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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.

SUBMITTER: Raymond EA

PROVIDER: S-EPMC4380986 | biostudies-literature | 2015 Apr

REPOSITORIES: biostudies-literature

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Design of an allosterically regulated retroaldolase.

Raymond Elizabeth A EA Mack Korrie L KL Yoon Jennifer H JH Moroz Olesia V OV Moroz Yurii S YS Korendovych Ivan V IV

Protein science : a publication of the Protein Society 20150113 4

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 ...[more]

PMID: 25516403

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