- N-acetyltransferases from three different organisms displaying distinct selectivity toward hexosamines and N-terminal amine of peptides
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N-acetyltransferases are a family of enzymes that catalyze the transfer of the acetyl moiety (–COCH3) from acetyl coenzyme A (Acetyl-CoA) to a primary amine of acceptor substrates from small molecules such as aminoglycoside to macromolecules of various proteins. In this study, the substrate selectivity of three N-acetyltransferases falling into different phylogenetic groups was probed against a series of hexosamines and synthetic peptides. GlmA from Clostridium acetobutylicum and RmNag from Rhizomucor miehei, which have been defined as glucosamine N-acetyltransferases, were herein demonstrated to be also capable of acetylating the free amino group on the very first glycine residue of peptide in spite of varied catalytic efficiency. The human recombinant N-acetyltransferase of Naa10p, however, prefers primary amine groups in the peptides as opposed to glucosamine. The varied preference of GlmA, RmNag and Naa10p probably arose from the divergent evolution of these N-acetyltransferases. The expanded knowledge of acceptor specificity would as well facilitate the application of these N-acetyltransferases in the acetylation of hexosamines or peptides.
- Zhang, Peiru,Liu, Pei,Xu, Yangyang,Liang, Yulu,Wang, Peng George,Cheng, Jiansong
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- Structure and antioxidant activity study of sulfated acetamido- polysaccharide from Radix Hedysari
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A new sulfated acetamido-heteropolysaccharide, HPS4-2A, was obtained by aqueous extraction followed by precipitation with ethanol and fractionation with DEAE column chromatography from Radix Hedysari. It was composed of rhamnose, arabinose, glucose, galactose and 2-acetamido-2-deoxy-D-galactose in the molar ratio of 10.09%:25.90%:25.90%:25.0%:12.30%. Elemental analysis indicated that HPS4-2A was a sulfated polysaccharide containing small amount of sulfate groups (1.87%). Partial acid hydrolysis, GC, GC-MS, 1D and 2D NMR spectroscopy analysis of the HPS4-2A revealed a predominance of glucose, galactose and 2-acetamido-2-deoxy-D-galactose linked in a highly-branched structure. Themolecular weight of HPS4-2Awas determined by HPSEC and HPSEC-MALLS. AFMstudy indicated that HPS4-2A took a highly branched conformation, which in consistent with the result studied by SEC-MALLS. Structural features of HPS4-2A were also investigated by SEM and TEM. Antioxidant assays demonstrated that HPS4-2A possessed of strong DPPH and hydroxyl radicals scavenging activities, suggesting that HPS4-2A could potentially be used as natural antioxidant.
- Dang, Zilong,Feng, Demei,Liu, Xiaohua,Yang, Tao,Guo, Long,Liang, Jin,Liang, Jiandi,Hu, Fangdi,Cui, Fang,Feng, Shilan
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- Structural Insights into the Recovery of Aldolase Activity in N-Acetylneuraminic Acid Lyase by Replacement of the Catalytically Active Lysine with γ-Thialysine by Using a Chemical Mutagenesis Strategy
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Chemical modification has been used to introduce the unnatural amino acid γ-thialysine in place of the catalytically important Lys165 in the enzyme N-acetylneuraminic acid lyase (NAL). The Staphylococcus aureus nanA gene, encoding NAL, was cloned and expressed in E. coli. The protein, purified in high yield, has all the properties expected of a class I NAL. The S. aureus NAL which contains no natural cysteine residues was subjected to site-directed mutagenesis to introduce a cysteine in place of Lys165 in the enzyme active site. Subsequently chemical mutagenesis completely converted the cysteine into γ-thialysine through dehydroalanine (Dha) as demonstrated by ESI-MS. Initial kinetic characterisation showed that the protein containing γ-thialysine regained 17% of the wild-type activity. To understand the reason for this lower activity, we solved X-ray crystal structures of the wild-type S. aureus NAL, both in the absence of, and in complex with, pyruvate. We also report the structures of the K165C variant, and the K165-γ-thialysine enzyme in the presence, or absence, of pyruvate. These structures reveal that γ-thialysine in NAL is an excellent structural mimic of lysine. Measurement of the pH-activity profile of the thialysine modified enzyme revealed that its pH optimum is shifted from 7.4 to 6.8. At its optimum pH, the thialysine-containing enzyme showed almost 30% of the activity of the wild-type enzyme at its pH optimum. The lowered activity and altered pH profile of the unnatural amino acid-containing enzyme can be rationalised by imbalances of the ionisation states of residues within the active site when the pKa of the residue at position 165 is perturbed by replacement with γ-thialysine. The results reveal the utility of chemical mutagenesis for the modification of enzyme active sites and the exquisite sensitivity of catalysis to the local structural and electrostatic environment in NAL.
