ABSTRACT
UDP-galactofuranose (UDP-Galf) is a substrate for two types of enzymes, UDP-galactopyranose mutase and galactofuranosyltransferases, which are present in many pathogenic organisms but absent from mammals. In particular, these enzymes are involved in the biosynthesis of cell wall galactan, a polymer essential for the survival of the causative agent of tuberculosis, Mycobacterium tuberculosis. We describe here the synthesis of derivatives of UDP-Galf modified at C-5 and C-6 using a chemoenzymatic route. In cell-free assays, these compounds prevented the formation of mycobacterial galactan, via the production of short "dead-end" intermediates resulting from their incorporation into the growing oligosaccharide chain. Modified UDP-furanoses thus constitute novel probes for the study of the two classes of enzymes involved in mycobacterial galactan assembly, and studies with these compounds may ultimately facilitate the future development of new therapeutic agents against tuberculosis.
Subject(s)
Antitubercular Agents/chemistry , Enzyme Inhibitors/chemistry , Galactans/biosynthesis , Galactose/analogs & derivatives , Galactosyltransferases/antagonists & inhibitors , Uridine Diphosphate/analogs & derivatives , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/pharmacology , Galactans/antagonists & inhibitors , Galactose/biosynthesis , Galactose/chemistry , Galactose/pharmacology , Galactosyltransferases/genetics , Galactosyltransferases/metabolism , Intramolecular Transferases/antagonists & inhibitors , Intramolecular Transferases/metabolism , Klebsiella pneumoniae/enzymology , Mycobacterium smegmatis/enzymology , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Uridine Diphosphate/biosynthesis , Uridine Diphosphate/chemistry , Uridine Diphosphate/pharmacologyABSTRACT
UDP-galactopyranose mutase (UGM) catalyzes the isomerization of UDP-galactopyranose (UDP-Galp) into UDP-galactofuranose (UDP-Galf), an essential step of the mycobacterial cell wall biosynthesis. UDP-(6-deoxy-6-fluoro)-D-galactofuranose 1 was tested as substrate of UGM. Turnover could be observed by HPLC. The k(cat) (7.4s(-1)) and the K(m) (24 mM) of 1 were thus measured and compared with those of UDP-Galf and other fluorinated analogs. The presence of the fluorine atom at the 6-position had a moderate effect on the rate of the reaction but a huge one on the interactions between the enzyme and its substrate. This result demonstrated that key interactions occur at the vicinity of the 6-position of UDP-galactose in the Michaelis complex.
Subject(s)
Galactose/analogs & derivatives , Intramolecular Transferases/chemistry , Uridine Diphosphate/analogs & derivatives , Carbohydrates/chemistry , Catalysis , Cell Wall/metabolism , Chemistry, Pharmaceutical/methods , Chromatography, High Pressure Liquid , Drug Design , Fluorine/chemistry , Furans/chemistry , Galactose/chemistry , Kinetics , Molecular Conformation , Mycobacterium/metabolism , Protein Binding , Uridine Diphosphate/chemistryABSTRACT
Nature's glycosylation catalysts, glycosyltransferases, indirectly manipulate and control many important biological processes by transferring sugar nucleotide donors onto acceptors. Challenging chemical synthesis impedes synthetic access to sugar nucleotides and limits the study of many glycosyltransferases. Enzymatic access to sugar nucleotides is a rapidly expanding avenue of research, limited only by the substrate specificity of the enzyme. We have explored the promiscuous thymidylyltransferase from Streptococcus pneumoniae, Cps2L, and enhanced its uridylyltransferase and guanidyltransferase activities by active site engineering. Mutagenesis at position Q24 resulted in a variant with 10-, 3-, and 2-fold enhancement of UDP-glucosamine, UDP-mannose, and UDP- N-acetylglucosamine production, respectively. New catalytic activities were observed for the Cps2L variant over the wild-type enzyme, including the formation of GDP-mannose. The variant was evaluated as a catalyst for the formation of a series of dTDP- and UDP-furanoses and notably produced dTDP-Gal f in 90% yield and UDP-Ara f in 30% yield after 12 h. A series of 3- O-alkylglucose 1-phosphates were also evaluated as substrates, and notable conversions to UDP-3- O-methylglucose and UDP-3- O-dodecylglucose were achieved with the variant but not the wild-type enzyme. The Q24S variant also enhanced essentially all thymidylyltransferase activities relative to the wild-type enzyme. Comparison of active sites of uridylyltransferases and thymidylyltransferases with products bound indicate the Q24S variant to be a new approach in broadening nucleotidylyltransferase activity.
