Crystal structure of the retaining galactosyltransferase LgtC from Neisseria meningitidis in complex with donor and acceptor sugar analogs

Many bacterial pathogens express lipooligosaccharides that mimic human cell surface glycoconjugates, enabling them to attach to host receptors and to evade the immune response. In Neisseria meningitidis, the galactosyltransferase LgtC catalyzes a key step in the biosynthesis of lipooligosaccharide structure by transferring alpha-d-galactose from UDP-galactose to a terminal lactose. The product retains the configuration of the donor sugar glycosidic bond; LgtC is thus a retaining glycosyltranferase. We report the 2 A crystal structures of the complex of LgtC with manganese and UDP 2-deoxy-2-fluoro-galactose (a donor sugar analog) in the presence and absence of the acceptor sugar analog 4'-deoxylactose. The structures, together with results from site-directed mutagenesis and kinetic analysis, give valuable insights into the unique catalytic mechanism and, as the first structure of a glycosyltransferase in complex with both the donor and acceptor sugars, provide a starting point for inhibitor design.     

Characterization and expression analysis of two cotton genes encoding putative UDP-Glycosyltransferases

UDP-Glycosyltransferases (UGT) are a large family of enzymes, which catalyze the transfer of a sugar from an activated sugar donor to an acceptor molecule. Both in plant and in mammalian, they are important in maintenance of cellular homeostasis. In this study, two genes (designated GhUGT1 and GhUGT2, respectively) encoding putative UGT were isolated from cotton fiber cDNA library. The deduced proteins contain the signature sequences of plant UGTs in the C-terminal region. The GhUGT1 gene encodes a polypeptide of 457 amino acids, and displays homology at amino acid levels with the known glucosyltransferase genes. Sequence analysis revealed that the GhUGT2 merely encodes a small protein, as there is a nucleotide substitution that results in formation of a stop codon in its open-reading frame. Real-time RT-PCR analysis revealed that the expression of GhUGT1 is higher in the fast growth tissues, such as in fibers and roots. GhUGT2 has also higher expression in roots, but with lower expression levels in fibers and other tissues. The result also showed that the expression of GhUGT1 is higher than GhUGT2. Further study showed that GhUGT1 and GhUGT2 expressions are regulated under osmotic stress, suggesting they may be involved in plants responding to osmotic stress.     

Structural evidence of a passive base-flipping mechanism for AGT, an unusual GT-B glycosyltransferase

The Escherichia coli T4 bacteriophage uses two glycosyltransferases to glucosylate and thus protect its DNA: the retaining alpha-glucosyltransferase (AGT) and the inverting beta-glucosyltransferase (BGT). They glucosylate 5-hydroxymethyl cytosine (5-HMC) bases of duplex DNA using UDP-glucose as the sugar donor to form an alpha-glucosidic linkage and a beta-glucosidic linkage, respectively. Five structures of AGT have been determined: a binary complex with the UDP product and four ternary complexes with UDP or UDP-glucose and oligonucleotides containing an A:G, HMU:G (hydroxymethyl uracyl) or AP:G (apurinic/apyrimidinic) mismatch at the target base-pair. AGT adopts the GT-B fold, one of the two folds known for GTs. However, while the sugar donor binding mode is classical for a GT-B enzyme, the sugar acceptor binding mode is unexpected and breaks the established consensus: AGT is the first GT-B enzyme that predominantly binds both the sugar donor and acceptor to the C-terminal domain. Its active site pocket is highly similar to four retaining GT-B glycosyltransferases (trehalose-6-phosphate synthase, glycogen synthase, glycogen and maltodextrin phosphorylases) strongly suggesting a common evolutionary origin and catalytic mechanism for these enzymes. Structure-guided mutagenesis and kinetic analysis do not permit identification of a nucleophile residue responsible for a glycosyl-enzyme intermediate for the classical double displacement mechanism. Interestingly, the DNA structures reveal partially flipped-out bases. They provide evidence for a passive role of AGT in the base-flipping mechanism and for its specific recognition of the acceptor base.     