- Timms, Nicole,Windle, Claire L.,Polyakova, Anna,Ault, James R.,Trinh, Chi H.,Pearson, Arwen R.,Nelson, Adam,Berry, Alan
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p. 474 - 481
(2013/05/08)
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- Structure elucidation of the O-antigen of Salmonella enterica O51 and its structural and genetic relation to the O-antigen of Escherichia coli O23
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The O-polysaccharide (O-antigen) of Salmonella enterica O51 was isolated by mild acid degradation of the lipopolysaccharide and its structure was established using sugar analysis and NMR spectroscopy. The O-antigen of Escherichia coli O23, whose structure was elucidated earlier, possesses a similar structure and differs only in the presence of an additional lateral α-D-Glcp residue at position 6 of the GlcNAc residue in the main chain. Sequencing of the O-antigen gene clusters of S. enterica O51 and E. coli O23 revealed the same genes with a high-level similarity. By comparison with opened gene databases, all genes expected for the synthesis of the common structure of the two O-antigens were assigned functions. It is suggested that the gene clusters of both bacteria originated from a common ancestor, whereas the O-antigen modification in E. coli O23, which, most probably, is induced by prophage genes outside the gene cluster, could be introduced after the species divergence.
- Perepelov,Liu, Bin,Guo, Dan,Senchenkova,Shahskov,Feng, Lu,Wang, Lei,Knirel
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experimental part
p. 774 - 779
(2012/01/19)
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- One-pot enzymatic production of 2-acetamido-2-deoxy-D-galactose (GalNAc) from 2-acetamido-2-deoxy-D-glucose (GlcNAc)
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2-Acetamido-2-deoxy-D-galactose (GalNAc) is a common monosaccharide found in biologically functional sugar chains, but its availability is often limited due to the lack of abundant natural sources. In order to produce GalNAc from abundantly available sugars, 2-acetamido-2-deoxy-D-glucose (GlcNAc) was converted to GalNAc by a one-pot reaction using three enzymes involved in the galacto-N-biose/lacto-N-biose I pathway of bifidobacteria. Starting the reaction with 600 mM GlcNAc, 170 mM GalNAc was produced at equilibrium in the presence of catalytic amounts of ATP and UDP-Glc under optimized conditions. GalNAc was separated from GlcNAc using water-eluting cation-exchange chromatography with a commonly available cation-exchange resin.
- Inoue, Kousuke,Nishimoto, Mamoru,Kitaoka, Motomitsu
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experimental part
p. 2432 - 2436
(2011/12/15)
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- Structural insights into substrate specificity in variants of N-acetylneuraminic acid lyase produced by directed evolution
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The substrate specificity of Escherichia coli N-acetylneuraminic acid lyase was previously switched from the natural condensation of pyruvate with N-acetylmannosamine, yielding N-acetylneuraminic acid, to the aldol condensation generating N-alkylcarboxamide analogues of N-acetylneuraminic acid. This was achieved by a single mutation of Glu192 to Asn. In order to analyze the structural changes involved and to more fully understand the basis of this switch in specificity, we have isolated all 20 variants of the enzyme at position 192 and determined the activities with a range of substrates. We have also determined five high-resolution crystal structures: the structures of wild-type E. coli N-acetylneuraminic acid lyase in the presence and in the absence of pyruvate, the structures of the E192N variant in the presence and in the absence of pyruvate, and the structure of the E192N variant in the presence of pyruvate and a competitive inhibitor (2R,3R)-2,3,4-trihydroxy-N,N-dipropylbutanamide. All structures were solved in space group P21 at resolutions ranging from 1.65 A to 2.2 A. A comparison of these structures, in combination with the specificity profiles of the variants, reveals subtle differences that explain the details of the specificity changes. This work demonstrates the subtleties of enzyme-substrate interactions and the importance of determining the structures of enzymes produced by directed evolution, where the specificity determinants may change from one substrate to another.
