Structural and catalytic effects of surface loop-helix transplantation within haloalkane dehalogenase family
| Autoři | |
|---|---|
| Rok publikování | 2020 |
| Druh | Článek v odborném periodiku |
| Časopis / Zdroj | Computational and Structural Biotechnology Journal |
| Fakulta / Pracoviště MU | |
| Citace | |
| www | https://www.sciencedirect.com/science/article/pii/S2001037020302828?via%3Dihub |
| Doi | http://dx.doi.org/10.1016/j.csbj.2020.05.019 |
| Klíčová slova | Haloalkane dehalogenase (HLD); Biocatalysis; Loop-helix transplantation; X-ray crystallography; Enantioselectivity; Access tunnel; Enzyme engineering; Protein design |
| Přiložené soubory | |
| Popis | Engineering enzyme catalytic properties is important for basic research as well as for biotechnological applications. We have previously shown that the reshaping of enzyme access tunnels via the deletion of a short surface loop element may yield a haloalkane dehalogenase variant with markedly modified substrate specificity and enantioselectivity. Here, we conversely probed the effects of surface loop-helix transplantation from one enzyme to another within the enzyme family of haloalkane dehalogenases. Precisely, we transplanted a nine-residue long extension of L9 loop and alpha 4 helix from DbjA into the corresponding site of DbeA. Biophysical characterization showed that this fragment transplantation did not affect the overall protein fold or oligomeric state, but lowered protein stability (Delta T-m = -5 to 6 degrees C). Interestingly, the crystal structure of DbeA mutant revealed the unique structural features of enzyme access tunnels, which are known determinants of catalytic properties for this enzyme family. Biochemical data confirmed that insertion increased activity of DbeA with various halogenated substrates and altered its enantioselectivity with several linear beta-bromoalkanes. Our findings support a protein engineering strategy employing surface loop-helix transplantation for construction of novel protein catalysts with modified catalytic properties. |
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