Ethylene formation in sugar beet leaves: evidence for the involvement of 3-hydroxytyramine and phenoloxidase after wounding

Ethylene production by sugar beet (Beta vulgaris L.) leaf discs is inhibited by white (or red, >610 nm) light or by wounding. In contrast, in wounded leaf discs, ethylene production is stimulated by light. The effect of light on wounded leaf discs has been studied by using an in vitro system which mimics the loss of compartmentation in the wounded leaf. Chlorophyll-free extracts from sugar beet leaves stimulate the production of the superoxide free radical ion (as a prerequisite for ethylene formation) by illuminated chloroplast lamellae. The substance from the crude leaf extracts which is active in stimulating the production of the superoxide free radical ion has been identified as 3-hydroxytyramine (dopamine). Exogenous dopamine between 5 mum and 100 mum stimulates ethylene formation by illuminated chloroplast lamellae from methional. It also stimulates the production of the superoxide free radical ion, the formation of which apparently involves both a lamellar phenoloxidase and photosynthetic electron transport as a 1-electron donor, and is cyanide-sensitive.     

Glycosyltransferase mechanisms: impact of a 5-fluoro substituent in acceptor and donor substrates on catalysis

In glycosyltransferase-catalyzed reactions a new carbohydrate-carbohydrate bond is formed between a carbohydrate acceptor and the carbohydrate moiety of either a sugar nucleotide donor or lipid-linked saccharide donor. It is currently believed that most glycosyltransferase-catalyzed reactions occur via an electrophilic activation mechanism with the formation of an oxocarbenium ion-like transition state, a hypothesis that makes clear predictions regarding the charge development on the donor (strong positive charge) and acceptor (minimal negative charge) substrates. To better understand the mechanism of these enzyme-catalyzed reactions, we have introduced a strongly electron-withdrawing group (fluorine) at C-5 of both donor and acceptor substrates in order to explore its effect on catalysis. In particular, we have investigated the effects of the 5-fluoro analogues on the kinetics of two glycosyltransferase-catalyzed reactions mediated by UDP-GlcNAc:GlcNAc-P-P-Dol N-acetylglucosaminyltransferase (chitobiosyl-P-P-lipid synthase, CLS) and beta-N-acetylglucosaminyl-beta-1,4 galactosyltransferase (GalT). The 5-fluoro group has a marked effect on catalysis when inserted into the UDP-GlcNAc donor, with the UDP(5-F)-GlcNAc serving as a competitive inhibitor of CLS rather than a substrate. The (5-F)-GlcNAc beta-octyl glycoside acceptor, however, is an excellent substrate for GalT. Both of these results support a weakly associative transition state for glycosyltransferase-catalyzed reactions that proceed with inversion of configuration.     

Protein in sugar films and in glycerol/water as examined by infrared spectroscopy and by the fluorescence and phosphorescence of tryptophan

Sugars are known to stabilize proteins. This study addresses questions of the nature of sugar and proteins incorporated in solid sugar films. Infrared (IR) and Raman spectroscopy was used to examine trehalose and sucrose films and glycerol/water solvent. Proteins and indole-containing compounds that are imbedded in the sugar films were studied by IR and optical (absorption, fluorescence, and phosphorescence) spectroscopy. Water is able to move in the sugar films in the temperature range of 20-300 K as suggested by IR absorption bands of HOH bending and OH stretching modes that shift continuously with temperature. In glycerol/water these bands reflect the glass transition at approximately 160 K. The fluorescence of N-acetyl-L-tryptophanamide and tryptophan of melittin, Ca-free parvalbumin, and staphylococcal nuclease in dry trehalose/sucrose films remains broad and red-shifted over a temperature excursion of 20-300 K. In contrast, the fluorescence of these compounds in glycerol/water solvent shift to the blue as temperature decreases. The fluorescence of the buried tryptophan in Ca-bound parvalbumin in either sugar film or glycerol/water remains blue-shifted and has vibronic resolution over the entire temperature range. The red shift for fluorescence of indole groups exposed to solvent in the sugars is consistent with the motion of water molecules around the excited-state molecule that occurs even at low temperature, although the possibility of static complex formation between the excited-state molecule and water or other factors is discussed. The phosphorescence yield for protein and model indole compounds is sensitive to the matrix glass transition. Phosphorescence emission spectra are resolved and shift little in different solvents or temperature, as predicted by the small dipole moment of the excited triplet state molecule. The conclusion is that the sugar film maintains the environment present at the glass formation temperature for surface Trp and amide groups over a wide temperature excursion. In glycerol/water these groups reflect local changes in the environment as temperature changes.     

