3615-17-6Relevant articles and documents
N-acetyltransferases from three different organisms displaying distinct selectivity toward hexosamines and N-terminal amine of peptides
Zhang, Peiru,Liu, Pei,Xu, Yangyang,Liang, Yulu,Wang, Peng George,Cheng, Jiansong
, p. 72 - 75 (2018/11/30)
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.
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
Timms, Nicole,Windle, Claire L.,Polyakova, Anna,Ault, James R.,Trinh, Chi H.,Pearson, Arwen R.,Nelson, Adam,Berry, Alan
, p. 474 - 481 (2013/05/08)
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.
One-pot enzymatic production of 2-acetamido-2-deoxy-D-galactose (GalNAc) from 2-acetamido-2-deoxy-D-glucose (GlcNAc)
Inoue, Kousuke,Nishimoto, Mamoru,Kitaoka, Motomitsu
experimental part, p. 2432 - 2436 (2011/12/15)
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.