Subject(s)
Ribonucleosides/metabolism , Transferases/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Binding Sites , Crystallography , Models, Molecular , Molecular Structure , Mutagenesis , Protein Engineering , Ribonucleosides/chemistry , Ribonucleosides/genetics , Streptococcus pneumoniae/enzymology , Substrate Specificity , Transferases/chemistry , Transferases/geneticsABSTRACT
Hexofuranosides are widely spread in nature, and notably in numerous pathogenic microorganisms. This particular five-membered ring for hexosides leads to novel biological properties and, as usual in glycochemistry, to completely different reactivity and selectivity. Far from being exhaustive, this review will first focus on the structure of the oligosaccharidic part of hexofuranosyl conjugates found in natural sources. Original syntheses will then be presented, stressing more particularly on the development of chemical and chemo-enzymatic tools for the access to 1,2-trans or 1,2-cis linkages. Finally, innovative applications related to biological, chemical and physicochemical fields for both natural and synthetic hexofuranosyl compounds will be described.
Subject(s)
Furans/chemistry , Glycosides/chemistry , Hexoses/chemistry , Animals , Anti-Bacterial Agents/chemical synthesis , Antineoplastic Agents/chemistry , Archaea/chemistry , Bacteria/chemistry , Carbohydrate Conformation , Carbohydrate Sequence , Eukaryota/chemistry , Fungi/chemistry , Glycoside Hydrolases/antagonists & inhibitors , Intramolecular Transferases/antagonists & inhibitors , Molecular Sequence Data , Mycobacterium tuberculosis/chemistry , Oligosaccharides/chemical synthesis , Oligosaccharides/chemistry , Plants/chemistry , Porifera/chemistry , Starfish/chemistry , Thioglycosides/chemistry , Thioglycosides/pharmacologyABSTRACT
In the title compound, C(12)H(15)NO(7)·0.5H(2)O, there are two independent mol-ecules in the asymmetric unit, together with one water molecule. The pyran-oside rings each have close to a (1)C(4) chair conformation and the nitro groups are almost coplanar with the benzene rings. The water mol-ecule links the two independent mol-ecules through O-Hâ¯O hydrogen bonds. All the hydroxyl groups are involved in hydrogen-bond inter-actions, giving rise to a three-dimensional network.
ABSTRACT
A bacterial alpha-d-glucopyranosyl-1-phosphate thymidylyltransferase was found to couple four hexofuranosyl-1-phosphates, as well as a pentofuranosyl-1-phosphate, with deoxythymidine 5'-triphosphate, providing access to furanosyl nucleotides. The enzymatic reaction mixtures were analyzed by electrospray ionization mass spectrometry and NMR spectroscopy to determine the anomeric stereochemistry of furanosyl nucleotide products. This is the first demonstration of a nucleotidylyltransferase discriminating between diastereomeric mixtures of sugar-1-phosphates to produce stereopure, biologically relevant furanosyl nucleotides.
Subject(s)
Nucleotides/chemical synthesis , Nucleotidyltransferases/metabolism , Catalysis , Molecular Structure , Nucleotides/chemistry , Stereoisomerism , Substrate SpecificityABSTRACT
Direct activation of unprotected thioimidoyl furanosides yielded in only one step and few minutes a panel of rare uridine 5'-diphospho-furanoses. Diastereoselectivity of the reaction was tightly connected with reaction time, temperature, and nature of the furanosyl donor. This approach was totally selective since no ring expansion from the initial five-membered ring to the more stable pyranose form was observed.