Structures of maltohexaose and maltoheptaose bound at the donor sites of cyclodextrin glycosyltransferase give insight into the mechanisms of transglycosylation activity and cyclodextrin size specificity

The enzymes from the alpha-amylase family all share a similar alpha-retaining catalytic mechanism but can have different reaction and product specificities. One family member, cyclodextrin glycosyltransferase (CGTase), has an uncommonly high transglycosylation activity and is able to form cyclodextrins. We have determined the 2.0 and 2.5 A X-ray structures of E257A/D229A CGTase in complex with maltoheptaose and maltohexaose. Both sugars are bound at the donor subsites of the active site and the acceptor subsites are empty. These structures mimic a reaction stage in which a covalent enzyme-sugar intermediate awaits binding of an acceptor molecule. Comparison of these structures with CGTase-substrate and CGTase-product complexes reveals three different conformational states for the CGTase active site that are characterized by different orientations of the centrally located residue Tyr 195. In the maltoheptaose and maltohexaose-complexed conformation, CGTase hinders binding of an acceptor sugar at subsite +1, which suggests an induced-fit mechanism that could explain the transglycosylation activity of CGTase. In addition, the maltoheptaose and maltohexaose complexes give insight into the cyclodextrin size specificity of CGTases, since they precede alpha-cyclodextrin (six glucoses) and beta-cyclodextrin (seven glucoses) formation, respectively. Both ligands show conformational differences at specific sugar binding subsites, suggesting that these determine cyclodextrin product size specificity, which is confirmed by site-directed mutagenesis experiments.     

Crystal structures of Pasteurella multocida sialyltransferase complexes with acceptor and donor analogues reveal substrate binding sites and catalytic mechanism

Sialyltransferases are key enzymes involved in the biosynthesis of biologically and pathologically important sialic acid-containing molecules in nature. Binary X-ray crystal structures of a multifunctional Pasteurella multocida sialyltransferase (Delta24PmST1) with a donor analogue CMP-3F(a)Neu5Ac or CMP-3F(e)Neu5Ac were determined at 2.0 and 1.9 A resolutions, respectively. Ternary X-ray structures of the protein in complex with CMP or a donor analogue CMP-3F(a)Neu5Ac and an acceptor lactose have been determined at 2.0 and 2.27 A resolutions, respectively. This represents the first sialyltransferase structure and the first GT-B-type glycosyltransferase structure that is bound to both a donor analogue and an acceptor simultaneously. The four structures presented here reveal that binding of the nucleotide-activated donor sugar causes a buried tryptophan to flip out of the protein core to interact with the donor sugar and helps define the acceptor sugar binding site. Additionally, key amino acid residues involved in the catalysis have been identified. Structural and kinetic data support a direct displacement mechanism involving an oxocarbenium ion-like transition state assisted with Asp141 serving as a general base to activate the acceptor hydroxyl group.     

DJ-1 null dopaminergic neuronal cells exhibit defects in mitochondrial function and structure: involvement of mitochondrial complex I assembly

DJ-1 is a Parkinson's disease-associated gene whose protein product has a protective role in cellular homeostasis by removing cytosolic reactive oxygen species and maintaining mitochondrial function. However, it is not clear how DJ-1 regulates mitochondrial function and why mitochondrial dysfunction is induced by DJ-1 deficiency. In a previous study we showed that DJ-1 null dopaminergic neuronal cells exhibit defective mitochondrial respiratory chain complex I activity. In the present article we investigated the role of DJ-1 in complex I formation by using blue native-polyacrylamide gel electrophoresis and 2-dimensional gel analysis to assess native complex status. On the basis of these experiments, we concluded that DJ-1 null cells have a defect in the assembly of complex I. Concomitant with abnormal complex I formation, DJ-1 null cells show defective supercomplex formation. It is known that aberrant formation of the supercomplex impairs the flow of electrons through the channels between respiratory chain complexes, resulting in mitochondrial dysfunction. We took two approaches to study these mitochondrial defects. The first approach assessed the structural defect by using both confocal microscopy with MitoTracker staining and electron microscopy. The second approach assessed the functional defect by measuring ATP production, O(2) consumption, and mitochondrial membrane potential. Finally, we showed that the assembly defect as well as the structural and functional abnormalities in DJ-1 null cells could be reversed by adenovirus-mediated overexpression of DJ-1, demonstrating the specificity of DJ-1 on these mitochondrial properties. These mitochondrial defects induced by DJ-1mutation may be a pathological mechanism for the degeneration of dopaminergic neurons in Parkinson's disease.     

