Karina Kizjakina

gfsA encodes a novel galactofuranosyltransferase involved in biosynthesis of galactofuranose antigen of O‐glycan in Aspergillus nidulans and Aspergillus fumigatus

The cells walls of filamentous fungi in the genus Aspergillus have galactofuranose (Galf)‐containing polysaccharides and glycoconjugates, including O‐glycans, N‐glycans, fungal‐type galactomannan and glycosylinositolphosphoceramide, which are important for cell wall integrity. Here, we attempted to identify galactofuranosyltransferases that couple Galf monomers onto other wall components in Aspergillus nidulans. Using reverse‐genetic and biochemical approaches, we identified that the AN8677 gene encoded a galactofuranosyltransferase, which we called GfsA, involved in Galf antigen biosynthesis. Disruption of gfsA reduced binding of β‐Galf‐specific antibody EB‐A2 to O‐glycosylated WscA protein and galactomannoproteins. The results of an in‐vitro Galf antigen synthase assay revealed that GfsA has β1,5‐ or β1,6‐galactofuranosyltransferase activity for O‐glycans in glycoproteins, uses UDP‐d‐Galf as a sugar donor, and requires a divalent manganese cation for activity. GfsA was found to be localized at the Golgi apparatus based on cellular fractionation experiments. ΔgfsA cells exhibited an abnormal morphology characterized by poor hyphal extension, hyphal curvature and limited formation of conidia. Several gfsA orthologues were identified in members of the Pezizomycotina subphylum of Ascomycota, including the human pathogen Aspergillus fumigatus. To our knowledge, this is the first characterization of a fungal β‐galactofuranosyltransferase, which was shown to be involved in Galf antigen biosynthesis of O‐glycans in the Golgi.

Targeting UDP-galactopyranose mutases from eukaryotic human pathogens.

UDP-Galactopyranose mutase (UGM) is a unique flavin-dependent enzyme that catalyzes the conversion of UDP-galactopyranose (UDP-Galp) to UDP-galactofuranose (UDP-Galf). The product of this reaction is the precursor to Galf, a major component of the cell wall and of cell surface glycoproteins and glycolipids in many eukaryotic and prokaryotic human pathogens. The function of UGM is important in the virulence of fungi, parasites, and bacteria. Its role in virulence and its absence in humans suggest that UGM is an ideal drug target. Significant structural and mechanistic information has been accumulated on the prokaryotic UGMs; however, in the past few years the research interest has shifted to UGMs from eukaryotic human pathogens such as fungi and protozoan parasites. It has become clear that UGMs from prokaryotic and eukaryotic organisms have different structural and mechanistic features. The amino acid sequence identity between these two classes of enzymes is low, resulting in differences in oligomeric states, substrate binding, active site flexibility, and interaction with redox partners. However, the unique role of the flavin cofactor in catalysis is conserved among this enzyme family. In this review, recent findings on eukaryotic UGMs are discussed and presented in comparison with prokaryotic UGMs.

Substrate-dependent dynamics of UDP-galactopyranose mutase: Implications for drug design

Trypanosoma cruzi is the causative agent of Chagas disease, a neglected tropical disease that represents one of the major health challenges of the Latin American countries. Successful efforts were made during the last few decades to control the transmission of this disease, but there is still no treatment for the 10 million adults in the chronic phase of the disease. In T. cruzi, as well as in other pathogens, the flavoenzyme UDP‐galactopyranose mutase (UGM) catalyzes the conversion of UDP‐galactopyranose to UDP‐galactofuranose, a precursor of the cell surface β‐galactofuranose that is involved in the virulence of the pathogen. The fact that UGM is not present in humans makes inhibition of this enzyme a good approach in the design of new Chagas therapeutics. By performing a series of computer simulations of T. cruzi UGM in the presence or absence of an active site ligand, we address the molecular details of the mechanism that controls the uptake and retention of the substrate. The simulations suggest a modular mechanism in which each moiety of the substrate controls the flexibility of a different protein loop. Furthermore, the calculations indicate that interactions with the substrate diphosphate moiety are especially important for stabilizing the closed active site. This hypothesis is supported with kinetics measurements of site‐directed mutants of T. cruzi UGM. Our results extend our knowledge of UGM dynamics and offer new alternatives for the prospective design of drugs.

