Directed evolution of a remarkably efficient Kdnase from a bacterial neuraminidase
| Autor: | Matthew C. Deen, Fahimeh S. Shidmoossavee, Saeideh Shamsi Kazem Abadi, Andrew J. Bennet, Jacqueline N. Watson |
|---|---|
| Rok vydání: | 2019 |
| Předmět: |
Glycan
Neuraminidase 010402 general chemistry 01 natural sciences Biochemistry Micromonospora 03 medical and health sciences chemistry.chemical_compound Carbohydrate Conformation Enzyme kinetics 030304 developmental biology chemistry.chemical_classification 0303 health sciences biology Mutagenesis Sugar Acids Glycosidic bond Directed evolution 0104 chemical sciences Sialic acid Enzyme chemistry biology.protein Biocatalysis Directed Molecular Evolution |
| Zdroj: | Glycobiology. 30(5) |
| ISSN: | 1460-2423 |
| Popis: | N-acetylneuraminic acid (5-acetamido-3,5-dideoxy-d-glycero-d-galacto-non-2-ulosonic acid), which is the principal sialic acid family member of the non-2-ulosonic acids and their various derivatives, is often found at the terminal position on the glycan chains that adorn all vertebrate cells. This terminal position combined with subtle variations in structure and linkage to the underlying glycan chains between humans and other mammals points to the importance of this diverse group of nine-carbon sugars as indicators of the unique aspects of human evolution and is relevant to understanding an array of human conditions. Enzymes that catalyze the removal N-acetylneuraminic acid from glycoconjugates are called neuraminidases. However, despite their documented role in numerous diseases, due to the promiscuous activity of many neuraminidases, our knowledge of the functions and metabolism of many sialic acids and the effect of the attachment to cellular glycans is limited. To this end, through a concerted effort of generation of random and site-directed mutagenesis libraries, subsequent screens and positive and negative evolutionary selection protocols, we succeeded in identifying three enzyme variants of the neuraminidase from the soil bacterium Micromonospora viridifaciens with markedly altered specificity for the hydrolysis of natural Kdn (3-deoxy-d-glycero-d-galacto-non-2-ulosonic acid) glycosidic linkages compared to those of N-acetylneuraminic acid. These variants catalyze the hydrolysis of Kdn-containing disaccharides with catalytic efficiencies (second-order rate constants: kcat/Km) of greater than 105 M−1 s−1; the best variant displayed an efficiency of >106 M−1 s−1 at its optimal pH. |
| Databáze: | OpenAIRE |
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