Rational Engineering of the Substrate Specificity of a Thermostable D-Hydantoinase (Dihydropyrimidinase)
| Autor: | Vehary Sakanyan, Pierre Weigel, Jean-Noël Hallet, Hovsep Alexandr Aganyants, A. A. Hambardzumyan, Haykanush Koloyan, Yeranuhi Hovhannisyan, Anichka S. Hovsepyan, Michèle Lecocq |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: |
biocatalyst
Stereochemistry substrate specificity thermophilic bacteria Biomedical Engineering Protein Data Bank (RCSB PDB) long inverse pcr Bioengineering Biochemistry dihydropyrimidinase Article lcsh:Chemistry 03 medical and health sciences Homology modeling Binding site Site-directed mutagenesis lcsh:Science d-hydantoinase lcsh:QH301-705.5 030304 developmental biology Alanine chemistry.chemical_classification 0303 health sciences 030302 biochemistry & molecular biology Substrate (chemistry) manganese-dependence Amino acid chemistry lcsh:Biology (General) lcsh:QD1-999 Dihydropyrimidinase lcsh:Q site-directed mutagenesis Biotechnology |
| Zdroj: | High-Throughput, Vol 9, Iss 1, p 5 (2020) High-Throughput Volume 9 Issue 1 |
| ISSN: | 2571-5135 |
| Popis: | D-hydantoinases catalyze an enantioselective opening of 5- and 6-membered cyclic structures and therefore can be used for the production of optically pure precursors for biomedical applications. The thermostable D-hydantoinase from Geobacillus stearothermophilus ATCC 31783 is a manganese-dependent enzyme and exhibits low activity towards bulky hydantoin derivatives. Homology modeling with a known 3D structure (PDB code: 1K1D) allowed us to identify the amino acids to be mutated at the substrate binding site and in its immediate vicinity to modulate the substrate specificity. Both single and double substituted mutants were generated by site-directed mutagenesis at appropriate sites located inside and outside of the stereochemistry gate loops (SGL) involved in the substrate binding. Substrate specificity and kinetic constant data demonstrate that the replacement of Phe159 and Trp287 with alanine leads to an increase in the enzyme activity towards D,L-5-benzyl and D,L-5-indolylmethyl hydantoins. The length of the side chain and the hydrophobicity of substrates are essential parameters to consider when designing the substrate binding pocket for bulky hydantoins. Our data highlight that D-hydantoinase is the authentic dihydropyrimidinase involved in the pyrimidine reductive catabolic pathway in moderate thermophiles. |
| Databáze: | OpenAIRE |
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