A UDP-glucose:isoflavone 7-O-glucosyltransferase from the roots of soybean (glycine max) seedlings. Purification, gene cloning, phylogenetics, and an implication for an alternative strategy of enzyme catalysis

Isoflavones, a class of flavonoids, play very important roles in plant-microbe interactions in certain legumes such as soybeans (Glycine max L. Merr.). G. max UDP-glucose:isoflavone 7-O-glucosyltransferase (GmIF7GT) is a key enzyme in the synthesis of isoflavone conjugates, which accumulate in large amounts in vacuoles and serve as an isoflavonoid pool that allows for interaction with microorganisms. In this study, the 14,000-fold purification of GmIF7GT from the roots of G. max seedlings was accomplished. The purified enzyme is a monomeric protein of 46 kDa, catalyzing regiospecific glucosyl transfer from UDP-glucose to isoflavones to produce isoflavone 7-O-beta-D-glucosides (k(cat) = 0.74 s(-1), K(m) for genistein = 3.6 microM, and K(m) for UDP-glucose = 190 microM). The GmIF7GT cDNA was isolated based on the amino acid sequence of the purified enzyme. Phylogenetic analysis showed that GmIF7GT is a novel member of glycosyltransferase family 1 and is distantly related to Glycyrrhiza echinata UDP-glucose:isoflavonoid 7-O-glucosyltransferase. The purified enzyme was unexpectedly devoid of the N-terminal 49-residue segment and thus lacks the histidine residue corresponding to the proposed catalytic residue of glycosyltransferases from Medicago truncatula (UGT71G1) and Vitis vinifera (VvGT1). The results of kinetic studies of site-directed mutants of GmIF7GT showed that both His-15 and Asp-125, which correspond to the catalytic residues of UGT71G1 and VvGT1, are not important for GmIF7GT activity. The results also suggest that an acidic residue at position 392 is very important for primary catalysis of GmIF7GT. These results led to the proposal that GmIF7GT utilizes a strategy of catalysis that is distinct from those proposed for UGT71G1 and VvGT1.     

Identification of residues that confer sugar selectivity to UDP-glycosyltransferase 3A (UGT3A) enzymes

Recent studies in this laboratory characterized the UGT3A family enzymes, UGT3A1 and UGT3A2, and showed that neither uses the traditional UDP-glycosyltransferase UGT co-substrate UDP-glucuronic acid. Rather, UGT3A1 uses GlcNAc as preferred sugar donor and UGT3A2 uses UDP-Glc. The enzymatic characterization of UGT3A mutants, structural modeling, and multispecies gene analysis have now been employed to identify a residue within the active site of these enzymes that confers their unique sugar preferences. An asparagine (Asn-391) in the UGT signature sequence of UGT3A1 is necessary for utilization of UDP-GlcNAc. Conversely, a phenylalanine (Phe-391) in UGT3A2 favors UDP-Glc use. Mutation of Asn-391 to Phe in UGT3A1 enhances its ability to utilize UDP-Glc and completely inhibits its ability to use UDP-GlcNAc. An analysis of homology models docked with UDP-sugar donors indicates that Asn-391 in UGT3A1 is able to accommodate the N-acetyl group on C2 of UDP-GlcNAc so that the anomeric carbon atom (C1) is optimally situated for catalysis involving His-35. Replacement of Asn with Phe at position 391 disrupts this catalytically productive orientation of UDP-GlcNAc but allows a more optimal alignment of UDP-Glc for sugar donation. Multispecies sequence analysis reveals that only primates possess UGT3A sequences containing Asn-391, suggesting that other mammals may not have the capacity to N-acetylglucosaminidate small molecules. In support of this hypothesis, Asn-391-containing UGT3A forms from two non-human primates were found to use UDP-GlcNAc, whereas UGT3A isoforms from non-primates could not use this sugar donor. This work gives new insight into the residues that confer sugar specificity to UGT family members and suggests a primate-specific innovation in glycosidation of small molecules.     

