Structural Snapshots of β-1,4-Galactosyltransferase-I Along the Kinetic Pathway (original) (raw)
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Journal of Molecular Biology, 2009
D-Galactofuranose (Galf) residues are found in the cell walls of pathogenic microbes such as Mycobacterium tuberculosis, and are essential for viability. UDP-galactopyranose mutase (UGM) is a unique flavo-enzyme that catalyzes the reversible conversion of UDP-galactopyranose (UDP-Galp) and UDP-galactofuranose (UDP-Galf). UDP-Galf is the active precursor of Galf residues found in cell walls. Despite the wealth of biochemical/ mechanistic data generated for UGM, the structural basis of substrate binding is still lacking. Here, we report the crystal structures of UGM from Deinococcus radiodurans (drUGM) in complex with its natural substrate (UDP-Galp) and UDP. Crystal structures of drUGM:UDP-Galp complexes with oxidized and reduced FAD were determined at 2.36 Å and 2.50 Å resolution, respectively. The substrate is buried in the active site in an unusual folded conformation and the anomeric carbon of the galactose is at a favorable distance (2.8 Å) from N5 of FAD to form an FAD-galactose adduct. The mobile loops in the substrate complex structure exist in a closed conformation. The drUGM-UDP complex structure was determined at 2.55 Å resolution and its overall structure is identical with that of the oxidized and reduced complexes, including the conformation of the mobile loops. Comparison with the recently reported UGM:UDP-glucose complex structure reveals key differences and the structures reported here are likely to represent the productive/active conformation of UGM. These structures provide valuable insights into substrate recognition and a basis for understanding the mechanism. These complex structures may serve as a platform for structure-guided design of inhibitors of UGM.
In Crystallo Capture of a Covalent Intermediate in the UDP-Galactopyranose Mutase Reaction
Biochemistry, 2016
UDP-galactopyranose mutase (UGM) plays an essential role in galactofuranose biosynthesis in pathogens by catalyzing the conversion of UDP-galactopyranose to UDP-galactofuranose. Here we report the first crystal structure of a covalent intermediate in the UGM reaction. The 2.3 Å resolution structure reveals UDP bound in the active site and galactopyranose linked to the FAD through a covalent bond between the anomeric C of galactopyranose and the FAD N5. The structure confirms the role of the flavin as nucleophile and supports the hypothesis that the proton destined for the galactofuranose O5 is shuttled from the FAD N5 via the FAD O4.
Journal of Molecular Biology, 2002
The crystal structure of the catalytic domain of bovine b1,4-galactosyltransferase (Gal-T1) co-crystallized with UDP-Gal and MnCl 2 has been solved at 2.8 Å resolution. The structure not only identifies galactose, the donor sugar binding site in Gal-T1, but also reveals an oligosaccharide acceptor binding site. The galactose moiety of UDP-Gal is found deep inside the catalytic pocket, interacting with Asp252, Gly292, Gly315, Glu317 and Asp318 residues. Compared to the native crystal structure reported earlier, the present UDP-Gal bound structure exhibits a large conformational change in residues 345-365 and a change in the sidechain orientation of Trp314. Thus, the binding of UDP-Gal induces a conformational change in Gal-T1, which not only creates the acceptor binding pocket for N-acetylglucosamine (GlcNAc) but also establishes the binding site for an extended sugar acceptor. The presence of a binding site that accommodates an extended sugar offers an explanation for the observation that an oligosaccharide with GlcNAc at the non-reducing end serves as a better acceptor than the monosaccharide, GlcNAc. Modeling studies using oligosaccharide acceptors indicate that a pentasaccharide, such as N-glycans with GlcNAc at their non-reducing ends, fits the site best. A sequence comparison of the human Gal-T family members indicates that although the binding site for the GlcNAc residue is highly conserved, the site that binds the extended sugar exhibits large variations. This is an indication that different Gal-T family members prefer different types of glycan acceptors with GlcNAc at their non-reducing ends.