- Campeotto, Ivan,Bolt, Amanda H.,Harman, Thomas A.,Dennis, Caitriona,Trinh, Chi H.,Phillips, Simon E.V.,Nelson, Adam,Pearson, Arwen R.,Berry, Alan
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scheme or table
p. 56 - 69
(2011/12/01)
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- COMPOSITIONS AND METHODS FOR TREATING HEPATITIS-C
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New compositions and methods for treating patients suffering from hepatitis-C, AIDS, aberrant apoptosis which include N-acetyl-D-glucosaminyl(β-1-4)-N-Acetyl-muramyl-L-ananyl-D-isoglutamine (GMDP) of at least 98% purity and provided either alone, as an active ingredient of blastolysine, or in combination with an aminosugar such as N-acetyl-glucosamine(NAG). The high purity GMDP has a decreased amount immunogenic impurities and demonstrates cell protection as opposed to solely immunostimulatory effects, while a synergistic cell protective effect is exhibited when GMDP in combined with NAG. The new compositions modulate FasL mediated apoptosis while simultaneously stimulating TNF-α production and further selectively inhibiting TNF-α receptor p55 (TNFR1), providing a treatment for patients suffering from hepatitis-C, AIDS or aberrant apoptosis.
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- An approach to the precise chemoenzymatic synthesis of 13C-labeled sialyloligosaccharide on an intact glycoprotein: A novel one-pot [3-13C]-labeling method for sialic acid analogues by control of the reversible aldolase reaction, enzymatic synthesis of [3-13C]-NeuAc-α-(2→3)-[U- 13C]-Gal-β-(1-4)-GlcNAc-β-sequence onto glycoprotein
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A one-pot enzymatic 13C-labeling method for the 3-position of sialic acid (NeuAc) analogues has been developed using NeuAc aldolase, lactate dehydrogenase (LDH), alcohol dehydrogenase (ADH), and nucleotide pyrophosphatase (NPP). This method consists of two steps, the first of which is degradation to 2-acetamido-2-deoxy-D-mannose (ManNAc) analogues. This degradation reaction was accelerated by a cofactor regeneration system which converts pyruvic acid into lactic acid using LDH, ADH, and β-nicotinamide adenine dinucleotide oxidized form (β-NAD+). The second step is condensation of the ManNAc analogue with [3-13C]-pyruvic acid newly added after decomposition of the cofactor by nucleotide pyrophosphatase which play a role like switch to stop conversion of pyruvic acid into lactic acid. Five different NeuAc analogues have been labeled in good yields using this newly developed one-pot enzymatic procedure. Following conversion of [3-13C]-NeuAc to CMP-[3-13C]-NeuAc, enzymatic synthesis of [3-13C]-NeuAc-α-(2→3)-[U- 13C]-Gal-β-(1→4)-GlcNAc-β-x-ovalbumin (x: hybrid type oligosaccharide) 23 and [3-13C]-NeuAc-α-(2→3)-[U- 13C]-Gal-β-(1→4)-GlcNAc-β-OMe 26 (sialyl LacNAc) was performed using bovine β-1,4-galactosyltransferase and rat recombinant α-2,3-sialyltransferase. The 1H chemical shifts of all protons in [3-13C]-NeuAc-α-(2→3)-[U-13C]-Gal-β-on a glycoprotein were assigned by 2D HMQC, 1D HSQC-TOCSY, and the herein described 1D and 2D HSQC-TOCSY-NOESY-TOCSY method. More specifically, the 7-, 8-, and 9-protons of NeuAc could be observed by this HSQC-TOCSY-NOESY-TOCSY method even with only a single 13C atom at the 3-position. In addition, 1D and 2D HMQC-NOESY spectra as well as carbon spin-lattice relaxation times (T1) were measured to compare the conformational properties and dynamic behavior of the sialylgalactoside as part of the sialyl LacNAc 26 and when bound to a glycoprotein 23. These analyses suggested that the conformational properties of sialyl LacNAc are similar for both the conjugated and unconjugated forms, and that the torsional angle of the sialyl linkage, i.e., COOH-C2NeuAc-O-C3Gal, is biased toward the anti (-146.7°) conformation. In addition, the flexibility of galactosyl ring when bound to a glycoprotein appears to be significantly restricted by the attachment of NeuAc as compared with unconjugated sialyl LacNAc.
- Miyazaki, Tatsuo,Sato, Hajime,Sakakibara, Tohru,Kajihara, Yasuhiro
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p. 5678 - 5694
(2007/10/03)
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- Structural studies of the O-specific side-chain of the lipopolysaccharide from Escherichia coli O 55.
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The structure of the O-specific side-chains of the lipopolysaccharide from Escherichia coli O 55 has been investigated, methylation analysis, specific degradations, and n.m.r. spectroscopy being the principal methods used. It is concluded that the O-specific side-chains are composed of pentasaccharide repeating-units having the following structure [where Col stands for colitose (3,6-dideoxy-L-xylo-hexose)].(See formula in text).
- Lindberg,Lindh,Loenngren
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p. 105 - 112
(2007/10/02)
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