Quench-flow analysis reveals multiple phases of GluT1-mediated sugar transport

Standard models for carrier-mediated nonelectrolyte transport across cell membranes do not explain sugar uptake by human red blood cells. This means that either (1) the models for sugar transport are incorrect or (2) measurements of sugar transport are flawed. Most measurements of red cell sugar transport have been made over intervals of 10 s or greater, a range which may be too long to measure transport accurately. In the present study, we examine the time course of sugar uptake over intervals as short as 5 ms to periods as long as 8 h. Using conditions where transport by a uniform population of cells is expected to be monophasic (use of subsaturating concentrations of a nonmetabolizable but transported sugar, 3-O-methylglucose), our studies demonstrate that red cell sugar uptake is comprised of three sequential, protein-mediated events (rapid, fast, and slow). The rapid phase is more strongly temperature-dependent than the fast and slow phases. All three phases are inhibited by extracellular (maltose or phloretin) or intracellular (cytochalasin B) sugar-transport inhibitors. The rate constant for the rapid phase of uptake is independent of the 3-O-methylglucose concentration. The magnitude (moles of sugar associated with cells) of the rapid phase increases in a saturable manner with [3-O-methylglucose] and is similar to (1) the amount of sugar that is retained by red cell membrane proteins upon addition of cytochalasin B and phloretin and (2) the d-glucose inhibitable cytochalasin B binding capacity of red cell membranes. These results are consistent with the hypothesis that previous studies have both under- and overestimated the rate of erythrocyte sugar transport. These data support a transport mechanism in which newly bound sugars are transiently sequestered within the translocation pathway where they become inaccessible to extra- and intracellular water.     

Conserved domains of glycosyltransferases.

Glycosyltransferases catalyze the synthesis of glycoconjugates by transferring a properly activated sugar residue to an appropriate acceptor molecule or aglycone for chain initiation and elongation. The acceptor can be a lipid, a protein, a heterocyclic compound, or another carbohydrate residue. A catalytic reaction is believed to involve the recognition of both the donor and acceptor by suitable domains, as well as the catalytic site of the enzyme. To elucidate the structural requirements for substrate recognition and catalytic reactions of glycosyltransferases, we have searched the databases for homologous sequences, identified conserved amino acid residues, and proposed potential domain motifs for these enzymes. Depending on the configuration of the anomeric functional group of the glycosyl donor molecule and of the resulting glycoconjugate, all known glycosyltransferases can be divided into two major types: retaining glycosyltransferases, which transfer sugar residue with the retention of anomeric configuration, and inverting glycosyltransferases, which transfer sugar residue with the inversion of anomeric configuration. One conserved domain of the inverting glycosyltransferases identified in the database is responsible for the recognition of a pyrimidine nucleotide, which is either the UDP or the TDP portion of a donor sugar-nucleotide molecule. This domain is termed "Nucleotide Recognition Domain 1 beta," or NRD1 beta, since the type of nucleotide is the only common structure among the sugar donors and acceptors. NRD1 beta is present in 140 glycosyltransferases. The central portion of the NRD1 beta domain is very similar to the domain that is present in one family of retaining glycosyltransferases. This family is termed NRD1 alpha to designate the similarity and stereochemistry of sugar transfer, and it consists of 77 glycosyltransferases identified thus far. In the central portion there is a homologous region for these two families and this region probably has a catalytic function. A third conserved domain is found exclusively in membrane-bound glycosyltransferases and is termed NRD2; this domain is present in 98 glycosyltransferases. All three identified NRDs are present in archaebacterial, eubacterial, viral, and eukaryotic glycosyltransferases. The present article presents the alignment of conserved NRD domains and also presents a brief overview of the analyzed glycosyltransferases which comprise about 65% of all known sugar-nucleotide dependent (Leloir-type) and putative glycosyltransferases in different databases. A potential mechanism for the catalytic reaction is also proposed. This proposed mechanism should facilitate the design of experiments to elucidate the regulatory mechanisms of glycosylation reactions. Amino acid sequence information within the conserved domain may be utilized to design degenerate primers for identifying DNA encoding new glycosyltransferases.     

