title: Design of an allosterically regulated retroaldolase.
abstract: We employed a minimalist approach for design of an allosterically controlled retroaldolase. Introduction of a single lysine residue into the nonenzymatic protein calmodulin led to a 15,000-fold increase in the second order rate constant for retroaldol reaction with methodol as a substrate. The resulting catalyst AlleyCatR is active enough for subsequent directed evolution in crude cell bacterial lysates. AlleyCatR's activity is allosterically regulated by Ca(2+) ions. No catalysis is observed in the absence of the metal ion. The increase in catalytic activity originates from the hydrophobic interaction of the substrate (∼2000-fold) and the change in the apparent pKa of the active lysine residue.
authors.affiliation: Department of Chemistry, Syracuse University, Syracuse, New York, 13244.
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Raymond
authors.forename: Korrie L
authors.initials: KL
authors.lastname: Mack
authors.forename: Jennifer H
authors.initials: JH
authors.lastname: Yoon
authors.forename: Olesia V
authors.initials: OV
authors.lastname: Moroz
authors.forename: Yurii S
authors.initials: YS
authors.lastname: Moroz
authors.forename: Ivan V
authors.initials: IV
authors.lastname: Korendovych
mesh_terms: D000495:Allosteric Site; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism; D004795:Enzyme Stability / Q000235:genetics; D005634:Fructose-Bisphosphate Aldolase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008239:Lysine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D015202:Protein Engineering / Q000379:methods*
chemical_list: D002147:Calmodulin; D005634:Fructose-Bisphosphate Aldolase; D008239:Lysine; D002118:Calcium
keywords: aldolase; calmodulin; enzyme catalysis; metalloproteins; protein design