99-20-7Relevant articles and documents
Deoxyfluoro-d-trehalose (FDTre) analogues as potential PET probes for imaging mycobacterial infection
Rundell, Sarah R.,Wagar, Zachary L.,Meints, Lisa M.,Olson, Claire D.,O'Neill, Mara K.,Piligian, Brent F.,Poston, Anne W.,Hood, Robin J.,Woodruff, Peter J.,Swarts, Benjamin M.
, p. 8598 - 8609 (2016)
Mycobacterium tuberculosis, the etiological agent of human tuberculosis, requires the non-mammalian disaccharide trehalose for growth and virulence. Recently, detectable trehalose analogues have gained attention as probes for studying trehalose metabolism and as potential diagnostic imaging agents for mycobacterial infections. Of particular interest are deoxy-[18F]fluoro-d-trehalose (18F-FDTre) analogues, which have been suggested as possible positron emission tomography (PET) probes for in vivo imaging of M. tuberculosis infection. Here, we report progress toward this objective, including the synthesis and conformational analysis of four non-radioactive deoxy-[19F]fluoro-d-trehalose (19F-FDTre) analogues, as well as evaluation of their uptake by M. smegmatis. The rapid synthesis and purification of several 19F-FDTre analogues was accomplished in high yield using a one-step chemoenzymatic method. Conformational analysis of the 19F-FDTre analogues using NMR and molecular modeling methods showed that fluorine substitution had a negligible effect on the conformation of the native disaccharide, suggesting that fluorinated analogues may be successfully recognized and processed by trehalose metabolic machinery in mycobacteria. To test this hypothesis and to evaluate a possible route for delivery of FDTre probes specifically to mycobacteria, we showed that 19F-FDTre analogues are actively imported into M. smegmatis via the trehalose-specific transporter SugABC-LpqY. Finally, to demonstrate the applicability of these results to the efficient preparation and use of short-lived 18F-FDTre PET radiotracers, we carried out 19F-FDTre synthesis, purification, and administration to M. smegmatis in 1 hour.
Chemoenzymatic synthesis of trehalose analogues: Rapid access to chemical probes for investigating mycobacteria
Urbanek, Bailey L.,Wing, Douglas C.,Haislop, Krystal S.,Hamel, Chelsey J.,Kalscheuer, Rainer,Woodruff, Peter J.,Swarts, Benjamin M.
, p. 2066 - 2070 (2014)
Trehalose analogues are emerging as valuable tools for investigating Mycobacterium tuberculosis, but progress in this area is slow due to the difficulty in synthesizing these compounds. Here, we report a chemoenzymatic synthesis of trehalose analogues that employs the heat-stable enzyme trehalose synthase (TreT) from the hyperthermophile Thermoproteus tenax. By using TreT, various trehalose analogues were prepared quickly (1 h) in high yield (up to >99 % by HPLC) in a single step from readily available glucose analogues. To demonstrate the utility of this method in mycobacteria research, we performed a simple "one-pot metabolic labeling" experiment that accomplished probe synthesis, metabolic labeling, and imaging of M. smegmatis in a single day with only TreT and commercially available materials. Trehalose tools for TB: A one-step chemoenzymatic method for the rapid and efficient synthesis of trehalose analogues was developed. This method enabled facile preparation and administration of a trehalose-based probe for detecting mycobacteria, which might enable the development of new diagnostic tools for tuberculosis (TB) research.
Construction of a recombinant thermostable β-amylase-trehalose synthase bifunctional enzyme for facilitating the conversion of starch to trehalose
Wang, Jia-Hung,Tsai, Meng-Yin,Lee, Guan-Chiun,Shaw, Jei-Fu
, p. 1256 - 1263 (2007)
A fusion gene that encoded a polypeptide of 1495 amino acids was constructed from the β-amylase (BA) gene of Clostridium thermosulfurogenes and trehalose synthase (TS) gene of Thermus thermophilus. The fused gene was overexpressed in Escherichia coli, and a recombinant bifunctional fusion protein with BA at the N-terminal (BATS) or C-terminal (TSBA) of TS having both β-amylase and trehalose synthase activities with an apparent molecular mass of 164 kDa was obtained. BATS or TSBA catalyzes the sequential reaction in which maltose is formed from starch and then is converted into trehalose. The Km values of the BATS and TSBA fusion enzymes for the reaction from starch to trehalose were smaller than those of an equimolar mixture of BA and TS (BA/TS). On the other hand, the kcat value of BATS approximated that of the BA/TS mixture, but that of TSBA exceeded it. TSBA showed much higher sequential catalytic efficiency than the separately expressed BA/TS mixture. The catalytic efficiency of TSBA or BATS was 3.4 or 2.4 times higher, respectively, than that of a mixture of individual enzymes, showing the kinetic advantage of the fusion enzyme. The thermal stability readings of the recombinant fusion enzymes BATS and TSBA were better than that of the mixture of individual recombinant enzymes. These results apparently demonstrate that fusion enzymes catalyzing sequential reactions have kinetic advantages over a mixture of both enzymes.
