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

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.