High Resolution Structures of the Human ABO(H) Blood Group Enzymes in Complex with Donor Analogs Reveal That the Enzymes Utilize Multiple Donor Conformations to Bind Substrates in a Stepwise Manner

Homologous glycosyltransferases α-(1→3)-N-acetylgalactosaminyltransferase (GTA) and α-(1→3)-galactosyltransferase (GTB) catalyze the final step in ABO(H) blood group A and B antigen synthesis through sugar transfer from activated donor to the H antigen acceptor. These enzymes have a GT-A fold type with characteristic mobile polypeptide loops that cover the active site upon substrate binding and, despite intense investigation, many aspects of substrate specificity and catalysis remain unclear. The structures of GTA, GTB, and their chimeras have been determined to between 1.55 and 1.39 Å resolution in complex with natural donors UDP-Gal, UDP-Glc and, in an attempt to overcome one of the common problems associated with three-dimensional studies, the non-hydrolyzable donor analog UDP-phosphono-galactose (UDP-C-Gal). Whereas the uracil moieties of the donors are observed to maintain a constant location, the sugar moieties lie in four distinct conformations, varying from extended to the “tucked under” conformation associated with catalysis, each stabilized by different hydrogen bonding partners with the enzyme. Further, several structures show clear evidence that the donor sugar is disordered over two of the observed conformations and so provide evidence for stepwise insertion into the active site. Although the natural donors can both assume the tucked under conformation in complex with enzyme, UDP-C-Gal cannot. Whereas UDP-C-Gal was designed to be “isosteric” with natural donor, the small differences in structure imposed by changing the epimeric oxygen atom to carbon appear to render the enzyme incapable of binding the analog in the active conformation and so preclude its use as a substrate mimic in GTA and GTB.     

Complement receptor 1 and malaria

Plasmodium falciparum malaria is an intracellular parasite that is transmitted by Anopheles mosquitoes. It is responsible for approximately 1 million deaths per year. Most deaths occur as a result of complications such as severe anaemia or cerebral malaria (coma). The complement receptor 1 is a key complement regulator found on the surface of red cells and most leucocytes. A growing body of evidence suggests that this molecule plays a critical role in the pathogenesis of P. falciparum malaria. Initial reports showed that CR1 enables the binding of infected red cells to uninfected red cells to form rosettes, which can potentially obstruct small capillaries. However, further evidence suggests that CR1 is also important in the control of complement activation and immune complex formation during malaria infection. Most recently, CR1 has also been shown to be a receptor for the invasion of red cells by the parasite. Its polymorphic nature almost certainly has allowed the selection of variants that have helped humankind survive the scurge of malaria. The finding of conflicting reports about the exact role of these variants in severe disease underlies the complexity of the parasite-host interactions and highlights the need for further studies.     

TIN2 binds TRF1 and TRF2 simultaneously and stabilizes the TRF2 complex on telomeres

Human telomeres contain two related telomeric DNA-binding proteins, TRF1 and TRF2. The TRF1 complex contains the TRF1 interacting partner, TIN2, as well as PIP1 and POT1 and regulates telomere-length homeostasis. The TRF2 complex is primarily involved in telomere protection and contains the TRF2 interacting partner human (h)Rap1 as well as several factors involved in the DNA damage response. A prior report showed that conditional deletion of murine TRF1 reduced the presence of TRF2 on telomeres. Here we showed that TRF2 is also lost from human telomeres upon TRF1 depletion with small interfering RNA prompting a search for the connection between the TRF1 and TRF2 complexes. Using mass spectrometry and co-immunoprecipitation, we found that TRF1, TIN2, PIP1, and POT1 are associated with the TRF2-hRap1 complex. Gel filtration identified a TRF2 complex containing TIN2 and POT1 but not TRF1 indicating that TRF1 is not required for this interaction. Co-immunoprecipitation, Far-Western assays, and two-hybrid assays showed that TIN2, but not POT1 or PIP1, interacts directly with TRF2. Furthermore, TIN2 was found to bind TRF1 and TRF2 simultaneously, showing that TIN2 can link these telomeric proteins. This connection appeared to stabilize TRF2 on the telomeres as the treatment of cells with TIN2 small interfering RNA resulted in a decreased presence of TRF2 and hRap1 at chromosome ends. The TIN2-mediated cooperative binding of TRF1 and TRF2 to telomeres has important implications for the mechanism of telomere length regulation and protection.     

