排序方式: 共有9条查询结果,搜索用时 15 毫秒
1
1.
An in situ transglycosylase assay has been developed using endogenously synthesized lipid II. The assay involves the preferential synthesis and accumulation of lipid II in a reaction mixture containing the cell wall membrane material isolated from Escherichia coli, exogenously supplied UDP-MurNAc-pentapeptide, and radiolabeled UDP-GlcNAc. In the presence of Triton X-100, the radiolabeled product formed is almost exclusively lipid II, while the subsequent formation of peptidoglycan is inhibited. Removal of the detergent resulted in the synthesis of peptidoglycan (25% incorporation of radiolabeled material) from the accumulated lipid II. This reaction was inhibited by moenomycin, a known transglycosylase inhibitor. In addition, tunicamycin, which affects an earlier step of the pathway by inhibiting MraY, had no effect on the formation of peptidoglycan in this assay, as expected. Similarly, ampicillin and bacitracin did not inhibit the formation of peptidoglycan under the conditions established. 相似文献
2.
Johan Larsbrink Atsushi Izumi Glyn R. Hemsworth Gideon J. Davies Harry Brumer 《The Journal of biological chemistry》2012,287(52):43288-43299
The metabolism of the storage polysaccharides glycogen and starch is of vital importance to organisms from all domains of life. In bacteria, utilization of these α-glucans requires the concerted action of a variety of enzymes, including glycoside hydrolases, glycoside phosphorylases, and transglycosylases. In particular, transglycosylases from glycoside hydrolase family 13 (GH13) and GH77 play well established roles in α-glucan side chain (de)branching, regulation of oligo- and polysaccharide chain length, and formation of cyclic dextrans. Here, we present the biochemical and tertiary structural characterization of a new type of bacterial 1,4-α-glucan 4-α-glucosyltransferase from GH31. Distinct from 1,4-α-glucan 6-α-glucosyltransferases (EC 2.4.1.24) and 4-α-glucanotransferases (EC 2.4.1.25), this enzyme strictly transferred one glucosyl residue from α(1→4)-glucans in disproportionation reactions. Substrate hydrolysis was undetectable for a series of malto-oligosaccharides except maltose for which transglycosylation nonetheless dominated across a range of substrate concentrations. Crystallographic analysis of the enzyme in free, acarbose-complexed, and trapped 5-fluoro-β-glucosyl-enzyme intermediate forms revealed extended substrate interactions across one negative and up to three positive subsites, thus providing structural rationalization for the unique, single monosaccharide transferase activity of the enzyme. 相似文献
3.
Particulate membrane preparations isolated from cambial cells and differentiating and differentiated xylem cells of pine (Pinus sylvestris L.) trees synthesised [14C]glucans using either guanosine 5-diphosphate (GDP)-D-[U-14C]glucose or uridine 5-diphosphate (UDP)-D-[U-14C]glucose as glycosyl donors. Although these glucans had -(13) and -(14) linkages in an approximate ratio 1:1, the distribution of the linkages in the glucan synthesised from GDP-D-glucose was different from that synthesised from UDP-D-glucose. The synthesis of the mixed -(13) and -(14) glucan from GDP-D-[U-14C]glucose was changed to that of -(14) glucomannan in the presence of increasing concentrations of GDP-D-mannose. The glucan formed from UDP-D-[U-14C]glucose was not affected by any concentration of GDP-D-mannose. The membrane preparations epimerized GDP-D-glucose to GDP-D-mannose; however, the low amount of GDP-D-mannose formed was not incorporated into the polymer becaus the affinity of the synthase for GDP-D-glucose was much greater than that for GDP-D-mannose. The glucan formed from GDP-D-glucose and the glucomannan formed from GDP-D-glucose together with GDP-D-mannose were characterized. The apparent K
m and V
max of the glucan synthase for GDP-D-glucose were 6.38 M and 5.08 M·min-1, respectively. No lipid intermediates were detected during the synthesis of either glucan or glucomannan. The results indicated that an enzyme complex for the formation of the glucomannan was bound to the membrane.Abbreviations GDP
guanosine 5-diphosphate
- GLC
gasliquid chromatography
- UDP
trridine 5-diphosphate 相似文献
4.
