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1.
糖及其衍生物在许多初级或次级代谢过程中发挥着重要作用。糖结构与功能的多样性和糖在疾病诊断与治疗中的重要性推动了糖生物学的快速发展。D型糖,尤其是D-六碳糖在糖中占据着主导地位,L-六碳糖也是许多重要糖蛋白复合物、多糖及抗生素的组成成分。了解L-六碳糖的形成机制有助于理性改造糖的结构并开发其应用价值。L-六碳糖通常由3,5位差向异构酶或5位差向异构酶催化D-六碳糖的C5位异构化形成,这种转变赋予了糖在构型上的多样性,并在许多天然产物中起决定生物活性的作用。对3,5位差向异构酶和5位差向异构酶的功能及晶体结构的研究揭示了L-六碳糖的形成机制。本文综述了L-六碳糖形成过程中不同类型的3,5-位差向异构酶和5-位差向异构酶的催化机制,揭示L-六碳糖在生理和医药领域的重要意义。 相似文献
2.
Several studies have shown that the native 7alpha-hydroxy-dehydroepiandrosterone (7alpha-hydroxy-DHEA) is a substrate for the human 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) which converts the 7alpha- into the 7beta-epimer through an oxido-reduction process. Research on the 11beta-HSD1 has investigated its function and structure through using native glucocorticoid substrates and known inhibitors. Other steroid substrates are also of interest. Among testosterone metabolites, 5alpha-androstane-3beta,17beta-diol (Adiol) is a substrate for the cytochrome P450 7B1 which produces 5alpha-androstane-3beta,7alpha,17beta-triol (7alpha-Adiol). This steroid may be a substrate for the 11beta-HSD1. We used recombinant yeast-expressed 11beta-HSD1 with NADP(H)-regenerating systems for examining the products obtained after incubation with 7alpha-Adiol, 7beta-Adiol or 7-oxo-Adiol. Oxidative conditions for the 11beta-HSD1 provided no trace of 7-oxo-Adiol but the inter-conversion of 7alpha- and 7beta-hydroxy-Adiol with V(max)/K(M) (pmol min(-1) microg(-1)/microM) values of 2 and 0.5, respectively. This state was maintained under reductive conditions. The use of a 7-oxo-Adiol substrate under reductive conditions led to the production of both 7alpha- and 7beta-hydroxy-Adiol with V(max)/K(M) values of 3.43 and 0.22, respectively. These findings support the hypothesis that the oxido-reductase and epimerase activities of 11beta-HSD1 depend on the positioning of the steroid substrates within the active site and may provide insight into its fine structure and mechanism of action. 相似文献
3.
Edith Buchinger Daniel H. Knudsen Manja A. Behrens Jan Skov Pedersen Olav A. Aarstad Anne T?ndervik Svein Valla Gudmund Skj?k-Br?k Reinhard Wimmer Finn L. Aachmann 《The Journal of biological chemistry》2014,289(45):31382-31396
The bacterium Azotobacter vinelandii produces a family of seven secreted and calcium-dependent mannuronan C-5 epimerases (AlgE1–7). These epimerases are responsible for the epimerization of β-d-mannuronic acid (M) to α-l-guluronic acid (G) in alginate polymers. The epimerases display a modular structure composed of one or two catalytic A-modules and from one to seven R-modules having an activating effect on the A-module. In this study, we have determined the NMR structure of the three individual R-modules from AlgE6 (AR1R2R3) and the overall structure of both AlgE4 (AR) and AlgE6 using small angle x-ray scattering. Furthermore, the alginate binding ability of the R-modules of AlgE4 and AlgE6 has been studied with NMR and isothermal titration calorimetry. The AlgE6 R-modules fold into an elongated parallel β-roll with a shallow, positively charged groove across the module. Small angle x-ray scattering analyses of AlgE4 and AlgE6 show an overall elongated shape with some degree of flexibility between the modules for both enzymes. Titration of the R-modules with defined alginate oligomers shows strong interaction between AlgE4R and both oligo-M and MG, whereas no interaction was detected between these oligomers and the individual R-modules from AlgE6. A combination of all three R-modules from AlgE6 shows weak interaction with long M-oligomers. Exchanging the R-modules between AlgE4 and AlgE6 resulted in a novel epimerase called AlgE64 with increased G-block forming ability compared with AlgE6. 相似文献
4.
