Y HD QG P K V E D H P V T K G R . . . . . . . . . . . . . . . . . . .G P D WV D R I G Y LR A L Y MV L V K E A I Q R V E K V R T L RC S G G C K E N R Y HD QG P K V E D Y P V T K G R . . . . . . . . . . . . . . . . . . .G P D WV D R I G Y LK A P Y MV L I K E A I Q R V E I V R T F TC C NG Y K E N R H HD Q R P I I Y D Y P F T K G R . . . . . . . . . . . . . . . . . . .

L.majorL.donovaniL.mexicanaTc_XP817720Tc_XP807317Tc_XP814596Tc_XP813514Tc_XP814250Tc_XP814920Tc_XP817334Tc_XP820070Tc_XP818858Tc_XP810069

L.majorL.donovaniL.mexicanaTc_XP817720Tc_XP807317Tc_XP814596Tc_XP813514Tc_XP814250Tc_XP814920Tc_XP817334Tc_XP820070Tc_XP818858Tc_XP810069

L.majorL.donovaniL.mexicanaTc_XP817720Tc_XP807317Tc_XP814596Tc_XP813514Tc_XP814250Tc_XP814920Tc_XP817334Tc_XP820070Tc_XP818858Tc_XP810069

L.majorL.donovaniL.mexicanaTc_XP817720Tc_XP807317Tc_XP814596Tc_XP813514Tc_XP814250Tc_XP814920Tc_XP817334Tc_XP820070Tc_XP818858Tc_XP810069

L.majorL.donovaniL.mexicanaTc_XP817720Tc_XP807317Tc_XP814596Tc_XP813514Tc_XP814250Tc_XP814920Tc_XP817334Tc_XP820070Tc_XP818858Tc_XP810069

L.majorL.donovaniL.mexicanaTc_XP817720Tc_XP807317Tc_XP814596Tc_XP813514Tc_XP814250Tc_XP814920Tc_XP817334Tc_XP820070Tc_XP818858Tc_XP810069

P D D C N P NG L L L LV L I V TM Y Q I KQ L I T V MT I M F I N MF R R S D R A V E L R D N F IN F F HC I A E R L S Y K DQH P A R I PY F Y P L D YV QP D D C N P NG L L L LV L I V TM Y Q I KQ L I T V MT I M F I N E F H R S N R A V E L K D N F IS F F R C V A D R L S Y N E QQ S A S I PY F Y P L D F V KP D D C N P NG L L L LV L I V TM Y Q I KQ L T T A MT I T F I N MF L R S D R A V E L G D N F IN F F HC V A E R L S Y K E E H P A R I PY F Y P L H YV KP D D C N P NG L L L LMV V LML LM FR RM I S V I R L V L VQ R E A S L Q E E R V Y G S R I VL L V D P K T RWMG GNN . G T D V I P L M L M Y . . W GP D D C N P NG L L L LMV V LML LM FR RM I S V I R L V L VQ R E A S L Q E E R V Y G S R V VL L V D P T T R Q L G GNN . G T D V I PM M L M Y . . W GP D D C N P NG L L L LMV V LML LM FR RM I NV I R L V L VQ R E A S L Q E E R V Y G S R V VL L V D P T T R R L G GNN . G T D V I P L M L M Y . . W GP D D C N P NG L L L LMV V LML LM FR RM I S V I R L V L VQ R E A S L Q E E R V Y G S R A VA S G G S ND T A A G GNNNG T D V I P L M L M C . . W GP D D C N P NG L L L LMV V LMF LM FR R T I S V I R L V L VQ R E A L L Q E E R V Y G S R V V. . . . . . . . . . . . . . . . . . . . . . M P M Y . . W GP D D C N P NG L L L LMV V LML LM FR RM I S V I R L V L VQ R E A S L Q E G R V Y G S R V V

.

.. . . . . . . . . . . . . . . . . . . . . . M L M C . . W GP D D C N P NG L L L LMV V LML LM FR RM I S V I R L V L VQ R E A S L Q E E R V Y G S R I V. . . . . . . . . . . . . . . . . . . . . . M L M C . . W GP D D C N P NG L L L LMV V LML LM FR RM I NV I R L V L VQ R E A S L Q E E R V Y G S R V VL L V D P T T RWMG GNN . G T D V I P L M L M Y . . W GP D D C N P NG L L L LMV V LML LM FR RM I S V I R L V L VQ R E A S L Q E E R V Y G S R V VL L V D P T T RWL G GNNNG T D V I P L M L M Y . . W GP D D C N P NG L L L LMV V LML LM FR RM I NV I R L V L VQ R E A S L Q E E R V Y G S R V VL L V D P T T RWL G GNNNG T D V I P L M L M Y . . W G

10 20 30 40 50 60

70 80 90 100 110 120 130 140

150 160 170 180 190 200 210 220

230 240 250 260 270 280 290

300 310 320 330 340 350

380 390 400 410 420 430

360 370

∗∗

Figure 7: Alignment of L. major LPG-1 (XP001683753), L. donovani LPG-1 (ADG26596), L. mexicana LPG-1 (CAB6682), and ten putativeT. cruzi GalfTs. Putative T. cruzi GalfTs were identified by BLAST search using L. major LPG-1 as the probe. The active site residues are shownin brackets, and the metal binding motif is represented with asterisks. The alignment was created as indicated in Figure 6.