The Essential Protein for Bacterial Flagella Formation FlgJ Functions as a β-N-Acetylglucosaminidase
Francesca A. Herlihey Patrick J. Moynihan Anthony J. Clarke 《The Journal of biological chemistry》2014,289(45):31029-31042
The flagellum is a major virulence factor of motile pathogenic bacteria. This structure requires more than 50 proteins for its biogenesis and function, one of which is FlgJ. Homologs of FlgJ produced by the β- and γ-proteobacteria, such as Salmonella enterica, Vibrio spp., and both Sphingomonas sp. and Pseudomonas spp. are bifunctional, possessing an N-terminal domain responsible for proper rod assembly and a C-terminal domain possessing peptidoglycan lytic activity. Despite the amount of research conducted on FlgJ from these and other bacteria over the past 15 years, no biochemical analysis had been conducted on any FlgJ and consequently confusion exists as to whether the enzyme is a peptidoglycan hydrolase or a lytic transglycosylase. In this study, we present the development of a novel assay for glycoside lytic enzymes and its use to provide the first enzymatic characterization of the lytic domain of FlgJ from S. enterica as the model enzyme. Surprisingly, FlgJ functions as neither a muramidase nor a lytic transglycosylases but rather as a β-N-acetylglucosaminidase. As such, FlgJ represents the first autolysin with this activity to be characterized from a Gram-negative bacterium. At its optimal pH of 4.0, the Michaelis-Menten parameters of Km and kcat for FlgJ from S. enterica were determined to be 0.64 ± 0.18 mg ml−1 and 0.13 ± 0.016 s−1, respectively, using purified PG as substrate. Its catalytic residues were identified as Glu184 and Glu223. 相似文献
5.
Mannan transglycosylases are cell wall enzymes able to transfer part of the mannan polysaccharide backbone to mannan-derived oligosaccharides (Schröder et al. in Planta 219:590–600, 2004). Mannan transglycosylase activity was purified to near homogeneity from ripe tomato fruit. N-terminal sequencing showed that the dominant band seen on SDS-PAGE was identical to LeMAN4a, a hydrolytic endo-β-mannanase found in ripe tomato fruit (Bewley et al. in J Exp Bot 51:529–538, 2000). Recombinant LeMAN4a protein expressed in Escherichia coli exhibited both mannan hydrolase and mannan transglycosylase activity. Western analysis of ripe tomato fruit tissue using an antibody raised against tomato seed endo-β-mannanase revealed four isoforms present after 2D-gel electrophoresis in the pH range 6–11. On separation by preparative liquid isoelectric focussing, these native isoforms exhibited different preferences for transglycosylation and hydrolysis. These results demonstrate that endo-β-mannanase has two activities: it can either hydrolyse mannan polysaccharides, or in the presence of mannan-derived oligosaccharides, carry out a transglycosylation reaction. We therefore propose that endo-β-mannanase should be renamed mannan transglycosylase/hydrolase, in accordance with the nomenclature established for xyloglucan endotransglucosylase/hydrolase. The role of endo-acting mannanases in modifying the structure of plant cell walls during cell expansion, seed germination and fruit ripening may need to be reinterpreted in light of their potential action as transglycosylating or hydrolysing enzymes. 相似文献
6.
