Role of lysine versus arginine in enzyme cold-adaptation: Modifying lysine to homo-arginine stabilizes the cold-adapted alpha-amylase from Pseudoalteramonas haloplanktis
[en] The cold-adapted alpha-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 homoarginine (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 a-amylase. The k(cat) of the modified enzyme decreased with a concomitant increase in Delta H-# and decrease in K-m. 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.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Siddiqui, K. S.
Poljak, A.
Guilhaus, M.
De Francisci, D.
Curmi, P. M. G.
Feller, Georges ; Université de Liège - ULiège > Département des sciences de la vie > Labo de biochimie
Gerday, Charles ; Université de Liège - ULiège > Services généraux (Faculté des sciences) > Relations académiques et scientifiques (Sciences)
Uversky, V. N.
Cavicchioli, R.
Language :
English
Title :
Role of lysine versus arginine in enzyme cold-adaptation: Modifying lysine to homo-arginine stabilizes the cold-adapted alpha-amylase from Pseudoalteramonas haloplanktis
Publication date :
01 August 2006
Journal title :
Proteins
ISSN :
0887-3585
eISSN :
1097-0134
Publisher :
Wiley Liss, Inc., Hoboken, United States - New Jersey
scite shows how a scientific paper has been cited by providing the context of the citation, a classification describing whether it supports, mentions, or contrasts the cited claim, and a label indicating in which section the citation was made.
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