8 Enzyme Research

is a homodimer that consists of two domains, an N-terminaldomain with the Rossmann fold and a C-terminal domainthat binds the substrate, UDP-Glc [88, 89]. The catalytic siteis located in the cleft between the two domains [88, 89].The mechanism is shown to be conserved across speciesand involves the deprotonation of the Glc O4′ hydroxyland hydride transfer from the C4 carbon of Gal to NAD+

[88, 89]. The intermediate 4-keto sugar rotates in the activesite and NADH transfers back the hydride to the oppositeface forming UDP-Gal [88, 89].

Mutant strains of T. brucei and T. cruzi with deletionof the galE gene have not been obtained suggesting thatGal metabolism is essential for parasite survival [49, 86,90, 91]. Conditional null mutants were created in T. bruceiusing tetracycline-regulated expression [49, 90]. Studies withthis strain showed that removal of tetracycline from thetrypomastigote parasite led to cell death and decreased Galsurface-expression levels by 30% [49, 90]. These studiesshowed that, upon Gal starvation, Gal was eliminated fromT. brucei variant surface glycoprotein (VSG) and from poly-N-acetyllactosamine-containing glycoproteins causing cellgrowth to cease and differentiation to a stumpy-like form,ultimately leading to cell death [91].

Single galE knockout mutants of T. cruzi epimastigoteswere also constructed [86]. These cell strains showed severalphenotypic differences including shortened flagella andagglutination, which is thought to be the result of a lackof surface mucins [86]. Interestingly, these cell strains showa preference for expressing high levels of Galf -containingGIPLs over Galp mucins, whose expression levels werereduced 6–9-fold, suggesting that Galf is preferentiallyexpressed in the glycocalyx over Galp [86]. In Leishmaniaspp., Gal can be obtained from extracellular sources, presum-ably by a family of hexose transporters [44, 92]. Thus, GalEis not essential in these parasites.

Studies have been undertaken to identify novel inhibitorsthat specifically target the GalE of T. brucei [93, 94].Using high-throughput screens and computer modelingexperiments, inhibitors that showed preference to T. bruceiGalE over human GalE were identified [93, 94]. However,when these compounds were tested in vivo with T. brucei andeither mammalian CHO cells or liver (MRC5) cells, thesecompounds either were cytotoxic to both the parasite andmammalian cells or the compound was ineffective againstT. brucei [93, 94]. These studies suggest that, while GalEremains a potential drug target, there will be many difficultiesin designing specific inhibitors for the treatment of thesediseases without unwanted side effects.

3.2. UDP-Galactopyranose Mutase (UGM). UGM is a flavo-dependent enzyme that catalyzes the conversion of UDP-Galp to UDP-Galf. UGM was first identified in Escherichiacoli K-12 in 1996, and since then it has been identifiedin several other pathogenic microorganisms including M.tuberculosis, L. major, T. cruzi, and A. fumigatus [5–8].Interestingly, while T. cruzi produces UGM the related T.brucei does not, and as a result, T. brucei does not produce

Galf [74]. UGM has been found to be the sole biosyntheticsource of Galf, since it is not found in mammals.

Deletion of the UGM gene in L. major shows that thisenzyme plays an important role in pathogenesis [25]. In theabsence of UGM, L. major mutants were completely depletedof Galf, lacked LPG PG repeats, and contained truncatedforms of GIPLs [25]. Furthermore, mice infection by L. majorlacking Galf was significantly attenuated [25]. As previouslymentioned, deletion of UGM also showed that Galf is avirulence factor in A. fumigatus and Aspergillus nidulans[23, 95]. These studies show the importance of UGM andvalidate this enzyme as a drug target in protozoan and othereukaryotic human pathogens.

Although the reaction catalyzed by UGM does notinvolve a net redox change for the conversion of UDP-Galpto UDP-Galf, the reaction requires the flavin cofactor to bein the reduced form [96, 97]. Structural and mechanisticstudies of the prokaryotic UGM have led to two proposals forthe ring contraction mechanism (Figure 5). One mechanismdepicts the reduced flavin acting as a nucleophile, attackingthe anomeric carbon (C1) of Gal to form a flavin N5-C1 Galadduct [98]. This adduct has been isolated and characterizedin the prokaryotic UGM from K. pneumoniae [98, 99].The other proposed mechanism involves a single-electrontransfer from the reduced flavin to Gal, which then formsthe sugar-flavin adduct [100]. 5

Several structures have been solved for prokaryoticUGMs, in both oxidized and reduced states with andwithout substrate bound, providing excellent groundworkfor the development of specific inhibitors [33, 99, 101]. Thestructure of prokaryotic UGMs show that it is a homodimerand a mixed α/β class protein with 3 domains: an FAD-binding domain with a typical Rossmann fold, a 5-helixbundle, and a 6-stranded antiparallel β-sheet [32, 101]. Thestructures of the reduced protein with substrate bound showthat Gal is properly positioned for interaction with the flavin[99, 101].

