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Role of lysine versus arginine in enzyme cold-adaptation: modifying lysine to homo-arginine stabilizes the cold-adapted α-amylase from Pseudoalteramonas haloplanktis
Siddiqui, K.S.; Poljak, A.; Guilhaus, M.; De Francisci, D.; Curmi, P.M.G.; Feller, G.; D'Amico, S.; Gerday, C.; Uversky, V.N.; Cavicchioli, R. (2006). Role of lysine versus arginine in enzyme cold-adaptation: modifying lysine to homo-arginine stabilizes the cold-adapted α-amylase from Pseudoalteramonas haloplanktis. Proteins-Structure Function and Bioinformatics 64(2): 486-501. https://dx.doi.org/10.1002/prot.20989
In: Proteins-Structure Function and Bioinformatics. Wiley-Blackwell: Hoboken. ISSN 0887-3585; e-ISSN 1097-0134, meer
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| Trefwoord |
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| Author keywords |
psychrophilic; guanidination; structure-function-stability relationship;enzyme kinetics; thermodynamics; bioinformatics |
| Auteurs | | Top |
- Siddiqui, K.S.
- Poljak, A.
- Guilhaus, M.
- De Francisci, D.
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- Curmi, P.M.G.
- Feller, G., meer
- D'Amico, S., meer
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- Gerday, C., meer
- Uversky, V.N.
- Cavicchioli, R.
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| Abstract |
The cold-adapted α-amylase from Pseudoalteromonas haloplanktis (AHA) is a multidomain enzyme capable of reversible unfolding. Cold-adapted proteins, including AHA, have been predicted to be structurally flexible and conformationally unstable as a consequence of a high lysine-to-arginine ratio. In order to examine the role of low arginine content in structural flexibility of AHA, the amino groups of lysine were guanidinated to form homo-arginine (hR), and the structure–function–stability properties of the modified enzyme were analyzed by transverse urea gradient-gel electrophoresis. The extent of modification was monitored by MALDI-TOF-MS, and correlated to changes in activity and stability. Modifying lysine to hR produced a conformationally more stable and less active α-amylase. The kcat of the modified enzyme decreased with a concomitant increase in ΔH# and decrease in Km. To interpret the structural basis of the kinetic and thermodynamic properties, the hR residues were modeled in the AHA X-ray structure and compared to the X-ray structure of a thermostable homolog. The experimental properties of the modified AHA were consistent with K106hR forming an intra-Domain B salt bridge to stabilize the active site and decrease the cooperativity of unfolding. Homo-Arg modification also appeared to alter Ca2+ and Cl− binding in the active site. Our results indicate that replacing lysine with hR generates mesophilic-like characteristics in AHA, and provides support for the importance of lysine residues in promoting enzyme cold adaptation. These data were consistent with computational analyses that show that AHA possesses a compositional bias that favors decreased conformational stability and increased flexibility. |
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