Structural analysis of sialyltransferase PM0188 from Pasteurella multocida complexed with donor analogue and acceptor sugar

PM0188 is a newly identified sialyltransferase from P. multocida which transfers sialic acid from cytidine 5'-monophosphonuraminic acid (CMP-NeuAc) to an acceptor sugar. Although sialyltransferases are involved in important biological functions like cell-cell recognition, cell differentiation and receptor-ligand interactions, little is known about their catalytic mechanism. Here, we report the X-ray crystal structures of PM0188 in the presence of an acceptor sugar and a donor sugar analogue, revealing the precise mechanism of sialic acid transfer. Site-directed mutagenesis, kinetic assays, and structural analysis show that Asp141, His311, Glu338, Ser355 and Ser356 are important catalytic residues; Asp141 is especially crucial as it acts as a general base. These complex structures provide insights into the mechanism of sialyltransferases and the structure-based design of specific inhibitors.     

Phosphate-guanosine interactions. A model for the involvement of guanine derivatives in autocatalytic reactions of ribonucleic acids

Proton magnetic resonance was used to study the interactions between nucleosides and phosphate monoanion in dimethyl sulfoxide. Ribose was able to form two mutually exclusive 1:1 complexes involving either OH3' and OH5' or OH3' and OH2' as hydrogen bond donor groups. Deoxyribose could form only one of these complexes. A specific interaction of phosphate with the base moiety of nucleosides was observed only with guanosine. A 1:1 complex was formed involving the N(1)H and NH2(2) of guanine. Association constants for both the base and sugar complexes were determined to be in the range 50-60 M-1 at 21 degrees C in dimethyl sulfoxide. This value is more than 1 order of magnitude higher than that measured for guanine-cytosine base pair formation under the same conditions. Water addition to dimethyl sulfoxide led to a decrease of all association constants but the guanine-phosphate "pair" remained more stable than the guanine-cytosine base pair.     

Dynamics and equilibrium for the formation of fluorescent Lewis acid-base exciplexes and triplexes between 9-aminophenanthrene and aliphatic amines.

The excited singlet states of 9-aminophenanthrene and its N-aminoalkyl derivatives are strongly fluorescent in cyclohexane. Addition of low concentrations of Et(3)N, Pr(2)NH, or PrNH(2) results in a red shift of the emission maximum and moderately decreased fluorescence intensity. Analysis of the fluorescence behavior using a combination of singular value decomposition with self-modeling and kinetic analysis provides evidence for the sequential formation of 1 : 1 (exciplex) and 1 : 2 (triplex) complexes between the excited 9-aminophenanthrene and ground-state alkylamine, both of which are strongly fluorescent. Both the formation and decay of the exciplex and triplex are dependent upon the extent of amine N-alkylation. Rate constants and equilibrium constants for complex formation follow the order 1 degree approximately 2 degree > 3 degree, analogous to that for the formation of ground-state complexes between amines and the soft Lewis base HgBr(2). Similarly, N-aminoalkyl derivatives of 9-aminophenanthrene form intramolecular exciplexes. Excited-state complex formation is attributed to a Lewis acid-base interaction between the excited aminophenanthrene (lone-pair acceptor) and ground-state amine (lone-pair donor). The factors which determine the stability of excited-state Lewis acid-base complexes are characteristic of the specific excited-state acceptor. No universal scale of lone-pair donor strength can be expected to describe the formation of such complexes.     

Structure of the Escherichia coli ThiS-ThiF complex, a key component of the sulfur transfer system in thiamin biosynthesis

We have determined the crystal structure of the Escherichia coli ThiS-ThiF protein complex at 2.0 A resolution. ThiS and ThiF are bacterial proteins involved in the synthesis of the thiazole moiety of thiamin. ThiF catalyzes the adenylation of the carboxy terminus of ThiS and the subsequent displacement of AMP catalyzed by ThiI-persulfide to give a ThiS-ThiI acyl disulfide. Disulfide interchange, involving Cys184 on ThiF, then generates the ThiS-ThiF acyl disulfide, which functions as the sulfur donor for thiazole formation. ThiS is a small 7.2 kDa protein that structurally resembles ubiquitin and the molybdopterin biosynthetic protein MoaD. ThiF is a 27 kDa protein with distinct sequence and structural similarity to the ubiquitin activating enzyme E1 and the molybdopterin biosynthetic protein MoeB. The ThiF-ThiS structure clarifies the mechanism of the sulfur transfer chemistry involved in thiazole biosynthesis.