Much less is known about the mechanism and structureof eukaryotic UGMs. These enzymes share low sequenceidentity, and the presence of inserts in the primary structurepredicts significant structural differences (Figure 6). In fact,comparison of the oligomeric states between prokaryotic andeukaryotic UGMs indicates that quaternary structures varyamong species (Table 1) [34]. Furthermore, our group, aswell as others, has demonstrated that known inhibitors ofeukaryotic UGM are not effective or have decreased potencyagainst L. major, A. fumigatus, and T. cruzi UGMs [7] (Qiand Sobrado unpublished results). Therefore, mechanisticand structural work is urgently needed on the eukaryoticenzymes.

3.3. UDP-Galactofuranose Transferases. UDP-α-Galf is syn-thesized in the cytosol by UGM and is transported intothe Golgi where it is attached to the LPG and GIPLs bygalactofuranosyl transferases (GalfTs) [102]. Currently, allknown linkages of Galf in T. cruzi and Leishmania spp.are in the β anomer conformation. The most extensivelystudied GalfT is LPG-1 from L. major and L. donovani.

Enzyme Research 9

Studies on LPG-1 have revealed that it is localized to theGolgi apparatus, where it adds the β-Galf to the core LPGstructure [102, 103]. LPG-1 is a metal glycosyltranferasewith typical conserved motifs including a cytoplasmic tail,a transmembrane domain, and a DXD metal-binding motif[104]. LPG-1 has been shown to only be responsible for theaddition of Galf to LPG and to not play a role in the additionof Galf in the GIPLs [103, 105]. Mutants with the deletionof lpg-1 gene in both L. major and L. donovani show LPG-1to be important for LPG formation. Due to the lack of LPG,the mutant strains with lpg-1 gene deleted in L. major displayattenuated virulence [103, 105]. These studies showed thatLPG-1 could serve as a drug target in L. major.

There are no published studies on the GalfT from T.cruzi. In order to identify GalfTs in T. cruzi, a BLAST searchwas conducted using LPG-1 from L. major as a template,and more than 30 putative proteins annotated as β-GalfTsin the T. cruzi genome were identified [106, 107]. The top10 putative GalfT sequences from the T. cruzi BLAST searchwere aligned with the L. major and L. donovani LPG-1showing high sequence identity between these sequences(Figure 7). These sequences all contain the proposed catalyticsite and demonstrate redundancy of the genes [104]. Thehigh number of GalfTs is typical for organisms, as oftendifferent transferases are used for each linkage type basedon anomericity, bond linkage, and the substrate acceptorsfor Galf [108]. Due to the high number of GalfTs within T.cruzi, targeting GalfTs for drug design most likely would notbe effective.

4. Concluding Remarks

To cause infection, protozoan parasites must recognize themammalian host environment, bind and infect the targetcells, and evade the immune system. Undoubtedly, the cellsurface of these pathogens plays important roles in theseprocesses. Current drugs are able to kill most of the parasitesduring treatment; however, these treatments do not elimi-nate all the parasites, presumably because they can “hide” inthe intracellular forms. Modification of the cell surface sugarcomposition will alter the mechanism of infection. Enzymesinvolved in the biosynthesis of Galf have been shown to playa role in parasite growth and pathogenesis. GalE is essentialfor growth in T. cruzi and T. brucei, while UGM, and LPG-1are important virulence factors in L. major [25, 86, 103]. Dueto the presence of a GalE homolog in humans, compoundsthat inhibit this enzyme have toxic side effects. Furthermore,this enzyme is not important for virulence in Leishmaniaspp. UGM plays a central role in Galf biosynthesis and isthe only source of UDP-Galf, which is the substrate forall the GalfT that attach Galf to the final sugar-acceptormolecules. Consequently, UGM emerges as an attractivedrug candidate, as no homolog is found in humans [109].The unique chemical structure of UGM suggests that specificinhibitors can be identified. Targeting UGM in T. cruzi andL. major will affect their virulence in humans and perhapsallow the immune system to effectively clear the parasite.Alternatively, inhibition of UGM will enhance the activity of

other antiparasitic drugs. Such combination therapy mightbe necessary to combat these complex eukaryotic humanpathogens.

Acknowledgments

This work was supported in part by NIH Grants RO1GM094468 (P. Sobrado, PI) and RO1 AI082542 (R. Tarleton,PI). M. Oppenheimer was supported by a fellowship from theAmerican Heart Association. A. L. Valenciano was supportedby a fellowship from the Ministry of Science and Technology(MICIT) from Costa Rica.

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