title: Role of lysine versus arginine in enzyme cold-adaptation: modifying lysine to homo-arginine stabilizes the cold-adapted alpha-amylase from Pseudoalteramonas haloplanktis.
abstract: 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 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 alpha-amylase. The k(cat) of the modified enzyme decreased with a concomitant increase in deltaH# 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.
authors.affiliation: School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, NSW, Australia.
authors.forename: Khawar Sohail
authors.initials: KS
authors.lastname: Siddiqui
authors.forename: Anne
authors.initials: A
authors.lastname: Poljak
authors.forename: Michael
authors.initials: M
authors.lastname: Guilhaus
authors.forename: Davide
authors.initials: D
authors.lastname: De Francisci
authors.forename: Paul M G
authors.initials: PM
authors.lastname: Curmi
authors.forename: Georges
authors.initials: G
authors.lastname: Feller
authors.forename: Salvino
authors.initials: S
authors.lastname: D'Amico
authors.forename: Charles
authors.initials: C
authors.lastname: Gerday
authors.forename: Vladimir N
authors.initials: VN
authors.lastname: Uversky
authors.forename: Ricardo
authors.initials: R
authors.lastname: Cavicchioli
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D003080:Cold Temperature; D006709:Homoarginine / Q000737:chemistry*; D006801:Humans; D008239:Lysine / Q000737:chemistry*; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D011487:Protein Conformation; D031161:Pseudoalteromonas / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013552:Swine; D000516:alpha-Amylases / Q000737:chemistry*
chemical_list: D006709:Homoarginine; D000516:alpha-Amylases; D008239:Lysine