Anomeric Selectivity of Trehalose Transferase with Rare l -Sugars
Bento, Isabel,Hagedoorn, Peter-Leon,Hanefeld, Ulf,Jeffries, Cy M.,Laustsen, Jesper U.,Marsden, Stefan R.,Mestrom, Luuk,Svergun, Dmitri I.,Van Der Eijk, Hessel
, p. 8835 - 8839 (2020/09/18)
Retaining LeLoir glycosyltransferases catalyze the formation of glycosidic bonds between nucleotide sugar donors and carbohydrate acceptors. The anomeric selectivity of trehalose transferase from Thermoproteus uzoniensis was investigated for both d- and l-glycopyranose acceptors. The enzyme couples a wide range of carbohydrates, yielding trehalose analogues with conversion and enantioselectivity of >98%. The anomeric selectivity inverts from α,α-(1 → 1)-glycosidic bonds for d-glycopyranose acceptors to α,β-(1 → 1)-glycosidic bonds for l-glycopyranose acceptors, while (S)-selectivity was retained for both types of sugar acceptors. Comparison of protein crystal structures of trehalose transferase in complex with α,α-trehalose and an unnatural α,β-trehalose analogue highlighted the mechanistic rationale for the observed inversion of anomeric selectivity.
Structures of trehalose-6-phosphate phosphatase from pathogenic fungi reveal the mechanisms of substrate recognition and catalysis
Miaoa, Yi,Tenorb, Jennifer L.,Toffalettib, Dena L.,Washingtona, Erica J.,Liuc, Jiuyu,Shadrickc, William R.,Schumachera, Maria A.,Leec, Richard E.,Perfectb, John R.,Brennana, Richard G.
, p. 7148 - 7153 (2016/07/07)
Trehalose is a disaccharide essential for the survival and virulence of pathogenic fungi. The biosynthesis of trehalose requires trehalose-6-phosphate synthase, Tps1, and trehalose-6-phosphate phosphatase, Tps2. Here, we report the structures of the N-terminal domain of Tps2 (Tps2NTD) from Candida albicans, a transition-state complex of the Tps2 C-terminal trehalose-6-phosphate phosphatase domain (Tps2PD) bound to BeF3 and trehalose, and catalytically dead Tps2PD(D24N) from Cryptococcus neoformans bound to trehalose-6-phosphate (T6P). The Tps2NTD closely resembles the structure of Tps1 but lacks any catalytic activity. The Tps2PD-BeF3 -trehalose and Tps2PD(D24N)-T6P complex structures reveal a "closed" conformation that is effected by extensive interactions between each trehalose hydroxyl group and residues of the cap and core domains of the protein, thereby providing exquisite substrate specificity. Disruption of any of the direct substrate-protein residue interactions leads to significant or complete loss of phosphatase activity. Notably, the Tps2PD-BeF3 -trehalose complex structure captures an aspartyl-BeF3 covalent adduct, which closely mimics the proposed aspartyl-phosphate intermediate of the phosphatase catalytic cycle. Structures of substrate-free Tps2PD reveal an "open" conformation whereby the cap and core domains separate and visualize the striking conformational changes effected by substrate binding and product release and the role of two hinge regions centered at approximately residues 102-103 and 184-188. Significantly, tps2Δ, tps2NTDAΔ, and tps2D705N strains are unable to grow at elevated temperatures. Combined, these studies provide a deeper understanding of the substrate recognition and catalytic mechanism of Tps2 and provide a structural basis for the future design of novel antifungal compounds against a target found in three major fungal pathogens.