Yi Qin Jiyuan Ke Xin Gu Jianping Fang Wucheng Wang Qifei Cong Jie Li Jinzhi Tan Joseph S. Brunzelle Chenghai Zhang Yi Jiang Karsten Melcher Jin-ping Li H. Eric Xu Kan Ding 《The Journal of biological chemistry》2015,290(8):4620-4630
Heparan sulfate (HS) is a glycosaminoglycan present on the cell surface and in the extracellular matrix, which interacts with diverse signal molecules and is essential for many physiological processes including embryonic development, cell growth, inflammation, and blood coagulation. d-Glucuronyl C5-epimerase (Glce) is a crucial enzyme in HS synthesis, converting d-glucuronic acid to l-iduronic acid to increase HS flexibility. This modification of HS is important for protein ligand recognition. We have determined the crystal structures of Glce in apo-form (unliganded) and in complex with heparin hexasaccharide (product of Glce following O-sulfation), both in a stable dimer conformation. A Glce dimer contains two catalytic sites, each at a positively charged cleft in C-terminal α-helical domains binding one negatively charged hexasaccharide. Based on the structural and mutagenesis studies, three tyrosine residues, Tyr468, Tyr528, and Tyr546, in the active site were found to be crucial for the enzymatic activity. The complex structure also reveals the mechanism of product inhibition (i.e. 2-O- and 6-O-sulfation of HS keeps the C5 carbon of l-iduronic acid away from the active-site tyrosine residues). Our structural and functional data advance understanding of the key modification in HS biosynthesis. 相似文献
5.
Jeffrey S. Rush Cristina Alaimo Riccardo Robbiani Michael Wacker Charles J. Waechter 《The Journal of biological chemistry》2010,285(3):1671-1680
Escherichia coli strain O157 produces an O-antigen with the repeating tetrasaccharide unit α-d-PerNAc-α-l-Fuc-β-d-Glc-α-d-GalNAc, preassembled on undecaprenyl pyrophosphate (Und-P-P). These studies were conducted to determine whether the biosynthesis of the lipid-linked repeating tetrasaccharide was initiated by the formation of GalNAc-P-P-Und by WecA. When membrane fractions from E. coli strains K12, O157, and PR4019, a WecA-overexpressing strain, were incubated with UDP-[3H]GalNAc, neither the enzymatic synthesis of [3H]GlcNAc-P-P-Und nor [3H]GalNAc-P-P-Und was detected. However, when membrane fractions from strain O157 were incubated with UDP-[3H]GlcNAc, two enzymatically labeled products were observed with the chemical and chromatographic properties of [3H]GlcNAc-P-P-Und and [3H]GalNAc-P-P-Und, suggesting that strain O157 contained an epimerase capable of interconverting GlcNAc-P-P-Und and GalNAc-P-P-Und. The presence of a novel epimerase was demonstrated by showing that exogenous [3H]GlcNAc-P-P-Und was converted to [3H]GalNAc-P-P-Und when incubated with membranes from strain O157. When strain O157 was metabolically labeled with [3H]GlcNAc, both [3H]GlcNAc-P-P-Und and [3H]GalNAc-P-P-Und were detected. Transformation of E. coli strain 21546 with the Z3206 gene enabled these cells to synthesize GalNAc-P-P-Und in vivo and in vitro. The reversibility of the epimerase reaction was demonstrated by showing that [3H]GlcNAc-P-P-Und was reformed when membranes from strain O157 were incubated with exogenous [3H]GalNAc-P-P-Und. The inability of Z3206 to complement the loss of the gne gene in the expression of the Campylobacter jejuni N-glycosylation system in E. coli indicated that it does not function as a UDP-GlcNAc/UDP-GalNAc epimerase. Based on these results, GalNAc-P-P-Und is synthesized reversibly by a novel GlcNAc-P-P-Und epimerase after the formation of GlcNAc-P-P-Und by WecA in E. coli O157. 相似文献
6.
