Purification, crystallization and structural elucidation of d-galactaro-1,4-lactone cycloisomerase from Agrobacterium tumefaciens involved in pectin degradation

Matthew W. Vetting, Jason T. Bouvier, John A. Gerlt, Steven C. Almo

Research output: Contribution to journalArticle

Abstract

Pectin is found in the cell wall of plants and is often discarded as waste. A number of research groups are interested in redirecting this biomass waste stream for the production of fuel and bulk chemicals. The primary monomeric subunit of this polysaccharide is d-galacturonate, a six-carbon acid sugar that is degraded in a five-step pathway to central metabolic intermediates by some bacteria, including Agrobacterium tumefaciens. In the third step of the pathway, d-galactaro-1,4-lactone is converted to 2-keto-3-deoxy-l-threo-hexarate by a member of the mandelate racemase subgroup of the enolase superfamily with a novel activity for the superfamily. The 1.6Å resolution structure of this enzyme was determined, revealing an overall modified (β/α)7β TIM-barrel domain, a hallmark of the superfamily. d-Galactaro-1,4-lactone was manually docked into the active site located at the interface between the N-terminal lid domain and the C-terminal barrel domain. On the basis of the position of the lactone in the active site, Lys166 is predicted to be the active-site base responsible for abstraction of the α proton. His296 on the opposite side of the active site is predicted to be the general acid that donates a proton to the β carbon as the lactone ring opens. The lactone ring appears to be oriented within the active site by stacking interactions with Trp298.

Original languageEnglish (US)
Pages (from-to)36-41
Number of pages6
JournalActa Crystallographica Section:F Structural Biology Communications
Volume72
DOIs
StatePublished - Jan 1 2016

    Fingerprint

Keywords

  • Agrobacterium tumefaciens
  • d-galactaro-1,4-lactone cycloisomerase
  • d-galacturonate
  • pectin

ASJC Scopus subject areas

  • Biophysics
  • Structural Biology
  • Biochemistry
  • Genetics
  • Condensed Matter Physics

Cite this