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《Angewandte Chemie (Weinheim an der Bergstrasse, Germany)》2017,129(48):15398-15408
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《Angewandte Chemie (Weinheim an der Bergstrasse, Germany)》2017,129(47):15193-15197
Carbohydrate‐based vaccines are among the safest and most effective vaccines and represent potent tools for prevention of life‐threatening bacterial infectious diseases, like meningitis and pneumonia. The chemical conjugation of a weak antigen to protein as a source of T‐cell epitopes generates a glycoconjugate vaccine that results more immunogenic. Several methods have been used so far to characterize the resulting polysaccharide–protein conjugates. However, a reduced number of methodologies has been proposed for measuring the degree of saccharide conjugation at the possible protein sites. Here we show that detailed information on large proteins conjugated with large polysaccharides can be achieved by a combination of solution and solid‐state NMR spectroscopy. As a test case, a large protein assembly, l ‐asparaginase II, has been conjugated with Neisseria meningitidis serogroup C capsular polysaccharide and the pattern and degree of conjugation were determined. 相似文献
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《Angewandte Chemie (Weinheim an der Bergstrasse, Germany)》2017,129(47):15049-15053
Glycosyltransferases (GTs) are a key family of enzymes that catalyze the synthesis of glycosidic bonds in all living organisms. The reaction involves the transfer of a glycosyl moiety and can proceed with retention or inversion of the anomeric configuration. To date, the catalytic mechanism of retaining GTs is a topic of great controversy, particularly for those enzymes containing a putative nucleophilic residue in the active site, for which the occurrence of a double‐displacement mechanism has been suggested. We report native ternary complexes of the retaining glycosyltransferase α‐1,3‐galactosyltransferase (α3GalT) from Bos taurus , which contains such a nucleophile in the active site, in a productive mode for catalysis in the presence of its sugar donor UDP‐Gal, the acceptor substrate lactose, and the divalent cation cofactor. This new experimental evidence supports the occurrence of a front‐side substrate‐assisted SNi‐type reaction for α3GalT, and suggests a conserved common catalytic mechanism among retaining GTs. 相似文献
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《Angewandte Chemie (Weinheim an der Bergstrasse, Germany)》2017,129(30):8823-8826
Bacteria possess cytosolic proteins (Csp3s) capable of binding large quantities of copper and preventing toxicity. Crystal structures of a Csp3 plus increasing amounts of CuI provide atomic‐level information about how a storage protein loads with metal ions. Many more sites are occupied than CuI equiv added, with binding by twelve central sites dominating. These can form [Cu4(S‐Cys)4] intermediates leading to [Cu4(S‐Cys)5]−, [Cu4(S‐Cys)6]2−, and [Cu4(S‐Cys)5(O‐Asn)]− clusters. Construction of the five CuI sites at the opening of the bundle lags behind the main core, and the two least accessible sites at the opposite end of the bundle are occupied last. Facile CuI cluster formation, reminiscent of that for inorganic complexes with organothiolate ligands, is largely avoided in biology but is used by proteins that store copper in the cytosol of prokaryotes and eukaryotes, where this reactivity is also key to toxicity. 相似文献
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Fumihiro Ishikawa Akimasa Miyanaga Hinano Kitayama Shinya Nakamura Isao Nakanishi Fumitaka Kudo Tadashi Eguchi Genzoh Tanabe 《Angewandte Chemie (Weinheim an der Bergstrasse, Germany)》2019,131(21):6980-6984
Adenylation (A) domains act as the gatekeepers of non‐ribosomal peptide synthetases (NRPSs), ensuring the activation and thioesterification of the correct amino acid/aryl acid building blocks. Aryl acid building blocks are most commonly observed in iron‐chelating siderophores, but are not limited to them. Very little is known about the reprogramming of aryl acid A‐domains. We show that a single asparagine‐to‐glycine mutation in an aryl acid A‐domain leads to an enzyme that tolerates a wide range of non‐native aryl acids. The engineered catalyst is capable of activating non‐native aryl acids functionalized with nitro, cyano, bromo, and iodo groups, even though no enzymatic activity of wild‐type enzyme was observed toward these substrates. Co‐crystal structures with non‐hydrolysable aryl‐AMP analogues revealed the origins of this expansion of substrate promiscuity, highlighting an enlargement of the substrate binding pocket of the enzyme. Our findings may be exploited to produce diversified aryl acid containing natural products and serve as a template for further directed evolution in combinatorial biosynthesis. 相似文献