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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
Author(s) -
Siddiqui Khawar Sohail,
Poljak Anne,
Guilhaus Michael,
De Francisci Davide,
Curmi Paul M. G.,
Feller Georges,
D'Amico Salvino,
Gerday Charles,
Uversky Vladimir N.,
Cavicchioli Ricardo
Publication year - 2006
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/prot.20989
Subject(s) - lysine , arginine , salt bridge , chemistry , enzyme , biochemistry , cooperativity , active site , protein structure , amino acid , amylase , crystallography , stereochemistry , mutant , gene
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 k cat of the modified enzyme decreased with a concomitant increase in ΔH # 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 Ca 2+ 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. Proteins 2006. © 2006 Wiley‐Liss, Inc.