The O antigen unit of Pseudomonas aeruginosa serotype O5 is a complex trisaccharide containing 2-acetamido-3-acetiminido-2, 3-dideoxy-beta-D-mannuronic acid, 2-acetimido-3-acetimido-2, 3-dideoxy-beta-D-mannuronic acid, and 2-acetimido-2, 6-deoxy-beta-D-galactosamine. Specific knockout mutations in the putative UDP-D-N-acetylglucosamine (UDP-D-GlcNAc) epimerase gene, wbpI, or the putative UDP-D-N-acetylmannosamine dehydrogenase gene, wbpA, resulted in strains that no longer produced B-band lipopolysaccharide, confirming the essential roles of these genes in B-band O antigen synthesis. Despite approximately 50% similarity of wbpI and wbpA to the Escherichia coli genes wecB (rffE) and wecC (rffD) involved in enterobacterial common antigen synthesis, cross-complementation experiments were not successful. These results imply that the P. aeruginosa UDP-D-GlcNAc precursor may be di-N-acetylated prior to further modification, preventing the E. coli enzymes from recognizing it as a substrate. 相似文献
7.
本实验用液体静置法体外培养小鼠骨髓基质细胞。用细胞化学法观察处于不同分化阶段基质细胞中的尿嘧啶核苷二磷酸半乳糖--4-表异构酶(Uridinediphosphogalactose-4-epimerase,UDPGal,EC5.1.3.a)活性。结果在各类骨髓基质细胞中均可见到很强的酶反应。在由原始网状细胞向成熟星状细胞分化过程中,酶活性逐渐增强。随着培养时间和延长(培养5、7、10天),星状细胞和成纤维细胞中酶反应的阳性率和阳性度逐渐增高。这证明在体外培养骨髓基质细胞中由UDPGal催化的代谢非常活跃 相似文献
8.
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
9.
Aquilini E Azevedo J Merino S Jimenez N Tomás JM Regué M 《The Journal of biological chemistry》2010,285(51):39739-39749
The core lipopolysaccharides (LPS) of Proteus mirabilis as well as those of Klebsiella pneumoniae and Serratia marcescens are characterized by the presence of a hexosamine-galacturonic acid disaccharide (αHexN-(1,4)-αGalA) attached by an α1,3 linkage to L-glycero-D-manno-heptopyranose II (L-glycero-α-D-manno-heptosepyranose II). In K. pneumoniae, S. marcescens, and some P. mirabilis strains, HexN is D-glucosamine, whereas in other P. mirabilis strains, it corresponds to D-galactosamine. Previously, we have shown that two enzymes are required for the incorporation of D-glucosamine into the core LPS of K. pneumoniae; the WabH enzyme catalyzes the incorporation of GlcNAc from UDP-GlcNAc to outer core LPS, and WabN catalyzes the deacetylation of the incorporated GlcNAc. Here we report the presence of two different HexNAc transferases depending on the nature of the HexN in P. mirabilis core LPS. In vivo and in vitro assays using LPS truncated at the level of galacturonic acid as acceptor show that these two enzymes differ in their specificity for the transfer of GlcNAc or GalNAc. By contrast, only one WabN homologue was found in the studied P. mirabilis strains. Similar assays suggest that the P. mirabilis WabN homologue is able to deacetylate both GlcNAc and GalNAc. We conclude that incorporation of d-galactosamine requires three enzymes: Gne epimerase for the generation of UDP-GalNAc from UDP-GlcNAc, N-acetylgalactosaminyltransferase (WabP), and LPS:HexNAc deacetylase. 相似文献
10.
Sugar nucleotide–modifying enzymes of the short-chain dehydrogenase/reductase type use transient oxidation–reduction by a tightly bound nicotinamide cofactor as a common strategy of catalysis to promote a diverse set of reactions, including decarboxylation, single- or double-site epimerization, and dehydration. Although the basic mechanistic principles have been worked out decades ago, the finely tuned control of reactivity and selectivity in several of these enzymes remains enigmatic. Recent evidence on uridine 5'-diphosphate (UDP)-glucuronic acid decarboxylases (UDP-xylose synthase, UDP-apiose/UDP-xylose synthase) and UDP-glucuronic acid-4-epimerase suggests that stereo-electronic constraints established at the enzyme's active site control the selectivity, and the timing of the catalytic reaction steps, in the conversion of the common substrate toward different products. The mechanistic idea of stereo-electronic control is extended to epimerases and dehydratases that deprotonate the Cα of the transient keto-hexose intermediate. The human guanosine 5'-diphosphate (GDP)-mannose 4,6-dehydratase was recently shown to use a minimal catalytic machinery, exactly as predicted earlier from theoretical considerations, for the β-elimination of water from the keto-hexose species. 相似文献