Mannan transglycosylase is a novel cell wall enzyme activity acting on mannan-based plant polysaccharides in primary cell walls of monocotyledons and dicotyledons. The enzyme activity was detected by its ability to transfer galactoglucomannan (GGM) polysaccharides to tritium-labelled GGM-derived oligosaccharides generating tritium-labelled GGM polysaccharides. Mannan transglycosylase was found in a range of plant species and tissues. High levels of the enzyme activity were present in flowers of some kiwifruit (Actinidia) species and in ripe tomato (Solanum lycopersicum L.) fruit. Low levels were detected in mature green tomato fruit and activity increased during tomato fruit ripening up to the red ripe stage. Essentially all activity was found in the tomato skin and outermost 2 mm of tissue. Mannan transglycosylase activity in tomato skin and outer pericarp is specific for mannan-based plant polysaccharides, including GGM, galactomannan, glucomannan and mannan. The exact structural requirements for valid acceptors remain to be defined. Nevertheless, a mannose residue at the second position of the sugar chain and the absence of a galactose substituent on the fourth residue (counting from the non-reducing end) appear to be minimal requirements. Mannan-based polysaccharides in the plant cell wall may have a role analogous to that of xyloglucans, introducing flexibility and forming growth-restraining networks with cellulose. Thus mannan transglycosylase and xyloglucan endotransglycosylase, the only other known transglycosylase activity in plant cell walls, may both be involved in remodelling and refining the cellulose framework in developmental processes throughout the life of a plant.Abbreviations EBM Endo--mannanase - GGM galactoglucomannan - GGMO Galactoglucomannan-derived oligosaccharide - G2M5 Di-galactosyl mannopentaitol - M2–M5 Mannobiitol to mannopentaitol oligosaccharides - SK+OP Skin plus outer pericarp - XET Xyloglucan endotransglucosylase - XG Xyloglucan 相似文献
7.
Satoru Suzuki Tatsuo Fukagawa 《Journal of industrial microbiology & biotechnology》1995,14(2):154-158
Summary A tributyltin chloride (TBTCl)-resistant bacterium,Alteromonas sp. M-1, was isolated from coastal seawater. This bacterium grew in medium containing 125 M TBTCl. TBTCl added to the medium was taken up by this bacterium, however, the amount of TBTCl in the cellular fraction was low after the logarithmic phase, suggesting the existence of a TBTCl-efflux system. A genetic library was constructed using plasmid vector pUC 19. Three positive clones were obtained, by whichE. coli was transformed to TBTCl resistance. Of the three clones, the shortest fragment fromHindIII-library was analyzed. This fragment was 1.8 kb long and contained one complete open reading frame. The predicted amino acid sequence of this open reading frame had a homologous domain to transglycosylases of bacteriophage andE. coli. TBTCl-tolerant marine bacteria other thanAlteromonas sp. M-1 were obtained from natural seawater to which TBTCl was added. DNA-DNA hybridization was performed between the three cloned fragments fromAlteromonas sp. M-1 and chromosomal DNA of the TBTCl-tolerant bacteria. Some strains hybridized with the fragments and some did not, suggesting that several genes are responsible for TBTCl tolerance. 相似文献
8.
Xiaolei Wang Larissa Krasnova Kevin Binchia Wu Wei-Shen Wu Ting-Jen Cheng Chi-Huey Wong 《Bioorganic & medicinal chemistry letters》2018,28(16):2708-2712
Described here is the asymmetric synthesis of iminosugar 2b, a Lipid II analog, designed to mimic the transition state of transglycosylation catalyzed by the bacterial transglycosylase. The high density of functional groups, together with a rich stereochemistry, represents an extraordinary challenge for chemical synthesis. The key 2,6-anti- stereochemistry of the iminosugar ring was established through an iridium-catalyzed asymmetric allylic amination. The developed synthetic route is suitable for the synthesis of focused libraries to enable the structure–activity relationship study and late-stage modification of iminosugar scaffold with variable lipid, peptide and sugar substituents. Compound 2b showed 70% inhibition of transglycosylase from Acinetobacter baumannii, providing a basis for further improvement. 相似文献
9.
The lack of novel antimicrobial drugs under development coupled with the increasing occurrence of resistance to existing antibiotics by community and hospital acquired infections is of grave concern. The targeting of biosynthesis of the peptidoglycan component of the bacterial cell wall has proven to be clinically valuable but relatively little therapeutic development has been directed towards the transglycosylase step of this process. Advances towards the isolation of new antimicrobials that target transglycosylase activity will rely on the development of the enzymological tools required to identify and characterise novel inhibitors of these enzymes. Therefore, in this article, we review the assay methods developed for transglycosylases and review recent novel chemical inhibitors discovered in relation to both the lipidic substrates and natural product inhibitors of the transglycosylase step. 相似文献
1