Journal of Molecular Biology, 2007
O-Glycan biosynthesis is initiated by the transfer of N-acetylgalactosamine (GalNAc) from a nucleotide sugar donor (UDP-GalNAc) to Ser/Thr residues of an acceptor substrate. The detailed transfer mechanism, catalyzed by the UDP-GalNAc polypeptide:N-acetyl-alpha-galactosaminyltransferases (ppGalNAcTs), remains unclear despite structural information available for several isoforms in complex with substrates at various stages along the catalytic pathway. We used all-atom molecular dynamics simulations with explicit solvent and counterions to study the conformational dynamics of ppGalNAcT-2 in several enzymatic states along the catalytic pathway. ppGalNAcT-2 is simulated both in the presence and in the absence of substrates and reaction products to examine the role of conformational changes in ligand binding. In multiple 40-ns-long simulations of more than 600 ns total run time, we studied systems ranging from 45,000 to 95,000 atoms. Our simulations accurately identified dynamically active regions of the protein, as previously revealed by the X-ray structures, and permitted a detailed, atomistic description of the conformational changes of loops near the active site and the characterization of the ensemble of structures adopted by the transferase complex on the transition pathway between the ligand-bound and ligand-free states. In particular, the conformational transition of a functional loop adjacent to the active site from closed (active) to open (inactive) is correlated with the rotameric state of the conserved residue W331. Analysis of water dynamics in the active site revealed that internal water molecules have an important role in enhancing the enzyme flexibility. We also found evidence that charged side chains in the active site rearrange during site opening to facilitate ligand binding. Our results are consistent with the single-displacement transfer mechanism previously proposed for ppGalNAcTs based on X-ray structures and mutagenesis data and provide new evidence for possible functional roles of certain amino acids conserved across several isoforms.
Biochemistry, 2008
R-1,3-Galactosyltransferase (R3GT) catalyzes the transfer of galactose from UDP-galactose to form an R 1-3 link with -linked galactosides; it is part of a family of homologous retaining glycosyltransferases that includes the histo-blood group A and B glycosyltransferases, Forssman glycolipid synthase, iGb3 synthase, and some uncharacterized prokaryotic glycosyltransferases. In mammals, the presence or absence of active forms of these enzymes results in antigenic differences between individuals and species that modulate the interplay between the immune system and pathogens. The catalytic mechanism of R3GT is controversial, but the structure of an enzyme complex with the donor substrate could illuminate both this and the basis of donor substrate specificity. We report here the structure of the complex of a low-activity mutant R3GT with UDP-galactose (UDP-gal) exhibiting a bent configuration stabilized by interactions of the galactose with multiple residues in the enzyme including those in a highly conserved region (His315 to Ser318). Analysis of the properties of mutants containing substitutions for these residues shows that catalytic activity is strongly affected by His315 and Asp316. The negative charge of Asp316 is crucial for catalytic activity, and structural studies of two mutants show that its interaction with Arg202 is needed for an active site structure that facilitates the binding of UDP-gal in a catalytically competent conformation. † H.J. was supported by a postgraduate studentship by the Ministry of Higher Education Malaysia and Universiti Teknologi, Malaysia.
Studies on the metal binding sites in the catalytic domain of 1,4-galactosyltransferase
Glycobiology, 2002
The catalytic domain of bovine β1,4-galactosyltransferase (β4Gal-T1) has been shown to have two metal binding sites, each with a distinct binding affinity. Site I binds Mn 2+ with high affinity and does not bind Ca 2+ , whereas site II binds a variety of metal ions, including Ca 2+. The catalytic region of β4Gal-T1 has DXD motifs, associated with metal binding in glycosyltransferases, in two separate sequences: D 242 YDY-NCFVFSDVD 254 (region I) and W 312 GWGGEDDD 320 (region II). Recently, the crystal structure of β4Gal-T1 bound with UDP, Mn 2+ , and α-lactalbumin was determined in our laboratory. It shows that in the primary metal binding site of β4Gal-T1, the Mn 2+ ion, is coordinated to five ligands, two supplied by the phosphates of the sugar nucleotide and the other three by Asp254, His347, and Met344. The residue Asp254 in the D 252 VD 254 sequence in region I is the only residue that is coordinated to the Mn 2+ ion. Region II forms a loop structure and contains the E 317 DDD 320 sequence in which residues Asp318 and Asp319 are directly involved in GlcNAc binding. This study, using site-directed mutagenesis, kinetic, and binding affinity analysis, shows that Asp254 and His347 are strong metal ligands, whereas Met344, which coordinates less strongly, can be substituted by alanine or glutamine. Specifically, substitution of Met344 to Gln has a less severe effect on the catalysis driven by Co 2+. Glu317 and Asp320 mutants, when partially activated by Mn 2+ binding to the primary site, can be further activated by Co 2+ or inhibited by Ca 2+ , an effect that is the opposite of what is observed with the wild-type enzyme.