Molecular cloning and enzymological characterization of pyridoxal 5'-phosphate independent aspartate racemase from hyperthermophilic archaeon Thermococcus litoralis DSM 5473
| Autor: | Tsubasa Washio, Tadao Oikawa, Shiro Kato |
|---|---|
| Rok vydání: | 2015 |
| Předmět: |
0301 basic medicine
Hot Temperature endocrine system diseases Iodoacetic acid Archaeal Proteins 030106 microbiology Aspartate racemase Microbiology Substrate Specificity Serine 03 medical and health sciences chemistry.chemical_compound Aspartic acid Enzyme Stability Thermococcus litoralis Pyridoxal Conserved Sequence Amino Acid Isomerases Alanine biology nutritional and metabolic diseases Aspartate racemase activity General Medicine Hydrogen-Ion Concentration biology.organism_classification Thermococcus 030104 developmental biology chemistry Biochemistry Amino Acid Substitution Pyridoxal Phosphate Molecular Medicine hormones hormone substitutes and hormone antagonists |
| Zdroj: | Extremophiles : life under extreme conditions. 20(5) |
| ISSN: | 1433-4909 |
| Popis: | We succeeded in expressing the aspartate racemase homolog gene from Thermococcus litoralis DSM 5473 in Escherichia coli Rosetta (DE3) and found that the gene encodes aspartate racemase. The aspartate racemase gene consisted of 687 bp and encoded 228 amino acid residues. The purified enzyme showed aspartate racemase activity with a specific activity of 1590 U/mg. The enzyme was a homodimer with a molecular mass of 56 kDa and did not require pyridoxal 5′-phosphate as a coenzyme. The enzyme showed aspartate racemase activity even at 95 °C, and the activation energy of the enzyme was calculated to be 51.8 kJ/mol. The enzyme was highly thermostable, and approximately 50 % of its initial activity remained even after incubation at 90 °C for 11 h. The enzyme showed a maximum activity at a pH of 7.5 and was stable between pH 6.0 and 7.0. The enzyme acted on l-cysteic acid and l-cysteine sulfinic acid in addition to d- and l-aspartic acids, and was strongly inhibited by iodoacetic acid. The site-directed mutagenesis of the enzyme showed that the essential cysteine residues were conserved as Cys83 and Cys194. d-Forms of aspartic acid, serine, alanine, and valine were contained in T. litoralis DSM 5473 cells. |
| Databáze: | OpenAIRE |
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