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Exploring allosteric activation of LigAB from Sphingobium sp. strain SYK-6 through kinetics, mutagenesis and computational studies
Archives of Biochemistry and Biophysics (2015)
  • Kevin P Barry, Wesleyan University
  • Jason P Gerbino, Wesleyan University
  • Abraham Ngu, Wesleyan University
  • Erin F Cohn, Wesleyan University
  • Joy M Cote, Wesleyan University
  • A. Maxwell Burroughs
  • Erika A Taylor, Wesleyan University
Abstract

The protocatechuate 4,5-dioxygenase (LigAB) from Sphingobium sp. strain SYK-6 is the defining member of the Type II extradiol dioxygenase superfamily (a.k.a. PCA Dioxygenase Superfamily or PCADSF) and plays a key aromatic ring-opening role in the metabolism of several lignin derived aromatic compounds. In our search for alternate substrates and inhibitors of LigAB, we discovered allosteric rate enhancement in the presence of non-substrate protocatechuate-like aldehydes such as vanillin. LigAB has the broadest substrate utilization profile of all protocatechuate (PCA) 4,5-dioxygenase described in the literature, however, the rate enhancement is only observed with PCA, with vanillin increasing kcat for LigAB by 36%. Computational docking has identified a potential site of allosteric binding near the entrance to the active site. Examination of a multiple sequence alignment reveals that many of the residues contributing to this newly identified allosteric pocket are highly conserved within the LigB family of the PCADSF. Point mutants of Phe103α and Ala18β, two residues located in the putative allosteric pocket, display altered rate enhancement as compared to LigAB-WT, providing support for the computationally identified allosteric binding site. Further investigation of this binding site may provide insight into the mechanism of this never before observed allosteric activation in extradiol dioxygenases.

Keywords
  • Allosteric activation,
  • Dioxygenase,
  • Lignin,
  • PCAD superfamily,
  • Protocatechuate 4,
  • 5-dioxygenase,
  • Vanillin
Publication Date
February 1, 2015
Citation Information
Kevin P Barry, Jason P Gerbino, Abraham Ngu, Erin F Cohn, et al.. "Exploring allosteric activation of LigAB from Sphingobium sp. strain SYK-6 through kinetics, mutagenesis and computational studies" Archives of Biochemistry and Biophysics Vol. 567 (2015)
Available at: http://works.bepress.com/eataylor/3/