Design of an allosterically regulated retroaldolase

Elizabeth A. Raymond, Korrie L. Mack, Jennifer H. Yoon, Olesia V. Moroz, Yurii S. Moroz, Ivan Korendovych

Research output: Contribution to journalArticle

15 Scopus citations

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 Ca2+ 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.

Original languageEnglish (US)
Pages (from-to)561-570
Number of pages10
JournalProtein Science
Volume24
Issue number4
DOIs
StatePublished - Apr 1 2015

Keywords

  • aldolase
  • calmodulin
  • enzyme catalysis
  • metalloproteins
  • protein design

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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    Raymond, E. A., Mack, K. L., Yoon, J. H., Moroz, O. V., Moroz, Y. S., & Korendovych, I. (2015). Design of an allosterically regulated retroaldolase. Protein Science, 24(4), 561-570. https://doi.org/10.1002/pro.2622