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

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 ± 0.4s(-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.

Contributions of Unique Active Site Residues of Eukaryotic UDP-Galactopyranose Mutases to Substrate Recognition and Active Site Dynamics

UDP-galactopyranose mutase (UGM) catalyzes the interconversion between UDP-galactopyranose and UDP-galactofuranose. Absent in humans, galactofuranose is found in bacterial and fungal cell walls and is a cell surface virulence factor in protozoan parasites. For these reasons, UGMs are targets for drug discovery. Here, we report a mutagenesis and structural study of the UGMs from Aspergillus fumigatus and Trypanosoma cruzi focused on active site residues that are conserved in eukaryotic UGMs but are absent or different in bacterial UGMs. Kinetic analysis of the variants F66A, Y104A, Q107A, N207A, and Y317A (A. fumigatus numbering) show decreases in kcat/KM values of 200–1000-fold for the mutase reaction. In contrast, none of the mutations significantly affect the kinetics of enzyme activation by NADPH. These results indicate that the targeted residues are important for promoting the transition state conformation for UDP-galactofuranose formation. Crystal structures of the A. fumigatus mutant enzymes were determined in the presence and absence of UDP to understand the structural consequences of the mutations. The structures suggest important roles for Asn207 in stabilizing the closed active site, and Tyr317 in positioning of the uridine ring. Phe66 and the corresponding residue in Mycobacterium tuberculosis UGM (His68) play a role as the backstop, stabilizing the galactopyranose group for nucleophilic attack. Together, these results provide insight into the essentiality of the targeted residues for realizing maximal catalytic activity and a proposal for how conformational changes that close the active site are temporally related and coupled together.

Flavin-N5 Covalent Intermediate in a Nonredox Dehalogenation Reaction Catalyzed by an Atypical Flavoenzyme

The flavin‐dependent enzyme 2‐haloacrylate hydratase (2‐HAH) catalyzes the conversion of 2‐chloroacrylate, a major component in the manufacture of acrylic polymers, to pyruvate. The enzyme was expressed in Escherichia coli, purified, and characterized. 2‐HAH was shown to be monomeric in solution and contained a non‐covalent, yet tightly bound, flavin adenine dinucleotide (FAD). Although the catalyzed reaction was redox‐neutral, 2‐HAH was active only in the reduced state. A covalent flavin‐substrate intermediate, consistent with the flavin‐acrylate iminium ion, was trapped with cyanoborohydride and characterized by mass spectrometry. Small‐angle X‐ray scattering was consistent with 2‐HAH belonging to the succinate dehydrogenase/fumarate reductase family of flavoproteins. These studies establish 2‐HAH as a novel noncanonical flavoenzyme.

Steric Control of the Rate-Limiting Step of UDP-Galactopyranose Mutase

Galactose is an abundant monosaccharide found exclusively in mammals as galactopyranose (Gal p), the six-membered ring form of this sugar. In contrast, galactose appears in many pathogenic microorganisms as the five-membered ring form, galactofuranose (Gal f). Gal f biosynthesis begins with the conversion of UDP-Gal p to UDP-Gal f catalyzed by the flavoenzyme UDP-galactopyranose mutase (UGM). Because UGM is essential for the survival and proliferation of several pathogens, there is interest in understanding the catalytic mechanism to aid inhibitor development. Herein, we have used kinetic measurements and molecular dynamics simulations to explore the features of UGM that control the rate-limiting step (RLS). We show that UGM from the pathogenic fungus Aspergillus fumigatus also catalyzes the isomerization of UDP-arabinopyranose (UDP-Ara p), which differs from UDP-Gal p by lacking a -CH2-OH substituent at the C5 position of the hexose ring. Unexpectedly, the RLS changed from a chemical step for the natural substrate to product release with UDP-Ara p. This result implicated residues that contact the -CH2-OH of UDP-Gal p in controlling the mechanistic path. The mutation of one of these residues, Trp315, to Ala changed the RLS of the natural substrate to product release, similar to the wild-type enzyme with UDP-Ara p. Molecular dynamics simulations suggest that steric complementarity in the Michaelis complex is responsible for this distinct behavior. These results provide new insight into the UGM mechanism and, more generally, how steric factors in the enzyme active site control the free energy barriers along the reaction path.